Disruption of PMR1, Encoding a Ca2+-ATPase Homolog in Yarrowia lipolytica, Affects Secretion and Processing of Homologous and Heterologous Proteins

The Yarrowia lipolytica PMR1 gene (YlPMR1) is a Saccharomyces cerevisiae PMR1 homolog which encodes a putative secretory pathway Ca²⁺-ATPase. In this study, we investigated the effects of a YlPMR1 disruption on the processing and secretion of native and foreign proteins in Y. lipolytica and found variable responses by the YlPMR1-disrupted mutant depending on the protein. The secretion of 32-kDa mature alkaline extracellular protease (AEP) was dramatically decreased, and incompletely processed precursors were observed in the YlPMR1-disrupted mutant. A 36- and a 52-kDa premature AEP were secreted, and an intracellular 52-kDa premature AEP was also detected. The acid extracellular protease activity of the YlPMR1-disrupted mutant was increased by 60% compared to that of the wild-type strain. The inhibitory effect of mutations in secretory pathway Ca²⁺-ATPase genes on the secretion of rice α-amylase was also observed in the Y. lipolytica and S. cerevisiae PMR1-disrupted mutants. Unlike rice α-amylase, the secretion of Trichoderma reesei endoglucanase I (EGI) was not influenced by the YlPMR1 disruption. However, the secreted EGI from the YlPMR1-disrupted mutant had different characteristics than that of the control. While wild-type cells secreted the hyperglycosylated form of EGI, hyperglycosylation was completely absent in the YlPMR1-disrupted mutant. Our results indicate that the effects of the YlPMR1 disruption as manifested by the phenotypic response depend on the characteristics of the reporter protein in the recombinant yeast strain evaluated.     

A tomato ER-type Ca-ATPase, LCA1, has a low thapsigargin-sensitivity and can transport manganese

Recombinant Ca²⁺-ATPase from tomato (i.e. LCA1 for Lycopersicon esculentum [Since the identification and naming of LCA1, the scientific name for the tomato has been changed to Solanum lycopersicum.] Ca-ATPase) was heterologously expressed in yeast for structure-function characterization. We investigate the differences between plant and animal Ca pumps utilizing comparisons between chicken and rabbit SERCA-type pumps with Arabidopsis (ECA1) and tomato plant (LCA1) Ca²⁺-ATPases. Enzyme function was confirmed by the ability of each Ca²⁺-ATPase to rescue K616 growth on EGTA-containing agar and directly via in vitro ATP hydrolysis. We found LCA1 to be ∼300-fold less sensitive to thapsigargin than animal SERCAs, whereas ECA1 was thapsigargin-resistant. LCA1 showed typical pharmacological sensitivities to cyclopiazonic acid, vanadate, and eosin, consistent with it being a P_IIA-type Ca²⁺-ATPase. Possible amino acid changes responsible for the reduced plant thapsigargin-sensitivity are discussed. We found that LCA1 also complemented K616 yeast growth in the presence of Mn²⁺, consistent with moving Mn²⁺ into the secretory pathway and functionally compensating for the lack of secretory pathway Ca-ATPases (SPCAs) in plants.     

Enhancement of polysaccharide production in suspension cultures of protocorm-like bodies from Dendrobium huoshanense by optimization of medium compositions and feeding of sucrose

