Carbachol increases Na+-HCO3− cotransport activity in murine colonic crypts in a M3−, Ca2+/calmodulin-, and PKC-dependent manner

2006 ◽  
Vol 291 (4) ◽  
pp. G650-G657 ◽  
Author(s):  
O. Bachmann ◽  
D. Reichelt ◽  
B. Tuo ◽  
M. P. Manns ◽  
U. Seidler
Keyword(s):  
Proximal Colon ◽  
Significant Inhibition ◽  
Dependent Manner ◽  
Activity Increase ◽  
Anion Secretion ◽  
Uptake Pathway ◽  
Calmodulin Kinase ◽  
Colonic Crypts ◽  
Acid Load ◽  
Intracellular Buffering Capacity

The Na+-HCO3− cotransporter (NBC) mediates HCO3− import into the colonocyte via its pNBC1 isoform. Whereas renal kNBC1 is inhibited by increased cAMP levels, pNBC1 is stimulated. Cholinergic stimulation activates renal NBC, but the effect on intestinal NBC is unknown. Therefore, crypts were isolated from the murine proximal colon by Ca2+ chelation and loaded with the pH-sensitive dye 2′,7′-bis-carboxyethyl-5,6-carboxyfluorescein. Na+-HCO3− cotransport activity was calculated from the dimethylamiloride-insensitive (500 μM) intracellular pH recovery from an acid load in the presence of CO2-HCO3− and the intracellular buffering capacity. Carbachol strongly increased Na+-HCO3− cotransport activity compared with control rates. Ca2+ chelation with BAPTA-AM, blockade of the M3 subtype of muscarinergic receptors with 4-diphenylacetoxy- N-methylpiperidine methiodide, and inhibition of Ca2+/calmodulin kinase II with KN-62 all caused significant inhibition of the carbachol-induced NBC activity increase. Furthermore, PKC inhibition with Gö-6976 and Gö-6850 significantly reduced the carbachol effect, which may be related to the unique NH2-terminal consensus site for PKC-dependent phosphorylation of pNBC1. We conclude that NBC in the murine colon is thus activated by carbachol, consistent with its presumed function as an anion uptake pathway during intestinal anion secretion, but that the signal transductions pathways are distinct from those involved in the cholinergic activation of renal NBC1.

scholarly journals Bacterial lipopolysaccharides variably modulate in vitro biofilm formation of Candida species

2010 ◽  
Vol 59 (10) ◽  
pp. 1225-1234 ◽  
Author(s):  
H. M. H. N. Bandara ◽  
O. L. T. Lam ◽  
R. M. Watt ◽  
L. J. Jin ◽  
L. P. Samaranayake
Keyword(s):  
Biofilm Formation ◽  
Laser Scanning ◽  
Significant Inhibition ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Bacterial Lipopolysaccharides ◽  
Species Specific ◽  
Biofilm Development

