KCNQ1 G219E and TRPM4 T160M polymorphisms are involved in the pathogenesis of long QT syndrome: A case report

Rationale:Long QT syndrome (LQTS) is an inheritable disease characterized by prolonged QT interval on the electrocardiogram. The pathogenesis of LQTS is related to mutations in LQTS-susceptible genes encoding cardiac ion channel proteins or subunits.Patient concerns:Here, we reported a 37-year-old female Uygur patient with palpitation and loss of consciousness.Diagnoses:At the time of admission, a 12-lead electrocardiogram showed a QTc interval of 514 ms. Genetic analysis revealed KCNQ1 G219E and TRPM4 T160M mutations.Interventions:Although beta-blockers remain the mainstay in treating LQTS, the patient underwent implantation of an automatic cardioverter defibrillator due to life-threatening arrhythmias.Outcomes:To explore the effect of the calcium ion antagonist verapamil on ion channels, we generated human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) from the peripheral blood mononuclear cells of the patient. The changes of action potential duration in response to verapamil were observed.Lessons:Our results showed that patient-derived hiPSC-CMs could recapitulate the electrophysiological features of LQTS and display pharmaceutical responses to verapamil.     

Functional interaction between native G protein-coupled purinergic receptors in Xenopus follicles

Purinergic receptors are expressed in the membrane of the follicular cell layer that communicates with the Xenopus oocyte. Adenosine (Ado) generates a cAMP-dependent K⁺ current (I_K,cAMP), whereas ATP activates a Cl⁻ current (F_Cl) and has a dual effect on I_K,cAMP, provoking both its activation and inhibition. Here, purinergic responses were studied electrophysiologically, first in the whole follicle (w.f.), and then in the same follicle after removal of its epithelium/theca layers (e.t.r. follicle). Responses were analyzed as the ratio of the current amplitudes (i_etr/i_wf) in the two preparations. For ATP activation of I_K,cAMP and F_Cl, the ratios i_etr/i_wf were 0.053 and 22, respectively, whereas that for Ado was 0.75. Thus, epithelium/theca removal drastically altered the ATP response, suggesting a change in the signaling pathway that correlated with changes in the pharmacological characteristics: the half-maximal effective concentration for activation of the main current in w.f. (I_K,cAMP) was 14 ± 3.8 μM [Hill coefficient (nH) = 2.7 ± 0.61], and that in e.t.r. follicles (F_Cl) was 1.8 ± 0.68 μM (nH = 0.76 ± 0.09), whereas Ado-response parameters did not change. Responses to UTP and β,γ-methylene-ATP, specific agonists for I_K,cAMP inhibition and activation, respectively, indicated that in e.t.r. follicles inhibition increased and activation decreased drastically. Thus, purinergic responses were not independent; instead, they were functionally linked. We hypothesize that this property was due to direct interactions between receptors for Ado (A2 subtype) and ATP (P2Y subtype) in the Xenopus follicle.     

Adenosine triggers preconditioning through MEK/ERK1/2 signalling pathway during hypoxia/reoxygenation in neonatal rat cardiomyocytes

Three subtypes of adenosine receptors (A(1), A(2A) and A(3) ARs) are functionally expressed in cardiomyocytes. Adenosine released during ischemia and ischemia/reperfusion plays a major role in cardioprotection. Phosphatidylinositol 3-kinase (PI-3K)/protein kinase B (PKB) and MEK/ERK1/2 pathways are involved in cell survival. Since the role of these pathways in AR-mediated preconditioning is poorly understood, we have investigated whether PI-3K/PKB and/or MEK1/ERK1/2 pathways are involved in AR-induced cardioprotection in neonatal rat cardiomyocytes. Cells were pre-treated (15 min) with adenosine (non-selective), CPA (A(1)), CGS 21680 (A(2A)) or Cl-IB-MECA (A(3)) before 4 h hypoxia (0.5% O(2)) and 18 h reoxygenation (HX4/R). HX4/R-induced increase in LDH release was significantly reduced by adenosine (70%), CPA (59%) and Cl-IB-MECA (46%). The MEK1 inhibitor PD 98059 suppressed the effects of adenosine, CPA, and Cl-IB-MECA on LDH release, whereas the PI-3K inhibitor wortmannin did not reverse this cardioprotection. Western blotting of phosphorylated ERK1/2 and PKB during HX4/R supported the involvement of ERK1/2 and not PKB in A(1) and A(3) agonist-mediated cardioprotection. In addition, adenosine, CPA and Cl-IB-MECA inhibited HX4/R-induced caspase 3 activity by 75%, 70% and 59%, respectively, and this inhibition was abolished by PD 98059. Interestingly, wortmannin inhibited by 66% the anti-apoptotic response triggered by Cl-IB-MECA but had no effect on adenosine or CPA-induced inhibition of caspase 3. CGS 21680 did not modify cell survival or caspase 3 activity. In conclusion, these data show that the preconditioning effect of adenosine requires A(1) and A(3) but not A(2A) ARs and involves an anti-apoptotic effect via MEK1/ERK1/2 pathway in neonatal rat cardiomyocytes. In addition, A(3)AR-induced preconditioning also involves a PI-3K dependent pathway.     

