Centrosomal control of microtubule dynamics

In many animal cells, minus ends of microtubules (MTs) are thought to be capped by the centrosome whereas plus ends are free and display dynamic instability. We tested the role of the centrosome by examining MT behavior in cytoplasts from which the centrosome was removed. Cells were injected with Cy3–tubulin to fluorescently label MTs and were enucleated by using a centrifugation procedure. Enucleation resulted in a mixture of cytoplasts containing or lacking the centrosome. Fibroblast (CHO-K1) and epithelial (BSC-1) cells were investigated. In fibroblast cytoplasts containing the centrosome, MTs showed dynamic instability indistinguishable from that in intact cells. In contrast, in cytoplasts lacking the centrosome, MTs treadmilled—shortened at the minus end at about 12 μm/min while growing at the plus end at the same rate. The change in behavior of the plus end from dynamic instability to persistent growth correlated with an elevated level of free tubulin subunits (78% in centrosome-free cytoplasts vs. 44% in intact cells) generated by minus-end depolymerization. In contrast to fibroblast cells, in centrosome-free cytoplasts prepared from epithelial cells, MTs displayed dynamic instability at plus ends and relative stability at minus ends presumably because of specific minus-end stability factors distributed throughout the cytoplasm. We suggest that, in fibroblast cells, a minus-end depolymerization mechanism functions to eliminate errors in MT organization and that dynamic instability of MT plus ends is a result of capping of minus ends by the centrosome.     

Sequence-dependent cytotoxic effects due to combinations of cisplatin and the antimicrotubule agents taxol and vincristine

The antineoplastic activity that taxol has demonstrated in advanced ovarian cancer and other neoplasms in which the platinum analogues are among the most active agents has been the impetus for the development of taxol/platinum combination regimens. Since both classes of agents are known to induce cell-cycle-dependent effects and to delay cell-cycle traverse in specific phases of the cycle, an evaluation of drug sequence dependence was incorporated into initial clinical studies of the drug combination. To complement clinical studies, sequence-dependent interactions were assessed in vitro using L1210 leukemia. Cytotoxicity resulting from the combination of taxol and cisplatin was significantly increased over that achieved with cisplatin alone only when cisplatin was administered after taxol. This sequence was significantly superior to both the reverse sequence and to simultaneous drug treatment. Results achieved with sequence iterations of vincristine and cisplatin were nearly identical. In addition, alkaline-elution studies, using the optimal sequence of cisplatin and either taxol or vincristine, demonstrated that these antimicrotubule agents do not increase the formation of cisplatin-induced DNA interstrand and DNA-protein crosslinking over that produced by cisplatin alone. Although the mechanisms for the sequence-dependent cytotoxic interactions between cisplatin and the antimicrotubule agents have not been determined, it is likely that antagonistic interactions occur with the suboptimal sequences, probably because of cell-cycle-dependent phenomena.     

Multidrug resistance protein 3 R652G may reduce susceptibility to idiopathic infant cholestasis

AIM: To evaluate the role of genetic factors in the pathogenesis of idiopathic infant cholestasis. METHODS: We performed a case-control study, including 78 infants with idiopathic infant cholestasis and 113 healthy infants as controls. Genomic DNA was extracted from peripheral venous blood leukocytes using phenol chloroform methodology. Polymerase chain reaction was used to amplify the multidrug resistance protein 3 (MDR3) R652G fragment, and products were sequenced using the ABI 3100 Sequencer. RESULTS: The R652G single nucleotide polymorphism (SNP) was signi(O)cantly more frequent in healthy infants (allele frequency 8.0%) than in patients (allele frequency 2.60%) ( P < 0.05), odds ratio, 0.29; 95% confidence interval, 0.12-0.84. The conjugated bilirubin in patients with the AG genotype was significantly lower than in those with the AA genotype (44.70 ± 6.15 μmol/L vs 95.52 ± 5.93 μmol/L, P < 0.05). CONCLUSION: MDR3 R652G is negatively correlated with idiopathic infant cholestasis. Children with the R652G SNP in Guangxi of China may have reduced susceptibility to infant intrahepatic cholestasis.     

SIRT3与胰岛素抵抗

沉默信息调节因子(SIRT)3是哺乳动物类NAD+依赖性组蛋白去乙酰化酶家族中的一员.研究表明,SIRT3可以改善胰岛素抵抗、增加胰岛素敏感性.其通过保护胰岛β细胞、促进骨骼肌葡萄糖摄取、调节骨骼肌代谢、减轻氧化应激、抵抗高糖诱导的细胞毒性等途径发挥作用.SIRT3为治疗2型糖尿病、肥胖、线粒体功能障碍等疾病带来了新的研究方向.     

