ABSTRACT
Myotonic dystrophy type 1 (DM1) involves misregulated alternative splicing for specific genes. We used exon or nucleotide deletion to mimic altered splicing of genes central to muscle excitation-contraction coupling in mice. Mice with forced skipping of exon 29 in the CaV1.1 calcium channel combined with loss of ClC-1 chloride channel function displayed markedly reduced lifespan, whereas other combinations of splicing mimics did not affect survival. The Ca2+/Cl- bi-channelopathy mice exhibited myotonia, weakness, and impairment of mobility and respiration. Chronic administration of the calcium channel blocker verapamil rescued survival and improved force generation, myotonia, and respiratory function. These results suggest that Ca2+/Cl- bi-channelopathy contributes to muscle impairment in DM1 and is potentially mitigated by common clinically available calcium channel blockers.
Subject(s)
Channelopathies , Myotonia , Myotonic Dystrophy , Mice , Animals , Myotonic Dystrophy/drug therapy , Myotonic Dystrophy/genetics , Myotonic Dystrophy/metabolism , Calcium/metabolism , Chlorides/metabolism , Myotonia/metabolism , Verapamil/pharmacology , Verapamil/metabolism , Channelopathies/genetics , Channelopathies/metabolism , Alternative Splicing , Chloride Channels/genetics , Chloride Channels/metabolism , Muscle, Skeletal/metabolismABSTRACT
Induction of mycobacterial efflux pumps is a cause of Mycobacterium tuberculosis (Mtb) drug tolerance, a barrier to shortening antitubercular treatment. Verapamil inhibits Mtb efflux pumps that mediate tolerance to rifampin, a cornerstone of tuberculosis (TB) treatment. Verapamil's mycobacterial efflux pump inhibition also limits Mtb growth in macrophages in the absence of antibiotic treatment. These findings suggest that verapamil could be used as an adjunctive therapy for TB treatment shortening. However, verapamil is rapidly and substantially metabolized when co-administered with rifampin. We determined in a dose-escalation clinical trial of persons with pulmonary TB that rifampin-induced clearance of verapamil can be countered without toxicity by the administration of larger than usual doses of verapamil. An oral dosage of 360 mg sustained-release (SR) verapamil given every 12 hours concomitantly with rifampin achieved median verapamil exposures of 903.1 ng.h/mL (area under the curve (AUC)0-12 h ) in the 18 participants receiving this highest studied verapamil dose; these AUC findings are similar to those in persons receiving daily doses of 240 mg verapamil SR but not rifampin. Moreover, norverapamil:verapamil, R:S verapamil, and R:S norverapamil AUC ratios were all significantly greater than those of historical controls receiving SR verapamil in the absence of rifampin. Thus, rifampin administration favors the less-cardioactive verapamil metabolites and enantiomers that retain similar Mtb efflux inhibitory activity to verapamil, increasing overall benefit. Finally, rifampin exposures were 50% greater after verapamil administration, which may also be advantageous. Our findings suggest that a higher dosage of verapamil can be safely used as adjunctive treatment in rifampin-containing treatment regimens.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis , Humans , Antitubercular Agents/pharmacology , Rifampin , Tuberculosis/drug therapy , Tuberculosis/microbiology , Verapamil/metabolismABSTRACT
ABCC9-related intellectual disability and myopathy syndrome (AIMS) arises from loss-of-function (LoF) mutations in the ABCC9 gene, which encodes the SUR2 subunit of ATP-sensitive potassium (KATP ) channels. KATP channels are found throughout the cardiovascular system and skeletal muscle and couple cellular metabolism to excitability. AIMS individuals show fatigability, muscle spasms, and cardiac dysfunction. We found reduced exercise performance in mouse models of AIMS harboring premature stop codons in ABCC9. Given the roles of KATP channels in all muscles, we sought to determine how myopathy arises using tissue-selective suppression of KATP and found that LoF in skeletal muscle, specifically, underlies myopathy. In isolated muscle, SUR2 LoF results in abnormal generation of unstimulated forces, potentially explaining painful spasms in AIMS. We sought to determine whether excessive Ca2+ influx through CaV 1.1 channels was responsible for myopathology but found that the Ca2+ channel blocker verapamil unexpectedly resulted in premature death of AIMS mice and that rendering CaV 1.1 channels nonpermeable by mutation failed to reverse pathology; results which caution against the use of calcium channel blockers in AIMS.
