Effect of GBE50 on L-type calcium current and cytosolic[Ca~(2+)]_i in ischemic guinea pig ventricular myocytes / 中国药理学通报

Aim To observe the effect of Ginkgo biloba extract 50(GBE50)on L-type calcium current and cytosolic[Ca~(2+)]_i in ischemic guinea pig ventricular myocytes.Methods Single ventricular myocytes were isolated by enzymatic dissociation. I Ca-L was recorded by whole-cell patch clamp technique in voltage clamp mode.[Ca~(2+)]_i was detected by laser confocal micros-copy and represented by relative fluorescent intensity (FI).Results During ischemia, the peak Ca~(2+) current was reduced, and the I-U curve of I Ca-L was shifted upward.50 mg·L~(-1) GBE50 reversed the change induced by ischemia(n =6, P >0.05).After perfusing ischemic solution for 12 min, intracellular calcium concentration was increased(n =10, P <0.01).After perfusion with ischemic solution containing 50 mg·L~(-1) GBE50, the increase of intracellular calcium concentration was markedly inhibited(n =10, P >0.05).Conclusion GBE50 can reverse the decrease of I Ca-L and partially inhibit calcium overloading during ischemia.

Forskolin Changes the Relationship between Cytosolic Ca2+ and Contraction in Guinea Pig Ileum

This study was designed to clarify the mechanism of the inhibitory effect of forskolin on contraction, cytosolic Ca2+ level ([Ca2+]i), and Ca2+ sensitivity in guinea pig ileum. Forskolin (0.1 nM~10 micrometer) inhibited high K+ (25 mM and 40 mM)- or histamine (3 micrometer)-evoked contractions in a concentration-dependent manner. Histamine-evoked contractions were more sensitive to forskolin than high K+-evoked contractions. Spontaneous changes in [Ca2+]i and contractions were inhibited by forskolin (1 micrometer) without changing the resting [Ca2+]i. Forskoln (10 micrometer) inhibited muscle tension more strongly than [Ca2+]i stimulated by high K+, and thus shifted the [Ca2+]i-tension relationship to the lower-right. In histamine-stimulated contractions, forskolin (1 micrometer) inhibited both [Ca2+]i and muscle tension without changing the [Ca2+]i-tension relationship. In alpha-toxin-permeabilized tissues, forskolin (10 micrometer) inhibited the 0.3 micrometer Ca2+-evoked contractions in the presence of 0.1 mM GTP, but showed no effect on the Ca2+-tension relationship. We conclude that forskolin inhibits smooth muscle contractions by the following two mechanisms: a decrease in Ca2+ sensitivity of contractile elements in high K+-stimulated muscle and a decrease in [Ca2+]i in histamine-stimulated muscle.

Resistin Binding Peptide Stimulates Basal Insulin Secretion of RINm5F Insulinoma Cells / 实用儿科临床杂志

Objective A resistin binding peptide (RBP) was selected by phage display in our previous work. Studies had shown that RBP could antagonize the role of resistin on the lipid metabolism and endocrine function of adipose tissue, but whether RBP affects the insulin secretion of pancreatic cells is still unknown. The aim of this study is to assess the effect of RBP on basal insulin secretion in RINm5F insulinoma cells. Methods The cell viability was measured by 3-[4,5-dimethyhhiazol-2-yl]-2,5-diphenyltetra-zolium bromide (MTT) cytotoxicity assay. The supernatants were assayed for insulin content by enzyme linked immunosorbent assay (ELISA). Reverse transcriptase-PCR assay and Western blotting were used to determine the expression of glucose transporter 2 (GLUT2) involved in insulin secretion. Cytosolic Ca2+, the trigger of insulin exocytosis, was analyzed with the fluorescent probe FURA-3/AM. Results RBP did no effect on the cell viability with a concentration of 10-8-10-12mol/L of 2 hours intervention. But it stimulated basal insulin secretion of RINm5F cells, accompanied by up-regulated increased expression of GLUT2 and elevated concentration of cytosolic Ca2+. Conclusion RBP could stimulate basal insulin secretion without affecting the cell viability.

Changes of Cytosolic Ca2+ under Metabolic Inhibition in Isolated Rat Ventricular Myocytes

To characterize cytosolic Ca2+ fluctuations under metabolic inhibition, rat ventricular myocytes were exposed to 200microM 2, 4-dinitrophenol (DNP), and mitochondrial Ca2+, mitochondrial membrane potential (delta psi m), and cytosolic Ca2+ were measured, using Rhod-2 AM, TMRE, and Fluo-4 AM fluorescent dyes, respectively, by Laser Scanning Confocal Microscopy (LSCM). Furthermore, the role of sarcolemmal Na+/Ca2+ exchange (NCX) in cytosolic Ca2+ efflux was studied in KB-R7943 and Na+-free normal Tyrode's solution (143 mM LiCl ). When DNP was applied to cells loaded with Fluo-4 AM, Fluo-4 AM fluorescence intensity initially increased by 70+/-10% within 70+/-10 s, and later by 400+/-200% at 850+/-46 s. Fluorescence intensity of both Rhod-2 AM and TMRE were initially decreased by DNP, coincident with the initial increase of Fluo-4 AM fluorescence intensity. When sarcoplasmic reticulum (SR) Ca2+ was depleted by 1microM thapsigargin plus 10microM ryanodine, the initial increase of Fluo-4 AM fluorescence intensity was unaffected, however, the subsequent progressive increase was abolished. KB-R7943 delayed both the first and the second phases of cytosolic Ca2+ overload, while Na+-free solution accelerated the second. The above results suggest that: 1) the initial rise in cytosolic Ca2+ under DNP results from mitochondrial depolarization; 2) the secondary increase is caused by progressive Ca2+ release from SR; 3) NCX plays an important role in transient cytosolic Ca2+ shifts under metabolic inhibition with DNP.