The role of leukotriene B4 in Clostridium difficile toxin A-induced ileitis in rats

BACKGROUND & AIMS: Clostridium difficile toxin A is a potent intestinal inflammatory agent that has been shown to act at least partially by neurogenic mechanisms involving activation of the transient receptor potential vanilloid 1 (TRPV1) (capsaicin) receptor. We tested the hypothesis that leukotriene B4 (LTB4) mediates the effects of toxin A via activation of the TRPV1 receptor. METHODS: Isolated rat ileal segments were pretreated with pharmacologic agents before intraluminal injection of toxin A or LTB4. After 3 hours, the treated segments were removed and inflammation was assessed by luminal fluid accumulation, myeloperoxidase activity, and histology. RESULTS: LTB4 caused ileitis similar to that caused by toxin A and antagonism of TRPV1 receptors but not LTB4 receptors inhibited LTB4-induced inflammation. LTB4 also stimulated TRPV1-mediated substance P release and pretreatment with a specific substance P-receptor antagonist blocked LTB4-induced substance P action and ileitis. Inhibition of the LTB4 biosynthetic enzyme 5-lipoxygenase inhibited toxin A-induced increases in ileal LTB4 levels and toxin A- but not LTB4-induced ileitis. CONCLUSIONS: LTB4 mediates the inflammatory effects of toxin A via activation of TRPV1 receptors.     

The Concept about the Regeneration of Spent Borohydrides and Used Catalysts from Green Electricity

Currently, the Brown-Schlesinger process is still regarded as the most common and mature method for the commercial production of sodium borohydride (NaBH₄). However, the metallic sodium, currently produced from the electrolysis of molten NaCl that is mass-produced by evaporation of seawater or brine, is probably the most costly raw material. Recently, several reports have demonstrated the feasibility of utilizing green electricity such as offshore wind power to produce metallic sodium through electrolysis of seawater. Based on this concept, we have made improvements and modified our previously proposed life cycle of sodium borohydride (NaBH₄) and ammonia borane (NH₃BH₃), in order to further reduce costs in the conventional Brown-Schlesinger process. In summary, the revision in the concept combining the regeneration of the spent borohydrides and the used catalysts with the green electricity is reflected in (1) that metallic sodium could be produced from NaCl of high purity obtained from the conversion of the byproduct in the synthesis of NH₃BH₃ to devoid the complicated purification procedures if produced from seawater; and (2) that the recycling and the regeneration processes of the spent NaBH₄ and NH₃BH₃ as well as the used catalysts could be simultaneously carried out and combined with the proposed life cycle of borohydrides.     

Expression of the P2X2 receptor in different classes of ileum myenteric neurons in the female obese ob/ob mouse