A strategy that optimization of medium compositions for maximum biomass followed by feeding of sucrose for maximum polysaccharide synthesis was developed for enhancing polysaccharide production in suspension culture of protocorm-like bodies (PLBs) of Dendrobium huoshanense C.Z. Tang et S.J. Cheng. In growth stage, the original half-strength MS medium was optimized with carbon sources, nitrogen sources and metal ion combinations. The effects of different carbon sources on PLBs growth were remarkable and sucrose at 35 g l⁻¹ was the most suitable. Sole nitrate nitrogen of 30 mmol l⁻¹ was the best for PLBs growth. Metal ions (Ca²⁺, Fe²⁺, Mn²⁺ and Zn²⁺) showed different influences on PLBs growth. The optimal concentration of Ca²⁺, Fe²⁺, Mn²⁺ and Zn²⁺ was 4.5 mmol l⁻¹, 0.1 mmol l⁻¹, 0.5 mmol l⁻¹ and 0.06 mmol l⁻¹, respectively. In the optimized medium (sucrose, nitrate, Ca²⁺, Fe²⁺, Mn²⁺ and Zn²⁺ concentration as described above, the other component concentration seen in half-strength MS), 33.9 g DW l⁻¹ PLBs were harvested after 30 days of culture and biomass increase was improved 245% as compared with that in the original medium. In production stage, polysaccharide synthesis was significantly improved by the feeding sucrose. The maximum polysaccharide production (22 g l⁻¹) was obtained in the case of 50 g l⁻¹ sucrose feeding at day 30 of culture, which was about 109-fold higher than that in the original medium without feeding of sucrose.     

Investigating the function of Gdt1p in yeast Golgi glycosylation

The Golgi ion homeostasis is tightly regulated to ensure essential cellular processes such as glycosylation, yet our understanding of this regulation remains incomplete. Gdt1p is a member of the conserved Uncharacterized Protein Family (UPF0016). Our previous work suggested that Gdt1p may function in the Golgi by regulating Golgi Ca^2 +/Mn^2 + homeostasis. NMR structural analysis of the polymannan chains isolated from yeasts showed that the gdt1Δ mutant cultured in presence of high Ca^2 + concentration, as well as the pmr1Δ and gdt1Δ/pmr1Δ strains presented strong late Golgi glycosylation defects with a lack of α-1,2 mannoses substitution and α-1,3 mannoses termination. The addition of Mn^2 + confirmed the rescue of these defects. Interestingly, our structural data confirmed that the glycosylation defect in pmr1Δ could also completely be suppressed by the addition of Ca^2 +. The use of Pmr1p mutants either defective for Ca^2 + or Mn^2 + transport or both revealed that the suppression of the observed glycosylation defect in pmr1Δ strains by the intraluminal Golgi Ca^2 + requires the activity of Gdt1p. These data support the hypothesis that Gdt1p, in order to sustain the Golgi glycosylation process, imports Mn^2 + inside the Golgi lumen when Pmr1p exclusively transports Ca^2 +. Our results also reinforce the functional link between Gdt1p and Pmr1p as we highlighted that Gdt1p was a Mn^2 + sensitive protein whose abundance was directly dependent on the nature of the ion transported by Pmr1p. Finally, this study demonstrated that the aspartic residues of the two conserved motifs E-x-G-D-[KR], likely constituting the cation binding sites of Gdt1p, play a crucial role in Golgi glycosylation and hence in Mn^2 +/Ca^2 + transport.     

Metal ion dependence of DNA cleavage by SepMI and EhoI restriction endonucleases

Most of type II restriction endonucleases show an absolute requirement for divalent metal ions as cofactors for DNA cleavage. While Mg²⁺ is the natural cofactor other metal ions can substitute it and mediate the catalysis, however Ca²⁺ (alone) only supports DNA binding. To investigate the role of Mg²⁺ in DNA cleavage by restriction endonucleases, we have studied the Mg²⁺ and Mn²⁺ concentration dependence of DNA cleavage by SepMI and EhoI. Digestion reactions were carried out at different Mg²⁺ and Mn²⁺ concentrations at constant ionic strength. These enzymes showed different behavior regarding the ions requirement, SepMI reached near maximal level of activity between 10 and 20 mM while no activity was detected in the presence of Mn²⁺ and in the presence of Ca²⁺ cleavage activity was significantly decreased. However, EhoI was more highly active in the presence of Mn²⁺ than in the presence of Mg²⁺ and can be activated by Ca²⁺. Our results propose the two-metal ion mechanism for EhoI and the one-metal ion mechanism for SepMI restriction endonuclease. The analysis of the kinetic parameters under steady state conditions showed that SepMI had a K_m value for pTrcHisB DNA of 6.15 nM and a V_max of 1.79 × 10^?2 nM min^?1, while EhoI had a K_m for pUC19 plasmid of 8.66 nM and a V_max of 2 × 10^?2 nM min^?1.     