Subject(s)
Muscular Diseases , Potassium Channels, Inwardly Rectifying , Animals , Mice , Adenosine Triphosphate , Muscle, Skeletal/metabolism , Muscular Diseases/chemically induced , Muscular Diseases/genetics , Potassium Channels, Inwardly Rectifying/genetics , Potassium Channels, Inwardly Rectifying/metabolism , Sulfonylurea Receptors/genetics , Sulfonylurea Receptors/metabolism , Verapamil/metabolismABSTRACT
Tuberculosis (TB) is caused by Mycobacterium tuberculosis, still one of the most common life-threatening infectious diseases worldwide. Although drug resistance in M.tuberculosis is mainly due to spontaneous chromosomal mutations in genes encoding drug target or drug activating enzymes, the resistance cannot be explained only by these mutations. Low permeability of the cell wall, drug inactivating enzymes and especially efflux pumps (EPs) are other mechanisms of drug resistance in mycobacteria. Efflux pump inhibitors (EPIs) binding to M.tuberculosis EPs were shown to inhibit efflux of anti-TB drugs, to enhance M.tuberculosis killing, to reduce drug resistance and to produce synergistic effects with first line anti-TB drugs. In this study, we aimed to determine the minimum inhibitory concentration (MIC) of first-line anti-TB drugs in the presence of verapamil (VER) and the expression of 21 putative EP genes belonged to the ATP-binding cassette (ABC), major facilitator superfamily (MFS) and resistance-nodulation-division (RND) families which might have caused the resistance in nine M.tuberculosis complex clinical isolates resistant to all of the first line anti-TB drugs. MIC values of the isolates were determined in 96-well U-bottom plates by the resazurin microtiter test (REMA) method based on the color change principle. According to the determined MIC values of each isolate, freshly grown cultures in Middlebrook 7H9 broth were exposed to first-line anti-TB drugs and MIC of first-line anti-TB drugs in the presence of VER (½ MIC) at 37°C for 48 hours for RNA extraction. The non-drug exposed cultures were used as control. Total RNA was extracted using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) and then treated with DNase I (Thermo Fischer Scientific Inc., Waltham, MA). Complementary DNA (cDNA) from the extracted RNAs was synthesized with the "First strand cDNA synthesis kit" (Thermo Fischer Scientific Inc., Waltham, MA) using oligo primers. The expression levels of efflux pump genes by quantitative realtime polymerase chain reaction (qRt-PCR) were performed using the QuantiTect SYBR Green Rt-PCR Kit (Qiagen, Germany). The housekeeping sigma factor gene sigA (Rv2703) was used as internal control in qRtPCR assays. Relative quantification of the clinical isolates was determined by the 2-∆∆Ct method by comparing the expression levels of efflux genes in cultures exposed to primary anti-TB drugs and VER with those of non-drug exposed cultures. MIC values of nine isolates by REMA method was determined between 32-512 µg/mL, 1-128 µg/mL, 2-32 µg/mL, 4-16 µg/mL and 15.62-250 µg/mL for streptomycin (SM), isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and VER, respectively. In the presence of ½ MIC VER, it was determined that the MIC of SM decreased 2-32 fold in eight isolates, the MIC of INH decreased by 2-8 fold in nine isolates, the MIC of RIF decreased by 2-16 fold in eight isolates, and the MIC of EMB decreased 2-4 fold in only five isolates. There was an increase in the expression of Rv1273c, Rv1456c, Rv1457 and Rv1819 efflux pump genes from the ABC family, Rv1634 and Rv0842 from the MFS family and Rv3823 efflux from the RND family in isolates exposed to ½ MIC of first-line anti-TB drugs stress. Rv1456c and Rv1819 were found to be associated with SM resistance, Rv1273c with EMB resistance, Rv1457, Rv0842 and Rv3823 with both RIF and EMB resistance, and Rv1634 with INH, RIF and EMB resistance. It was determined that there was a decrease in the expression levels of eight efflux pump genes from the ABC family (Rv1456c, Rv1457c, Rv1458c, Rv0194, Rv1272c, Rv1686c, Rv1687c, Rv1819c), six from MFS family (Rv0842, Rv0849, Rv1634, Rv2265, Rv2456c, Rv0876c) and two from RND family (Rv0507, Rv0676c) in isolates exposed to MIC of first-line anti-TB drugs in the presence of VER (½ MIC). Further studies with clinical isolates are needed to investigate the EPIs that can be used in alternative therapy and to determine the contribution of EPs to the development of resistance due to the increasing TB resistance.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis, Multidrug-Resistant , Tuberculosis , Humans , Antitubercular Agents/pharmacology , Antitubercular Agents/therapeutic use , Verapamil/pharmacology , Verapamil/metabolism , DNA, Complementary/metabolism , DNA, Complementary/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Tuberculosis/microbiology , Isoniazid/pharmacology , Rifampin/pharmacology , Microbial Sensitivity Tests , Tuberculosis, Multidrug-Resistant/drug therapyABSTRACT
We examined whether the α1L-adrenoceptor (AR), which shows low affinity (pA2 < 9) for prazosin (an α1-AR antagonist) and high affinity (pA2 ≈ 10) for tamsulosin/silodosin (α1A-AR antagonists), is involved in phenylephrine-induced contractions in the guinea pig (GP) thoracic aorta (TA). Intracellular signaling induced by α1L-AR activation was also examined by focusing on Ca2+ influx pathways. Tension changes of endothelium-denuded TAs were isometrically recorded and mRNA encoding α-ARs/Ca2+ channels and their related molecules were measured using RT-quantitative PCR. Phenylephrine-induced contractions were competitively inhibited by prazosin/tamsulosin, and their pA2 value were calculated to be 8.53/9.74, respectively. These contractions were also inhibited by silodosin concentration-dependently. However, the inhibition was not competitive fashion with the apparent pA2 value being 9.48. In contrast, phenylephrine-induced contractions were not substantially suppressed by L-765314 (an α1B-AR antagonist), BMY 7378 (an α1D-AR antagonist), yohimbine, and idazoxan (α2-AR antagonists). Phenylephrine-induced contractions were markedly inhibited by YM-254890 (a Gq protein inhibitor) or removal of extracellular Ca2+, and partially inhibited by verapamil (a voltage-dependent Ca2+ channel (VDCC) inhibitor). The residual contractions in the presence of verapamil were slightly inhibited by LOE 908 (a receptor-operated Ca2+ channel (ROCC) inhibitor) and strongly inhibited by SKF-96365 (a store-operated Ca2+ channel (SOCC) and ROCC inhibitor). Among the mRNA encoding α-ARs/SOCC-related molecules, α1A-AR (Adra1a)/Orai3, Orai1, and Stim2 were abundant in this tissue. In conclusion, phenylephrine-induced contractions in the GP TA can be triggered by stimulation of Gq protein-coupled α1L-AR, followed by activation of SOCCs and VDCCs.
