Differential regulation of the lysosomal, Ca2+-dependent and ubiquitin/proteasome-dependent proteolytic pathways in fast-twitch and slow-twitch rat muscle following hyperinsulinaemia.

In order to characterize the poorly defined mechanisms that account for the anti-proteolytic effects of insulin in skeletal muscle, we investigated in rats the effects of a 3 h systemic euglycaemic hyperinsulinaemic clamp on lysosomal, Ca(2+)-dependent proteolysis, and on ubiquitin/proteasome-dependent proteolysis. Proteolysis was measured in incubated fast-twitch mixed-fibre extensor digitorum longus (EDL) and slow-twitch red-fibre soleus muscles harvested at the end of insulin infusion. Insulin inhibited proteolysis (P<0.05) in both muscles. This anti-proteolytic effect disappeared in the presence of inhibitors of the lysosomal/Ca(2+)-dependent proteolytic pathways in the soleus, but not in the EDL, where only the proteasome inhibitor MG 132 (benzyloxycarbonyl-leucyl-leucyl-leucinal) was effective. Furthermore, insulin depressed ubiquitin mRNA levels in the mixed-fibre tibialis anterior, but not in the red-fibre diaphragm muscle, suggesting that insulin inhibits ubiquitin/proteasome-dependent proteolysis in mixed-fibre muscles only. However, depressed ubiquitin mRNA levels in such muscles were not associated with significant decreases in the amount of ubiquitin conjugates, or in mRNA levels or protein content for the 14 kDa ubiquitin-conjugating enzyme E2 and 20 S proteasome subunits. Thus alternative, as yet unidentified, mechanisms are likely to contribute to inhibit the ubiquitin/proteasome system in mixed-fibre muscles.     

Molecular mechanisms for large conductance Ca2+-activated K+ channel activation by a novel opener, 12,14-dichlorodehydroabietic acid

Our recent study has revealed that 12,14-dichlorodehydroabietic acid (diCl-DHAA), which is synthetically derived from a natural product, abietic acid, is a potent opener of large conductance Ca(2+)-activated K(+) (BK) channel. Here, we examined, by using a channel expression system in human embryonic kidney 293 cells, the mechanisms underlying the BK channel opening action of diCl-DHAA and which subunit of the BK channel (alpha or beta1) is the site of action for diCl-DHAA. BK channel activity was significantly enhanced by diCl-DHAA at concentrations of 0.1 microM and higher in a concentration-dependent manner. diCl-DHAA enhanced the activity of BKalpha by increasing sensitivity to both Ca(2+) and membrane potential without changing the single channel conductance. It is notable that the increase in BK channel open probability by diCl-DHAA showed significant inverse voltage dependence, i.e., larger potentiation at lower potentials. Since coexpression of beta1 subunit with BKalpha did not affect the potency of diCl-DHAA, the site of action for diCl-DHAA is suggested to be BKalpha subunit. Moreover, kinetic analysis of single channel currents indicates that diCl-DHAA opens BKalpha mainly by decreasing the time staying in a long closed state. Although reconstituted voltage-dependent Ca(2+) channel current was significantly reduced by 1 microM diCl-DHAA, BK channels were selectively activated at lower concentrations. These results indicate that diCl-DHAA is one of the most potent BK channel openers acting on BKalpha and a useful prototype compound to develop a novel BK channel opener.     

Endothelin action in rat liver. Receptors, free Ca2+ oscillations, and activation of glycogenolysis.