The objective of this study was to evaluate the effect of the bacterial endotoxin LPS on Candida biofilm formation in vitro. The effect of the LPS of Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia marcescens and Salmonella typhimurium on six different species of Candida, comprising Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, Candida tropicalis ATCC 13803, Candida parapsilosis ATCC 22019 and Candida dubliniensis MYA 646, was studied using a standard biofilm assay. The metabolic activity of in vitro Candida biofilms treated with LPS at 90 min, 24 h and 48 h was quantified by XTT reduction assay. Viable biofilm-forming cells were qualitatively analysed using confocal laser scanning microscopy (CLSM), while scanning electron microscopy (SEM) was employed to visualize the biofilm structure. Initially, adhesion of C. albicans was significantly stimulated by Pseudomonas and Klebsiella LPS. A significant inhibition of Candida adhesion was noted for the following combinations: C. glabrata with Pseudomonas LPS, C. tropicalis with Serratia LPS, and C. glabrata, C. parapsilosis or C. dubliniensis with Salmonella LPS (P<0.05). After 24 h of incubation, a significant stimulation of initial colonization was noted for the following combinations: C. albicans/C. glabrata with Klebsiella LPS, C. glabrata/C. tropicalis/C. krusei with Salmonella LPS. In contrast, a significant inhibition of biofilm formation was observed in C. glabrata/C. dubliniensis/C. krusei with Pseudomonas LPS, C. krusei with Serratia LPS, C. dubliniensis with Klebsiella LPS and C. parapsilosis/C. dubliniensis /C. krusei with Salmonella LPS (P<0.05). On further incubation for 48 h, a significant enhancement of biofilm maturation was noted for the following combinations: C. glabrata/C. tropicalis with Serratia LPS, C. dubliniensis with Klebsiella LPS and C. glabrata with Salmonella LPS, and a significant retardation was noted for C. parapsilosis/C. dubliniensis/C. krusei with Pseudomonas LPS, C. tropicalis with Serratia LPS, C. glabrata/C. parapsilosis/C. dubliniensis with Klebsiella LPS and C. dubliniensis with Salmonella LPS (P<0.05). These findings were confirmed by SEM and CLSM analyses. In general, the inhibition of the biofilm development of LPS-treated Candida spp. was accompanied by a scanty architecture with a reduced numbers of cells compared with the profuse and densely colonized control biofilms. These data are indicative that bacterial LPSs modulate in vitro Candida biofilm formation in a species-specific and time-dependent manner. The clinical and the biological relevance of these findings have yet to be explored.

Fatty acids and cytokine mRNA expression in human osteoblastic cells: a specific effect of arachidonic acid

2002 ◽  
Vol 102 (4) ◽  
pp. 403-409 ◽  
Author(s):  
G. PRIANTE ◽  
L. BORDIN ◽  
E. MUSACCHIO ◽  
G. CLARI ◽  
B. BAGGIO
Keyword(s):  
Fatty Acids ◽  
Arachidonic Acid ◽  
Mrna Expression ◽  
Specific Effect ◽  
Significant Inhibition ◽  
Cytokine Gene Expression ◽  
Dependent Manner ◽  
Human Osteoblast ◽  
Cytokine Mrna ◽  
Cytokine Mrna Expression

Epidemiological, clinical and experimental evidence suggests that fatty acids have a modulatory effect on bone metabolism in animals and humans. To investigate this hypothesis, we evaluated the effects of three different fatty acids, arachidonic acid (AA), eicosapentaenoic acid (EPA) and oleic acid (OA), on the expression of cytokines involved in bone remodelling. Cytokine mRNAs in the human osteoblast-like cell line MG-63 were quantified by reverse transcription-PCR. AA induced increased expression of interleukin-1α, interleukin-1β, tumour necrosis factor-α and macrophage colony-stimulating factor mRNAs in a time- and dose-dependent manner. EPA and OA had no stimulatory effects, but instead caused a significant inhibition of AA-induced cytokine mRNA expression. Cell treatment with calphostin C, an inhibitor of protein kinase C (PKC), and cellular PKC down-regulation experiments independently resulted in significant inhibition of AA-induced cytokine expression, suggesting that a PKC-dependent mechanism accounts for the effects of AA on cytokine production. In conclusion, our study demonstrates specific effects of fatty acids on cytokine gene expression in human osteoblast-like cells. The clinical relevance of our findings requires further investigation.

scholarly journals Inhibition of Cytochrome P450 (CYP450) Isoforms by Isoniazid: Potent Inhibition of CYP2C19 and CYP3A

2001 ◽  
Vol 45 (2) ◽  
pp. 382-392 ◽  
Author(s):  
Zeruesenay Desta ◽  
Nadia V. Soukhova ◽  
David A. Flockhart
Keyword(s):  
Cytochrome P450 ◽  
Liver Microsomes ◽  
Cost Effective ◽  
Competitive Inhibitor ◽  
Inhibitory Effect ◽  
Significant Inhibition ◽  
Specific Substrate ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
The Mean

