Vasopressin-induced calcium increases in smooth muscle cells from spontaneously hypertensive rats

Cytosolic free Ca2+ concentrations [( Ca2+]i) were measured in smooth muscle cells (SMC) from spontaneously hypertensive rats (SHR) and age and sex matched Wistar-Kyoto rats (WKY). Resting levels of [Ca2+]i were 114 +/- 6 nM and 116 +/- 5 nM in SMC from WKY and SHR, respectively. Angiotensin II (AII) induced a dose-dependent large increases in [Ca2+]i in SMC. There were no significant differences in resting or AII-stimulated levels of [Ca2+]i when SMC from WKY and SHR were compared. Arg-vasopressin (AVP) caused a similar but smaller [Ca2+]i increase than AII in SMC. AVP caused larger [Ca2+]i increases in SMC from SHR than in SMC from WKY. Although concentrations of AVP higher than those ordinarily detected in plasma were necessary to obtain different responses between SHR and WKY, these differences may be related to the pathogenesis of hypertension.     

Decreased Ca2+ extrusion via Na+/Ca2+ exchange in epicardial left ventricular myocytes during compensated hypertrophy

Hypertension-induced cardiac hypertrophy alters the amplitude and time course of the systolic Ca2+ transient of subepicardial and subendocardial ventricular myocytes. The present study was designed to elucidate the mechanisms underlying these changes. Myocytes were isolated from the left ventricular subepicardium and subendocardium of 20-wk-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats (WKY; control). We monitored intracellular Ca2+ using fluo 3 or fura 2; caffeine (20 mmol/l) was used to release Ca2+ from the sarcoplasmic reticulum (SR), and Ni2+ (10 mM) was used to inhibit Na+/Ca2+ exchange (NCX) function. SHR myocytes were significantly larger than those from WKY hearts, consistent with cellular hypertrophy. Subepicardial myocytes from SHR hearts showed larger Ca2+ transient amplitude and SR Ca2+ content and less Ca2+ extrusion via NCX compared with subepicardial WKY myocytes. These parameters did not change in subendocardial myocytes. The time course of decline of the Ca2+ transient was the same in all groups of cells, but its time to peak was shorter in subepicardial cells than in subendocardial cells in WKY and SHR and was slightly prolonged in subendocardial SHR cells compared with WKY subendocardial myocytes. It is concluded that the major change in Ca2+ cycling during compensated hypertrophy in SHR is a decrease in NCX activity in subepicardial cells; this increases SR Ca2+ content and hence Ca2+ transient amplitude, thus helping to maintain the strength of contraction in the face of an increased afterload.     

Transport of univalent cations across the erythrocyte membrane of hypertensive rats of various ages

In the erythrocytes incubated at low temperature (3-6 degrees C), the uptake of Li+ in 6- and 16-week old spontaneously hypertensive rats (SHR) was significantly higher than in the normotensive rats (WKY) of the same age. During the incubation of cells at 37 degrees C no difference occurred in either ouabain-sensitive or ouabain-resistant fluxes of Rb+, Na+ and Li+ between the 16-week old SHR and the WKY. K+ efflux from the erythrocytes at 3 degrees C was consistently stimulated after addition to the incubation medium of 1 mmol/l Ca2+. The value of Ca2+-dependent K+-transport was significantly elevated in 16-week old SHR than in the WKY, but there was no difference in 6-week old rats. Propranolol-induced Ca2+-dependent K+ efflux from the cells at 22 degrees C was markedly higher in 6- and 16-week old SHR as compared with the WKY. The results provide a further evidence in favor of the hypothesis on the existence of a "membrane defect" in red blood cells in the SHR.     