High affinity binding sites for endothelin (ET) were identified on rat liver plasma membranes. Binding of 125I-ET-1 with its site was specific, saturable, and time dependent (kobs = 0.019 +/- 0.001 min-1), but dissociation of receptor-bound ligand was minimal. A single class of high affinity binding sites for 125I-ET-1 was identified with an apparent Kd of 32.4 +/- 9.8 pM and a Bmax of 1084 +/- 118 fmol/mg protein. ET-3 and big-ET-1 (1-38) (human) inhibited 125I-ET-1 binding with IC50 values of 1.85 +/- 1.03 nM and 43 +/- 6 nM, respectively. Aequorin measurements of cytosolic free Ca2+ in single, isolated rat hepatocytes showed that ET-1 at subnanomolar concentrations induced a series of repetitive, sustained Ca2+ transients. ET-1 had no effect on cAMP production. Finally, ET-1 caused a rapid and sustained stimulation of glycogenolysis in rat hepatocytes. A 1.8-fold maximal increase in glycogen phosphorylase alpha was observed at 1 pM ET-1, with an EC50 of 0.03 pM. Stimulation of the enzyme was specific for ET-1 since the order of potency of related peptides was similar to that in binding experiments (ET-1 greater than ET-3 greater than big ET-1). These data constitute the first demonstration of the presence of ET-1 binding sites in liver which is associated with a rise in cytosolic free Ca2+ and a potent glycogenolytic effect. We conclude that ET-1 behaves as a typical Ca2+ mobilizing hormone in liver.     

马桑内酯对大鼠海马锥体神经元钙激活钾通道的影响

背景:神经细胞的电活动以细胞膜离子通道的活动为基础.神经元异常放电是癫痫的基本特征,其基础是细胞膜离子通道的激活与离子的跨膜运动,然而马桑内酯致痫机制中是否存在钙激活钾通道的激活还不清楚.目的:以大鼠海马锥体神经元为靶细胞,了解马桑内酯在其致痫机制中对神经元钙激活钾通道的作用.设计:非随机对照实验.单位:四川大学华西医院神经内科和泸州医学院心肌电教研室.材料:实验于2000-05/12在四川省泸州医学院完成.选择出生24h之内Wistar乳鼠100只.方法:Wistar乳鼠在麻醉状态下和无菌条件下分离出海马组织,以培养第7～10天,生长良好、形态典型的锥体神经元进行膜片钳试验.将培养皿随机分成9组:①正常对照组,19皿,加入DMEM培养基,给以不同的钳制电压,以后加入四乙基胺.②10-8,10-7,10-6mol/L钙浓度组;加入含不同浓度钙离子的DMEM培养基,以后加入四乙基胺,每一浓度8皿,共24皿;马桑内酯0,0.25,0.5,1.0,2.0 mL/L致痫组,加入不同浓度含马桑内酯的DMEM培养基,以后加入四乙基胺.每一浓度26皿,共130皿.运用膜片钳制技术贴附式和内面向外式方法记录神经元单通道电活动,并分析通道活动的开放概率、平均开放时间、平均关闭时间、电流幅值等.主要观察指标:①观察并记录正常,不同钳制电压、不同钙离子浓度对锥体神经元钙激活钾通道的激活作用和四乙基胺的影响.②观察并记录致痫剂马桑内酯对锥体神经元细胞膜钙激活钾通道的激活作用及四乙基胺的影响.结果:①在钳制电压为0 mV时,锥体神经元钙激活钾通道有少量的随机开放,具有明显的电压依赖性,通道电导值为(122.79±21.68)pS,可被钾通道阻断剂四乙基胺所阻断.②在内面向外式膜片下,钙激活钾通道表现出钙离子的浓度依赖性.当钙浓度为10-8,10-7,10-6 mol/L时,平均开放概率分别为0.022±0.006,0.040±0.007,0.142±0.049(P＜0.01).③在细胞贴附式膜片下,浴液中游离钙离子浓度10-8 mol/L,膜电位在20 mV时,发现马桑内酯能明显增加钙激活钾通道的开放概率.④与马桑内酯0 mL/L比较,马桑内酯1.0 mL/L能增加钙激活钾通道平均开放时间(1.867±0.210,6.900±0.120,P＜0.01),减少平均关闭时间(78.505±7.192,6.233±0.854,P＜0.01).结论:在马桑内酯诱导的癫痫发病中,钙激活钾通道活化可能起重要的负反馈调节作用.     

Regulation of Ca(2+)-activated K+ currents by ciglitazone in rat pituitary GH3 cells.

