Protein phosphatase inhibitors okadaic acid and calyculin A alter cell shape and F-actin distribution and inhibit stimulus-dependent increases in cytoskeletal actin of human neutrophils.

The phosphatase inhibitors okadaic acid and calyculin A were found to elicit or to modify several neutrophil responses, suggesting that dephosphorylation plays a regulatory role. The concentrations of okadaic acid (> or = 1 mumol/L) that were effective on neutrophil functions (shape changes and marginal stimulation of pinocytosis) were shown to stimulate the incorporation of 32PO4 into many neutrophil proteins several-fold. Calyculin A was effective at 50-fold lower concentrations. In the presence of the inhibitors, the cells exhibited a nonpolar shape and the polarization response induced by chemotactic peptide was inhibited. Both phosphatase inhibitors also induced the association of F-actin with the cell membrane. A steady-state phosphatase activity is thus involved in maintaining shape and F-actin localization of resting cells. Inhibitors alone had no significant effect on the amount of cytoskeleton-associated actin. The increase in cytoskeletal actin observed at 30 minutes of stimulation with phorbol ester or 5 to 30 minutes of stimulation with chemotactic peptide, however, was abolished by okadaic acid or calyculin A, suggesting an important role of a phosphatase. In contrast, the early increase in cytoskeleton-associated actin observed at 1 minute of stimulation with peptide was not affected. This finding indicates that the increased association of actin with the cytoskeleton in the early and the later stages of neutrophil activation may be mediated by different signalling pathways.     

Protein phosphatase inhibitors and bone resorption: inhibition by okadaic acid and biphasic actions of calyculin-A.

PTH receptors on osteoblasts and calcitonin receptors on osteoclasts are coupled to adenylate cyclase. Despite similar transduction mechanisms, these hormones have opposing physiological actions. We investigated the consequences of persistent protein phosphorylation on bone resorption in neonatal mouse calvariae using okadaic acid (OA) and calyculin-A, two inhibitors of protein phosphatase-1 and -2A. These two inhibitors caused different responses in bone at picomolar and low nanomolar concentrations. OA inhibited, in a dose-dependent manner, bone resorption stimulated by PTH, 1,25-Dihydroxyvitamin D3, phorbol ester, and prostaglandin E2 (PGE2). OA did not inhibit the generation of the second messengers cAMP or PGs and did not have nonspecific toxic effects, as measured by protein and RNA synthesis. Thus, OA appeared to mimic the global inhibitory action of calcitonin on bone resorption. Unlike OA, calyculin-A elicited a biphasic dose response. At concentrations of 3.3 nM and greater, calyculin-A inhibited, in a dose-dependent manner, stimulated bone resorption. However, calyculin-A alone, at 0.625 and 2.5 nM, stimulated bone resorption via a PG-independent pathway. In calvariae, OA and calyculin-A increased phosphorylation of a 58- to 60-kilodalton protein. A protein of similar molecular mass was hyperphosphorylated in OA-treated ROS 17/2.8 osteoblast-like cells. We conclude that in addition to hormonal regulation of protein kinase activity, protein dephosphorylation plays a functionally important role in the modulation of bone resorption.     

Ultrastructural studies of metabolically active isolated adult rat heart myocytes.

Myocytes from adult rat heart were isolated by a method recently developed by us in which hearts were pre-perfused with calcium-free phosphate-buffered saline medium containing collagenase and hyaluronidase. This was followed by incubation in the enzyme medium and cellular sedimentation through 3% Ficoll. Myocytes isolated in this manner were metabolically active and morphologically intact. In the present study, scanning and transmission electron microscopy of isolated myocytes showed long cylindrical cells with transverse microridges which corresponded directly with sarcomere lengths. Most cells appeared to be in a fully contracted state. Contractile elements and associated membranes, other intracellular compartments and sarcolemmae were indistinguishable from their in vivo counterparts. Although myocytic basal laminae and other structurally identifiable surface coats were removed by our isolation procedure, sarcolemmae remained remarkably unaffected. Cyclic AMP assays in control and epinephrine- or glucagon-stimulated cells strongly suggested that membranebound receptors were present and the functional integrity of the sarcolemmae was maintained in our preparations.     