Changes in mitochondrial functionality and calcium uptake in hypertensive rats as a function of age

We studied whether mitochondrial functions and Ca2+ metabolism were altered in Wistar Kyoto normotensive (WKY) and spontaneous hypertensive rats (SHR). Ca2+ uptake was decreased in SHR compared to WKY rats. Accumulation of Ca2+ was more efficient in WKY than in SHR rats. mDeltaPsi was lower in SHR compared to WKY rats. Basal complex IV activity was higher in SHR than WKY rats, whereas basal L-citrulline production, an indicator of nitric oxide synthesis, was decreased in SHR and dependent on Ca2+ concentration (p<0.05). Impact of Ca2+ was counteracted by EGTA. These data show an age-dependent decreased mitochondrial functions in brain mitochondria during hypertension.     

Properties of ventricular myocytes isolated from the hypertrophied and failing hearts of spontaneously hypertensive rats.

OBJECTIVE: To investigate how the morphological and physiological properties of single myocytes isolated from the hypertrophied, failing left ventricles (LV) differ from those of normal or hypertrophied not failing ventricles. METHOD: Single myocytes were isolated separately from right (RV) and left ventricles (LV) of male spontaneously hypertensive rats (SHR) or Wistar-Kyoto (WKY) rats at the age of 6 and 12 months and of SHRs which developed or not developed heart failure at the age of 20-24 months. We measured cells dimensions, range and kinetics of electrically stimulated or initiated by caffeine contractions and Ca2+ transients, and investigated the response of cells to thapsigargin. RESULTS: The transversal dimensions of the LV myocytes of 6 months old SHRs showed approximately 20% increase with respect to transversal dimensions of their RV myocytes and LV and RV myocytes of WKY rats. The difference did not change with progressing age and in the heart failure. The LV myocytes of 6 or 12 months old SHRs showed slowed kinetics of the Ca2+ transients and of contraction and relaxation and decreased contractile response to 2 s superfusion with 15 mM caffeine preceded by 5 mM Ni2+ used as an index of the sarcoplasmic reticulum (SR) Ca2+ content. Despite of this the range of shortening and relative contribution of the SR to contraction (assessed by measuring of the residual contractile response to electrical stimulation in cells poisoned with thapsigargin) or relaxation (assessed by calculation of the ratio of rate constants of the electrically stimulated and stimulated by 30 s superfusion with caffeine Ca2+ transients) was not altered in the hypertrophied myocytes. Properties of the LV myocytes of the 20-24 old SHRs with or without heart failure did not differ from those of LV myocytes of younger SHRs. The contractile response to caffeine of their RV myocytes dropped to the level of that in the LV myocytes. CONCLUSION: Our results suggest that transition from the compensated hypertrophy to the heart failure in 20-24 months old SHRs did not result from the further changes in properties of the surviving myocytes. Data from literature suggest that myocyte apoptosis and remodeling of the extramyocyte space is the more likely reason.     

Effect of 1,25(OH)2 vitamin D3 and ionized Ca2+ on 45Ca uptake by primary cultures of aortic myocytes of spontaneously hypertensive and Wistar Kyoto normotensive rats

The effect of several regulators of whole animal Ca2+ homeostasis on 45Ca uptake by primary cultures of aortic myocytes isolated from spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats was examined. Exposure of confluent cells to 1.0, 1.25 or 1.50 mM ionized Ca2+ in serum-free medium for seven days resulted in increased 45Ca uptake at the higher concentrations of Ca2+ in cells of the SHR but not the WKY. 1,25 (OH)2 vitamin D3 (1 ng/ml) for 7 days caused enhanced influx in cells from both the SHR and WKY while parathyroid hormone (1-34) (1 ng/ml) was without effect. The data indicate that humoral factors that serve to regulate whole animal Ca2+ homeostasis may also play a role in the regulation of Ca2+ metabolism of the vascular smooth muscle cell.     