Isolation and divalent-metal activation of a β-xylosidase,RUM630-BX

The gene encoding RUM630-BX, a β-xylosidase/arabinofuranosidase, was identified from activity-based screening of a cow rumen metagenomic library. The recombinant enzyme is activated as much as 14-fold (k_cat) by divalent metals Mg²⁺, Mn²⁺ and Co²⁺ but not by Ca²⁺, Ni²⁺, and Zn²⁺. Activation of RUM630-BX by Mg²⁺ (t_0.5 144 s) is slowed two-fold by prior incubation with substrate, consistent with the X-ray structure of closely related xylosidase RS223-BX that shows the divalent-metal activator is at the back of the active-site pocket so that bound substrate could block its entrance. The enzyme is considerably more active on natural substrates than artificial substrates, with activity (k_cat/K_m) of 299 s⁻¹ mM⁻¹ on xylotetraose being the highest reported.     

Arginase from Bacillus thuringiensis SK 20.001: Purification,characteristics, and implications for l-ornithine biosynthesis

An l-ornithine high producing strain Bacillus thuringiensis SK20.001 was screened by our laboratory. An intracellular arginase used to biosynthesize l-ornithine from the strain was purified and characterized. The final specific arginase activity was 589.2 units/mg, with 70.1 fold enrichment and 22.4% recovery. The molecular weight of the enzyme was approximately 33,000 Da as evaluated by SDS-PAGE and 191,000 Da as determined by gel filtration. The enzyme had an optimum pH of 10.0 and an optimum temperature of 40 °C. It was stable from pH 8.0–12.0 and <50 °C without Mn²⁺. The presence of Mn²⁺ and Ni²⁺ had strong effects on the enzyme activity, and Mn²⁺ significantly increased the thermal stability of the enzyme. The arginase was slightly inhibited by Ca²⁺, Fe²⁺ and Zn²⁺. Trp, Asp, Glu, Tyr, and Arg residues were directly involved in the arginase activity evaluated by chemical modifications. The K_m and V_max for l-arginine were estimated to be 15.6 mM and 538.9 μmol/min/mg. The biosynthesis yield of l-ornithine was 72.7 g/L with the enzyme.     

Caloxin 1b3: A novel plasma membrane Ca2+-pump isoform 1 selective inhibitor that increases cytosolic Ca2+ in endothelial cells

The purpose of this study was to invent an extracellular inhibitor selective for the plasma membrane Ca²⁺ pump(s) (PMCA) isoform 1. PMCA extrude Ca²⁺ from cells during signalling and homeostasis. PMCA isoforms are encoded by 4 genes (PMCA1–4). Pig coronary artery endothelium and smooth muscle express the genes PMCA1 and 4. We showed that the endothelial cells contained mostly PMCA1 protein while smooth muscle cells had mostly PMCA4. A random peptide phage display library was screened for binding to synthetic extracellular domain 1 of PMCA1. The selected phage population was screened further by affinity chromatography using PMCA from rabbit duodenal mucosa which expressed mostly PMCA1. The peptide displayed by the selected phage was termed caloxin 1b3. Caloxin 1b3 inhibited PMCA Ca²⁺–Mg²⁺-ATPase in the rabbit duodenal mucosa (PMCA1) with a greater affinity (inhibition constant = 17 ± 2 μM) than the PMCA in the human erythrocyte ghosts (PMCA4, inhibition constant = 45 ± 4 μM). The affinity of caloxin 1b3 was also higher for PMCA1 than for PMCA2 and 3 indicating its selectivity for PMCA1. Consistent with an inhibition of PMCA1, caloxin 1b3 addition to the medium increased cytosolic Ca²⁺ concentration in endothelial cells. Caloxin 1b3 is the first known PMCA1 selective inhibitor. We anticipate caloxin 1b3 to aid in understanding PMCA physiology in endothelium and other tissues.     