Dynamic interplay between nitration and phosphorylation of tubulin cofactor B in the control of microtubule dynamics

Tubulin cofactor B (TCoB) plays an important role in microtubule dynamics by facilitating the dimerization of α- and β-tubulin. Recent evidence suggests that p21-activated kinase 1 (Pak1), a major signaling nodule in eukaryotic cells, phosphorylates TCoB on Ser-65 and Ser-128 and plays an essential role in microtubule regrowth. However, to date, no upstream signaling molecules have been identified to antagonize the functions of TCoB, which might help in maintaining the equilibrium of microtubules. Here, we discovered that TCoB is efficiently nitrated, mainly on Tyr-64 and Tyr-98, and nitrated-TCoB attenuates the synthesis of new microtubules. In addition, we found that nitration of TCoB antagonizes signaling-dependent phosphorylation of TCoB, whereas optimal nitration of TCoB requires the presence of functional Pak1 phosphorylation sites, thus providing a feedback mechanism to regulate phosphorylation-dependent MT regrowth. Together these findings identified TCoB as the third cytoskeleton protein to be nitrated and suggest a previously undescribed mechanism, whereby growth factor signaling may coordinately integrate nitric oxide signaling in the regulation of microtubule dynamics.     

The Dam1 kinetochore complex harnesses microtubule dynamics to produce force and movement

Kinetochores remain attached to microtubule (MT) tips during mitosis even as the tips assemble and disassemble under their grip, allowing filament dynamics to produce force and move chromosomes. The specific proteins that mediate tip attachment are uncertain, and the mechanism of MT-dependent force production is unknown. Recent work suggests that the Dam1 complex, an essential component of kinetochores in yeast, may contribute directly to kinetochore-MT attachment and force production, perhaps by forming a sliding ring encircling the MT. To test these hypotheses, we developed an in vitro motility assay where beads coated with pure recombinant Dam1 complex were bound to the tips of individual dynamic MTs. The Dam1-coated beads remained tip-bound and underwent assembly- and disassembly-driven movement over approximately 3 microm, comparable to chromosome displacements in vivo. Dam1-based attachments to assembling tips were robust, supporting 0.5-3 pN of tension applied with a feedback-controlled optical trap as the MTs lengthened approximately 1 microm. The attachments also harnessed energy from MT disassembly to generate movement against tension. Reversing the direction of force (i.e., switching to compressive force) caused the attachments to disengage the tip and slide over the filament, but sliding was blocked by areas where the MT was anchored to a coverslip, consistent with a coupling structure encircling the filament. Our findings demonstrate how the Dam1 complex may contribute directly to MT-driven chromosome movement.     