ABSTRACT Isoniazid (INH) remains the most safe and cost-effective drug for the treatment and prophylaxis of tuberculosis. The use of INH has increased over the past years, largely as a result of the coepidemic of human immunodeficiency virus infection. It is frequently given chronically to critically ill patients who are coprescribed multiple medications. The ability of INH to elevate the concentrations in plasma and/or toxicity of coadministered drugs, including those of narrow therapeutic range (e.g., phenytoin), has been documented in humans, but the mechanisms involved are not well understood. Using human liver microsomes (HLMs), we tested the inhibitory effect of INH on the activity of common drug-metabolizing human cytochrome P450 (CYP450) isoforms using isoform-specific substrate probe reactions. Incubation experiments were performed at a single concentration of each substrate probe at its Km value with a range of INH concentrations. CYP2C19 and CYP3A were inhibited potently by INH in a concentration-dependent manner. At 50 μM INH (∼6.86 μg/ml), the activities of these isoforms decreased by ∼40%. INH did not show significant inhibition (<10% at 50 μM) of other isoforms (CYP2C9, CYP1A2, and CYP2D6). To accurately estimate the inhibition constants (Ki values) for each isoform, four concentrations of INH were incubated across a range of five concentrations of specific substrate probes. The meanKi values (± standard deviation) for the inhibition of CYP2C19 by INH in HLMs and recombinant human CYP2C19 were 25.4 ± 6.2 and 13 ± 2.4 μM, respectively. INH showed potent noncompetitive inhibition of CYP3A (Ki = 51.8 ± 2.5 to 75.9 ± 7.8 μM, depending on the substrate used). INH was a weak noncompetitive inhibitor of CYP2E1 (Ki = 110 ± 33 μM) and a competitive inhibitor of CYP2D6 (Ki = 126 ± 23 μM), but the mean Ki values for the inhibition of CYP2C9 and CYP1A2 were above 500 μM. Inhibition of one or both CYP2C19 and CYP3A isoforms is the likely mechanism by which INH slows the elimination of coadministered drugs, including phenytoin, carbamazepine, diazepam, triazolam, and primidone. Slow acetylators of INH may be at greater risk for adverse drug interactions, as the degree of inhibition was concentration dependent. These data provide a rational basis for understanding drug interaction with INH and predict that other drugs metabolized by these two enzymes may also interact.

scholarly journals Neural FFA3 activation inversely regulates anion secretion evoked by nicotinic ACh receptor activation in rat proximal colon

2016 ◽  
Vol 594 (12) ◽  
pp. 3339-3352 ◽  
Author(s):  
Izumi Kaji ◽  
Yasutada Akiba ◽  
Kohtarou Konno ◽  
Masahiko Watanabe ◽  
Shunsuke Kimura ◽  
...  
Keyword(s):  
Receptor Activation ◽  
Proximal Colon ◽  
Anion Secretion

scholarly journals Comparative Evaluation of Analgesic, Anti-inflammatory and Antipyretic Effects of Synthetic Derivatives of Organoantimony (?) Ferocenyl Benzoate with Piroxicam

2016 ◽  
Vol 19 (1) ◽  
pp. 15-24
Author(s):  
Muhammad Shoaib Akhtar ◽  
Zulfiqar Khan ◽  
Muhammad Naveed Mushtaq ◽  
Muhammad Salman Akhtar
Keyword(s):  
Pharmaceutical Journal ◽  
Significant Inhibition ◽  
Diffusion Method ◽  
Dependent Manner ◽  
Standard Drug ◽  
Compound A ◽  
Paw Oedema ◽  
Anti Inflammatory ◽  
Synthetic Derivatives ◽  
Dose Dependent