Improved high thermal stability of pullulanase from a newly isolated thermophilic Bacillus sp. AN-7

A thermophilic Bacillus sp. strain AN-7, isolated from a soil in India, produced an extracellular pullulanase upon growth on starch–peptone medium. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The optimum temperature and pH for activity was 90 °C and 6.0. With half-life time longer than one day at 80 °C the enzyme proves to be thermostable in the pH range 4.5–7.0. The pullulanase from Bacillus strain lost activity rapidly when incubated at temperature higher than 105 °C or at pH lower than 4.5. Pullulanase was completely inhibited by the Hg²⁺ ions. Ca²⁺, dithiothreitol, and Mn²⁺ stimulated the pullulanase activity. Kinetic experiments at 80 °C and pH 6.0 gave V_max and K_m values of 154 U mg⁻¹ and 1.3 mg ml⁻¹. The products of pullulan were maltotriose and maltose. This proved that the purified pullulanase (pullulan-6-glucanohydrolase, EC 3.2.1.41) from Bacillus sp. AN-7 is classified under pullulanase type I. To our knowledge, this Bacillus pullulanase is the most highly thermostable type I pullulanase known to date.     

Effects of chronic exposure to ammonia concentrations on brain monoamines and ATPases of Nile tilapia (Oreochromis niloticus)

The effects of chronic exposure to total ammonia nitrogen (TAN) concentrations on the brain monoamines and ATPases of Nile tilapia, Oreochromis niloticus fingerlings, were studied. The period of exposure was 70 consecutive days, and the initial weight of the fingerlings was 18 ± 2.1 g. In addition to the control, three treatment groups exposed to 2.5 (low), 5 (medium), and 10 (high) mg TAN L^?1 concentrations were tested. The unionized ammonia nitrogen (NH₃) levels calculated in mg L^?1 were 0.059, 0.185, and 0.575 in aquaria at 26 °C. The brain monoamines were serotonin (5-HT), dopamine (DA), and norepinephrine (NE), as well as their derivatives, 5-hydroxyindoleacetic acid (5-HIAA) and dihydroxyphenylacetic acid (DOPAC). Compared with the controls, the levels of brain monoamines and Na⁺/K⁺- and Ca²⁺-ATPase activities were not significantly altered in fish exposed to low TAN concentration. However, there was a significant decrease in 5-HT, DA, and NE levels, and a significant increase in both serotonergic (5-HIAA/5-HT) and dopaminergic (DOPAC/DA) activities of fish exposed to medium TAN and high TAN concentrations. The activities of brain Na⁺/K⁺- and Ca²⁺-ATPases of fish exposed to medium TAN and high TAN concentrations significantly increased, while Mg²⁺-ATPase did not significantly change compared with that of the controls. The quantity of the detected alterations increased in fish exposed to high TAN concentration.     

Extracellular cold-active lipase of Microbacterium luteolum isolated from Gangotri glacier,western Himalaya: Isolation,partial purification and characterization

A psychrophilic bacterium producing cold-active lipase upon growth at low temperature was isolated from the soil samples of Gangotri glacier and identified as Microbacterium luteolum. The bacterial strain produced maximum lipase at 15 °C, at a pH of 8.0. Beef extract served as the best organic nitrogen source and ammonium nitrate as inorganic for maximum lipase production. Castor oil served as an inducer and glucose served as an additional carbon source for production of cold-active lipase. Ferric chloride as additional mineral salt in the medium, highly influenced the lipase production with an activity of 8.01 U ml^?1. The cold-active lipase was purified to 35.64-fold by DEAE-cellulose column chromatography. It showed maximum activity at 5 °C and thermostability up to 35 °C. The purified lipase was stable between pH 5 and 9 and the optimal pH for enzymatic hydrolysis was 8.0. Lipase activity was stimulated in presence of all the solvents (5%) tested except with acetonitrile. Lipase activity was inhibited in presence of Mn²⁺, Cu²⁺, and Hg²⁺; whereas Fe⁺, Na⁺ did not have any inhibitory effect on the enzyme activity. The purified lipase was stable in the presence of SDS; however, EDTA and dithiothreitol inhibited enzyme activity. Presence of Ca²⁺ along with inhibitors stabilized lipase activity. The cold active lipase thus exhibiting activity and stability at a low temperature and alkaline pH appears to be practically useful in industrial applications especially in detergent formulations.     

