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.

Histamine stimulates ion transport by dog pancreatic duct epithelial cells through H1receptors

1998 ◽  
Vol 275 (1) ◽  
pp. G76-G84 ◽  
Author(s):  
Toan D. Nguyen ◽  
Charles N. Okolo ◽  
Mark W. Moody
Keyword(s):  
Epithelial Cells ◽  
Ion Transport ◽  
Receptor Antagonist ◽  
Pancreatic Duct ◽  
Direct Action ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ussing Chambers

Histamine affects pancreatic secretion, but its direct action on ion transport by pancreatic duct epithelial cells (PDEC) has not been defined. We now characterize the secretory effects of histamine on cultured, well-differentiated, and nontransformed dog PDEC. Histamine stimulated, in a concentration-dependent manner (1–100 μM), a cellular125I−efflux that was inhibited by 500 μM 5-nitro-2-(3-phenylpropylamino)benzoic acid, 2.5 mM diphenylamine-2-carboxylate, and 500 μM DIDS and thus mediated through Ca2+-activated Cl− channels. Histamine-stimulated125I−efflux was 1) inhibited by 100 μM diphenhydramine, an H1receptor antagonist, 2) resistant to 1 mM cimetidine, an H2 receptor antagonist, 3) not reproduced by 1 mM dimaprit, an H2 agonist, and 4) inhibited by 50 μM 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM, a Ca2+ chelator, suggesting that it was mediated through H1 receptors acting via increased cytosolic Ca2+. Histamine also stimulated a86Rb+efflux that was sensitive to 100 nM charybdotoxin and thus mediated through Ca2+-activated K+ channels. When PDEC monolayers were studied in Ussing chambers, a short-circuit current of 21.7 ± 3.1 μA/cm2 was stimulated by 100 μM histamine. This effect was inhibited by diphenhydramine but not cimetidine, was not reproduced with dimaprit, and was observed only after serosal addition of histamine, suggesting that it was mediated by basolateral H1 receptors on PDEC. In conclusion, histamine, acting through basolateral H1 receptors, activates both Ca2+-activated Cl− and K+ channels; in this manner, it may regulate PDEC secretion in normal or inflamed pancreas.

Potassium secretion by rabbit descending colon: effects of adrenergic stimuli

1986 ◽  
Vol 250 (4) ◽  
pp. G432-G439 ◽  
Author(s):  
P. L. Smith ◽  
R. D. McCabe
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Maximal Effect ◽  
Dependent Manner ◽  
Adrenergic Stimulation ◽  
Adrenergic Agents ◽  
Ussing Chambers ◽  
K Secretion ◽  
Potassium Secretion

Stripped rabbit distal colonic mucosa was studied in vitro in Ussing chambers to investigate the effects of adrenergic stimuli on Na+, K+, and Cl- transport. The adrenergic stimuli epinephrine and norepinephrine decrease short-circuit current in a dose-dependent manner, with a half-maximal effect at 5 X 10(-7) M and a maximal effect between 10(-5) and 10(-4) M. The effects produced by norepinephrine and epinephrine can also be elicited by the beta 1-agonist dobutamine, but not by the beta 2-agonist terbutaline or the alpha-agonist phenylephrine. In addition, the effects of adrenergic stimulation can be inhibited by the beta-antagonist propranolol but not by the muscarinic antagonist atropine, the alpha 2-antagonist yohimbine, or tetrodotoxin. The decrease in short-circuit current elicited by adrenergic stimuli is accompanied by an increase in net K+ secretion with no change in net Cl- or Na+ transport. This increase in net K+ secretion elicited by beta-adrenergic stimulation can be inhibited by trifluoperazine but not by indomethacin. These studies suggest that K+ transport by the colon can be regulated by adrenergic agents acting via beta 1-receptors.

Desensitization of P2Y2receptor-activated transepithelial anion secretion

1999 ◽  
Vol 276 (4) ◽  
pp. C777-C787 ◽  
Author(s):  
Lane L. Clarke ◽  
Matthew C. Harline ◽  
Miguel A. Otero ◽  
Geraldine G. Glover ◽  
Richard C. Garrad ◽  
...  
Keyword(s):  
Receptor Agonist ◽  
Inositol Phosphate ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Extracellular Nucleotides ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Anion Secretion ◽  
Ussing Chambers ◽  
Intracellular Ca