The current study was planned to evaluate comparative anti-inflammatory, analgesic and anti-pyretic activities of two newly synthesized organo-antimony (v) ferrocenyl benzoate derivatives with piroxicam. Anti-microbial activity of these compounds was also screened against two microorganisms. Analgesic effect of test compounds was evaluated by formalin-induced paw licking test in mice. The test compounds at 50 and 100 mg/kg b.w. doses exhibited significant (p<0.001) reduction of paw licking in treated mice comparable with standard drug piroxicam. Anti-inflammatory activity was assessed against carrageenan-induced paw oedema. The compound A produced anti-inflammatory effects comparable with standard piroxicam in dose dependent manner whereas compound B showed better effects than piroxicam at dose of 100 mg/kg body weight. To investigate anti-pyretic activity, fever was induced by administration of Brewer’s yeast in mice. Compound A showed highly significant inhibition of pyrexia (p<0.001) comparable to piroxicam after 3 hours while compound B (50 and 100 mg/kg) produced relatively lower anti-pyretic effect than standard drug. Antibacterial activity determined by disc diffusion method showed that compound B was relatively more effective than compound A against Staphylococcus aureus and Klebsiella pneumoniae. It is conceivable that both the tested compounds possessed anti-inflammatory, analgesic, anti-pyretic and anti-microbial effects even after the structural modification of parent compound.Bangladesh Pharmaceutical Journal 19(1): 15-24, 2016

Protein kinase-dependent and Ca2+-independent cAMP inhibition of ANP release in beating rabbit atria

2002 ◽  
Vol 282 (5) ◽  
pp. R1477-R1489 ◽  
Author(s):  
Xun Cui ◽  
Jin Fu Wen ◽  
Jing Yu Jin ◽  
Wen Xie Xu ◽  
Sung Zoo Kim ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Mechanical Stretch ◽  
Dependent Manner ◽  
Calmodulin Kinase ◽  
Minor Portion ◽  
Channel Dependent ◽  
A Minor ◽  
The Relationship ◽  
Rabbit Atria

Regulation of atrial release of atrial natriuretic peptide (ANP) is coupled to changes in atrial dynamics. However, the mechanism by which mechanical stretch controls myocytic ANP release must be defined. The purpose of this study was to define the mechanism by which cAMP controls myocytic ANP release in perfused, beating rabbit atria. The cAMP-elevating agents forskolin and 3-isobutyl-1-methylxanthine (IBMX) inhibited myocytic ANP release. The activation of adenylyl cyclase with forskolin inhibited ANP release, which was a function of an increase in cAMP production. Inhibitors for L-type Ca2+ channels and protein kinase A (PKA) attenuated a minor portion of the forskolin-induced inhibition of ANP release. Gö-6976 and KN-62, which are specific inhibitors for protein kinase C-α and Ca2+/calmodulin kinase, respectively, failed to modulate forskolin-induced inhibition of ANP release. The nonspecific protein kinase inhibitor staurosporine blocked forskolin-induced inhibition of ANP release in a dose-dependent manner. Staurosporine but not nifedipine shifted the relationship between cAMP and ANP release. Inhibitors for L-type Ca2+ channels and PKA and staurosporine blocked forskolin-induced accentuation of atrial dynamics. These results suggest that cAMP inhibits atrial myocytic release of ANP via protein kinase-dependent and L-type Ca2+-channel-dependent and -independent signaling pathways.

scholarly journals TMIC-43. ROLE OF EXTRACELLULAR FERRITIN HEAVY CHAIN PROTEIN IN GLIOBLASTOMA MULTIFORME

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi257-vi257
Author(s):  
Bhavyata Pandya ◽  
Vagisha Ravi ◽  
James Connor
Keyword(s):  
Glioblastoma Multiforme ◽  
Heavy Chain ◽  
Dependent Manner ◽  
Cellular Viability ◽  
Ferritin Heavy Chain ◽  
Uptake Pathway ◽  
Iron Delivery ◽  
Human Oligodendrocytes ◽  
Dose Dependent