Redox-sensitive stimulation of type-1 ryanodine receptors by the scorpion toxin maurocalcine

The scorpion toxin maurocalcine acts as a high affinity agonist of the type-1 ryanodine receptor expressed in skeletal muscle. Here, we investigated the effects of the reducing agent dithiothreitol or the oxidizing reagent thimerosal on type-1 ryanodine receptor stimulation by maurocalcine. Maurocalcine addition to sarcoplasmic reticulum vesicles actively loaded with calcium elicited Ca²⁺ release from native vesicles and from vesicles pre-incubated with dithiothreitol; thimerosal addition to native vesicles after Ca²⁺ uptake completion prevented this response. Maurocalcine enhanced equilibrium [³H]-ryanodine binding to native and to dithiothreitol-treated reticulum vesicles, and increased 5-fold the apparent K_i for Mg²⁺ inhibition of [³H]-ryanodine binding to native vesicles. Single calcium release channels incorporated in planar lipid bilayers displayed a long-lived open sub-conductance state after maurocalcine addition. The fractional time spent in this sub-conductance state decreased when lowering cytoplasmic [Ca²⁺] from 10 μM to 0.1 μM or at cytoplasmic [Mg²⁺] ≥ 30 μM. At 0.1 μM [Ca²⁺], only channels that displayed poor activation by Ca²⁺ were readily activated by 5 nM maurocalcine; subsequent incubation with thimerosal abolished the sub-conductance state induced by maurocalcine. We interpret these results as an indication that maurocalcine acts as a more effective type-1 ryanodine receptor channel agonist under reducing conditions.     

Characterization of a thermophilic l-arabinose isomerase from Thermoanaerobacterium saccharolyticum NTOU1

l-Arabinose isomerase (EC 5.3.1.4, l-AI) mainly catalyzes the reversible aldose–ketose isomerization between l-arabinose and l-ribulose. l-AIs can also catalyze other reactions, such as the conversion of d-galactose to d-tagatose. In this study, the araA gene encoding l-AI was PCR-cloned from Thermoanaerobacterium saccharolyticum NTOU1 and then expressed in Escherichia coli. The recombinant l-AI was purified from the cell-free extract using nickel nitrilotriacetic acid metal-affinity chromatography. The purified enzyme showed an optimal activity at 70 °C and pH 7–7.5. The enzyme was stable at pHs ranging from 6.5 to 9.5 and the activity was fully retained after 2 h incubation at 55–65 °C. The low concentrations of divalent metal ions, either 0.1 mM Mn²⁺ or 0.05 mM Co²⁺, could improve both catalytic activity and thermostability at higher temperatures. The recombinant T. saccharolyticum NTOU1 l-AI has the lowest demand for metal ions among all characterized thermophilic l-AIs. This thermophilic l-AI shows a potential to be used in industry to produce d-tagatose from d-galactose.     

