scholarly journals Anandamide inhibits transport-related oxygen consumption in the loop of Henle by activating CB1 receptors

2013 ◽  
Vol 304 (4) ◽  
pp. F376-F381 ◽  
Author(s):  
Guillermo B. Silva ◽  
Douglas K. Atchison ◽  
Luis I. Juncos ◽  
Néstor H. García
Keyword(s):  
Oxygen Consumption ◽  
Receptor Antagonist ◽  
Cannabinoid Receptors ◽  
Cb1 Receptors ◽  
Dependent Manner ◽  
Cb2 Receptor ◽  
Na Transport ◽  
Concentration Dependent Manner ◽  
Selective Agonist

The energy required for active Na chloride reabsorption in the thick ascending limb (TAL) depends on oxygen consumption and oxidative phosphorylation (OXP). In other cells, Na transport is inhibited by the endogenous cannabinoid anandamide through the activation of the cannabinoid receptors (CB) type 1 and 2. However, it is unclear whether anandamide alters TAL transport and the mechanisms that could be involved. We hypothesized that anandamide inhibits TAL transport via activation of CB1 receptors and NO. For this, we measured oxygen consumption (QO2) in TAL suspensions to monitor the anandamide effects on transport and OXP. Anandamide reduced QO2 in a concentration-dependent manner. During Na-K-2Cl cotransport and Na/H exchange inhibition, anandamide did not inhibit TAL QO2. To test the role of the cannabinoid receptors, we used specific agonists and antagonists of CB1 and CB2 receptors. The CB1-selective agonist WIN55212–2 reduced QO2 in a concentration-dependent manner. Also, the CB1 receptor antagonist rimonabant blocked the effect of anandamide on QO2. In contrast, the CB2-selective agonist JHW-133 had no effect on QO2, while the CB2 receptor antagonist AM-630 failed to block the anandamide effects on QO2. To confirm these results, we measured CB1 and CB2 receptor expression and only CB1 expression was detected. Because CB1 receptors are strong nitric oxide synthase (NOS) stimulators and NO inhibits transport in TALs, we evaluated the role of NO. Anandamide stimulated NO production and the NOS inhibitor NG-nitro-l-arginine methyl ester blocked the anandamide effects on QO2. We conclude that anandamide inhibits TAL Na transport-related QO2 via activation of CB1 receptor and NOS.

scholarly journals Extracellular ATP inhibits transport in medullary thick ascending limbs: role of P2X receptors

2009 ◽  
Vol 297 (5) ◽  
pp. F1168-F1173 ◽  
Author(s):  
Guillermo B. Silva ◽  
Jeffrey L. Garvin
Keyword(s):  
Nitric Oxide ◽  
Oxygen Consumption ◽  
Oxidative Phosphorylation ◽  
Receptor Antagonist ◽  
P2x Receptors ◽  
Extracellular Atp ◽  
Thick Ascending Limb ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Nitric Oxide Synthase Inhibitor

Absorption of NaCl by the thick ascending limb (TAL) involves active transport and therefore depends on oxidative phosphorylation. Extracellular ATP has pleiotropic effects, including both stimulation and inhibition of transport and inhibition of oxidative phosphorylation. However, it is unclear whether ATP alters TAL transport and how this occurs. We hypothesized that ATP inhibits TAL Na absorption by reducing Na entry. We measured oxygen consumption in TAL suspensions. ATP reduced oxygen consumption in a concentration-dependent manner. The purinergic (P2) receptor antagonist suramin (300 μM) blocked the effect of ATP on TAL oxygen consumption (147 ± 15 vs. 146 ± 16 nmol O2·min−1·mg protein−1). In contrast, the adenosine receptor antagonist theophylline did not block the effect of ATP on oxygen consumption. When Na-K-2Cl cotransport and Na/H exchange were blocked with furosemide (100 μM) plus dimethyl amiloride (100 μM), ATP did not inhibit TAL oxygen consumption (from 78 ± 13 to 98 ± 5 nmol O2·min−1·mg protein−1). The Na ionophore nystatin (200 U/ml) increased TAL oxygen consumption to a similar extent in both ATP- and vehicle-treated samples (368 ± 41 vs. 397 ± 47 nmol O2·min−1·mg protein−1). The nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester (3 mM) blocked the ATP effects on TAL oxygen consumption (157 ± 10 vs. 165 ± 15 nmol O2·min−1·mg protein−1). The P2X-selective receptor antagonist NF023 blocked the effect of ATP on oxygen consumption, whereas the P2X-selective agonist β-γ-Me-ATP reduced oxygen consumption in a concentration-dependent manner. We conclude that ATP inhibits Na transport-related oxygen consumption in TALs by reducing Na entry and P2X receptors and nitric oxide mediate this effect.