Abstract Increased expression of Ferritin heavy chain (FHC) protein has been associated with poor prognosis in Glioblastoma Multiforme (GBM) which is one of the most aggressive and common types of brain cancer. GBM patients have also been found to have increased extracellular ferritin levels, in their serum and cerebrospinal fluid (CSF), which are lowered once the source/tumor has been resected. Extracellular FHC can function as an iron delivery protein, and increasing amount of iron has been known to contribute to tumor initiation and proliferation. To study the effect of extracellular FHC in GBM cells we used patient derived GBM, CD133+ cancer stem cells (GSCs) from the pro-neural (T3691) and mesenchymal (T387) subtypes. Using recombinant FHC, conjugated with quantum dots (QD), we observed significant increase in cellular viability and intracellular uptake of FHC by the GSCs in a dose dependent manner. Our lab has previously shown that extracellular FHC interacts with T-Cell Immunoglobulin Mucin Receptor 1 (Tim-1) in the human oligodendrocytes. In order to determine if GSCs express the Tim-1 receptor we first confirmed its expression on GSCs using immunoblotting and immunocytochemistry. To test if FHC interacts with Tim-1, we performed knockdown of Tim-1 using siRNAs. However, the siRNA was not able to downregulate the Tim-1 receptors. Next, we exposed the GSCs to Sema4A, which has been shown in our previous studies to interact with Tim-1 receptor on human oligodendrocytes and is toxic to oligodendrocytes. The GSCs however were not affected by the saturable concentration of Sema4A. Thus, through this study we have shown the expression of potential FHC receptors on GSCs and a robust effect of H-ferritin on GSCs proliferation. Further experiments are warranted in this direction to understand this extracellular FHC uptake pathway and its role in GBM cell proliferation.

scholarly journals Anti-Nociceptive and Anti-Inflammation Effect Mechanisms of Mutants of Syb-prII, a Recombinant Neurotoxic Polypeptide

Toxins ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 699
Author(s):  
Chunli Li ◽  
Mengqi Ban ◽  
Fei Bai ◽  
Jianzhao Chen ◽  
Xiaoquan Jin ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Inhibitory Effect ◽  
Significant Inhibition ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Analgesic Effects ◽  
Medicinal Value ◽  
Inflammatory Nociception ◽  
Channel Currents

Syb-prII, a recombinant neurotoxic polypeptide, has analgesic effects with medicinal value. Previous experiments indicated that Syb-prII displayed strong analgesic activities. Therefore, a series of in vivo and vitro experiments were designed to investigate the analgesic and anti-inflammatory properties and possible mechanisms of Syb-prII. The results showed that administered Syb-prII-1 and Syb-prII-2 (0.5, 1, 2.0 mg/kg, i.v.) to mice significantly reduced the time of licking, biting, or flicking of paws in two phases in formalin-induced inflammatory nociception. Syb-prII-1 inhibited xylene-induced auricular swelling in a dose-dependent manner. The inhibitory effect of 2.0 mg/kg Syb-prII-1 on the ear swelling model was comparable to that of 200 mg/kg aspirin. In addition, the ELISA and Western blot analysis suggested that Syb-prII-1 and Syb-prII-2 may exert an analgesic effect by inhibiting the expression of Nav1.8 and the phosphorylation of ERK, JNK, and P38. Syb-prII-1 markedly suppressed the expression of IL-1β, IL-6, and TNF-α of mice in formalin-induced inflammatory nociception. We used the patch-clamp technique and investigated the effect of Syb-prII-1 on TTX-resistant sodium channel currents in acutely isolated rat DRG neurons. The results showed that Syb-prII-1 can significantly down regulate TTX-resistant sodium channel currents. In conclusion, Syb-prII mutants may alleviate inflammatory pain by significantly inhibiting the expression of Nav1.8, mediated by the phosphorylation of MAPKs and significant inhibition of TTX-resistant sodium channel currents.

scholarly journals Activation of TRPA1 by luminal stimuli induces EP4-mediated anion secretion in human and rat colon