Protective effect of β-casomorphin-7 on cardiomyopathy of streptozotocin-induced diabetic rats via inhibition of hyperglycemia and oxidative stress

β-Casomorphin-7 (β-CM-7) is regarded as the most representative milk-derived bioactive peptide. The present work studies the efficacy of β-CM-7 against myocardial injury in streptozotocin-induced diabetic rats, focusing on the following assays: (1) the level of blood glucose and advanced glycosylation end product (AGE), the activity of lactate dehydrogenase (LDH) in serum; (2) the level of hydrogen peroxide (H₂O₂), the activity of Na⁺K⁺-ATPase, Ca²⁺Mg²⁺-ATPase and enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) in myocardial tissue; (3) the protein expression of glucose transporter-4 (GLUT-4) in myocardial tissue. It showed that with the influence of β-CM-7, the levels of blood glucose of β-CM-7 treatment group decreased markedly compared with model group (P < 0.01) accompanied with their alleviated symptoms of diabetes. In the antioxidant and oxidant levels, β-CM-7 treatment group signified a remarkable increase in the activity of GSH-Px, SOD and CAT of the anti-oxidation system and meanwhile demonstrated a considerable reduction in H₂O₂ content (all P < 0.05) in comparison with model group. We also found both the content of AGE and the activity of LDH of β-CM-7 treated group considerably reduced while the content of GLUT-4 and the activity of Na⁺K⁺-ATPase and Ca²⁺Mg²⁺-ATPase of β-CM-7 treated group increased obviously (P < 0.05). Meanwhile the cardiac indexes were significantly lessened. Thus our assay validates that the remedy employing β-CM-7 may treat diabetic cardiomyopathy with high efficacy predominantly associated with the mechanism that β-CM-7 ameliorates myocardial energy metabolism and abates free-radical-mediated oxidative stress in blood and myocardium.     

The effects of clove oil on the enzyme activity of Varroa destructor Anderson and Trueman (Arachnida: Acari: Varroidae)

Varroa destructor, a key biotic threat to the Western honey bee, has played a major role in colony losses over the past few years worldwide. Overuse of traditional acaricides, such as tau-fluvalinate and flumethrin, on V. destructor has only increased its tolerance to them. Therefore, the application of essential oils in place of traditional pesticides is an attractive alternative, as demonstrated by its high efficiency, lack of residue and tolerance resistance. To study the acaricidal activity of essential oils, we used clove oil (Syzygium aromaticum L.), a typical essential oil with a wide range of field applications, and examined its effects on the enzyme activities of Ca²⁺-Mg²⁺-ATPase, glutathione-S-transferase (GST) and superoxide dismutase (SOD) and its effects on the water-soluble protein content of V. destructor body extracts after exposure to 0.1 μl and 1.0 μl of clove oil for 30 min. Our results showed that the water-soluble protein content significantly decreased after the treatments, indicating that the metabolism of the mites was adversely affected. The bioactivity of GSTs increased significantly after a low dosage (0.1 μl) exposure but decreased at a higher dosage (1.0 μl), while the activities of SOD and Ca²⁺-Mg²⁺-ATPase were significantly elevated after treatments. These results suggest that the protective enzyme SOD and detoxifying enzymes Ca²⁺-Mg²⁺-ATPase and GST contributed to the stress reaction of V. destructor to the essential oils and that the detoxification ability of V. destructor via GST was inhibited at higher dosages. Our findings are conducive to understanding the physiological reactions of V. destructor to treatment with essential oils and the underlying mechanisms behind the acaricidal activities of these natural products.     

Highly thermostable carbonic anhydrase from Persephonella marina EX-H1: Its expression and characterization for CO2-sequestration applications

The codon-optimized carbonic anhydrase gene of Persephonella marina EX-H1 (PMCA) was expressed and characterized. The gene with the signal peptide removed, PMCA(sp−), resulted in the production of approximately five times more purified protein than from the intact gene PMCA using an Escherichia coli expression system. PMCA(sp−) is formed as homo-dimer complex. PMCA(sp−) has a wide pH tolerance (optimum pH 7.5) and a high thermostability even at 100 °C (88 min of thermal deactivation half-life). The melting temperature for PMCA(sp−) was 84.5 °C. The apparent k_cat and K_m values for CO₂ hydration were 3.2 × 10⁵ s⁻¹ and 10.8 mM. The activity of the PMCA(sp−) enzyme was enhanced by Zn²⁺, Co²⁺, and Mg²⁺, but was strongly inhibited by Cu²⁺, Fe³⁺, Al³⁺, Pb²⁺, Ag⁺, and Hg²⁺. PMCA(sp−) readily catalyzed the hydration of CO₂, precipitating CaCO₃ as calcite in the presence of Ca²⁺.     