Pharmacological characterization of synthetic tetrahydroisoquinoline alkaloids, YS 51 and YS 55, on the cardiovascular system

Tetrahydroisoquinoline (THI) alkaloids can be considered as cyclized derivatives of simple phenylethylamines, and many of them, especially with 6,7-disubstitution, demonstrate relatively high affinity for catecholamines. Two -OH groups at 6 and 7 positions are supposed to be essential to exert beta-receptor activities. However, it is not clear whether -OH at 6,7 substitution of THIs also shows alpha-adrenoceptor activities. In the present study, we investigated whether -OH or -OCH3 substitutions of 6,7 position of THIs differently affect the alpha1-adrenoceptor affinity. We synthesized two 1-naphthylmethyl THI alkaloids, 1-beta-naphthylmethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline HBr (YS 51) and 1-beta-naphthylmethyl-6, 7-dimethoxy-1,2,3,4-tetrahydroisoquinoline HCl (YS 55), and their pharmacological actions on alpha1-adrenoceptor were compared. YS 51 and YS 55, concentration-dependently relaxed endothelium-denuded rat thoracic aorta precontracted with phenylephrine (PE, 0.1 micrometer) in which pEC50 were 5.89+0.21 and 5.93+ 0.19, respectively. Propranolol (30 nM) did not affect the relaxation-response curves to YS 51 and YS 55. Concentration-response curves to PE were shifted to right by the pretreatment with YS 51 or YS 55. The pA2 values of YS 51 and YS 55 showed 6.05 + 0.24 and 5.88 + 0.16, respectively. Both probes relaxed KCl (65.4 mM)-contacted aorta and inhibited CaCl2-induced contraction of PE-stimulated endothelium-denuded rat thoracic aorta in Ca2+-free solutions. In isolated guinea pig papillary muscle, 1 and 10 micrometer YS 51 increased contractile force about 4- and 8- fold over the control, respectively, along with the concentration-dependent increment of cytosolic Ca2+ ions. While, 10 micrometer YS 55 reduced the contractile force about 50 % over the control and lowered the cytosolic Ca2+ level, in rat brain homogenates, YS 51 and YS 55 displaced (3H)prazosin binding competitively with Ki 0.15 and 0.12 micrometer, respectively. However, both probes were ineffective on (3H)nitrendipine binding. Therefore, it is concluded that two synthetic naphthylmethyl-THI alkaloids have considerable affinity to alpha1-adrenenoceptors in rat aorta and brain.

Changes in Cytosolic Ca2+ Concentration of Single Rabbit Coronary Artery Smooth Muscle Cell during Ischemic Cardioplegic Period

BACKGROUND: No-reflow is a specific type of vascular damage occuring when removal of coronary occlusion dose not lead to restoration of coronary flow. There are three major explanations for the no-reflow phenomenon such as endothelial cell edema, microvascular plugging by platelets or thrombi and coronary occlusion by ischemic contracture of the myocardium. But detailed mechanisms of no-reflow phenomenon are not known. The objects of this study are to elucidate the possibility whether elevation of cytosolic Ca2+ concentration during ischemic cardioplegic period is mechanism of no-reflow phenomenon or not. METHODS: Changes in cytosolic Ca2+ concentration were measured under varying experimental condition. Free [Ca2+] in the cytosole [Ca2+]i of single rabbit coronary artery cells was measured with fluorescent Ca2+ indicator, Fura-2. RESULTS: Resting [Ca2+]i was 134.2+/-34 nM (n=43). When single cells were perfused with cardioplegic or ischemic cardioplegic solution, [Ca2+]i was significantly increased and degree of [Ca2+]i elevation was further augmented by ischemic cardioplegic solution. Pretreatment of sarcoplasmic reticulum emptying agent (20mM caffeine) had no effect on cardioplegia-induced [Ca2+]i change, but application of Ca2+ channel blocker (5x10-7M nifedipine) or an antagonist of Na+/Ca2+ exchange (5mM Ni2+ ) partially (nifedipine) or completely (nickel) inhibited the [Ca2+]i elevation. Pretreament of caffeine had no effect on ischemic cardioplegia-induced [Ca2+]i change, but application of nifedipine or nickel partially inhibited the [Ca2+]i elevation. Magnitude of ischemic cardioplegia-induced [Ca2+]i elevation was dependent on the Ca2+ concentration of perfusate from 0 to 2.5mM. When Ni2+ was added to reperfusion solution, recovery of ischemic cardioplegia-induced [Ca2+]i elevation was very rapid compared with control. CONCLUSIONS: From the above results, it may be speculated that ischemic cardioplegia-induced [Ca2+]i elevation may act as one of the mechanism of no-reflow phenomenon in rabbit coronary artery.