The Endocannabinoid 2-Arachidonoylglycerol Regulates Platelet Function.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3904-3904
Author(s):  
Samantha Baldassarri ◽  
Alessandra Bertoni ◽  
Paolo Lova ◽  
Stefania Reineri ◽  
Chiara Sarasso ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Cannabinoid Receptors ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Dependent Manner ◽  
Cb2 Receptor ◽  
Thromboxane A2 Receptor

Abstract 2-Arachidonoylglycerol (2-AG) is a naturally occurring monoglyceride that activates cannabinoid receptors and meets several key requisites of an endogenous cannabinoid substance. It is present in the brain and hematopoietic cells, including macrophages, lymphocytes and platelets. 2-AG is released from cells in a stimulus-dependent manner and is rapidly eliminated by uptake into cells and enzymatic hydrolysis in arachidonic acid and glycerol. 2-AG might exert a very fine control on platelet function either through mechanisms intertwining with the signal transduction pathways used by platelet agonists or through mechanisms modulating specific receptors. The aim of this study was to define the role of 2-AG in human platelets and characterize the mechanisms by which it performs its action. Platelets from healthy donors were isolated from plasma by differential centrifugations and gel-filtration on Sepharose 2B. The samples were incubated with 2-AG (10–100 μM) under constant stirring in the presence or absence of various inhibitors. Platelet aggregation was measured by Born technique. We have found that stimulation of human platelets with 2-AG induced irreversible aggregation, which was significantly enhanced by co-stimulation with ADP (1–10 μM). Furthermore, 2-AG-dependent platelet aggregation was completely inhibited by ADP scavengers, aspirin, and Rho kinase inhibitor, as well as by antagonists of the 2-AG receptor (CB2), of the ADP P2Y12 receptor, and of the thromboxane A2 receptor. We further investigated the role of endocannabinoids on calcium mobilization. Intracellular [Ca2+] was measured using FURA-2-loaded platelets prewarmed at 37°C under gentle stirring in a spectrofluorimeter. 2-AG induced rapid increase of cytosolic [Ca2+] in a dose-dependent manner. This effect was partially blocked by ADP scavengers and CB2 receptor antagonists. Furthermore, 2-AG-induced [Ca2+] mobilization was totally suppressed by aspirin or the thromboxane A2 receptor antagonist. These results suggest that 2-AG is able to trigger platelet activation, and that this action is partially mediated by CB2 receptor and ADP. Furthmore, 2-AG-dependent platelet activation is totally dependent on thromboxane A2 generation.

The P-element-induced silencing effect of KP transposons is dose dependent in Drosophila melanogaster

Genome ◽  
2011 ◽  
Vol 54 (9) ◽  
pp. 752-762 ◽  
Author(s):  
Alireza Sameny ◽  
John Locke
Keyword(s):  
Drosophila Melanogaster ◽  
Critical Role ◽  
Mutagenic Effect ◽  
P Element ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
P Elements ◽  
Single Well ◽  
Dose Dependent

Transposable elements are found in the genomes of all eukaryotes and play a critical role in altering gene expression and genome organization. In Drosophila melanogaster, transposable P elements are responsible for the phenomenon of hybrid dysgenesis. KP elements, a deletion-derivative of the complete P element, can suppress this mutagenic effect. KP elements can also silence the expression of certain other P-element-mediated transgenes in a process called P-element-dependent silencing (PDS), which is thought to involve the recruitment of heterochromatin proteins. To explore the mechanism of this silencing, we have mobilized KP elements to create a series of strains that contain single, well-defined KP insertions that show PDS. To understand the quantitative role of KP elements in PDS, these single inserts were combined in a series of crosses to obtain genotypes with zero, one, or two KP elements, from which we could examine the effect of KP gene dose. The extent of PDS in these genotypes was shown to be dose dependent in a logarithmic rather than linear fashion. A logarithmic dose dependency is consistent with the KP products interacting with heterochromatic proteins in a concentration-dependent manner such that two molecules are needed to induce gene silencing.