AIM:To examine whether the ob/ob mouse model of obesity is accompanied by enteric nervous system ab-normalities such as altered motility METHODS:The study examined the distribution of the P2X 2 receptor (P2X 2 R) in myenteric neurons of female ob/ob mice. Specifically, we used immunohistochemistry to analyze the co-expression of the P2X 2 R with neuronal nitric oxide synthase (nNOS), choline acetyltrans-ferase (ChAT), and calretinin (CalR) in neurons of the small intestine myenteric plexus in ob/ob and control female mice In these sections, we used scanning confocal microscopy to analyze the co-localization of these markers as well as the neuronal density (cm 2 ) and area profile (μm2) of P2X 2 R-positive neurons In addition, enteric neurons were labeled using the nicotinamide adenine dinucleotide (NA H) diaphorase method and analyzed with light microscopy as an alternate means by which to analyze neuronal density and areaRESULTS:In the present study, we observed a 29 6% increase in the body weight of the ob/ob animals (OG) compared to the control group (CG) In addition, the average small intestine area was increased by approxi-mately 29 6% in the OG compared to the CG Immu-noreactivity (IR) for the P2X 2 R, nNOS, ChAT and CalR was detectable in the myenteric plexus, as well as in the smooth muscle, in both groups This IR appeared to be mainly cytoplasmic and was also associated with the cell membrane of the myenteric plexus neurons, where it outlined the neuronal cell bodies and their processes P2X 2 R-IR was observed to co-localize 100% with that for nNOS, ChAT and CalR in neurons of both groups In the ob/ob group, however, we observed that the neuronal density (neuron/cm 2 ) of P2X 2 R-IR cells was in-creased by 62% compared to CG, while that of NOS-IR and ChAT-IR neurons was reduced by 49% and 57%, respectively, compared to control mice The neuronal density of CalR-IR neurons was not different between the groups Morphometric studies further demonstrated that the cell body profile area (μm2) of nNOS-IR, ChAT-IR and CalR-IR neurons was increased by 34%, 20% and 55%, respectively, in the OG compared to controls Staining for NA H diaphorase activity is widely used to detect alterations in the enteric nervous system; however, our qualitative examination of NA H-diaphorase positive neurons in the myenteric ganglia revealed an overall similarity between the two groups CONCLUSION:We demonstrate increases in P2X2R expression and alterations in nNOS, ChAT and CalR IR in ileal myenteric neurons of female ob/ob mice compared to wild-type controls.     

Uridine adenosine tetraphosphate is a novel neurogenic P2Y1 receptor activator in the gut

Enteric purinergic motor neurotransmission, acting through P2Y1 receptors (P2Y1R), mediates inhibitory neural control of the intestines. Recent studies have shown that NAD⁺ and ADP ribose better meet criteria for enteric inhibitory neurotransmitters in colon than ATP or ADP. Here we report that human and murine colon muscles also release uridine adenosine tetraphosphate (Up4A) spontaneously and upon stimulation of enteric neurons. Release of Up4A was reduced by tetrodotoxin, suggesting that at least a portion of Up4A is of neural origin. Up4A caused relaxation (human and murine colons) and hyperpolarization (murine colon) that was blocked by the P2Y1R antagonist, MRS 2500, and by apamin, an inhibitor of Ca²⁺-activated small-conductance K⁺ (SK) channels. Up4A responses were greatly reduced or absent in colons of P2ry1^−/− mice. Up4A induced P2Y1R–SK-channel–mediated hyperpolarization in isolated PDGFRα⁺ cells, which are postjunctional targets for purinergic neurotransmission. Up4A caused MRS 2500-sensitive Ca²⁺ transients in human 1321N1 astrocytoma cells expressing human P2Y1R. Up4A was more potent than ATP, ADP, NAD⁺, or ADP ribose in colonic muscles. In murine distal colon Up4A elicited transient P2Y1R-mediated relaxation followed by a suramin-sensitive contraction. HPLC analysis of Up4A degradation suggests that exogenous Up4A first forms UMP and ATP in the human colon and UDP and ADP in the murine colon. Adenosine then is generated by extracellular catabolism of ATP and ADP. However, the relaxation and hyperpolarization responses to Up4A are not mediated by its metabolites. This study shows that Up4A is a potent native agonist for P2Y1R and SK-channel activation in human and mouse colon.Uridine adenosine tetraphosphate (Up4A) is, to the authors’ knowledge, the first dinucleotide isolated from living organisms that contains both purine and pyrimidine moieties. Up4A is a recently-identified, nonpeptide, endothelium-derived vasoconstrictor (1, 2). Up4A is likely associated with blood pressure regulation, because its levels in plasma are elevated in hypertensive subjects (3) and it causes vasoconstriction in deoxycorticosterone acetate-salt hypertensive rats (4) and type 2 diabetic rats (5). Up4A also contracts rat and human airways (6) and rat gastric smooth muscles (7). Pharmacological studies suggest that Up4A causes vasoconstriction via activation of P2X1, P2Y2, and P2Y4 receptors (1) and endothelium-dependent vasodilatation via activation of P2Y1 and P2Y2 receptors (8). In porcine coronary artery Up4A causes vasodilatation via adenosine (P1) receptors (9). Furthermore, Up4A causes vascular smooth muscle cell proliferation and migration (10), stimulates monocyte and lymphocyte oxidative burst activities (11), is a potent proinflammatory agent in the vascular wall (12), and may contribute to the proinflammatory status in patients with chronic kidney disease (11). Plasma of healthy human subjects contains ∼50 nmol/L Up4A, which is sufficient to elicit vascular effects (1). The role of Up4A in the gastrointestinal (GI) tract is unknown.Enteric neural regulation of GI motility includes motor neurotransmission mediated by inhibitory neurons releasing purines that act via P2Y1 receptors (P2Y1Rs) (13–17) and apamin-sensitive small-conductance Ca²⁺-activated K⁺ (SK) channels (13, 14, 18, 19). ATP (20), NAD⁺ (14, 15, 21), and adenosine 5′-diphosphate ribose (ADPR) (17) activate P2Y1R and SK channels and might be inhibitory neurotransmitters in the colon (22). Because Up4A appears to stimulate P2Y1Rs in endothelium, and P2Y1Rs are important for purinergic signaling in the colon, we investigated whether Up4A is released in colonic muscle, whether Up4A affects membrane potentials and contractions of colonic muscles, whether Up4A is an agonist for P2Y1R, whether cells expressing PDGF receptor α (PDGFRα) are targets of Up4A, and how Up4A is metabolized in colons of humans and mice.     