Subject(s)
Adrenergic alpha-Antagonists , Aorta, Thoracic , Guinea Pigs , Animals , Phenylephrine/pharmacology , Adrenergic alpha-Antagonists/metabolism , Adrenergic alpha-Antagonists/pharmacology , Tamsulosin/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Prazosin/pharmacology , Verapamil/pharmacology , Verapamil/metabolism , RNA, Messenger/metabolism , Muscle ContractionABSTRACT
OBJECTIVES: To explore the inhibitory effect of verapamil, a calcium channel blocker, on the growth of human airway granulation fibroblasts to provide an experimental basis for the clinical use of calcium channel blockers in preventing and treating benign airway stenosis. METHODS: Primary human airway normal fibroblasts and human airway granulation fibroblasts were cultured by tissue block attachment culture method, and the experimental studies were carried out using 3-8 generation cells. Cell Counting Kit-8 (CCK-8) was used to test the proliferation of human normal airway fibroblasts and human airway granulation fibroblasts and the semi-inhibitory concentration of verapamil on normal airway fibroblasts and airway granulation fibroblasts. A scratch test detected the migration effect of verapamil on human airway granulation fibroblasts. The mRNA relative expression levels of related factors were detected by PCR to compare the differences between normal airway fibroblasts and airway granulation fibroblasts. Western blot was used to detect the relative amount of related proteins and compare the differences between normal airway fibroblasts and granulation airway fibroblasts. After 48 hours of treatment with half of the inhibitory concentration of Vera Pammy for granulation airway fibroblasts, the relative expression levels of related factors on mRNA and protein were observed. RESULTS: Human normal airway fibroblasts and human airway granulation fibroblasts with a purity of more than 95% could be obtained from primary culture by tissue block adherence method. CCK8 results showed that the proliferation rate of human airway granulation fibroblasts was faster than that of the normal human airway fibroblasts. The semi-inhibitory concentration of verapamil on human normal airway fibroblasts was 92.81 ug/ml, while the semi-inhibitory concentration on human airway granulation fibroblasts was 69.57 ug/ml. The scratch test indicated that the cell migration rate of human airway granulation fibroblasts treated with verapamil decreased significantly (P < 0.05). PCR results showed that the mRNA relative expression levels of TGFß1, COL1A1, Smad2/3, VEGFA, IL6, and IL8 in human airway granulation fibroblasts were significantly higher than those in normal human airway fibroblasts (P < 0.05). The mRNA relative expressions of TGFß1, smad2/3, and COL1A1 in human airway granulation fibroblasts treated with semi-inhibited verapamil for 48h were down-regulated (P < 0.05), while the mRNA relative expressions of VEGFA, IL6 and IL8 had no significant changes (P > 0.05). WB test showed that the relative protein expressions of TGFß1, Smad2, and VEGFC in human airway granulation fibroblasts were upregulated (P < 0.05) but downregulated after verapamil treatment compared with before treatment (P < 0.05). CONCLUSION: Calcium channel blockers can inhibit the proliferation of human airway granulation fibroblasts through TGFß1/ Smad pathway, which may be a method to prevent and treat benign airway stenosis.
Subject(s)
Calcium Channel Blockers , Verapamil , Humans , Verapamil/pharmacology , Verapamil/metabolism , Calcium Channel Blockers/pharmacology , Calcium Channel Blockers/metabolism , Constriction, Pathologic/metabolism , Interleukin-6/metabolism , Interleukin-8/metabolism , Interleukin-8/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Fibroblasts/metabolism , Cells, CulturedABSTRACT
BACKGROUND: Verapamil enhances the sensitivity of Mycobacterium tuberculosis to anti-tuberculosis (TB) drugs, promotes the macrophage anti-TB ability, and reduces drug resistance, but its mechanism is unclear. Herein, we have investigated the effect of verapamil on cytokine expression in mouse peritoneal macrophages. METHODS: Macrophages from mice infected with M. tuberculosis or S. aureus were cultured with verapamil, the cytokines were detected by enzyme-linked immunosorbent assay, and the RNA was measured with quantitative real-time polymerase chain reaction and agarose gel electrophoresis. The intracellular calcium signaling was measured by confocal microscopy. RESULTS: Significantly higher levels of NF-κB, IL-12, TNF-α, and IL-1ß were observed after TB infection. The levels of NF-κB and IL-12 increased when verapamil concentration was less than 50 µg/ml, but decreased when verapamil concentration was greater than 50µg/ml. With the increase in verapamil concentration, TNF-α and IL-1ß expressed by macrophages decreased. The L-type calcium channel transcription significantly increased in M. tuberculosis rather than S. aureus-infected macrophages. Furthermore, during bacillus Calmette-Guerin (BCG) infection, verapamil stimulated a sharp peak in calcium concentration in macrophages, while calcium concentration increased mildly and decreased smoothly over time in the absence of verapamil. CONCLUSION: Verapamil enhanced macrophage immunity via the NF-κB pathway, and its effects on cytokine expression may be achieved by its regulation of intracellular calcium signaling.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis , Mice , Animals , NF-kappa B/metabolism , Tumor Necrosis Factor-alpha/metabolism , Verapamil/pharmacology , Verapamil/metabolism , Calcium/metabolism , Staphylococcus aureus/metabolism , Macrophages/metabolism , Tuberculosis/drug therapy , Tuberculosis/metabolism , Cytokines/metabolism , Interleukin-12/metabolismABSTRACT