Effect of okadaic acid and calyculin-A, two protein phosphatase inhibitors, on thyrotropin-stimulated triiodothyronine secretion in cultured sheep thyroid cells

We have studied the effect of two protein phosphatase inhibitors on thyrotropin (TSH)-stimulated triiodothyronine (T₃) production by sheep thyroid cells grown in primary culture. Incubation of sheep thyrocytes with okadaic acid (OA) and calyculin-A (CL-A), two potent inhibitors of type 1 (PP1) and type 2A (PP2A) protein phosphatases, resulted in an increase of TSH-stimulated T₃ production. This effect was detected using concentrations as low as 0.1 pM with OA and 1 fM with CL-A. An inhibitory effect on T₃ production, due to cellular death, was observed with 6 nM OA and 1 nM CL-A. In the absence of TSH, OA or CL-A had no effect on T₃ production by thyrocytes. Forskoline (10 μM), an activator of adenylate cyclase, increased the basal and TSH-stimulated T₃ release by sheep thyroid cells; this effect was increased by OA in cells grown in the basal state but not in the presence of TSH. These results suggest that the marine toxins OA and CL-A, two potent inhibitors of PP-1 and PP-2A, have significant stimulatory effects on T₃ secretion promoted by TSH and FK. These observations indicate that these proteins could be important mediators of thyroid hormone production.     

Effects of calmodulin and okadaic acid on myofibrillar Ca2+ sensitivity in cardiac myocytes

Whereas it has been established that the phosphorylation of 20 kD regulatory myosin light chain (MLC₂₀) is a key regulator of contraction in smooth muscle, troponin complex has been thought to be that of myofibrillar Ca²⁺ sensitivity in cardiac muscle. To elucidate the role of the phosphorylation of cardiac regulatory myosin light chain (MLC2) in the regulation of cardiac muscle contraction, we observed effects of calmodulin and okadaic acid, a protein phosphatase inhibitor, on myofibrillar Ca²⁺ sensitivity as estimated by pCa₅₀ values obtained from pCa-tension relationships using β-escin-skinned cardiomyocytes from Wistar rat hearts, in relation to changes in the phosphorylation of myofibrillar regulatory proteins. Whereas myofibrillar Ca²⁺ sensitivity tended to be progressively decreased by repeated Ca²⁺-activation in the absence of calmodulin (pCa₅₀; from 5.91 to 5.86, n = 5), calmodulin (2.5 μM) significantly increased myofibrillar Ca²⁺ sensitivity (pCa₅₀; from 5.92 to 6.03, n = 5, p < 0.05). Okadaic acid over 3 μM enhanced Ca²⁺-activated force, which was inhibited by 50 μM trifluoperazine, a calmodulin antagonist. Okadaic acid (3 μM) significantly increased myofibrillar Ca²⁺ sensitivity (pCa₅₀; from 5.96 to 6.11, n = 6, p < 0.05). Whereas the phosphorylation level of troponin I was not changed by 3 μM okadaic acid, that of MLC2 was significantly increased by the same dose of okadaic acid (from 12 to 31%, n = 4, p < 0.05). These results suggest that MLC2 phosphorylation plays a partial role in the regulation of myofibrillar Ca²⁺ sensitivity in cardiac muscle. Received: 25 May 2001, Returned for revision: 19 June 2001, Revision received: 13 September 2001, Accepted: 24 September 2001     

Induction of apoptosis in cultured retinoblastoma cells by the protein phosphatase inhibitor,okadaic acid

The induction of apoptosis in cultured retinoblastoma cells by diverse drugs was examined by analyzing DNA fragmentation, a hallmark of apoptosis. First, the ability of six retinoblastoma cell lines to undergo apoptosis was surveyed using etoposide (30 g/ml, 20 h exposure). The NCC-RbC-60 cell line, established in this laboratory, showed DNA fragmentation clearly, whereas the other cell lines tested, including the representative retinoblastoma cell line, Y-79, did not show distinct DNA fragmentation. Biochemical modulators, such as A23187, forskolin, retinoic acid, phorbol 12-myristate 13-acetate and okadaic acid, were examined to ascertain whether they could induced apoptosis in NCC-RbC-60 and Y-79 cells after exposure for 20 h. Only okadaic acid induced DNA fragmentation in all the retinoblastoma cell lines tested and it induced DNA fragmentation in Y-79 cells in a time- and concentration-dependent manner. Flow-cytometric analysis and microscopic examination revealed that Y-79 cells treated with okadaic acid for 24–48 h accumulated at the G2/M, especially M, phases, before undergoing DNA fragmentation. Other mitotic poisons, nocodazole, colcemid and taxol, also induced apoptosis in Y-79 cells. In the K1034 cell line, established from non-malignant retinal pigmental epithelium, okadaic acid failed to induce both G₂/M arrest and DNA fragmentation. These findings suggest that okadaic-acid-induced apoptosis occurs as a result of metaphase arrest.Abbreviations OA okadaic acid - TPA phorbol 12-myristate 13-acetate - FBS fetal bovine serum This study was supported, in part, by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare, Japan.     