Loss of Vac14, a regulator of the signaling lipid phosphatidylinositol 3,5-bisphosphate, results in neurodegeneration in mice

The signaling lipid, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P(2)), likely functions in multiple signaling pathways. Here, we report the characterization of a mouse mutant lacking Vac14, a regulator of PI(3,5)P(2) synthesis. The mutant mice exhibit massive neurodegeneration, particularly in the midbrain and in peripheral sensory neurons. Cell bodies of affected neurons are vacuolated, and apparently empty spaces are present in areas where neurons should be present. Similar vacuoles are found in cultured neurons and fibroblasts. Selective membrane trafficking pathways, especially endosome-to-TGN retrograde trafficking, are defective. This report, along with a recent report on a mouse with a null mutation in Fig4, presents the unexpected finding that the housekeeping lipid, PI(3,5)P(2), is critical for the survival of neural cells.     

Alterations in the myocardial creatine kinase system precede the development of contractile dysfunction in beta(1)-adrenergic receptor transgenic mice

The beta-adrenergic receptor system not only plays a central role in modulating heart rate and left-ventricular (LV) contractility, but is also involved in the development of heart failure. We have, recently, shown that heart-specific overexpression of the beta(1)-adrenergic receptor in transgenic mice (TG) initially leads to increased contractility, followed by LV hypertrophy and heart failure. Since one feature for all forms of heart failure are characteristic changes in myocardial energy metabolism, we asked whether alterations in energetics are detectable in these mice before signs of LV impairment are present. Myocardial energetics ((31)P NMR spectroscopy) and LV performance were measured simultaneously in isolated perfused hearts at different workloads. LV performance as well as contractile reserve was identical for hearts of 4-month-old TG and wild-type mice. The ratio of phosphocreatine to ATP (1.16 +/- 0.05 vs. 1.46 +/- 0.10) and total creatine content (17.6 +/- 1.2 vs. 22.6 +/- 0.9 mmol/l) were significantly reduced in TG. Furthermore, there was a significant decrease in creatine transporter content (-43%), mitochondrial (-44%) and total creatine kinase (CK) activity (-21%) as well as citrate synthase activity (-25%), indicating impaired oxidative energy generation in TG. In conclusion, these findings of alterations in the CK system, creatine metabolism and mitochondrial proteins in TG hearts prior to the development of LV dysfunction provide further evidence that changes in myocardial energetics play a central role in the deterioration of cardiac function after chronic beta-adrenergic stimulation.     