The inwardly rectifying potassium current of the cardiomyocyte (IK1) is the main determinant of the resting potential. Ion channels Kir2.1, Kir2.2, and Kir2.3 form tetramers and are the molecular correlate of macroscopic IK1 current. Verapamil is an antiarrhythmic drug used to suppress atrial and ventricular arrhythmias. Its primary mechanism of action is via blocking calcium channels. In addition, it has been demonstrated to block IK1 current and the Kir2.1 subunit. Its effect on other subunits that contribute to IK1 current has not been studied to date. We therefore analyzed the effect of verapamil on the Kir channels 2.1, 2.2, and 2.3 in the Xenopus oocyte expression system. Kir2.1, Kir2.2, and Kir2.3 channels were heterologously expressed in Xenopus oocytes. Respective currents were measured with the voltage clamp technique and the effect of verapamil on the current was measured. At a concentration of 300 µM, verapamil inhibited Kir2.1 channels by 41.36% ± 2.7 of the initial current, Kir2.2 channels by 16.51 ± 3.6%, and Kir2.3 by 69.98 ± 4.2%. As a verapamil effect on kir2.3 was a previously unknown finding, we analyzed this effect further. At wash in with 300 µM verapamil, the maximal effect was seen within 20 min of the infusion. After washing out with control solution, there was only a partial current recovery. The current reduction from verapamil was the same at - 120 mV (73.2 ± 3.7%), - 40 mV (85.5 ± 6.5%), and 0 mV (61.5 ± 10.6%) implying no voltage dependency of the block. Using site directed mutations in putative binding sites, we demonstrated a decrease of effect with pore mutant E291A and absence of verapamil effect for D251A. With mutant I214L, which shows a stronger affinity for PIP2 binding, we observed a normalized current reduction to 61.9 ± 0.06% of the control current, which was significantly less pronounced compared to wild type channels. Verapamil blocks Kir2.1, Kir2.2, and Kir2.3 subunits. In Kir2.3, blockade is dependent on sites E291 and D251 and interferes with activation of the channel via PIP2. Interference with these sites and with PIP2 binding has also been described for other Kir channels blocking drugs. As Kir2.3 is preferentially expressed in atrium, a selective Kir2.3 blocking agent would constitute an interesting antiarrhythmic concept.
Subject(s)
Anti-Arrhythmia Agents , Verapamil , Verapamil/pharmacology , Verapamil/metabolism , Anti-Arrhythmia Agents/pharmacology , Binding Sites , Oocytes/metabolismABSTRACT
Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are multidomain transmembrane proteins, which facilitate the transport of various substances across cell membranes using energy derived from ATP hydrolysis. They are important drug targets since they mediate decreased drug susceptibility during pharmacological treatments. For the methylotrophic yeast Pichia pastoris, a model organism that is a widely used host for protein expression, the role and function of its ABC transporters is unexplored. In this work, we investigated the Pichia ABC-B transporter STE6-2p. Functional investigations revealed that STE6-2p is capable of transporting rhodamines in vivo and is active in the presence of verapamil and triazoles in vitro. A phylogenetic analysis displays homology among multidrug resistance (MDR) transporters from pathogenic fungi to human ABC-B transporters. Further, we present high-resolution single-particle electron cryomicroscopy structures of an ABC transporter from P. pastoris in the apo conformation (3.1 Å) and in complex with verapamil and adenylyl imidodiphosphate (AMP-PNP) (3.2 Å). An unknown density between transmembrane helices 4, 5, and 6 in both structures suggests the presence of a sterol-binding site of unknown function.
Subject(s)
ATP-Binding Cassette Transporters , Sterols , Humans , ATP-Binding Cassette Transporters/metabolism , Adenylyl Imidodiphosphate/metabolism , Sterols/metabolism , Phylogeny , Adenosine Triphosphate/metabolism , Saccharomyces cerevisiae/metabolism , Verapamil/pharmacology , Verapamil/metabolism , Triazoles/metabolism , Rhodamines/metabolismABSTRACT
Elastin-derived peptides (EDPs) contain replications of the Val-Gly-Val-Ala-Pro-Gly (VGVAPG) hexapeptide. It has been described that the VGVAPG peptide induces reactive oxygen species (ROS) production in murine monocytes and astrocytes, human fibroblasts, and the human neuroblastoma (SH-SY5Y) cell line. To date, there is growing evidence that calcium channel blockers (CCBs) reduce oxidative stress and development of inflammation in the nervous system. Therefore, the aim of the present study was to evaluate the impact of such CCBs as Nifedipine, Verapamil, and MK-801 on the expression of peroxisome proliferator-activated receptor (Pparγ), i.e. ROS-related and inflammation-related proteins, in mouse astrocytes exposed in vitro to the VGVAPG peptide. The experiments showed that Nifedipine or MK-801 used in co-treatment with the VGVAPG peptide potentiated the effect of this peptide on the Pparγ level after the 24-h and 48-h treatment. Moreover, all studied compounds decreased the VGVAPG-induced caspase-1 activity in both time intervals. The data also showed that the VGVAPG peptide decreased the interleukin 1 beta (IL-1ß) level in both studied time intervals. Upon a short-time exposure, the use of CCBs intensified the decrease in IL-1ß stimulated by the VGVAPG peptide, opposite to the longer treatment. Moreover, the VGVAPG peptide decreased the IL-1ßR1 level in both studied time intervals. After 24 h, Nifedipine and Verapamil potentiated the effect of the VGVAPG peptide. The VGVAPG peptide decreased the catalase (Cat) protein expression only after 24 h, whereas CCBs did not affect the expression of Cat induced by the VGVAPG peptide. The VGVAPG peptide increased the expression of the superoxide dismutase 1 (Sod1) protein. After 24 h of exposure, Nifedipine and Verapamil potentiated the increase in the Sod1 protein expression. Finally, our data showed that VGVAPG did not change the level of estradiol (E2) in the astrocytes. Interestingly, Nifedipine and Verapamil in co-treatment with VGVAPG increased the E2 level. Summarizing, it can be assumed that increased amounts of the VGVAPG during lifetime can play a certain role in calcium channel functioning in neurodegenerative diseases.