Regulation of microtubule minus-end dynamics by CAMSAPs and Patronin

The microtubule (MT) cytoskeleton plays an essential role in mitosis, intracellular transport, cell shape, and cell migration. The assembly and disassembly of MTs, which can occur through the addition or loss of subunits at the plus- or minus-ends of the polymer, is essential for MTs to carry out their biological functions. A variety of proteins act on MT ends to regulate their dynamics, including a recently described family of MT minus-end binding proteins called calmodulin-regulated spectrin-associated protein (CAMSAP)/Patronin/Nezha. Patronin, the single member of this family in Drosophila, was previously shown to stabilize MT minus-ends against depolymerization in vitro and in vivo. Here, we show that all three mammalian CAMSAP family members also bind specifically to MT minus-ends and protect them against kinesin-13–induced depolymerization. However, these proteins differ in their abilities to suppress tubulin addition at minus-ends and to dissociate from MTs. CAMSAP1 does not interfere with polymerization and tracks along growing minus-ends. CAMSAP2 and CAMSAP3 decrease the rate of tubulin incorporation and remain bound, thereby creating stretches of decorated MT minus-ends. By using truncation analysis, we find that somewhat different minimal domains of CAMSAP and Patronin are involved in minus-end localization. However, we find that, in both cases, a highly conserved C-terminal domain and a more variable central domain cooperate to suppress minus-end dynamics in vitro and that both regions are required to stabilize minus-ends in Drosophila S2 cells. These results show that members of the CAMSAP/Patronin family all localize to and protect minus-ends but have evolved distinct effects on MT dynamics.Microtubules (MTs) are cellular polymers that are important for a variety of functions, including cargo transport and mitotic spindle formation. MTs are composed of dimers of α- and β-tubulin that assemble head-to-tail, creating a polar protofilament. Protofilaments then assemble laterally to form the canonical 13-protofilament MT structure, with β-tubulin exposed at fast-growing plus-ends and α-tubulin exposed at slow-growing minus-ends (1). MTs exhibit an intriguing property termed “dynamic instability,” whereby the polymer can abruptly switch between episodes of net growth and shrinkage (2). The rates of growth and shrinkage as well as the frequency of transitions between these two states are regulated by numerous MT-associated proteins, many of which bind to the ends of the polymer (3, 4).The dynamics of MT plus-ends are regulated by a well-characterized network of plus-end tracking proteins (+TIPs) (5). End-binding proteins recognize a tubulin conformation unique to the growing ends of MTs and can affect the dynamics of plus-ends by intrinsically altering the structure of the MT end (6–8) as well as recruiting other interacting proteins (9). In contrast, TOG domain-containing proteins, such as XMAP215, promote MT growth and have been suggested to act as MT “polymerases” (10, 11). Conversely, kinesin-13s [e.g., mitotic centromere-associated kinesin (MCAK) from hamster] increase instability of MT ends, leading to increased catastrophe frequency (12, 13). Thus, regulation of these and other +TIPs can dramatically affect the stability and turnover of the MT network (14, 15).In comparison with the well-characterized +TIPs, much less is known about regulation of MT minus-ends. In many cells, minus-ends in vivo are anchored at the centrosome by the γ-tubulin ring complex (γ-TuRC). However, cells such as epithelial cells and neurons have noncentrosomal MT arrays, and many mitotic spindle MTs are not directly connected to centrosomes. Minus-ends that are not connected to the centrosome appear to be highly stable, in contrast to the behavior of minus-ends composed of pure tubulin. For example, newly created minus-ends formed by breakage or laser severing tend to neither grow nor shrink, whereas newly created plus-ends tend to rapidly depolymerize (16–20). It is unclear how this stability is mediated and whether minus-end stability is regulated to control MT turnover in cells.Previous work in our laboratory identified the Drosophila protein Patronin (from the Latin patronus, protect) in a whole-genome RNAi screen for mitotic spindle formation (21) (originally named ssp4) and showed that this protein binds to and protects the MT minus-end against depolymerization by kinesin-13 in vitro and in vivo (22). Further work in flies has shown that Patronin antagonizes kinesin-13 during mitosis and that regulation of Patronin activity facilitates spindle elongation in anaphase B (23). In mammals, Takeichi and coworkers (24, 25) have shown that a homologous protein, which they termed Nezha, anchors minus-ends of MTs in cell–cell adherens junctions. The protein family was also identified through a spectrin binding activity and named calmodulin-regulated spectrin-associated protein (CAMSAP) (26, 27). Bioinformatic analyses suggest that this protein family first evolved in metazoans; invertebrates possess a single gene whereas vertebrates possess three CAMSAP genes (CAMSAP1, CAMSAP2, and CAMSAP3/Nezha; Fig. 1A) (24, 26). Recent work has demonstrated that CAMSAP2 and CAMSAP3 bind MT minus-ends and that depletion of these proteins causes a reduction in MT numbers and changes the MT organization in epithelial cells (28, 29). The Caenorhabditis elegans homolog of Patronin/CAMSAP (PTRN-1) stabilizes MTs in neurons and promotes neurite and synapse stability (30, 31).Open in a separate windowFig. 1.CAMSAPs have a conserved function of binding MT minus-ends. (A) Most vertebrate species possess three CAMSAP/Patronin homologs, whereas Drosophila and C. elegans each have only one. (B) Motor gliding assay for determining the end of the MT bound by CAMSAPs; in a kinesin gliding assay, the minus-end of the GMPCPP-stabilized MT is the leading end; polarity is reversed in a dynein gliding assay. Kinesin assay for GFP-CAMSAP2 is shown. Arrows show GFP-CAMSAP2 on MT minus-ends. (Scale bar: 5 μm.) Movement of an MT–CAMSAP2 complex over time (Scale bar: Inset, 2 μm.) (C) Quantification of number of MT with GFP-CAMSAPs on their ends. Results compiled from two independent experiments (>100 MTs scored per assay).Although these studies demonstrate that CAMSAPs and Patronin stabilize MT minus-ends in vivo, many questions remain as to how these proteins recognize minus-ends and affect their dynamics. Here, we show that all vertebrate CAMSAP family members bind to MT minus-ends in vitro. However, we find differences between the CAMSAPs and Patronin with regard to their effects on minus-end dynamics. These results indicate that minus-end binding and regulation is universal for all CAMSAP members but that this activity is tunable in ways that might be exploited by cells to regulate MT organization. While the present paper was in submission, Jiang et al. (32) reported similar findings for CAMSAP proteins in vitro; we compare our findings in the Discussion.     