大鼠高血压相关基因表达蛋白抑制血管平滑肌细胞增殖

大鼠高血压相关基因 ( r HRG- 1 )编码一新细胞内信号传递蛋白 .体外转染 r HRG- 1表达蛋白发现 r HRG- 1表达蛋白能抑制自发性高血压大鼠血管平滑肌细胞内 Raf蛋白 ( Raf- 1 )和丝裂素活化蛋白激酶 ( MAPK)活性 ,抑制抗细胞凋亡基因 ( bcl- 2 )和增殖细胞核抗原 ( PCNA)基因 m RNA表达 ,同时还抑制该细胞 DNA的合成 .r HRG- 1是一正常血压大鼠血管平滑肌细胞内高度表达的基因 ,由此推测在自发性高血压大鼠血管平滑肌细胞内转染 r HRG- 1表达蛋白抑制其细胞 DNA合成的作用可能是抑制细胞内 Raf- 1活性与 MAPK活性及抑制 PCNA和 bcl- 2基因表达的结果     

Effect of nifedipine on calcium-induced contractions to potassium in the aorta and mesenteric arteries of spontaneously hypertensive and normotensive rats

Graded contractions to cumulative additions of calcium in the presence of KCl were obtained in strips of aorta and mesenteric arteries of normotensive (WKY) and spontaneously hypertensive (SHR) rats. In calcium-free medium, a maximally effective concentration of KCl produced a response that was larger in the mesenteric arteries (43-51% of control) than in the aorta (12-14% of control). The calcium channel blocker nifedipine (NFD, up to 10(-7) M) did not significantly alter these calcium-insensitive responses. The Ca2+-induced responses were inhibited by NFD, in a concentration-dependent fashion, in both vessel types of WKY and SHR rats. The aortic responses were more sensitive to inhibition by NFD than the responses of mesenteric arteries. Moreover, the aortic responses of WKY were inhibited to a greater extent than those of the SHR. The results suggest: (a) a differential calcium dependence of contractions to KCl in the vessels studied; (b) that aortic responses are dependent on NFD-sensitive voltage-sensitive Ca2+ channels to a greater extent than the responses of mesenteric arteries; and (c) that hypertension results in a decreased sensitivity of the aorta Ca2+ channels to NFD.     

Atypical Ca2+ currents in chromaffin cells from SHR and WKY rat strains result from the deficient expression of a splice variant of the α1D Ca2+ channel

Ca(2+) currents (I(Ca)) recorded from adrenal chromaffin cells (CCs) of spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats are similar to one another, but different from those recorded in other rodent species. I(Ca) in WKY/SHR CCs comprises an early, transient (I(Ca(e))) and a late, sustained component (I(Ca(s))). In Wistar CCs, I(Ca(e)) is absent, and I(Ca(s)) is of greater amplitude. Activation and steady-state inactivation of I(Ca(e)) and I(Ca(s)) in WKY/SHR CCs suggest the recruitment of at least two populations of Ca(2+) channels with different voltage dependence and kinetics. In WKY/SHR CCs, I(Ca(e)) is inhibited by nifedipine, enhanced by BAY K 8644, is not blocked by the mibefradil analog NNC 55-0396, and displays Ca(2+)-dependent inactivation and fast deactivation kinetics, suggesting that it results from the opening of L-type rather than T-type Ca(2+) channels. I(Ca(e)) properties suggest that it originates from the opening of Ca(2+) channels formed with the short splice variant (Ca(V)1.3(42A)). RT-PCR showed that expression of Ca(V)1.3(42A) mRNA is similar in both Wistar and WKY/SHR, but that the long variant (Ca(V)1.3(42)) is virtually absent in WKY/SHR. Thus I(Ca(e)) corresponds to the recruitment of Ca(V)1.3(42A) channels, unmasked by the absence of Ca(V)1.3(42) channels. Studies in WKY CCs do not report major functional alterations, despite the unusual expression pattern of Ca(V)1.3 splice variants. It remains to be established if more subtle functional alterations exist, and if the atypical splicing pattern of Ca(V)1.3 could be related to the functional and behavioral alterations reported in WKY/SHR rats, including their susceptibility to develop hypertension.     