Loss of the inactive myotubularin-related phosphatase Mtmr13 leads to a Charcot-Marie-Tooth 4B2-like peripheral neuropathy in mice

Charcot-Marie-Tooth disease type 4B (CMT4B) is a severe, demyelinating peripheral neuropathy characterized by slowed nerve conduction velocity, axon loss, and distinctive myelin outfolding and infolding. CMT4B is caused by recessive mutations in either myotubularin-related protein 2 (MTMR2; CMT4B1) or MTMR13 (CMT4B2). Myotubularins are phosphoinositide (PI) 3-phosphatases that dephosphorylate phosphatidylinositol 3-phosphate (PtdIns3P) and PtdIns(3,5)P(2), two phosphoinositides that regulate endosomal-lysosomal membrane traffic. Interestingly, nearly half of the metazoan myotubularins are predicted to be catalytically inactive. Both active and inactive myotubularins have essential functions in mammals and in Caenorhabditis elegans. MTMR2 and MTMR13 are active and inactive PI 3-phosphatases, respectively, and the two proteins have been shown to directly associate, although the functional significance of this association is not well understood. To establish a mouse model of CMT4B2, we disrupted the Mtmr13 gene. Mtmr13-deficient mice develop a peripheral neuropathy characterized by reduced nerve conduction velocity and myelin outfoldings and infoldings. Dysmyelination is evident in Mtmr13-deficient nerves at 14 days and worsens throughout life. Thus, loss of Mtmr13 in mice leads to a peripheral neuropathy with many of the key features of CMT4B2. Although myelin outfoldings and infoldings occur most frequently at the paranode, our morphological analyses indicate that the ultrastructure of the node of Ranvier and paranode is intact in Mtmr13-deficient nerve fibers. We also found that Mtmr2 levels are decreased by approximately 50% in Mtmr13-deficient sciatic nerves, suggesting a mode of Mtmr2 regulation. Mtmr13-deficient mice will be an essential tool for studying how the loss of MTMR13 leads to CMT4B2.     

The essential role of insulin-like growth factor-1 in the intestinal tropic effects of glucagon-like peptide-2 in mice

BACKGROUND & AIMS: Glucagon-like peptide-2 (GLP-2) is an intestinal hormone that acts through unknown pathways to induce intestinal growth. We investigated the role of the insulin-like growth factors (IGF-1 and IGF-2) as mediators of GLP-2-enhanced growth in the murine intestine. METHODS: IGF-1 expression and secretion were determined in GLP-2-responsive primary intestinal cultures treated with GLP-2. Parameters of intestinal growth were assessed in wild-type (CD1, Igf1(+/+) and Igf2+), heterozygous (Igf1(+/-)), and null (Igf1(-/-) and Igf2(-P)) mice treated chronically with saline, GLP-2, IGF-1, or R-Spondin1. RESULTS: GLP-2 increased IGF-1 messenger RNA expression and IGF-1 secretion in intestinal cultures and increased expression of IGF-1 messenger RNA in mouse small intestine in vivo. Igf1(+/+) and Igf2+ mice responded to .1 microg/g(-1) per day(-1) GLP-2 with increased intestinal weights, morphometric parameters, and proliferative indices. In contrast, Igf1(-/-) mice were unresponsive to the same dose of GLP-2, failing to demonstrate changes in intestinal weight, morphometry, or proliferation. However, a significant effect of 1 microg/g(-1) per day(-1) GLP-2 was observed in Igf1(-/-) mice, but only in terms of small intestinal weight when normalized for body weight. Furthermore, Igf2(-P) mice demonstrated a partially impaired response in terms of small intestinal growth. Both Igf1(-/-) and Igf2(-P) mice exhibited normal-enhanced intestinal growth in response to IGF-1 and/or R-Spondin1. CONCLUSIONS: GLP-2 enhances intestinal IGF-1 expression and secretion, and IGF-1 is required for small and large intestinal growth in response to GLP-2. These findings identify IGF-1 as an essential mediator of the intestinotropic actions of GLP-2.     

UGT1A1*28 is associated with greater decrease in serum K+ levels following oral intake of procaterol

Background and objective: Procaterol is a potent β₂-agonist frequently used for the management of asthma and chronic obstructive pulmonary disease. The efficacy and adverse effects of β₂-agonists are heterogeneous in individual patients, which may be partly caused by genetic variations in metabolizing enzymes and receptor molecules. The present study was designed to analyze the relationship between gene polymorphisms and physiological effects of procaterol in healthy subjects. Methods: Ninety-two non-smoking healthy volunteers were given 1?µg/kg body weight (max 50?µg) of procaterol as a dry syrup preparation, and the serum concentrations of procaterol, serum K⁺, and the physical responses were monitored for 240?min. We genotyped β₂-adrenergic receptor (ADRB2) (Arg16Gly and Gln27Glu), cytochrome P450 3A4 (rs2246709, rs4646437), and uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) (rs4148323 [allele A, *6], rs12479045, rs4148328, rs4663971, rs12052787, rs4148329, A (TA)_6/7 TAA [seven-repeat allele, *28]). Procaterol concentrations in serum were measured by liquid chromatography-tandem mass spectrometry. Results: No gene polymorphisms affected serum procaterol concentrations. Meanwhile, overall serum K⁺ level changes were significantly lower in carriers of UGT1A1*28 than in non-carriers after correcting for strong effects of serum procaterol concentrations and baseline K⁺ levels. No other polymorphisms were associated with serum K⁺ levels. None of polymorphisms of ADRB2 were associated with any physical responses. Conclusion: The present study indicates that significant hypokalemia may occur in carriers of UGT1A1*28 by systemic administration of procaterol and potentially by other β₂-agonists metabolized in the liver.     