Role of mitochondrial re-energization and Ca2+ influx in reperfusion injury of metabolically inhibited cardiac myocytes

OBJECTIVE: We used isolated myocytes to investigate the role of mitochondrial re-energization and Ca2+ influx during reperfusion on hypercontracture, loss of Ca2+ homeostasis and contractile function. METHODS: Isolated adult rat ventricular myocytes were exposed to metabolic inhibition (NaCN and iodoacetate) and reperfusion injury was assessed from hypercontracture, loss of Ca2+ homeostasis ([Ca2+]i measured with fura-2) and failure of contraction in response to electrical stimulation. Mitochondrial membrane potential was followed using the potentiometric dye tetramethylrhodamine ethyl ester. RESULTS: Metabolic inhibition led to contractile failure and rigor accompanied by a sustained increase in [Ca2+]i. Reperfusion after 10 min metabolic inhibition led to an abrupt repolarization of the mitochondrial membrane potential (after 25.5+/-1.2 s), a transient fall in [Ca(2+]i followed by an abrupt hypercontracture (37.1+/-1.8 s) in 84% of myocytes. Ca2+ homeostasis (diastolic [Ca2+]i < 250 nM) recovered in only 23.3+/-5.1% of cells and contractions recovered in 15.3+/-2.2%. Oligomycin abolished the hypercontracture on reperfusion, but mitochondrial repolarization was unaffected. Preventing Ca2+ influx during reperfusion with [Ca2+]i-free Tyrode or with an inhibitor of Na(+)/Ca2+ exchange did not prevent the hypercontracture, but increased the percentage of cells recovering Ca2+ homeostasis and contractile function. The presence of 0.5 microM cyclosporin A did not prevent hypercontracture but increased the percentage of cells recovering Ca2+ homeostasis to 56.2+/-3.6% and contractile function to 52+/-4.3%. CONCLUSIONS: Reperfusion-induced hypercontracture, and loss of Ca2+ homeostasis and contractile function are initiated following mitochondrial re-energization. The hypercontracture requires the production of oxidative ATP but not Ca2+ influx during reperfusion. Loss of Ca2+ homeostasis and contractile function are linked to Ca2+ influx during reperfusion, probably via opening of mitochondrial permeability transition pores.     

Impaired long-chain fatty acid utilization by cardiac myocytes isolated from mice lacking the heart-type fatty acid binding protein gene.

Heart-type fatty acid binding protein (H-FABP), abundantly expressed in cardiac myocytes, has been postulated to facilitate the cardiac uptake of long-chain fatty acids (LCFAs) and to promote their intracellular trafficking to sites of metabolic conversion. Mice with a disrupted H-FABP gene were recently shown to have elevated plasma LCFA levels, decreased cardiac deposition of a LCFA analogue, and increased cardiac deoxyglucose uptake, which qualitatively establishes a requirement for H-FABP in cardiac LCFA utilization. To study the underlying defect, we developed a method to isolate intact, electrically stimulatable cardiac myocytes from adult mice and then studied substrate utilization under defined conditions in quiescent and in contracting cells from wild-type and H-FABP(-/-) mice. Our results demonstrate that in resting and in contracting myocytes from H-FABP(-/-) mice, both uptake and oxidation of palmitate are markedly reduced (between -45% and -65%), whereas cellular octanoate uptake, and the capacities of heart homogenates for palmitate oxidation and for octanoate oxidation, and the cardiac levels of mRNAs encoding sarcolemmal FA transporters remain unaltered. In contrast, in resting H-FABP(-/-) cardiac myocytes, glucose oxidation is increased (+80%) to a level that would require electrical stimulation in wild-type cells. These findings provide a physiological demonstration of a crucial role of H-FABP in uptake and oxidation of LCFAs in cardiac muscle cells and indicate that in H-FABP(-/-) mice the diminished contribution of LCFAs to cardiac energy production is, at least in part, compensated for by an increase in glucose oxidation.     