1,25 (OH)2 vitamin D3-induced 45CA uptake in vascular myocytes cultured from spontaneously hypertensive and normotensive rats

The effect of 1,25 (OH)2 vitamin D3 on basal 45Ca uptake was examined in vascular smooth muscle cells cultured from mesenteric arteries of spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) normotensive rats. Basal uptake of 45Ca was significantly greater in myocytes of WKY than SHR at 5, 10, 30 and 60 min incubation with the isotope. Incubation with 1 ng/ml 1,25 (OH)2 vitamin D3 for 48 hr increased basal 45Ca uptake between 1-10 min in SHR and between 5-10 min in WKY. The dose-response relationship indicated that cells from both strains are equally sensitive to the calciotropic effects of 1,25 (OH)2 vitamin D3 with half-maximal stimulation occurring at approximately 0.3-0.4 ng/ml. In cells of both strains maximal stimulation of 45Ca uptake was achieved only after a 12-24 hr period of incubation with hormone and pretreatment with cycloheximide inhibited 1,25 (OH)2 vitamin D3-enhanced 45Ca uptake. Although 45Ca binding by extracellular matrix material was significantly greater in WKY than SHR, 1,25 (OH)2 vitamin D3 had no effect on the amount of matrix 45Ca binding in either strain. These results suggest that 1,25 (OH)2 vitamin D3 induces an increase in intracellular protein synthesis that results in enhanced 45Ca uptake. The similar responses of the two strains indicate that hypertensive smooth muscle is not more sensitive to 1,25 (OH)2 vitamin D3 and the Ca2+ response is a general property of vascular muscle.     

成年SHR动脉平滑肌细胞端粒酶活性和周期蛋白D1的研究

为探索自发性高血压大鼠动脉平滑肌细胞(SMC)增生的机理,采用3H-TdR标记、端粒酶活性以及细胞周期蛋白D1基因RT-PCR检测分别对10周龄SHR、WKY大动脉及其体外分离的SMC进行研究。成年、高血压状态的SHR胸、腹主动脉段端粒酶有高的活性,而同龄、同源WKY大鼠者则没有。从成年SHR腹主动脉段分离的SMC3H-TdR的掺入率比WKY者约提高了43％。成年高血压状态下的SHR腹主动脉SMC细胞周期蛋白D1基因的RT-PCR的产物与WKY者相差不明显。     

Intestinal calcium transport in spontaneously hypertensive rats (SHR) and their genetically matched WKY rats

Calcium transport across the basolateral membranes of the enterocyte represents the active step in calcium translocation. This step occurs by two mechanisms, an ATP-dependent pump and a Ca2+/Na+ exchange process. These studies were designed to investigate these two processes in jejunal basolateral membrane vesicles (BLMV) of the spontaneously hypertensive rats (SHR) and their genetically matched controls, Wistar-Kyoto (WKY) rats. The ATP-dependent calcium uptake was stimulated several-fold compared with no ATP condition in both SHR and WKY, but no differences were noted between rate of calcium uptake in SHR and WKY. Kinetics of ATP-dependent calcium uptake at concentrations between 0.01 and 1.0 microM revealed a Vmax of 0.67 +/- 0.03 nmol/mg protein/20 sec and a Km of 0.2 +/- 0.03 microM in SHR and Vmax of 0.69 +/- 0.12 and a Km of 0.32 +/- 0.14 microM in WKY rats. Ca2+/Na+ exchange in jejunal BLMV of SHR and WKY was investigated in two ways. First, sodium was added to the incubation medium (cis-Na+). Second, Ca2+ efflux from BLMV was studied in the presence of extravesicular Na+ (trans-Na+). Both studies suggest a decreased exchange of calcium and Na+. Kinetic parameters of Na(+)-dependent Ca2+ uptake at concentrations between 0.01 and 1.0 microM exhibited Vmax of 0.05 +/- 0.01 nanmol/mg protein/5 sec and a Km of 0.21 +/- 0.13 microM in SHR and Vmax of 0.11 +/- 0.02 nanmol/mg protein/5 sec and a Km of 0.09 +/- 0.05 in WKY, respectively. These results confirm that the intestinal BLMV of SHR and WKY rats have two mechanisms for calcium extrusion, an ATP-dependent Ca2+ transport process and a Na+/Ca2+ exchange process. The ATP-dependent process appears to be functional in SHR; however, the Ca2+/Na+ exchange mechanism appears to have a marked decrease in its maximal capacity. These findings suggest that calcium extrusion via Ca2+/Na+ is impaired in the SHR, which may lead to an increase in intracellular calcium concentration. These findings may have relevance to the development of hypertension.     