Desensitization of P2Y2 receptor-activated anion secretion may limit the usefulness of extracellular nucleotides in secretagogue therapy of epithelial diseases, e.g., cystic fibrosis (CF). To investigate the desensitization process for endogenous P2Y2 receptors, freshly excised or cultured murine gallbladder epithelia (MGEP) were mounted in Ussing chambers to measure short-circuit current ( I sc), an index of electrogenic anion secretion. Luminal treatment with nucleotide receptor agonists increased the I sc with a potency profile of ATP = UTP > 2-methylthioATP >> α,β-methylene-ATP. RT-PCR revealed the expression of P2Y2 receptor mRNA in the MGEP cells. The desensitization of anion secretion required a 10-min preincubation with the P2Y2receptor agonist UTP and increased in a concentration-dependent manner (IC50 ≈ 10−6 M). Approximately 40% of the anion secretory response was unaffected by maximal desensitizing concentrations of UTP. Recovery from UTP-induced desensitization was rapid (<10 min) at preincubation concentrations less than the EC50 (1.9 × 10−6 M) but required progressively longer time periods at greater concentrations. UTP-induced total inositol phosphate production and intracellular Ca2+ mobilization desensitized with a concentration dependence similar to that of anion secretion. In contrast, maximal anion secretion induced by Ca2+ ionophore ionomycin was unaffected by preincubation with a desensitizing concentration of UTP. It was concluded that 1) desensitization of transepithelial anion secretion stimulated by the P2Y2 receptor agonist UTP is time and concentration dependent; 2) recovery from desensitization is prolonged (>90 min) at UTP concentrations >10−5 M; and 3) UTP-induced desensitization occurs before the operation of the anion secretory mechanism.

Colonic potassium and chloride secretion: role of cAMP and calcium

1985 ◽  
Vol 248 (1) ◽  
pp. G103-G109 ◽  
Author(s):  
R. D. McCabe ◽  
P. L. Smith
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Bathing Solution ◽  
Ionophore A23187 ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Intracellular Ca

Stripped rabbit colonic mucosa was studied in vitro in Ussing chambers to further investigate the role of Ca in regulating K and Cl secretion stimulated by the divalent cation ionophore A23187, prostaglandin E1 (PGE1), or 8-bromo-cAMP (8BrcAMP). To assess the effects of these secretagogues on the paracellular shunt permeability, we measured the Na concentration dependence of the serosal-to-mucosal Na flux in the absence or presence of these stimuli. Results from these studies reveal that changes in net K and Cl secretion produced by secretory stimuli cannot be accounted for by a change in shunt permeability. The possible involvement of Ca in the secretory response of the colon to these stimuli was investigated by measuring the changes in Cl and K transport elicited by A23187, PGE1, or 8BrcAMP in the absence or presence of trifluoperazine (10(-4) M) added to the serosal bathing solution. Trifluoperazine alone did not significantly alter basal Na or Cl fluxes or short-circuit current (Isc) but did decrease transepithelial conductance (Gt) and the serosal-to-mucosal K flux. Pretreatment of the tissues with trifluoperazine significantly reduced or abolished the changes in K fluxes elicited by A23187, 8BrcAMP, or PGE1 without altering the changes in Cl transport, Isc, and Gt. These results suggest that K secretion induced by these secretagogues involves an increase in intracellular Ca concentration and may be mediated by calmodulin.

Activation of proteinase-activated receptor-1 inhibits neurally evoked chloride secretion in the mouse colon in vitro

2005 ◽  
Vol 288 (2) ◽  
pp. G337-G345 ◽  
Author(s):  
Michelle C. Buresi ◽  
Nathalie Vergnolle ◽  
Keith A. Sharkey ◽  
Catherine M. Keenan ◽  
Patricia Andrade-Gordon ◽  
...  
Keyword(s):  
Short Circuit ◽  
Cholinergic Neurons ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Bathing Medium ◽  
Ussing Chambers ◽  
Mouse Colon ◽  
Deficient Mice

The proteinase-activated thrombin receptor-1 (PAR-1) belongs to a unique family of G protein-coupled receptors activated by proteolytic cleavage. We studied the effect of PAR-1 activation in the regulation of ion transport in mouse colon in vitro. Expression of PAR-1 in mouse colon was assessed by RT-PCR and immunohistochemistry. To study the role of PAR-1 activation in chloride secretion, mouse colon was mounted in Ussing chambers. Changes in short-circuit current ( Isc) were measured in tissues exposed to either thrombin, saline, the PAR-1-activating peptide TFLLR-NH2, or the inactive reverse peptide RLLFT-NH2, before electrical field stimulation (EFS). Experiments were repeated in the presence of either a PAR-1 antagonist or in PAR-1-deficient mice to assess receptor specificity. In addition, studies were conducted in the presence of chloride-free buffer or the muscarinic antagonist atropine to assess chloride dependency and the role of cholinergic neurons in the PAR-1-induced effect. PAR-1 mRNA was expressed in full-thickness specimens and mucosal scrapings of mouse colon. PAR-1 immunoreactivity was found on epithelial cells and on neurons in submucosal ganglia where it was colocalized with both VIP and neuropeptide Y. After PAR-1 activation by thrombin or TFLLR-NH2, secretory responses to EFS but not those to forskolin or carbachol were significantly reduced. The reduction in the response to EFS was not observed in the presence of the PAR-1 antagonist, in PAR-1-deficient mice, when chloride was excluded from the bathing medium, or when atropine was present. PAR-1 is expressed in submucosal ganglia in the mouse colon and its activation leads to a decrease in neurally evoked epithelial chloride secretion.