Subject(s)
Elastin , Neuroblastoma , Animals , Astrocytes/metabolism , Calcium Channel Blockers/metabolism , Calcium Channel Blockers/pharmacology , Dizocilpine Maleate/pharmacology , Elastin/chemistry , Elastin/metabolism , Elastin/pharmacology , Humans , Inflammation/metabolism , Mice , Neuroblastoma/metabolism , Nifedipine/metabolism , Nifedipine/pharmacology , Oligopeptides , PPAR gamma/metabolism , Peptides/metabolism , Reactive Oxygen Species/metabolism , Superoxide Dismutase-1/metabolism , Verapamil/metabolism , Verapamil/pharmacologyABSTRACT
Although tannins are widely distributed in broad beans and alfalfa, the pea aphid (Acyrthosiphon pisum) can still destroy them. The ATP binding cassette (ABC) transporters participate in the metabolism of plant secondary metabolites and pesticides in insects. However, whether ABC transporter genes play a role in the metabolism of tannins in the pea aphid is unclear. Here, we found that verapamil (an ABC transporter inhibitor) significantly increased the mortality of tannic acid to pea aphid, which indicated that ABC transporter gene was related to the metabolism of tannic acid by pea aphid. Then, we identified 54 putative ABC transporter genes from the genome database of A. pisum. These genes were divided into eight subfamilies, ApABCA to ApABCH, of which subfamily G has the largest number of genes with 19, followed by the subfamily C with 14. RT-qPCR results show that the expression levels of ApABCA2, ApABCC7, ApABCG2, and ApABCG3 were highly expressed in the first instar, while those of ApABCA3, ApABCG6, ApABCG7, ApABCH3, and ApABCH4 were highly expressed in adults. Furthermore, transcription levels of many ABC transporter genes were induced by tannic acid. Especially, ApABCG17 and ApABCH2 were obviously induced after being exposed to tannic acid. Meanwhile, knockdown of ApABCG17 by RNA interference resulted in increased sensitivity of pea aphid to tannic acid. These results suggest that ApABCG17 may be involved in tannic acid metabolism in pea aphid. This study will help us to understand the mechanism of tannic acid metabolism in pea aphid, and provides a basis for further research on the physiological function of ABC transporter genes in pea aphid.
Subject(s)
Aphids , Pesticides , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphate/metabolism , Animals , Aphids/genetics , Medicago sativa , Pisum sativum/metabolism , Pesticides/metabolism , Tannins/metabolism , Verapamil/metabolismABSTRACT
BACKGROUND: Ureteral stricture (US) is a fibrotic process that leads to urinary tract obstruction and even kidney damage, with the characteristic of reduced extracellular matrix (ECM) degradation and increased collagen synthesis. Verapamil, as a calcium channel blocker, was reported to prevent scar formation. Our work aimed to investigate the biological effects and mechanism of verapamil in US. METHODS: Fibroblasts were subjected to transforming growth factor-beta 1 (TGF-ß1) to stimulate collagen synthesis, and the messenger ribonucleic acid (mRNA) and protein expressions in fibroblasts were assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The location of phosphorylation-signal transducer and activator of transcription 3 (p-STAT3) and Jund proto-oncogene subunit (JunD) in fibroblasts were determined by immunofluorescence (IF). The binding relationship between signal transducer and activator of transcription 3 (STAT3) and collagen type I alpha1 (COL1A1)/collagen type III alpha 1 chain (COL3A1) and the binding relationship between JunD and tissue inhibitor of metalloproteinases-1 (TIMP-1) were verified by dual luciferase reporter gene and chromatin Immunoprecipitation (ChIP) assays. RESULTS: Herein, we found that verapamil could inhibit TGF-ß1/Ca2 + /calmodulin-dependent protein kinase II (CaMK II)-mediated STAT3 activation in fibroblasts, and STAT3 inhibition repressed collagen production. In addition, verapamil could inhibit TGF-ß1/CaMK II-mediated Mothers against DPP homolog 3 (Smad3)/JunD pathway activation in fibroblasts, and JunD silencing inhibited TIMP1 (a matrix metalloproteinase inhibitor) expression. Our subsequent experiments revealed that STAT3 bound with COL1A1 promoter and COL3A1 promoter and activated their transcription, and JunD bound with TIMP1 promoter and activated its transcription. Moreover, as expected, STAT3 activation could eliminate the inhibitory effect of verapamil treatment on TGF-ß1-induced collagen production in fibroblasts, and JunD overexpression reversed the inhibitory effect of verapamil treatment on TGF-ß1-induced TIMP1 expression in fibroblasts. CONCLUSION: Verapamil inhibited collagen production and TIMP-1 expression in US by blocking CaMK II-mediated STAT3 and Smad3/JunD pathways.