Strain and site dependence of polyploidization of cultured rat smooth muscle

Smooth muscle cell (SMC) growth may play an important role in the pathogenesis of vascular diseases such as atherosclerosis and hypertension. Recent studies have demonstrated that, under different growth stimuli in vivo, SMC may respond by proliferation of diploid cells, polyploidization to the tetraploid (or even octaploid) state, or both. In this study, we used flow cytometry to evaluate the intrinsic tendencies of aortic SMC and nonarterial cells from rats of different strains, ages, and blood pressures to polyploidize in response to in vitro growth stimulation. Significant strain-related differences in polyploidization of aortic SMC were found (P less than 0.001): highest in WKY (normotensive inbred rat related to SHR), intermediate in SHR (genetically hypertensive rat), and lowest in Sprague-Dawley and Fischer (normotensive outbred and inbred rats). Animal age had less or no effect on the degree of polyploidization. Nonarterial cells (venous SMC and lung cells) from WKY and SHR remained essentially diploid, suggesting tissue specificity of in vitro polyploidization. Studies of the growth kinetics of uncloned and clonal populations of aortic SMC revealed decreased proliferation as the ploidy increased in WKY, SHR, and Sprague-Dawley. These findings suggest that genetic strain factors as well as cell type/site of origin significantly influence in vitro polyploidization, whereas animal age and blood pressure do not. The findings also emphasize the need to consider ploidy changes when evaluating in vitro SMC growth kinetics. Further studies will improve understanding of SMC growth regulation and the functional significance of vascular polyploidy.     

Incidence of multinucleated and polyploid aortic smooth muscle cells cultured from different age groups of spontaneously hypertensive rats.

Cell size and incidence of multinucleated, polyploid cells in cultured aortic smooth muscle cells from different age groups of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) were compared. Smooth muscle cells from SHR were generally larger than those from WKY, and the percentage of multinucleated smooth muscle cells was always higher in SHR than WKY in the three age groups of rats studied (3-4, 10-12, and 28-30 weeks). In smooth muscle cells from the 3- to 4-week group, there was a positive correlation between cell diameter and the percentage of multinucleated smooth muscle cells. Microdensitometric measurements also showed that the incidence of polyploid smooth muscle cells was always higher in SHR than WKY in the three age groups. There was a positive correlation between DNA density and nuclear area measurements in all the age groups of SHR and WKY. We conclude that cultured aortic smooth muscle cells from different age groups of SHR and WKY contained heterogeneous populations of cells and that, under our culture conditions, the polyploidy of the smooth muscle cells found in vivo was maintained in the SHR and WKY.     

Brain synaptosomal Ca2+ uptake: comparison of Sprague-Dawley, Wistar-Kyoto and spontaneously hypertensive rats

1. K(+)-stimulated 45Ca2+ uptake by synaptosomes was measured with respect to the strain differences between Sprague-Dawley (SD), Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). 2. 45Ca2+ uptake by synaptosomes isolated from cerebral cortex of SD, WKY and SHR was measured at 15, 30, 60, 120 and 240 sec time periods. 3. The sequence of both the magnitude and rate of resting and depolarization-dependent 45Ca2+ uptake was SHR greater than WKY greater than SD. 4. The fastest rates of resting and depolarization-dependent 45Ca2+ uptake occurred in each rat during the first 15 sec and uptake rates dropped off quickly in both resting and depolarization states. 5. At 15 sec, there were significant differences between SHR and WKY, while there were no significant differences between WKY and SD. 6. The results suggest that an important alteration in Ca2+ channel characteristics may occur in SHR brain synaptosomes.     