Antiproliferative mechanism of action of cryptophycin-52: Kinetic stabilization of microtubule dynamics by high-affinity binding to microtubule ends

Cryptophycin-52 (LY355703) is a new synthetic member of the cryptophycin family of antimitotic antitumor agents that is currently undergoing clinical evaluation. At high concentrations (≥10 times the IC₅₀), cryptophycin-52 blocked HeLa cell proliferation at mitosis by depolymerizing spindle microtubules and disrupting chromosome organization. However, low concentrations of cryptophycin-52 inhibited cell proliferation at mitosis (IC₅₀ = 11 pM) without significantly altering spindle microtubule mass or organization. Cryptophycin-52 appears to be the most potent suppressor of microtubule dynamics found thus far. It suppressed the dynamic instability behavior of individual microtubules in vitro (IC₅₀ = 20 nM), reducing the rate and extent of shortening and growing without significantly reducing polymer mass or mean microtubule length. Using [³H]cryptophycin-52, we found that the compound bound to microtubule ends in vitro with high affinity (K_d, 47 nM, maximum of ≈19.5 cryptophycin-52 molecules per microtubule). By analyzing the effects of cryptophycin-52 on dynamics in relation to its binding to microtubules, we determined that ≈5–6 molecules of cryptophycin-52 bound to a microtubule were sufficient to decrease dynamicity by 50%. Cryptophycin-52 became concentrated in cells 730-fold, and the resulting intracellular cryptophycin-52 concentration was similar to that required to stabilize microtubule dynamics in vitro. The data suggest that cryptophycin-52 potently perturbs kinetic events at microtubule ends that are required for microtubule function during mitosis and that it acts by forming a reversible cryptophycin-52-tubulin stabilizing cap at microtubule ends.     

Resistance to Taxol in lung cancer cells associated with increased microtubule dynamics

Microtubule dynamics are crucial for mitotic spindle assembly and chromosome movement. Suppression of dynamics by Taxol appears responsible for the drug's potent ability to inhibit mitosis and cell proliferation. Although Taxol is an important chemotherapeutic agent, development of resistance limits its efficacy. To examine the role of microtubule dynamics in Taxol resistance, we measured the dynamic instability of individual rhodamine-labeled microtubules in Taxol-sensitive and -resistant living human cancer cells. Taxol-resistant A549-T12 and -T24 cell lines were selected from a human lung carcinoma cell line, A549. They are, respectively, 9- and 17-fold resistant to Taxol and require low concentrations of Taxol for proliferation. We found that microtubule dynamic instability was significantly increased in the Taxol-resistant cells. For example, with A549-T12 cells in the absence of added Taxol, microtubule dynamicity increased 57% as compared with A549 cells. The length and rate of shortening excursions increased 75 and 59%, respectively. These parameters were further increased in A549-T24 cells, with overall dynamicity increasing by 167% compared with parental cells. Thus, the decreased Taxol-sensitivity of these cells can be explained by their increased microtubule dynamics. When grown without Taxol, A549-T12 cells were blocked at the metaphase/anaphase transition and displayed abnormal mitotic spindles with uncongressed chromosomes. In the presence of 2-12 nM Taxol, the cells grew normally, suggesting that mitotic block resulted from excessive microtubule dynamics. These results indicate that microtubule dynamics play an important role in Taxol resistance, and that both excessively rapid dynamics and suppressed dynamics impair mitotic spindle function and inhibit proliferation.     