BACKGROUND: Ciglitazone, an antidiabetic agent of the thiazolidinedione family, is known to be an activator of the peroxisome-proliferator activator receptor (PPAR)-gamma. The underlying mechanism of ciglitazone actions on ionic currents in neuroendocrine cells remains unclear. METHODS: The effects of ciglitazone on ionic currents were investigated in rat pituitary GH3 cells using the whole-cell and inside-out configurations of the patch-clamp technique. RESULTS: In GH3 cells, ciglitazone at 3-300 mumol/L caused a reversible increase in the amplitude of the Ca(2+)-activated K+ current (IK(Ca)) with a half-maximal concentration of 16 mumol/L. Under the inside-out patch recording mode, ciglitazone applied intracellularly increased the activity of the large-conductance Ca(2+)-activated K+ (BKCa) channels, but did not affect their single-channel conductance. However, troglitazone (30 mumol/L) caused a reduction in the channel activity. The ciglitazone-induced change in the kinetic behavior of BKCa channels is due to an increase in mean open time and a decrease in mean closed time, whereas the troglitazone-induced decrease in the channel activity is related to a decrease in mean open time and an increase in mean closed time. Ciglitazone caused a left shift in the midpoint for voltage-dependent opening. The ciglitazone-stimulated activity of BKCa channels is independent of internal Ca2+. Under the current clamp mode, ciglitazone (30 mumol/L) hyperpolarized the membrane potential. CONCLUSIONS: This study shows that in addition to its activation of PPAR-gamma, ciglitazone can stimulate the activity of BKCa channels expressed in GH3 cells. These effects may affect membrane potentials and contribute to the ciglitazone-induced change in the functional activity of neurons or neuroendocrine cells.     

Urocortin induces endothelium-dependent vasodilatation and hyperpolarization of rat mesenteric arteries by activating Ca2+-activated K+ channels

Urocortin, a member of corticotropin releasing factor (CRF) peptide family, has positive chronotropic and inotropic effects on heart and also shows a vasodilatory effect. However, the mechanism underlying its vasodilatory effect has yet to be elucidated. Endothelium-dependent relaxation of resistance arteries is mainly achieved by activation of K+ channels. Therefore, we investigated possible role of K+ channels and hyperpolarization for the vasodilatory effect of urocortin using the isolated perfused rat mesenteric arteries. Urocortin (0.2 nM) produced a slow-onset decrease in the perfusion pressure of the mesenteric vascular bed, which was elevated by an alpha1-adrenoceptor agonist, phenylephrine (2-4 microM). Urocortin also hyperpolarized the main mesenteric artery. Removal of endothelium with saponin treatment considerably inhibited the relaxation and hyperpolarization induced by urocortin. In contrast, the hyperpolarization was not significantly changed by cyclooxygenase inhibitor, indomethacin (1 microM) and/or nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine (100 microM). Urocortin-induced relaxation was not affected by the combination of a guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 microM), indomethacin and N(omega)-nitro-L-arginine. However, the relaxation and hyperpolarization were abolished by high extracellular potassium concentration (40 mM) or by a large conductance Ca(2+)-activated K+ channel blocker, charybdotoxin (1 nM). Glibenclamide (1 microM), an ATP-dependent K+ channel inhibitor, did not affect the relaxation and hyperpolarization. These results suggest that urocortin causes endothelium-dependent relaxation and hyperpolarization of rat mesenteric arteries, probably through the activation of charybdotoxin sensitive Ca2+-activated K+ channels. These findings also indicate an essential role of the endothelium for the urocortin-elicited vascular relaxation and hyperpolarization.     

Reversibility of gelsolin/actin interaction in macrophages. Evidence of Ca2+-dependent and Ca2+-independent pathways

We have developed an immunoadsorption technique for quantitating EGTA-resistant gelsolin/actin complexes in macrophages extracted with Triton X-100. We report here that the proportion of gelsolin complexed irreversibly to actin is low in freshly harvested macrophages. The amount of the EGTA-resistant complex increases spontaneously during incubation of the cells in suspension at 37 degrees C, or after exposure to the Ca2+ ionophore ionomycin. On the other hand, exposure of suspended cells to the chemotactic oligopeptide, FMLP, or plating of the cells onto tissue culture dishes causes the EGTA-resistant complex to dissociate rapidly. Plating even prevents Ca2+ ionomycin-treated cells with elevated intracellular Ca2+ from inducing this complex. Therefore, our results suggest that macrophages possess a mechanism, not directly involving Ca2+, for dissociating actin/gelsolin EGTA-resistant complexes. This mechanism may be a Ca2+-independent signal for leukocyte activation.     