Protein kinase C potentiates cAMP-stimulated mouse duodenal mucosal bicarbonate secretion in vitro

2004 ◽  
Vol 286 (5) ◽  
pp. G814-G821 ◽  
Author(s):  
Bi-Guang Tuo ◽  
Jimmy Y. C. Chow ◽  
Kim E. Barrett ◽  
Jon I. Isenberg
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Duodenal Mucosa ◽  
Bicarbonate Secretion ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ussing Chambers ◽  
Duodenal Bicarbonate Secretion

PKC has been shown to regulate epithelial Cl- secretion in a variety of models. However, the role of PKC in duodenal mucosal bicarbonate secretion is less clear. We aimed to investigate the role of PKC in regulation of duodenal mucosal bicarbonate secretion. Bicarbonate secretion by murine duodenal mucosa was examined in vitro in Ussing chambers using a pH-stat technique. PKC isoform expression and activity were assessed by Western blotting and in vitro kinase assays, respectively. PMA (an activator of PKC) alone had no effect on duodenal bicarbonate secretion or short-circuit current ( Isc). When PMA and dibutyryl-cAMP (db-cAMP) were added simultaneously, PMA failed to alter db-cAMP-stimulated duodenal bicarbonate secretion or Isc ( P > 0.05). However, a 1-h preincubation with PMA potentiated db-cAMP-stimulated duodenal bicarbonate secretion and Isc in a concentration-dependent manner (from 10-8 to 10-5M) ( P < 0.05). PMA preincubation had no effects on carbachol- or heat-stable toxin-stimulated bicarbonate secretion. Western blot analysis revealed that PKCα, -γ, -ϵ, -θ, -μ, and -ι/λ were expressed in murine duodenal mucosa. Ro 31–8220 (an inhibitor active against PKCϵ, -α, -β, and -γ), but not Gö 6983 (an inhibitor active against PKCα, -γ, -β, and -δ), reversed the potentiating effect of PMA on db-cAMP-stimulated bicarbonate secretion. PMA also time- and concentration-dependently increased the activity of PKCϵ, an effect that was prevented by Ro 31–8220 but not Gö 6983. These results demonstrate that activation of PKC potentiates cAMP-stimulated duodenal bicarbonate secretion, whereas it does not modify basal secretion. The effect of PKC on cAMP-stimulated bicarbonate secretion is mediated by the PKCϵ isoform.

scholarly journals Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

2004 ◽  
Vol 167 (1) ◽  
pp. 75-85 ◽  
Author(s):  
Brenton L. Scott ◽  
Jeffrey S. Van Komen ◽  
Hassan Irshad ◽  
Song Liu ◽  
Kirilee A. Wilson ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Fusion ◽  
Membrane Trafficking ◽  
Snare Complex ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Bud Neck ◽  
Precise Role

Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

scholarly journals Effect of Palmitic Acid on Exosome-Mediated Secretion and Invasive Motility in Prostate Cancer Cells

Molecules ◽  
2020 ◽  
Vol 25 (12) ◽  
pp. 2722
Author(s):  
Ivan V. Maly ◽  
Wilma A. Hofmann
Keyword(s):  
Prostate Cancer ◽  
Palmitic Acid ◽  
Cancer Cells ◽  
Dependent Manner ◽  
Prostate Cancer Cells ◽  
Concentration Dependent Manner ◽  
Fat Consumption ◽  
Progression Markers

High fat consumption can enhance metastasis and decrease survival in prostate cancer, but the picture remains incomplete on the epidemiological and cell-biological level, impeding progress toward individualized recommendations in the clinic. Recent work has highlighted the role of exosomes secreted by prostate cancer cells in the progression of the disease, particularly in metastatic invasion, and also the utility of targeting these extracellular vesicles for diagnostics, as carriers of disease progression markers. Here, we investigated the question of a potential impact of the chief nutritional saturated fatty acid on the exosome secretion. Palmitic acid decreased the secretion of exosomes in human prostate cancer cells in vitro in a concentration-dependent manner. At the same time, the content of some prospective metastatic markers in the secreted exosomal fraction was also reduced, as was the ability of the cells to invade across extracellular matrix barriers. While by themselves our in vitro results imply that on the cell level, palmitic acid may be beneficial vis-à-vis the course of the disease, they also suggest that, by virtue of the decreased biomarker secretion, palmitic acid has the potential to cause unjustified deprioritization of treatment in obese and lipidemic men.