Human prostatic acid phosphatase directly stimulates collagen synthesis and alkaline phosphatase content of isolated bone cells.

Human prostatic acid phosphatase (hPAP) directly enhances the differentiated characteristics of isolated bone cells in vitro. This enzyme, when added to cell cultures for 24 h in vitro stimulates collagen synthesis and the production of alkaline phosphatase. The effects are dose dependent, with statistically significant effects occurring from 0.1-100 nM hPAP. Concentrations higher than 100 nM do not evoke greater effects. The maximal effect of hPAP occurs between 12 and 24 h of exposure. The cells stimulated to the greatest degree are osteoprogenitor cells and osteoblasts. Fibroblasts isolated from the same tissue show a lesser sensitivity to hPAP. hPAP has no detectable effect on cell proliferation, as measured by radiolabeled thymidine incorporation or total DNA synthesis. None of the observations reported in this work can be attributed to contaminating proteins in the hPAP preparation. hPAP was radiolabeled with 125I and was used for affinity binding and cross-linking studies. Scatchard analysis of specific binding indicated the presence of 1.0 X 10(5) high affinity binding sites/cell, with a Kd of 6.5 nM. Cross-linking studies demonstrated the presence of one 320-kDa binding complex. The pH profile and kinetic determinations of Km and maximum velocity for hPAP were similar to those previously reported, except for the finding of positive cooperativity of the substrate with the enzyme under the conditions of our assay. We believe that the direct stimulation of bone-forming cells by hPAP may contribute to the sclerotic nature of skeletal bone around sites of neoplastic prostatic metastases and that the effect of the enzyme is probably mediated by a plasma membrane receptor.     

Effects of insulin treatment on the activities of phosphoprotein phosphatase and its inhibitors

An increase in glycogen synthase phosphatase (phosphoprotein phosphatase) activity was observed in the rat skeletal muscle extract following insulin administration. The phosphoprotein phosphatase activity present in the muscle extract from insulin treated rats was observed to remain elevated after the extract had been subjected to a molecular sieve chromatography. These results indicate that the stimulatory effects of insulin is due to modification of phosphatase itself or some macromolecular weight modifiers. The heat-stable protein inhibitors of the phosphoprotein phosphatase were isolated from skeletal muscle of insulin treated and control rats and their inhibitory potencies were compared over a wide range of protein concentrations. The inhibitory potency in the insulin treated rat skeletal muscle was found to be significantly less than that in the control muscle. Since type-1 inhibitor is well-known to be active only after being phosphorylated by cyclic AMP-dependent protein kinase, we suggest that the observed change in phosphoprotein phosphatase inhibitor potency is most likely mediated by an alteration in the phosphorylation state of type-1 inhibitor.     

蛋白磷酸酶2A抑制剂对胰腺癌细胞活力的影响及其机制

目的 研究蛋白磷酸酶2A(PP2A)抑制剂对胰腺癌细胞系PANC-1活力的影响及其机制.方法 用PP2A抑制剂斑蝥素和冈田酸处理PANC-1细胞.通过Western印迹检测核因子(NF) -κB通路的激活程度.通过转染PP2A活性亚基cα(PP2A cα)表达质粒、NF-κB抑制蛋白激酶(IKK)α和NF-κB抑制蛋白(IκB)α的显性负性突变体、p65干扰质粒,分别从各环节阻断NF-κB通路,通过四甲基偶氮唑盐比色法(MTT法)检测细胞活力.结果 PP2A抑制剂可使IKKa磷酸化,进一步使IκBa磷酸化并降解,继而释放p65入核.过表达PP2Acα、IKKα显性负性突变体、IκBα显性负性突变体或干扰p65可分别使斑蝥素对细胞活力的抑制率下降(31.85±13.37)％、(23.48±8.98)％、(22.63±5.81)％、(20.88±3.24)％,使冈田酸对细胞活力的抑制率下降(40.17±11.65)％、(27.34±14.28)％、(24.85±3.39)％、(27.08±3.81)％.结论 PP2A抑制剂通过PP2A/IKKα/IκBα/p65依赖性通路发挥抗胰腺癌作用.     