Intracellular free Ca2+ in the course of the Ca2+ paradox and during poisoning. Ca2+-selective microelectrode measurements in the perfused rat heart

The free intracellular Ca2+ concentration of perfused rat hearts was measured using Ca2+-selective microelectrodes. In Krebs-Ringer bicarbonate buffer + 0.1 mmol/l Ca2+ (controls) the intracellular Ca2+ concentration was 0.87 +/- 0.07 mumol/l and the membrane potential was -51.5 +/- 0.3 mV. Without glucose the membrane potential approached zero after ca. 60 min, whereas the Ca2+ concentration during 110 min increased only slowly to 10.0 mumol/l. During Ca2+-free perfusion (5 min) both parameters did not change significantly. With reintroduction of 2.0 mmol/l Ca2+ the membrane potential rapidly collapsed and the intracellular Ca2+ concentration was elevated above 0.1 mmol/l within two min. Reperfusion with only 0.1 mmol/l Ca2+ decelerated both changes. Poisoning by carbonyl cyanide-p-trifluoromethoxyphenylhydrazone or antimycin A in Ca2+-free Krebs-Ringer bicarbonate buffer increased the intracellular Ca2+ concentration to 30.0 and 25.0 mumol/l, and the membrane potential was collapsed after 16 and 10 min, respectively. In antimycin A- and Ca2+-containing sucrose medium the intracellular Ca2+ during 16 min increased above 1.0 mmol/l, and the membrane potential began to increase only after 10 min. The results are consistent with the postulate of a hypothetical mechanism of cell injury, in which noxious membrane-cytoskeleton interactions are induced by an elevated intracellular Ca2+ concentration. It is concluded that Ca2+ entry via Na/Ca exchange is not fundamentally involved with induction of injury.     

Renal Ca2+ wasting, hyperabsorption, and reduced bone thickness in mice lacking TRPV5

Ca2+ ions play a fundamental role in many cellular processes, and the extracellular concentration of Ca2+ is kept under strict control to allow the proper physiological functions to take place. The kidney, small intestine, and bone determine the Ca2+ flux to the extracellular Ca2+ pool in a concerted fashion. Transient receptor potential (TRP) cation channel subfamily V, members 5 and 6 (TRPV5 and TRPV6) have recently been postulated to be the molecular gatekeepers facilitating Ca2+ influx in these tissues and are members of the TRP family, which mediates diverse biological effects ranging from pain perception to male aggression. Genetic ablation of TRPV5 in the mouse allowed us to investigate the function of this novel Ca2+ channel in maintaining the Ca2+ balance. Here, we demonstrate that mice lacking TRPV5 display diminished active Ca2+ reabsorption despite enhanced vitamin D levels, causing severe hypercalciuria. In vivo micropuncture experiments demonstrated that Ca2+ reabsorption was malfunctioning within the early part of the distal convolution, exactly where TRPV5 is localized. In addition, compensatory hyperabsorption of dietary Ca2+ was measured in TRPV5 knockout mice. Furthermore, the knockout mice exhibited significant disturbances in bone structure, including reduced trabecular and cortical bone thickness. These data demonstrate the key function of TRPV5 in active Ca2+ reabsorption and its essential role in the Ca2+ homeostasis.     