Genetic control of susceptibility to osteoporosis

Osteoporosis is a common disease with a strong genetic component. Twin studies have shown that genetic factors play an important role in regulating bone mineral density (BMD), ultrasound properties of bone, skeletal geometry, and bone turnover as well as contributing to the pathogenesis of osteoporotic fracture itself. These phenotypes are determined by the combined effects of several genes and environmental influences, but occasionally, osteoporosis or unusually high bone mass can occur as the result of mutations in a single gene. Examples are the osteoporosis-pseudoglioma syndrome, caused by inactivating mutations in the lipoprotein receptor-related protein 5 gene and the high bone mass syndrome, caused by activating mutations of the same gene. Genome-wide linkage studies in man have identified loci on chromosomes 1p36, 1q21, 2p21, 5q33-35, 6p11-12, and 11q12-13 that show definite or probable linkage to BMD, but so far, the causative genes remain to be identified. Linkage studies in mice have similarly identified several loci that regulate BMD, and a future challenge will be to investigate the syntenic loci in humans. A great deal of research has been done on candidate genes; among the best studied are the vitamin D receptor and the collagen type I alpha 1 gene. Polymorphisms of vitamin D receptor have been associated with bone mass in several studies, and there is evidence to suggest that this association may be modified by dietary calcium and vitamin D intake. A functional polymorphism affecting an Sp1 binding site has been identified in the collagen type I alpha 1 gene that predicts osteoporotic fractures independently of bone mass by influencing collagen gene regulation and bone quality. An important problem with most candidate gene studies is small sample size, and this has led to conflicting results in different populations. Some researchers are exploring the use of meta-analysis to try and address this issue and gain an accurate estimate of effect size for different polymorphisms in relation to relevant clinical endpoints, such as BMD and fracture. From a clinical standpoint, advances in knowledge about the genetic basis of osteoporosis are important, because they offer the prospect of developing genetic markers for the assessment of fracture risk and the opportunity to identify molecules that will be used as targets for the design of new drugs for the prevention and treatment of bone disease.     

SIRT1 and SIRT3 deacetylate homologous substrates: AceCS1,2 and HMGCS1,2

SIRT1 and SIRT3 are NAD+-dependent protein deacetylases that are evolutionarily conserved across mammals. These proteins are located in the cytoplasm/nucleus and mitochondria, respectively. Previous reports demonstrated that human SIRT1 deacetylates Acetyl-CoA Synthase 1 (AceCS1) in the cytoplasm, whereas SIRT3 deacetylates the homologous Acetyl-CoA Synthase 2 (AceCS2) in the mitochondria. We recently showed that 3-hydroxy-3-methylglutaryl CoA synthase 2 (HMGCS2) is deacetylated by SIRT3 in mitochondria, and we demonstrate here that SIRT1 deacetylates the homologous 3-hydroxy-3-methylglutaryl CoA synthase 1 (HMGCS1) in the cytoplasm. This novel pattern of substrate homology between cytoplasmic SIRT1 and mitochondrial SIRT3 suggests that considering evolutionary relationships between the sirtuins and their substrates may help to identify and understand the functions and interactions of this gene family. In this perspective, we take a first step by characterizing the evolutionary history of the sirtuins and these substrate families.     

Cold temperatures reduce the sensitivity of stored platelets to disaggregating agents

In this study, we compared the effect of signal transduction inhibitors on fibrinogen binding, aggregation, the activation state of GPIIb-IIIa, and cytosolic calcium levels in cold and room temperature-stored platelets. Cold-stored platelets have a higher sensitivity to agonist-induced aggregation when compared to room temperature-stored platelets. We also found that cold-stored platelets had a significantly higher aggregation response to ADP and epinephrine, while platelets stored at room temperature responded poorly to these agonists (mean values of 61 vs. 18%, n = 14). Four inhibitors were selected to target various signaling pathways. Cold-stored platelets were more resistant to disaggregation by promethazine, prostaglandin D2, yohimbine, and echistatin. The effects of cold temperatures on stored platelets are targeted to activation pathways as there was no spontaneous aggregation or spontaneous fibrinogen binding as measured in this study. PAC-1 binding was not inhibited to the same degree as aggregation or fibrinogen binding responses, suggesting that the disaggregation was not caused by a change in the conformation of GPIIb-IIIa. Cytosolic calcium levels did not decrease in cold-stored platelets after inhibitor addition. The inhibitors are likely acting after the establishment of the GPIIb-IIIa activation state and may affect the post-occupancy signaling by the fibrinogen-occupied integrin. Differences between aggregation and disaggregation responses of cold- and room temperature-stored platelets suggest that cold-stored platelets may have different mechanisms to stabilize platelet aggregates during their formation.     