Changes of beta-adrenoceptors in the aortic muscle cells of spontaneously hypertensive rats

The characteristics of [125I]monoiodocyanopindolol (ICYP) binding to beta-adrenoceptors of cultured aortic smooth muscle cells derived from 4-week-old spontaneously hypertensive rats (SHR) and the Wistar-Kyoto normotensive rats (WKY) were examined. During optimization of the binding assays, we found that the specific binding of ICYP by intact cells was masked by a high level of nonspecific ICYP accumulation in intact cells presumably owing to the lipophilic nature of ICYP. Optimal specific ICYP binding requires that the cells be gently lysed with hypotonic dilution followed by a freeze-and-thaw cycle. Under most experimental conditions tested, the total number of ICYP binding sites in WKY aortic muscle cells was considerably and consistently smaller than that in SHR cells. There was no difference in the Kd values for ICYP binding to SHR and WKY cells. However, when ICYP binding was carried out using crude membrane fractions with well-defined plasma membrane content isolated from aortic muscle strips of adult rats, we found no difference in the number of beta-adrenoceptor sites between SHR and WKY. Morphological evidence indicated that cultured SHR aortic muscle cells contained a greater proportion of larger cells with multinuclear features. These results suggest that an increase in the number of beta-adrenoceptor density per cell in SHR may be associated with cellular hypertrophy of aortic smooth muscle cells. We conclude that under cultured conditions, a higher incidence of polyploid smooth muscle cells in the SHR as compared with WKY was expressed earlier than under in vivo conditions. Therefore, the interpretation of results obtained from cultured cell studies in relation to under in vivo conditions should be exercised with caution.     

Effect of swimming training on cardiac function and myosin ATPase activity in SHR

Male spontaneously hypertensive rats (SHR) and Wistar-Kyoto normotensive rats (WKY) were subjected to swimming training 6 times/wk, commencing at 4 wk of age, to determine whether this type of endurance exercise might alter contractile proteins and cardiac function in young adult SHR. The total duration of exercise was 190 h. Myofibrillar adenosinetriphosphatase (ATPase) activity was assayed at various free [Ca2+] ranging from 10(-7) to 10(-5) M. Ca2+-stimulated ATPase activity of actomyosin and purified myosin was determined at various Ca2+ concentrations both in the low and high ionic strength buffers. Actin-activated myosin ATPase activity of purified myosin was assayed at several concentrations of actin purified from rabbit skeletal muscle. Under all these conditions the contractile protein ATPase activity was comparable between trained and untrained WKY and SHR. Analysis of myosin isoenzymes on pyrophosphate gels showed a single band corresponding to V1 isoenzyme, and there were no differences between swimming-trained and nontrained WKY and SHR. Ventricular performance was assessed by measuring cardiac output and stroke volume after rapid intravenous volume overloading. Both cardiac index and stroke index were comparable in nontrained WKY and SHR but were significantly increased in the trained groups compared with their respective nontrained controls. These results suggest that myosin ATPase activity and distribution of myosin isoenzymes are not altered in the moderately hypertrophied left ventricle whether the hypertrophy is due to genetic hypertension (SHR) or to exercise training (trained WKY). Moreover, the data indicate that SHR, despite the persistence of a pressure overload, undergo similar increases in left ventricular mass and peak cardiac index after training, as do normotensive WKY.     

Intracellular free [Ca2+] in circulating lymphocytes of spontaneously hypertensive rats

In the light of previous reports suggesting a common abnormality of Ca handling in most tissues of hypertensive humans and rats, we applied a novel technique using the fluorescent probe Quin 2 for measurement of cytosolic free Ca2+ in lymphocytes of spontaneously hypertensive rats (SHR). (Ca2+)i is increased in SHR (122.1 +/- 7.4 nM) versus normotensive Wistar-Kyoto (WKY) control rats (81.1 +/- 6.3 nM) Membrane exchange, as challenged by varying the extracellular Ca concentration over a 10(5)-fold range proved to be relatively unimportant in regulating (Ca2+)i and did not significantly affect the difference between SHR and WKY. Catecholamines and ouabain had no appreciable effect on (Ca2+)i. The mechanisms of increased (Ca2+)i in SHR lymphocytes remain to be fully elucidated.