Increased leakage of sarcoplasmic reticulum Ca2+ contributes to abnormal myocyte Ca2+ handling and shortening in sepsis

OBJECTIVE: Changes in cardiac function due to sepsis have been widely reported. However, the underlying mechanisms remain poorly understood. In the mammalian heart, myocyte function and intracellular calcium homeostasis are closely coupled. In this study we tested the hypothesis that alterations in cardiac calcium homeostasis due to sepsis underlie the observed myocyte dysfunction. DESIGN: Randomized prospective animal study. SETTING: Research laboratory. SUBJECTS: Male Sprague-Dawley rats weighing 250-275 g. INTERVENTIONS: We induced sepsis by cecal ligation and puncture in the rat, which mimics the type of infection caused by perforation of the intestine in humans. MEASUREMENTS AND RESULTS: Forty-eight hours after cecal ligation and puncture, isolated cardiac ventricular cardiomyocytes demonstrated a 57% decreased peak systolic [Ca]. The time constant of the Ca transient increased 71% and 57% in myocytes obtained 24 hrs and 48 hrs after cecal ligation and puncture, respectively. The average shortening of cardiomyocytes 48 hrs after cecal ligation and puncture was significantly decreased. To investigate the cellular mechanisms of altered Ca transients and myocyte shortening, we measured Ca sparks, the spontaneous local Ca release events in cardiomyocytes at resting states. The Ca spark frequency progressively increased in myocytes 24 hrs and 48 hrs after cecal ligation and puncture. The total activity of sparks also increased compared with sham-operated animals. The overall leakage of sarcoplasmic reticulum Ca in resting states was increased in sepsis and resulted in reduced sarcoplasmic reticulum Ca content. CONCLUSIONS: Abnormal Ca leakage from the sarcoplasmic reticulum contributes significantly to the depressed myocyte shortening in sepsis. In the future, modalities that prevent this Ca leakage may prove beneficial in the treatment of sepsis-induced myocyte shortening.     

Depletion and repletion of Ca2+ in the perfused rat liver.

Repletion with Ca2+ often leads to damage of previously Ca(2+)-depleted hearts (the calcium paradox). The behavior of the liver under similar conditions is not well understood. With a perfused rat liver model, we examined liver cell damage and lipid peroxidation during Ca2+ depletion and repletion and used lucigenin-enhanced chemiluminescence as a measure of oxygen radicals. During 30 minutes of Ca2+ depletion, release of lactate dehydrogenase and thiobarbituric acid-reactive substance did not change significantly. When Ca2+ depletion was extended to 150 minutes, release of lactic acid dehydrogenase and thiobarbituric acid-reactive substance and tissue oxygen radical levels all increased progressively, accompanied by decrease in oxygen uptake. Ca2+ repletion after 30 minutes of Ca2+ depletion caused small increases in release of lactic acid dehydrogenase and thiobarbituric acid-reactive substance but significantly suppressed the changes described, compared with expression in depleted livers without Ca2+ repletion. There were large releases of sinusoidal glutathione and glutathione disulfide at the onset of Ca2+ depletion, which declined within 15 minutes. On Ca2+ repletion, sinusoidal glutathione level decreased to its baseline but glutathione disulfide level did not change significantly. During long-term Ca2+ depletion, sinusoidal glutathione level was significantly higher than baseline but glutathione disulfide level remained low. These results indicate that long-term Ca2+ depletion causes oxidative stress and liver damage. Ca(2+)-dependent release of sinusoidal glutathione appears to result from causes other than oxidative stress. There is no evidence for the calcium paradox in the liver; in fact, reexposure to Ca2+ protects the liver from the injury caused by Ca2+ depletion.     

Effects of Ca2+ sensitizers on contraction, [Ca2+]i transient and myofilament Ca2+ sensitivity in diabetic rat myocardium: potential usefulness as inotropic agents.