2012 ◽  
Vol 302 (7) ◽  
pp. G690-G701 ◽  
Author(s):  
Izumi Kaji ◽  
Yukiko Yasuoka ◽  
Shin-ichiro Karaki ◽  
Atsukazu Kuwahara
Keyword(s):  
Host Defense ◽  
Transient Receptor Potential ◽  
Defense Mechanism ◽  
Ussing Chamber ◽  
Pcr Analysis ◽  
Rat Colon ◽  
Receptor Subtype ◽  
Neural Pathways ◽  
Dependent Manner ◽  
Anion Secretion

In gastrointestinal (GI) physiology, anion and fluid secretion is an important function for host defense and is induced by changes in the luminal environment. The transient receptor potential A1 (TRPA1) channel is considered to be a chemosensor in several sensory tissues. Although the function of TRPA1 has been studied in GI motility, its contribution to the transepithelial ion transport system has rarely been discussed. In the present study, we investigated the secretory effect of the potential TRPA1 agonist allyl isothiocyanate (AITC) in rat and human colon using an Ussing chamber. The mucosal application of AITC (10−6-10−3 M) induced Cl− and HCO3− secretion in a concentration-dependent manner, whereas the serosal application induced a significantly weaker effect. AITC-evoked anion secretion was attenuated by tissue pretreatment with piroxicam and prostaglandin (PG) E2; however, this secretion was not affected by TTX, atropine, or extracellular Ca2+ depletion. These experiments indicate that TRPA1 activation induces anion secretion through PG synthesis, independent of neural pathways in the colon. Further analysis also indicates that AITC-evoked anion secretion is mediated mainly by the EP4 receptor subtype. The magnitude of the secretory response exhibited segmental heterogeneity in rat colon. Real-time PCR analysis showed the segmental difference was corresponding to the differential expression of EP4 receptor and cyclooxygenase-1 and -2. In addition, RT-PCR, in situ hybridization, and immunohistochemical studies showed TRPA1 expression in the colonic epithelia. Therefore, we conclude that the activation of TRPA1 in colonic epithelial cells is likely involved in the host defense mechanism through rapid anion secretion.

scholarly journals Regulation of electrolyte transport across cultured endometrial epithelial cells by prolactin

2008 ◽  
Vol 197 (3) ◽  
pp. 575-582 ◽  
Author(s):  
Chatsri Deachapunya ◽  
Sutthasinee Poonyachoti ◽  
Nateetip Krishnamra
Keyword(s):  
Epithelial Cells ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Disulfonic Acid ◽  
Maximum Effect ◽  
Channel Blockers ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Anion Secretion ◽  
Endometrial Epithelial Cells

The effect of prolactin (PRL) on ion transport across the porcine glandular endometrial epithelial cells was studied in primary cell culture using the short-circuit current technique. Addition of 1 μg/ml PRL either to the apical solution or to the basolateral solution produced a peak followed by a sustained increase in Isc, but with a lesser response when PRL was added apically. Basolateral addition of PRL increased the Isc in a concentration-dependent manner with a maximum effect at 1 μg/ml and an effective concentration value of 120 ng/ml. The PRL-stimulated Isc was significantly reduced by pretreatment with an apical addition of 5-nitro-2-(3-phenylpropylamino) benzoic acid (200 μM), diphenylamine-2-carboxylic acid (1 mM) or 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (200 μM), Cl− channel blockers, but not by amiloride (10 μM), a Na+ channel blocker. In addition, pretreatment with bumetanide (200 μM), a Na+–K+–2Cl− cotransporter inhibitor, in the basolateral solution significantly reduced the PRL-stimulated Isc. Replacement of Cl− or in the bathing solutions also decreased the Isc response to PRL. Pretreatment of the monolayer with AG490 (50 μM), an inhibitor of JAK2 activity significantly inhibited the PRL-induced increase in Isc. Western blot analysis of the porcine endometrial epithelial cells revealed the presence of short isoform of PRL receptor (PRLR-S) that could be regulated by 17β-estradiol. The results of this investigation showed that PRL acutely stimulated anion secretion across the porcine endometrial epithelial cells possibly through PRLR-S present in both apical and basolateral membranes. The PRL response appeared to be mediated by the JAK2-dependent pathway.

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