Elucidation of antimicrobial activity and mechanism of action by N-substituted carbazole derivatives

Compounds belonging to a carbazole series have been identified as potent fungal plasma membrane proton adenosine triphophatase (H⁺-ATPase) inhibitors with a broad spectrum of antifungal activity. The carbazole compounds inhibit the adenosine triphosphate (ATP) hydrolysis activity of the essential fungal H⁺-ATPase, thereby functionally inhibiting the extrusion of protons and extracellular acidification, processes that are responsible for maintaining high plasma membrane potential. The compound class binds to and inhibits the H⁺-ATPase within minutes, leading to fungal death after 1–3 h of compound exposure in vitro. The tested compounds are not selective for the fungal H⁺-ATPase, exhibiting an overlap of inhibitory activity with the mammalian protein family of P-type ATPases; the sarco(endo)plasmic reticulum calcium ATPase (Ca²⁺-ATPase) and the sodium potassium ATPase (Na⁺,K⁺-ATPase). The ion transport in the P-type ATPases is energized by the conversion of ATP to adenosine diphosphate (ADP) and phosphate and a general inhibitory mechanism mediated by the carbazole derivative could therefore be blocking of the active site. However, biochemical studies show that increased concentrations of ATP do not change the inhibitory activity of the carbazoles suggesting they act as allosteric inhibitors. Furthermore decreased levels of intracellular ATP would suggest that the compounds inhibit the H⁺-ATPase indirectly, but Candida albicans cells exposed to potent H⁺-ATPase-inhibitory carbazoles result in increased levels of intracellular ATP, indicating direct inhibition of H⁺-ATPase.     

C1q/tumor necrosis factor-related protein-3 enhances the contractility of cardiomyocyte by increasing calcium sensitivity

C1q/tumor necrosis factor-related protein-3 (CTRP3) is an adipokine that protects against myocardial infarction-induced cardiac dysfunction through its pro-angiogenic, anti-apoptotic, and anti-fibrotic effects. However, whether CTRP3 can directly affect the systolic and diastolic function of cardiomyocytes remains unknown. Adult rat cardiomyocytes were isolated and loaded with Fura-2AM. The contraction and Ca²⁺ transient data was collected and analyzed by IonOptix system. 1 and 2 μg/ml CTRP3 significantly increased the contraction of cardiomyocytes. However, CTRP3 did not alter the diastolic Ca²⁺ content, systolic Ca²⁺ content, Ca²⁺ transient amplitude, and L-type Ca²⁺ channel current. To reveal whether CTRP3 affects the Ca²⁺ sensitivity of cardiomyocytes, the typical phase-plane diagrams of sarcomere length vs. Fura-2 ratio was performed. We observed a left-ward shifting of the late relaxation trajectory after CTRP3 perfusion, as quantified by decreased Ca²⁺ content at 50% sarcomere relaxation, and increased mean gradient (μm/Fura-2 ratio) during 500–600 ms (-0.163 vs. −0.279), 500–700 ms (-0.159 vs. −0.248), and 500–800 ms (-0.148 vs. −0.243). Consistently, the phosphorylation level of cardiac troponin I at Ser23/24 was reduced by CTRP3, which could be eliminated by preincubation of okadaic acid, a type 2A protein phosphatase inhibitor. In summary, CTRP3 increases the contraction of cardiomyocytes by increasing the myofilament Ca²⁺ sensitivity. CTRP3 might be a potential endogenous Ca²⁺ sensitizer that modulates the contractility of cardiomyocytes.