Subject(s)
Transforming Growth Factor beta1 , Ureteral Obstruction , Calcium Channel Blockers/pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/pharmacology , Collagen/metabolism , Collagen Type I/genetics , Collagen Type III , Constriction, Pathologic , Fibroblasts/metabolism , Humans , Luciferases/metabolism , Matrix Metalloproteinase Inhibitors/metabolism , Matrix Metalloproteinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-jun , RNA , RNA, Messenger/metabolism , STAT3 Transcription Factor , Signal Transduction , Smad3 Protein/metabolism , Smad3 Protein/pharmacology , Tissue Inhibitor of Metalloproteinase-1/metabolism , Transforming Growth Factor beta1/metabolism , Transforming Growth Factors/metabolism , Ureteral Obstruction/metabolism , Verapamil/metabolism , Verapamil/pharmacologyABSTRACT
Background: Fetal brain development in the first half of pregnancy is dependent on maternal thyroid hormone (TH), highlighting the importance of trans-placental TH transport. It is yet unclear which transporters are involved in this process. We aimed to identify the major TH transporters in a human placental cell model (BeWo cells). Methods: Messenger RNA expression of the known TH transporters (the monocarboxylate transporter [MCT]8, MCT10, the L-type amino acid transporter [LAT]1, LAT2, the organic anion transporting peptide [OATP]1A2 and OATP4A1) in BeWo cells and human placenta were determined by quantitative PCR. To determine the specificity and efficacy of transporter inhibitors, we first determined TH uptake at different inhibitor concentrations in African green monkey kidney fibroblast-like cells (COS1 cells) overexpressing TH transporters. We then tested TH uptake in BeWo cells in the presence or absence of the optimal inhibitor concentrations. Results: All tested TH transporters were expressed in human term placentas, whereas MCT8 was absent in BeWo cells. Both 2-amino-2-norbornanecarboxylic acid (BCH) and L-tryptophan at 1 mM inhibited LATs, whereas at the highest concentration (10 mM) L-tryptophan also inhibited MCT10. Verapamil inhibited OATP1A2 and less efficiently both MCTs, but not LATs. Both rifampicin and naringin reduced OATP1A2 activity. Finally, silychristin inhibited MCT8 at submicromolar concentrations and OATP1A2 partially only at the highest concentration tested (10 µM). In BeWo cells, verapamil reduced triiodothyronine (T3) uptake by 24%, BCH by 31%, and 1 mM L-tryptophan by 41%. The combination of BCH and verapamil additively decreased T3 uptake by 53% and the combination of BCH and 10 mM L-tryptophan by 60%, suggesting a major role for MCT10 and LATs in placental T3 uptake. Indeed, transfection of BeWo cells with MCT10-specific small interfering RNA significantly reduced T3 uptake. Only the combination of BCH and verapamil significantly reduced thyroxine (T4) uptake in BeWo cells, by 32%. Conclusions: Using pharmacological inhibitors, we show that MCT10 and LATs play a major role in T3 uptake in BeWo cells. T4 uptake appears independent of known TH transporters, suggesting the presence of, currently unknown, alternative transporter(s).
Subject(s)
Amino Acid Transport Systems, Neutral , Symporters , Amino Acid Transport Systems, Neutral/genetics , Amino Acid Transport Systems, Neutral/metabolism , Animals , Chlorocebus aethiops , Female , Humans , Monocarboxylic Acid Transporters/genetics , Monocarboxylic Acid Transporters/metabolism , Peptides/metabolism , Placenta/metabolism , Pregnancy , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Rifampin/metabolism , Symporters/metabolism , Thyroid Hormones/metabolism , Thyroxine/metabolism , Thyroxine/pharmacology , Triiodothyronine/metabolism , Triiodothyronine/pharmacology , Tryptophan/metabolism , Verapamil/metabolismABSTRACT
The inhibitory effects of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and linoleic acid (LA) on the contractions induced by five prostanoids and U46619 (a TP receptor agonist) were examined in guinea pig gastric fundus smooth muscle (GFSM). Tension changes were isometrically measured, and the mRNA expression of prostanoid receptors was measured by RT-qPCR. DHA and EPA significantly inhibited contractions induced by the prostanoids and U46619, whereas LA inhibited those induced by prostaglandin D2 and U46619. The mRNA expression levels of the prostanoid receptors were TP ≈ EP3 >> FP > EP1 . The inhibition by DHA, EPA, and LA was positively correlated with that by SQ 29,548 (a TP receptor antagonist) but not with that by L-798,106 (an EP3 receptor antagonist). DHA and EPA suppressed high KCl-induced contractions by 35% and 25%, respectively, and the contractions induced by the prostanoids and U46619 were suppressed by verapamil, a voltage-dependent Ca2+ channel (VDCC) inhibitor, by 40%-85%. Although LA did not suppress high KCl-induced contractions, it suppressed U46619-induced contractions in the presence of verapamil. However, LA did not show significant inhibitory effects on U46619-induced Ca2+ increases in TP receptor-expressing cells. In contrast, LA inhibited U46619-induced contractions in the presence of verapamil, which was also suppressed by SKF-96365 (a store-operated Ca2+ channel [SOCC] inhibitor). These findings suggest that the TP receptor and VDCC are targets of DHA and EPA to inhibit prostanoid-induced contractions of guinea pig GFSM, and SOCCs play a significant role in LA-induced inhibition of U46619-induced contractions.