The Role Of Calcium In Platelet Microtubule Assembly- Disassembly

1981 ◽  
Author(s):  
M Kikuchi ◽  
Y Ikeda ◽  
M Handa ◽  
S Matsuda ◽  
H Muraki ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Dynamic Equilibrium ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Microtubule Assembly ◽  
Base Line ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Transient Decrease

Microtubules exist in a dynamic equilibrium between polymerized and depolymerized forms in human platelets, playing a major role to maintain the discoid shape of platelets. It has been previously shown that the interaction of aggregating agents with platelets leads to a rapid but transient disassembly of microtubules. ( Steiner and Ikeda, J.Clin. Invest. 63:443,1979 ) In this paper, the role of calcium in the equilibrium between assembled and disassembled microtubules was investigated. The respective pools of soluble and polymerized tubulin were “frozen” by addition of a glycerol-dimethyl sulfoxide-containing medium to platelet rich plasma, preincubated with 2 µM A23187 for various time intervals. The two pools of tubulin were estimated by measuring the colchicine binding activities of total and polymerized tubulin according to the method of Wilson.Resting platelets were found to contain 56.2 ± 2.7 µg tubulin per 109 platelets, of which 56.7 % was in polymerized form. Addition of A23187 to platelet rich plasma produced a transient decrease in the pool of polymerized tubulin within 30 sec., followed by a return to base-line values within 2 min.. TMB-8, a known intracellular calcium antagonist, abolished this transient decrease in polymerized tubulin induced by A23187 in a concentration dependent manner, while indomethacin or acetylsalycylic acid did not.These findings may indicate the important role of intracellular calcium in microtubule assembly-disassembly.

scholarly journals Cholecystokinin Octapeptide Increases Spontaneous Glutamatergic Synaptic Transmission to Neurons of the Nucleus Tractus Solitarius Centralis

2005 ◽  
Vol 94 (4) ◽  
pp. 2763-2771 ◽  
Author(s):  
V. Baptista ◽  
Z. L. Zheng ◽  
F. H. Coleman ◽  
R. C. Rogers ◽  
R. A. Travagli
Keyword(s):  
Receptor Antagonist ◽  
Nucleus Tractus Solitarius ◽  
Dependent Manner ◽  
Excitatory Effect ◽  
Concentration Dependent Manner ◽  
Whole Cell Patch Clamp ◽  
Glutamatergic Synaptic Transmission ◽  
Immunohistochemical Techniques ◽  
20 Nm ◽  
Brain Stem Slices

Cholecystokinin (CCK) is released from enteroendocrine cells after ingestion of nutrients and induces multiple effects along the gastrointestinal tract, including gastric relaxation and short-term satiety. We used whole cell patch-clamp and immunohistochemical techniques in rat brain stem slices to characterize the effects of CCK. In 45% of the neurons of nucleus tractus solitarius subnucleus centralis (cNTS), perfusion with the sulfated form of CCK (CCK-8s) increased the frequency of spontaneous excitatory currents (sEPSCs) in a concentration-dependent manner (1–300 nM). The threshold for the CCK-8s excitatory effect was 1 nM, the EC50 was 20 nM, and Emax was 100 nM. The excitatory effects of CCK-8s were still present when the slices were preincubated with tetrodotoxin or bicuculline or when the recordings were conducted with Cs+ electrodes. Pretreatment with the CCK-A receptor antagonist, lorglumide (1 μM), antagonized the effects of CCK-8s, whereas perfusion with the CCK-B preferring agonist CCK-8 nonsulfated (CCK-ns, 1 μM) did not affect the frequency of sEPSCs. Similarly, pretreatment with the CCK-B receptor antagonist, triglumide (1 μM), did not prevent the actions of CCK-8s. Although the majority (i.e., 76%) of CCK-8s unresponsive cNTS neurons had a bipolar somata shape and were TH-IR negative, no differences were found in either the morphological or the neurochemical phenotype of cNTS neurons responsive to CCK-8s. Our results suggest that the excitatory effects of CCK-8s on terminals impinging on a subpopulation of cNTS neurons are mediated by CCK-A receptors; these responsive neurons, however, do not have morphological or neurochemical characteristics that automatically distinguish them from nonresponsive neurons.