硫化氢对离体大鼠心室肌细胞ATP依赖的钾通道外向电流的影响

目的 观察内源性及外源性硫化氢(hydrogen sulfide,H2S)对大鼠离体心室肌细胞ATP依赖的钾通道(KATP)外向电流的影响,以探讨H2s对心窜肌细胞的作用.方法 对大鼠离体心脏采用胶原酶酶解法得到单个心室肌细胞,采用膜片钳全细胞技术记录DL-propargylglycine(PPG)及不同浓度硫氢化钠(NaHS,外源性H2S的供体)干预前后的KATP什电流.结果 经PPG(200 μmol/L)干预后,KATP峰电流密度(+70 mV)显著减小[干预前后分别为(5.3258±0.7556)pA/pF比(3.7856±0.4312)pA/pF,P＜0.01],且具有时间依赖性.经NaHS(9.375、18.75、37.5、75、150μmoL/L)干预后,KATP峰电流密度呈浓度依赖性增大,至150 μmol/L时峰电流密度明显增大[(6.6310±0.6092)pA/pF比(9.0949±1.0259)pA/pF,P＜0.01].结论 内源性及外源性H2s均可以开放大鼠离体心室肌KATP通道,使KATP电流增加.     

硫化氢对离体大鼠心室肌细胞ATP依赖的钾通道外向电流的影响

Objective To investigate the effects of endogenous and exogenous hydrogen sulfide (H2S) on the KATP current in isolated rat ventricular myoeytes. Methods Ventrieular myoeytes were isolated from rat heart by modified Langendoff perfusion with collagenase. KATP current of single rat ventricular myocytes was recorded by whole-cell patch-clamp technique. Results The density of KATP current was significantly reduced by 200 μmol/L DL-propargylglyeine (PPG, an irreversible inhibitor of the H2S) [(5.3258±0.7556) pA/pF vs. (3.7856±0.4312) pA/pF, P ＜ 0.01] in a time-dependent way. The density of KATP current could be significantly increased by NariS(a H2S donor, 9.375, 18.75, 37.5,75, 150 μmol/L) in a concentration-dependent manner [(6.6310±0.6092) pA/pF vs. (9.0949±1.0259)pA/pF at 150 μmol/L, P ＜ 0.01]. Conclusion Both endogenous and exogenous H2S could open KATP channels and enhance the KATP current in rat ventricular myocytes.     

硫化氢对离体大鼠心室肌细胞ATP依赖的钾通道外向电流的影响

Objective To investigate the effects of endogenous and exogenous hydrogen sulfide (H2S) on the KATP current in isolated rat ventricular myoeytes. Methods Ventrieular myoeytes were isolated from rat heart by modified Langendoff perfusion with collagenase. KATP current of single rat ventricular myocytes was recorded by whole-cell patch-clamp technique. Results The density of KATP current was significantly reduced by 200 μmol/L DL-propargylglyeine (PPG, an irreversible inhibitor of the H2S) [(5.3258±0.7556) pA/pF vs. (3.7856±0.4312) pA/pF, P ＜ 0.01] in a time-dependent way. The density of KATP current could be significantly increased by NariS(a H2S donor, 9.375, 18.75, 37.5,75, 150 μmol/L) in a concentration-dependent manner [(6.6310±0.6092) pA/pF vs. (9.0949±1.0259)pA/pF at 150 μmol/L, P ＜ 0.01]. Conclusion Both endogenous and exogenous H2S could open KATP channels and enhance the KATP current in rat ventricular myocytes.     

硫化氢对离体大鼠心室肌细胞ATP依赖的钾通道外向电流的影响

Objective To investigate the effects of endogenous and exogenous hydrogen sulfide (H2S) on the KATP current in isolated rat ventricular myoeytes. Methods Ventrieular myoeytes were isolated from rat heart by modified Langendoff perfusion with collagenase. KATP current of single rat ventricular myocytes was recorded by whole-cell patch-clamp technique. Results The density of KATP current was significantly reduced by 200 μmol/L DL-propargylglyeine (PPG, an irreversible inhibitor of the H2S) [(5.3258±0.7556) pA/pF vs. (3.7856±0.4312) pA/pF, P ＜ 0.01] in a time-dependent way. The density of KATP current could be significantly increased by NariS(a H2S donor, 9.375, 18.75, 37.5,75, 150 μmol/L) in a concentration-dependent manner [(6.6310±0.6092) pA/pF vs. (9.0949±1.0259)pA/pF at 150 μmol/L, P ＜ 0.01]. Conclusion Both endogenous and exogenous H2S could open KATP channels and enhance the KATP current in rat ventricular myocytes.     