The purpose of the present study was to investigate the effects of Ca2+ sensitizers EMD 57033, MCI-154, and EGIS-9377 in cardiac preparations from streptozotocin-induced diabetic rats. In enzymatically dissociated ventricular myocytes loaded with the Ca2+ probe indo 1, these Ca2+ sensitizers caused an increase in cell shortening without a significant effect on the intracellular Ca2+ ([Ca2+]i) transient. The contractile responses were substantially similar in myocytes from diabetic and age-matched control rats. In contrast, the contractile and [Ca2+]i responses to pimobendan and isoproterenol were significantly less in diabetic myocytes. The Ca2+ sensitivity of tension in beta-escin-skinned trabeculae from diabetic hearts was not significantly different from that of controls. The effect of EMD 57033 on myofilament responsiveness to Ca2+ was identical in control and diabetic preparations. The slower time course of relaxation observed in diabetic papillary muscles was further prolonged in the presence of EMD 57033. However, the extent of the increase in relaxation produced by EMD 57033 did not differ between control and diabetic muscles, and the detrimental effect on resting tension was less pronounced in the two groups. In anesthetized rats, echocardiography showed that intra-duodenal administration of EMD 57033 increased left ventricular systolic function without affecting variables of diastolic filling in both groups. Taken together, the present results suggest that Ca2+ sensitizers, unlike conventional inotropic agents, have the potential to increase in force of contraction to the same extent in nondiabetic and diabetic myocardium, possibly without exaggerating extremely the impairment of diastolic function in diabetes.     

Microvascular perfusion is impaired in a rat model of normotensive sepsis.

We hypothesized that normotensive sepsis affects the ability of the microcirculation to appropriately regulate microregional red blood cell (RBC) flux. An extensor digitorum longus muscle preparation for intravital study was used to compare the distribution of RBC flux and the functional hyperemic response in SHAM rats and rats made septic by cecal ligation and perforation (CLP). Using intravital microscopy, we found that sepsis was associated with a 36% reduction in perfused capillary density (from 35.3 +/- 1.5 to 22.5 +/- 1.0 capillaries/mm of test line) and a 265% increase in stopped-flow capillaries (from 0.9 +/- 0.2 to 3.3 +/- 0.4 capillaries/mm); the spatial distribution of perfused capillaries was also 72% more heterogeneous. Mean intercapillary distance (ICD) increased 30% (from 25.7 +/- 0.8 to 33.5 +/- 1.6 microns), and the proportion of capillary pairs with intercapillary distances > 33.8 microns (the 75th percentile of ICDSHAM) was greater with sepsis. Mean capillary RBC velocity increased 17% in CLP rats (391 vs 333 microns/s). Laser Doppler flowmetry was used to assess the functional hyperemic response of the extensor digitorum longus muscle before and after a period of maximal twitch contraction designed to increase oxygen demand. RBC flux was 36% lower in the CLP rats at rest. After contraction, RBC flux increased in both SHAM and CLP rats; however, the relative increase was less in the CLP group. We concluded that sepsis affects the ability of the skeletal muscle microcirculation to appropriately distribute RBC flux and to respond to increases in oxygen need.     

Selective inhibition of relaxation of guinea-pig trachea by charybdotoxin, a potent Ca(++)-activated K+ channel inhibitor.

Airway smooth muscle plasma membranes are rich in K+ channels of various types. Charybdotoxin (ChTX) is a potent blocker of the high-conductance Ca(++)-activated K+ channel in smooth muscle and produces a concentration-dependent contraction of guinea pig trachea. In the present study, pharmacologic experiments were performed on carbachol-contracted (0.34 microM) guinea-pig trachea contracted further with ChTX in order to determine if Ca(++)-activated K+ channels play a role in the responses to cAMP-dependent and cAMP-independent bronchodilators. Relaxation concentration response curves to the beta-agonists, isoproterenol and salbutamol; the phosphodiesterase inhibitor, aminophylline; the cAMP mimic, N6-2'-O-adenosine 3':5'-cyclic monophosphate the guanylate cyclase activator, sodium nitroprusside; and the K+ channel agonists, BRL-34915 and pinacidil, were obtained in the absence and presence of ChTX. The concentration response curves to isoproterenol and salbutamol were shifted to the right (approximately 27-fold and greater than 40-fold, respectively) by 180 nM ChTX, whereas concentration response curves to N6-2'-O-adenosine 3':5'-cyclic monophosphate and aminophylline were affected significantly less (shifted approximately 7.5-fold). Concentration response curves to the cGMP-dependent relaxant sodium nitroprusside were also altered by ChTX (17-fold rightward shift at 180 nM). In the presence of 60 nM ChTX, the concentration response curves to the above relaxants were shifted only 3- to 5-fold. In contrast, ChTX (60 and 180 nM) failed to produce a significant rightward shift in the concentration response curves to BRL-34915 or pinacidil. Relaxation to BRL-34915 was however, blocked by glybenclamide, suggesting differences in the mechanism of relaxation. Contraction of tissues with depolarizing concentrations of KCl (20-80 mM) inhibited responses to all bronchodilators. These results suggest that hyperpolarization of tracheal smooth muscle as a result of opening various types of K+ channels can lead to relaxation of carbachol-contracted tracheal smooth muscle.     