Subject(s)
Docosahexaenoic Acids , Eicosapentaenoic Acid , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/metabolism , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology , Animals , Calcium Channels, L-Type/metabolism , Docosahexaenoic Acids/metabolism , Docosahexaenoic Acids/pharmacology , Eicosapentaenoic Acid/metabolism , Eicosapentaenoic Acid/pharmacology , Gastric Fundus/metabolism , Guinea Pigs , Muscle, Smooth , Prostaglandins/metabolism , Prostaglandins/pharmacology , RNA, Messenger/metabolism , Receptors, Thromboxane/metabolism , Verapamil/metabolism , Verapamil/pharmacologyABSTRACT
PURPOSE: Sofosbuvir, a nucleotide antiviral drug, is a Biopharmaceutics Classification System (BCS) class III prodrug suffering from limited intestinal absorption due to its high hydrophilicity and low intestinal permeability. This research aims to investigate the luminal stability of Sofosbuvir, the influence of anatomical site on its intestinal absorption and the effects of verapamil on such absorption. METHOD: The study utilized in situ rabbit intestinal perfusion technique to examine absorption of Sofosbuvir from duodenum, jejunum, ileum and ascending colon. This was conducted both with and without verapamil. RESULTS: The luminal stability study showed that Sofosbuvir was subjected to premature degradation with varying fractions degraded from the different intestinal segments. The in situ perfusion data showed incomplete absorption of Sofosbuvir from small and large intestinal segments. The recorded values of the absorptive clearance per unit length (Pe.A/L) of Sofosbuvir were 0.026, 0.0075, 0.0026, & 0.054 ml/min.cm for duodenum, jejunum, ileum, and ascending colon, respectively. The Pe.A/L values were ordered as colon > duodenum > jejunum > ileum. This is the opposite rank of P-gp content in the different intestinal segments. The recorded values of the length required for complete Sofosbuvir absorption (L95%) were 29.58, 128.47, 949.2 and, 13.63 cm for duodenum, jejunum, ileum, and ascending colon, respectively. Co-perfusion with verapamil significantly increased Pe.A/L and reduced the L95% of Sofosbuvir from both jejunum and ileum (P-value < 0.05). CONCLUSION: The results indicated that the absorptive clearance of Sofosbuvir was site dependent and associated with the content of P-glycoprotein, in addition to the expected drug interactions that can occur in polymedicated hepatitis C virus (HCV) infected patients.
Subject(s)
Sofosbuvir , Verapamil , Animals , Humans , Intestinal Absorption , Intestines , Jejunum/metabolism , Rabbits , Sofosbuvir/pharmacology , Verapamil/metabolism , Verapamil/pharmacologyABSTRACT
Upon development of a workflow to analyze (±)-Verapamil and its metabolites using differential mobility spectrometry (DMS), we noticed that the ionogram of protonated Verapamil consisted of two peaks. This was inconsistent with its metabolites, as each exhibited only a single peak in the respective ionograms. The unique behaviour of Verapamil was attributed to protonation at its tertiary amino moiety, which generated a stereogenic quaternary amine. The introduction of additional chirality upon N-protonation of Verapamil renders four possible stereochemical configurations for the protonated ion: (R,R), (S,S), (R,S), or (S,R). The (R,R)/(S,S) and (R,S)/(S,R) enantiomeric pairs are diastereomeric and thus exhibit unique conformations that are resolvable by linear and differential ion mobility techniques. Protonation-induced chirality appears to be a general phenomenon, as N-protonation of 12 additional chiral amines generated diastereomers that were readily resolved by DMS.
Subject(s)
Protons , Verapamil/analysis , Ion Mobility Spectrometry , Verapamil/metabolismABSTRACT
1. Si-Ni-San (SNS) possesses extensive therapeutic effects, however, the extent to which main components are absorbed and the mechanisms involved are controversial. 2. In this study, MDCK cell model was used to determine the permeability characteristics and interaction between the major components of Si-Ni-San, including saikosaponin a, paeoniflorin, naringin and glycyrrhizic acid. 3. The transport of the major components was concentration-dependent in both directions. Moreover, the transport of paeoniflorin, naringin and glycyrrhizic acid was significantly reduced at 4 °C or in the presence of NaN3. Additionally, the efflux of paeoniflorin and naringin were apparently reduced in the presence of P-gp inhibitor verapamil. The transport of glycyrrhizic acid was clearly inhibited by the inhibitors of MRP2, indicating that MRP2 may be involved in the transport of glycyrrhizic acid. However, the results indicated that saikosaponin a was absorbed mainly by passive diffusion. Furthermore, the combined incubation of four major components had a powerful sorbefacient effect than a single drug used alone which may be regulated by tight junctions. 4. Taken together, our study provides useful information for pharmacological applications of Si-Ni-San and offers new insights into this ancient decoction for further researches, especially in drug synergism.