硫化氢对离体大鼠心室肌细胞ATP依赖的钾通道外向电流的影响

Objective To investigate the effects of endogenous and exogenous hydrogen sulfide (H2S) on the KATP current in isolated rat ventricular myoeytes. Methods Ventrieular myoeytes were isolated from rat heart by modified Langendoff perfusion with collagenase. KATP current of single rat ventricular myocytes was recorded by whole-cell patch-clamp technique. Results The density of KATP current was significantly reduced by 200 μmol/L DL-propargylglyeine (PPG, an irreversible inhibitor of the H2S) [(5.3258±0.7556) pA/pF vs. (3.7856±0.4312) pA/pF, P ＜ 0.01] in a time-dependent way. The density of KATP current could be significantly increased by NariS(a H2S donor, 9.375, 18.75, 37.5,75, 150 μmol/L) in a concentration-dependent manner [(6.6310±0.6092) pA/pF vs. (9.0949±1.0259)pA/pF at 150 μmol/L, P ＜ 0.01]. Conclusion Both endogenous and exogenous H2S could open KATP channels and enhance the KATP current in rat ventricular myocytes.     

硫化氢对离体大鼠心室肌细胞ATP依赖的钾通道外向电流的影响

Objective To investigate the effects of endogenous and exogenous hydrogen sulfide (H2S) on the KATP current in isolated rat ventricular myoeytes. Methods Ventrieular myoeytes were isolated from rat heart by modified Langendoff perfusion with collagenase. KATP current of single rat ventricular myocytes was recorded by whole-cell patch-clamp technique. Results The density of KATP current was significantly reduced by 200 μmol/L DL-propargylglyeine (PPG, an irreversible inhibitor of the H2S) [(5.3258±0.7556) pA/pF vs. (3.7856±0.4312) pA/pF, P ＜ 0.01] in a time-dependent way. The density of KATP current could be significantly increased by NariS(a H2S donor, 9.375, 18.75, 37.5,75, 150 μmol/L) in a concentration-dependent manner [(6.6310±0.6092) pA/pF vs. (9.0949±1.0259)pA/pF at 150 μmol/L, P ＜ 0.01]. Conclusion Both endogenous and exogenous H2S could open KATP channels and enhance the KATP current in rat ventricular myocytes.     

硫化氢对离体大鼠心室肌细胞ATP依赖的钾通道外向电流的影响

Objective To investigate the effects of endogenous and exogenous hydrogen sulfide (H2S) on the KATP current in isolated rat ventricular myoeytes. Methods Ventrieular myoeytes were isolated from rat heart by modified Langendoff perfusion with collagenase. KATP current of single rat ventricular myocytes was recorded by whole-cell patch-clamp technique. Results The density of KATP current was significantly reduced by 200 μmol/L DL-propargylglyeine (PPG, an irreversible inhibitor of the H2S) [(5.3258±0.7556) pA/pF vs. (3.7856±0.4312) pA/pF, P ＜ 0.01] in a time-dependent way. The density of KATP current could be significantly increased by NariS(a H2S donor, 9.375, 18.75, 37.5,75, 150 μmol/L) in a concentration-dependent manner [(6.6310±0.6092) pA/pF vs. (9.0949±1.0259)pA/pF at 150 μmol/L, P ＜ 0.01]. Conclusion Both endogenous and exogenous H2S could open KATP channels and enhance the KATP current in rat ventricular myocytes.     

硫化氢对离体大鼠心室肌细胞ATP依赖的钾通道外向电流的影响

Objective To investigate the effects of endogenous and exogenous hydrogen sulfide (H2S) on the KATP current in isolated rat ventricular myoeytes. Methods Ventrieular myoeytes were isolated from rat heart by modified Langendoff perfusion with collagenase. KATP current of single rat ventricular myocytes was recorded by whole-cell patch-clamp technique. Results The density of KATP current was significantly reduced by 200 μmol/L DL-propargylglyeine (PPG, an irreversible inhibitor of the H2S) [(5.3258±0.7556) pA/pF vs. (3.7856±0.4312) pA/pF, P ＜ 0.01] in a time-dependent way. The density of KATP current could be significantly increased by NariS(a H2S donor, 9.375, 18.75, 37.5,75, 150 μmol/L) in a concentration-dependent manner [(6.6310±0.6092) pA/pF vs. (9.0949±1.0259)pA/pF at 150 μmol/L, P ＜ 0.01]. Conclusion Both endogenous and exogenous H2S could open KATP channels and enhance the KATP current in rat ventricular myocytes.