Oxidation of ubiquinol by peroxynitrite: implications for protection of mitochondria against nitrosative damage

2000 ◽  
Vol 349 (1) ◽  
pp. 35-42 ◽  
Author(s):  
Francisco SCHÖPFER ◽  
Natalia RIOBÓ ◽  
María Cecilia CARRERAS ◽  
Beatriz ALVAREZ ◽  
Rafael RADI ◽  
...  
Keyword(s):  
Radical Scavenging ◽  
Redox Status ◽  
Experimental Models ◽  
Dependent Manner ◽  
Mitochondrial Membranes ◽  
Concentration Dependent Manner ◽  
Major Pathway ◽  
Membrane Bound ◽  
Spectroscopy Studies

A major pathway of nitric oxide utilization in mitochondria is its conversion to peroxynitrite, a species involved in biomolecule damage via oxidation, hydroxylation and nitration reactions. In the present study the potential role of mitochondrial ubiquinol in protecting against peroxynitrite-mediated damage is examined and the requirements of the mitochondrial redox status that support this function of ubiquinol are established. (1) Absorption and EPR spectroscopy studies revealed that the reactions involved in the ubiquinol/peroxynitrite interaction were first-order in peroxynitrite and zero-order in ubiquinol, in agreement with the rate-limiting formation of a reactive intermediate formed during the isomerization of peroxynitrite to nitrate. Ubiquinol oxidation occurred in one-electron transfer steps as indicated by the formation of ubisemiquinone. (2) Peroxynitrite promoted, in a concentration-dependent manner, the formation of superoxide anion by mitochondrial membranes. (3) Ubiquinol protected against peroxynitrite-mediated nitration of tyrosine residues in albumin and mitochondrial membranes, as suggested by experimental models, entailing either addition of ubiquinol or expansion of the mitochondrial ubiquinol pool caused by selective inhibitors of complexes III and IV. (4) Increase in membrane-bound ubiquinol partially prevented the loss of mitochondrial respiratory function induced by peroxynitrite. These findings are analysed in terms of the redox transitions of ubiquinone linked to both nitrogen-centred radical scavenging and oxygen-centred radical production. It may be concluded that the reaction of mitochondrial ubiquinol with peroxynitrite is part of a complex regulatory mechanism with implications for mitochondrial function and integrity.

scholarly journals Mucin inhibitsPseudomonas aeruginosabiofilm formation by significantly enhancing twitching motility

2014 ◽  
Vol 60 (3) ◽  
pp. 155-166 ◽  
Author(s):  
Cecily L. Haley ◽  
Cassandra Kruczek ◽  
Uzma Qaisar ◽  
Jane A. Colmer-Hamood ◽  
Abdul N. Hamood
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iv Pili ◽  
Dependent Manner ◽  
Twitching Motility ◽  
Strain Pao1 ◽  
Concentration Dependent Manner ◽  
Dependent Effect ◽  
Type Iv ◽  
Biofilm Development

In Pseudomonas aeruginosa, type IV pili (TFP)-dependent twitching motility is required for development of surface-attached biofilm (SABF), yet excessive twitching motility is detrimental once SABF is established. In this study, we show that mucin significantly enhanced twitching motility and decreased SABF formation in strain PAO1 and other P. aeruginosa strains in a concentration-dependent manner. Mucin also disrupted partially established SABF. Our analyses revealed that mucin increased the amount of surface pilin and enhanced transcription of the pilin structural gene pilA. Mucin failed to enhance twitching motility in P. aeruginosa mutants defective in genes within the pilin biogenesis operons pilGHI/pilJK-chpA-E. Furthermore, mucin did not enhance twitching motility nor reduce biofilm development by chelating iron. We also examined the role of the virulence factor regulator Vfr in the effect of mucin. In the presence or absence of mucin, PAOΔvfr produced a significantly reduced SABF. However, mucin partially complemented the twitching motility defect of PAOΔvfr. These results suggest that mucin interferes with SABF formation at specific concentrations by enhancing TFP synthesis and twitching motility, that this effect, which is iron-independent, requires functional Vfr, and only part of the Vfr-dependent effect of mucin on SABF development occurs through twitching motility.

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