Subject(s)
Drugs, Chinese Herbal/metabolism , Animals , Biological Transport , Dogs , Flavanones/metabolism , Glucosides/metabolism , Glycyrrhizic Acid/metabolism , Humans , Madin Darby Canine Kidney Cells , Models, Biological , Monoterpenes/metabolism , Oleanolic Acid/analogs & derivatives , Oleanolic Acid/metabolism , Permeability , Saponins/metabolism , Verapamil/metabolismABSTRACT
The human blood-brain barrier (BBB) transporter P-gp can efflux amyloid-ß (Aß) out of the central nervous system (CNS). Aß is thought to be the causative agent for Alzheimer's disease (AD). Using positron emission tomography imaging, we have shown that BBB P-gp activity is reduced in AD, as quantified by the in vivo brain distribution of the P-gp probe [11 C]-verapamil. Therefore, the aim of this study was to determine whether this reduced BBB P-gp activity in AD was due to decreased P-gp abundance at the BBB. Using targeted proteomics, we quantified the abundance of P-gp and other drug transporters in gray matter brain microvessels isolated from 43 subjects with AD and 38 age-matched controls (AMCs) from regions affected by AD (hippocampus and the parietal lobe of the brain cortex) and not affected by AD (cerebellum). First, P-gp abundance was decreased in the BBB of the hippocampus vs. the cerebellum in both subjects with AD and AMCs, and therefore was not AD-related. In addition, gray matter BBB abundance of P-gp (and of other transporters) in the hippocampus and the parietal lobe was not different between AD and AMC. The gray matter BBB abundance of all drug transporters decreased with age, likely due to age-dependent decrease in the density of brain microvessels. Collectively, the observed reduced in vivo cerebral BBB P-gp activity in AD cannot be explained by reduced abundance of P-gp at the BBB. Nevertheless, the drug transporter abundance at the human gray matter BBB data provided here can be used to predict brain distribution of drugs targeted to treat CNS diseases, including AD.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Alzheimer Disease/metabolism , Blood-Brain Barrier/metabolism , Gray Matter/blood supply , Microvessels/metabolism , Proteomics , ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism , Adult , Age Factors , Aged , Aged, 80 and over , Alzheimer Disease/diagnostic imaging , Amyloid beta-Peptides/metabolism , Biological Transport , Blood-Brain Barrier/diagnostic imaging , Case-Control Studies , Equilibrative Nucleoside Transporter 1/metabolism , Female , Humans , Low Density Lipoprotein Receptor-Related Protein-1/metabolism , Male , Microvessels/diagnostic imaging , Neoplasm Proteins/metabolism , Organic Anion Transporters/metabolism , Positron-Emission Tomography , Verapamil/metabolism , Young AdultABSTRACT
Radioresistance significantly decreases the efficacy of radiotherapy, which can ultimately lead to tumor recurrence and metastasis. As a novel type of nano-radiosensitizer, silver nanoparticles (AgNPs) have shown promising radiosensitizing properties in the radiotherapy of glioma, but their ability to efficiently enter and accumulate in tumor cells needs to be improved. In the current study, AS1411 and verapamil (VRP) conjugated bovine serum albumin (BSA) coated AgNPs (AgNPs@BSA-AS-VRP) were synthesized and characterized. Dark-field imaging and inductively coupled plasma mass spectrometry were applied to investigate the accumulation of AgNPs@BSA-AS and AgNPs@BSA-AS-VRP mixed in different ratios in U251 glioma cells. To assess the influences of 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP on the P-glycoprotein (P-gp) efflux activity, rhodamine 123 accumulation assay was carried out. Colony formation assay and tumor-bearing nude mice model were employed to examine the radiosensitizing potential of 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP. Thioredoxin Reductase (TrxR) Assay Kit was used to detect the TrxR activity in cells treated with different functionally modified AgNPs. Characterization results revealed that AgNPs@BSA-AS-VRP were successfully constructed. When AgNPs@BSA-AS and AgNPs@BSA-AS-VRP were mixed in a ratio of 19:1, the amount of intracellular nanoparticles increased greatly through AS1411-mediated active targeting and inhibition of P-gp activity. In vitro and in vivo experiments clearly showed that the radiosensitization efficacy of 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP was much stronger than that of AgNPs@BSA and AgNPs@BSA-AS. It was also found that 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP significantly inhibited intracellular TrxR activity. These results indicate that 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP can effectively accumulate in tumor cells and have great potential as high-efficiency nano-radiosensitizers in the radiotherapy of glioma.
Subject(s)
Aptamers, Nucleotide/metabolism , Brain Neoplasms/radiotherapy , Glioma/radiotherapy , Metal Nanoparticles/chemistry , Oligodeoxyribonucleotides/metabolism , Radiation Tolerance , Radiation-Sensitizing Agents/pharmacology , Silver/chemistry , Verapamil/metabolism , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/pharmacology , Brain Neoplasms/pathology , Cell Line, Tumor , Glioma/pathology , Humans , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/pharmacology , Verapamil/chemistry , Verapamil/pharmacologyABSTRACT
Multidrug resistance (MDR) caused by overexpressed permeability-glycoprotein (P-gp) in cancer cells is the main barrier for the cure of cancers. P-gp can pump many chemotherapeutic drugs, which is a viable target to overcome P-gp-mediated MDR by efficient inhibitors of P-gp. However, limited understanding of the efflux mechanism by human P-gp hinders the development of efficient inhibitors. Herein, the transport of a P-gp inhibitor, verapamil, by human P-gp has been investigated using targeted molecular dynamics simulations and energetics analysis based on our previous research on the transport of a drug (doxorubicin). The energetics analysis identifies that the driving forces for the transport of verapamil are electrostatic repulsions contributed by the positively charged residues in the initial stage and then hydrophobic interactions contributed by the important residues in the later stage. This scenario is generally consistent with that in the transport of doxorubicin. However, the positively charged residues and the important residues for the transport of verapamil are incompletely consistent with the relative residues for the transport of doxorubicin. Moreover, the binding free energy contributions of the positively charged residues for the transport of verapamil are generally higher than them for the transport of doxorubicin, while the important residues constitute significantly different binding free energy compositions in the transports of the two substrates. Consequently, the pathway for the transport of verapamil is identified, which shares only two residues (F336 and M986) with the pathway of doxorubicin. This may imply the weak competitiveness of verapamil with doxorubicin in the substrate efflux. Taken together, this work provided new insights into the efflux mechanisms by human P-gp and would be beneficial in the design of potent P-gp inhibitors.