次声作用后大鼠心肌细胞内钙离子变化与L型钙通道、Ca2＋-ATP酶及Na＋-K＋-ATP酶变化的关系

目的：观察不同时间次声作用后大鼠心肌细胞钙离子变化与L型钙通道、Ca^2＋-ATP酶及Na^＋-K^＋-ATP酶变化的关系. 方法：实验于2005-04/2006-08在解放军第四军医大学西京医院次声实验室完成.①实验分组：选择健康雄性SD大鼠32只,按随机数字表法分为4组,每组8只.次声暴露1,7,14 d组（大鼠置于5 Hz/130 dB次声舱中,次声作用2 h/次,1次/d）,正常对照组（不予次声暴露）.②实验评估：测定大鼠心肌细胞内钙离子荧光强度,作为反映钙离子浓度的指标;测定L型钙通道mRNA的表达;测定Ca^2＋-ATP酶及Na^＋-K^＋-ATP酶活性. 结果：①随着次声作用时间延长,大鼠心肌细胞钙离子浓度和L型钙通道mRNA的表达明显高于正常对照组（P＜0.01）.②次声暴露1 d组大鼠心肌细胞Ca^2＋-ATP酶及Na^＋-K^＋-ATP酶的活性显著高于正常对照组（P＜0.01）,次声暴露7 d与14 d组大鼠心肌细胞Ca^2＋-ATP酶及Na^＋-K^＋-ATP酶的活性显著低于正常对照组（P＜0.01）. 结论：5 Hz/130 dB次声引起大鼠心肌钙离子浓度的时间依赖性变化,这种变化可能与L型钙通道、Ca^2＋-ATP酶及Na^＋-K^＋-ATP酶的活性变化有关.     

Ca2+ channel activation by platelet-derived growth factor-induced tyrosine phosphorylation and Ras guanine triphosphate-binding proteins in rat glomerular mesangial cells.

We investigated the signaling pathways mediating 1-pS Ca2+ channel activation by PDGF in cultured rat mesangial cells. In cell-attached patches, intrapipette PDGF-BB (PDGF B chain homodimer isoform) (50 ng/ml) dramatically stimulates channel activity (P < 0.003, n = 6). Tyrosine kinase inhibition (100 microM genistein or 10 microM tryphostin 9) abolished PDGF-induced channel activation (P < 0.02, n = 6). In excised patches, the effect of tyrosine kinase inhibition could be reversed by 200 microM GTPgammaS (P < 0.02, n = 4). In contrast, 200 microM GDPbetaS inhibited PDGF-induced channel activity (P < 0.04, n = 6). Pertussis toxin (250 ng/ml) had no effect on PDGF-induced channel activity (P = 0.45, n = 6). When excised patches were exposed to anti-Ras antibody (5 microg/ml), PDGF-induced channel activity was abolished (P < 0.002, n = 11). Western immunoblots revealed that PDGF-BB binding stimulates the formation of a membrane-bound complex consisting of growth factor receptor-binding protein 2, son of sevenless, and the PDGF-beta receptor. Complex formation was abolished by genistein. In mesangial cells, the intrinsic tyrosine kinase activity of the PDGF-beta receptor stimulates the formation of a membrane-bound growth factor receptor-binding protein 2/son of sevenless/PDGF-beta receptor complex and activation of the pertussis toxin-insensitive GTP-binding protein, p21-Ras, which leads to the opening of 1-pS Ca2+ channels.