Cold temperatures reduce the sensitivity of stored platelets to disaggregating agents

In this study, we compared the effect of signal transduction inhibitors on fibrinogen binding, aggregation, the activation state of GPIIb-IIIa, and cytosolic calcium levels in cold and room temperature-stored platelets. Cold-stored platelets have a higher sensitivity to agonist-induced aggregation when compared to room temperature-stored platelets. We also found that cold-stored platelets had a significantly higher aggregation response to ADP and epinephrine, while platelets stored at room temperature responded poorly to these agonists (mean values of 61 vs. 18%, n = 14). Four inhibitors were selected to target various signaling pathways. Cold-stored platelets were more resistant to disaggregation by promethazine, prostaglandin D2, yohimbine, and echistatin. The effects of cold temperatures on stored platelets are targeted to activation pathways as there was no spontaneous aggregation or spontaneous fibrinogen binding as measured in this study. PAC-1 binding was not inhibited to the same degree as aggregation or fibrinogen binding responses, suggesting that the disaggregation was not caused by a change in the conformation of GPIIb-IIIa. Cytosolic calcium levels did not decrease in cold-stored platelets after inhibitor addition. The inhibitors are likely acting after the establishment of the GPIIb-IIIa activation state and may affect the post-occupancy signaling by the fibrinogen-occupied integrin. Differences between aggregation and disaggregation responses of cold- and room temperature-stored platelets suggest that cold-stored platelets may have different mechanisms to stabilize platelet aggregates during their formation.     

猪三毛滴虫对4种药物的体外药敏试验

目的探讨硝唑尼特、替硝唑、甲硝唑和制霉菌素4种药物对猪三毛滴虫的作用效果,以期为临床用药提供依据。方法在体外培养条件下,用24h敏感性试验确定4种药物对猪三毛滴虫24h的最低致死浓度(MLC);绘制时间-杀虫曲线,分析不同浓度受试药物作用不同时间对猪三毛滴虫的杀灭效果。结果 4种药物均具有不同程度的抑制或杀灭猪三毛滴虫的作用,其中硝唑尼特杀灭猪三毛滴虫的效果较好,24hMLC为12.50mg/L;替硝唑和甲硝唑24hMLC均为25.00mg/L,但时间-杀虫曲线显示替硝唑效果好于甲硝唑;制霉菌素的杀虫效果较差。结论猪三毛滴虫对硝唑尼特和替硝唑敏感。     

Aging increases pulmonary veins arrhythmogenesis and susceptibility to calcium regulation agents.

BACKGROUND: Aging and pulmonary veins (PVs) play a critical role in the pathophysiology of atrial fibrillation. Abnormal Ca(2+) regulation and ryanodine receptors are known to contribute to PV arrhythmogenesis. OBJECTIVE: The purpose of this study was to investigate whether aging alters PV electrophysiology, Ca(2+) regulation proteins, and responses to rapamycin, FK-506, ryanodine, and ouabain. METHODS: Conventional microelectrodes were used to record action potential and contractility in isolated PV tissue samples in 15 young (age 3 months) and 16 aged (age 3 years) rabbits before and after drug administration. Expression of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a), ryanodine receptor, and Na(+)/Ca(2+) exchanger was evaluated by western blot. RESULTS: Aged PVs had larger amplitude of delayed afterdepolarizations, greater depolarized resting membrane potential, longer action potential duration, and higher incidence of action potential alternans and contractile alternans with increased expression of Na(+)/Ca(2+) exchanger and ryanodine receptor and decreased expression of SERCA2a. Rapamycin (1,10,100 nM), FK-506 (0.01, 0.1, 1 microM), ryanodine (0.1, 1 microM), and ouabain (0.1, 1 microM) concentration-dependently increased PV spontaneous rates and the incidence of delayed afterdepolarizations in young and aged PVs. Compared with results in young PVs, rapamycin and FK-506 in aged PVs increased PV spontaneous rates to a greater extent and exhibited a larger delayed afterdepolarization amplitude. In PVs without spontaneous activity, rapamycin and FK-506 induced spontaneous activity only in aged PVs, but ryanodine and ouabain induced spontaneous activity in both young and aged PVs. CONCLUSION: Aging increases PV arrhythmogenesis via abnormal Ca(2+) regulation. These findings support the concept that ryanodine receptor dysfunction may result in high PV arrhythmogenesis and aging-related arrhythmogenic vulnerability.