Apigenin inhibits tumor necrosis factor alpha plus high glucose-induced LOX-1 expression in human endothelial cells

Although hyperglycemia can induce diabetic vascular disorders, the mechanisms responsible for the early stages of this process are unknown. To determine the factor(s) that initially stimulate hyperglycemia and the preventive effects of polyphenols, we examined the effects of high glucose (HG) conditions and several dietary polyphenols on human endothelial cells (EC). The purpose of the present study was to investigate the augmentation of the expression of angiotensin II type I receptor (AT1R), cyclooxygenase-2 (COX-2), lectin-like oxidized LDL receptor-1 (LOX-1), prostacyclin/prostaglandin I 2 synthase (PGIS), and thromboxane A2 synthase (TXA2S) by tumor necrosis factor-alpha (TNFα) in HG conditions (30 mM) in human EC over a short period, and we also investigated the regulatory effects of 10 dietary flavonoids. HG plus TNFα strongly induced LOX-1 and AT1R expression in the EC. Furthermore, apigenin, kaempferol, chrysin, and flavone significantly inhibited HG plus TNFα-induced LOX-1 expression. The inhibition of LOX-1 expression by apigenin was found to require a flavone skeleton, the double bond found in its C-ring, and the absence of a third hydroxyl group from its B- and C-rings. These findings suggest that TNFα and HG regulate diverse cellular processes and promote endothelial dysfunction via the expression of LOX-1 and AT1R. Conversely, the inhibitory action of apigenin may be beneficial for the treatment of diabetic endothelial dysfunction.     

The Synergistic Inhibitions of Tungstate and Molybdate Anions on Pitting Corrosion Initiation for Q235 Carbon Steel in Chloride Solution

In this work, the synergistic inhibitions of tungstate (WO₄²⁻) and molybdate (MoO₄²⁻) anions, including role and mechanism, on the initiation of pitting corrosion (PC) for Q235 carbon steel in chloride (Cl⁻) solution were investigated with electrochemical and surface techniques. The pitting potential (E_p) of the Q235 carbon steel in WO₄²⁻ + MoO₄^2- + Cl⁻ solution was more positive than that in WO₄²⁻ + Cl⁻ or MoO₄²⁻ + Cl⁻ solution; at each E_p, both peak potential and affected region of active pitting sites in WO₄²⁻ + MoO₄²⁻ + Cl⁻ solution were smaller than those in WO₄²⁻ + Cl⁻ or MoO₄²⁻ + Cl⁻ solution. WO₄²⁻ and MoO₄²⁻ showed a synergistic role to inhibit the PC initiation of the Q235 carbon steel in Cl⁻ solution, whose mechanism was mainly attributed to the influences of two anions on passive film. Besides iron oxides and iron hydroxides, the passive film of the Q235 carbon steel formed in WO₄²⁻ + Cl⁻, MoO₄²⁻ + Cl⁻, or WO₄²⁻ + MoO₄²⁻ + Cl⁻ solution was also composed of FeWO₄ plus Fe₂(WO₄)₃, Fe₂(MoO₄)₃, or Fe₂(WO₄)₃ plus Fe₂(MoO₄)₃, respectively. The film resistance and the defect quantity for Fe₂(WO₄)₃ plus Fe₂(MoO₄)₃ film were larger and smaller than those for FeWO₄ plus Fe₂(WO₄)₃ film and Fe₂(MoO₄)₃ film, respectively; for the inhibition of PC initiation, Fe₂(WO₄)₃ plus Fe₂(MoO₄)₃ film provided better corrosion resistance to Q235 carbon steel than FeWO₄ plus Fe₂(WO₄)₃ film and Fe₂(MoO₄)₃ film did.