Mechanisms of disposal of acid and alkali in rabbit duodenum

1975 ◽  
Vol 229 (6) ◽  
pp. 1641-1648 ◽  
Author(s):  
RG Fiddian-Green ◽  
W Silen
Keyword(s):  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Duodenal Mucosa ◽  
Ringer Solution ◽  
Co2 Production ◽  
Rapid Phase ◽  
Ussing Chambers

Stripped duodenal mucosa of rabbits was mounted in Ussing chambers containing a Ringer solution gassed with 100% O2. The disappearance of acid or alkali from the mucosal solution of short-circuited tissue was measured with a pH stat while the serosal pH was kept at 7.4. The duodenum rapidly disposed of both acid and alkali; neither property was altered by gassing with N2 while iodoacetate was in the perfusing solutions. Prevention of release of CO2 from the mucosal chamber obliterated the early rapid phase of acid disposal by the mucosa while a similar maneuver in the serosal chamber increased the appearance of serosal acid without altering the rate of acid disposal. Gut sacs of rabbit duodenum in vitro and in vivo showed a positive correlation between acid disposal and the rate of luminal CO2 production. While acid disposal progressively decreased with time for the in vitro gut sacs, the in vivo gut sac showed no fatigue in this respect. Luminal acidification in the Ussing chamber was associated with a profound reduction in short-circuit current (Isc), partially reversible by elevation of the mucosal pH but not by luminal glucose. Our data suggest that acid disposal occurs in part by intraluminal neutralization and in part by diffusion into the mucosa.

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.

Role of nutrient HCO3(-) in protection of amphibian gastric mucosa

1980 ◽  
Vol 239 (6) ◽  
pp. G536-G542
Author(s):  
R. Schiessel ◽  
A. Merhav ◽  
J. B. Matthews ◽  
L. A. Fleischer ◽  
A. Barzilai ◽  
...  
Keyword(s):  
Gastric Mucosa ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Experimental Conditions ◽  
Slow Decline ◽  
Alkaline Tide ◽  
Artificial Gastric Juice ◽  
Rapid Drop

In in vitro bullfrog fundic mucosa inhibited with 10(-3) M metiamide and exposed to a luminal pH of 2 a progressive slow decline in potential difference (PD) and short-circuit current (Isc) and a rise in resistance (R) were observed when the nutrient solution (N) contained 18 mM HCO3(-), but these changes were restored by an N containing 50 mM HCO3(-). Substitution of PO4(3-) or N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid for NHO3(-) in N caused a rapid drop in PD and Isc in inhibited tissues, changes that could be prevented by 10(-4) M histamine. Ulceration occurred more frequently in metiamide-inhibited gastric sacs exposed to artificial gastric juice with an N of 18 mMHCO3(-) than with 50 mM HCO3(-), but histamine prevented ulceration in the 18 mM HCO3(-) solution. JnetCl approximated Isc under most experimental conditions in inhibited mucosa and was reduced dramatically as were both Jn leads to sCl and Js leads to nCl when HCO3(-) was removed from N. In histamine-stimulated tissues, removal of nutrient HCO3(-) did not influence Cl- transport. Our results are consistent with the proposal that HCO3(-) in N supports normal Cl- flux and that the alkaline tide of actively secreting oxyntic cells can do the same in the absence of ambient HCO3(-).

Prostaglandin-induced recovery of barrier function in porcine ileum is triggered by chloride secretion

1999 ◽  
Vol 276 (1) ◽  
pp. G28-G36 ◽  
Author(s):  
Anthony T. Blikslager ◽  
Malcolm C. Roberts ◽  
Robert A. Argenzio
Keyword(s):  
Short Circuit ◽  
Second Messengers ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Ringer Solution ◽  
Signal Recovery ◽  
Osmotic Gradient ◽  
Current Increase ◽  
Ussing Chambers

We have previously shown that PGI2 and PGE2 have a synergistic role in restoring electrical transepithelial resistance ( R) in ischemia-injured porcine ileum via the second messengers Ca2+ and cAMP. Because Ca2+ and cAMP stimulate Cl− secretion, we assessed the role of PG-induced Cl−secretion in recovery of R. Mucosa from porcine ileum subjected to ischemia for 45 min was mounted in Ussing chambers and bathed in indomethacin and Ringer solution. Addition of PGs stimulated a twofold increase in R, which was preceded by elevations in short-circuit current (increase of 25 μA/cm2). The PG-induced effect on R was partially inhibited with bumetanide, an inhibitor of Cl− secretion. The remaining elevations in R were similar in magnitude to those induced in ischemic tissues by amiloride, an inhibitor of Na+ absorption. Treatment with 10−4 M 8-bromo-cGMP or 300 mosM mucosal urea resulted in elevations in R similar to those attained with PG treatment. PGs signal recovery of Rvia induction of Cl−secretion and inhibition of Na+absorption, possibly by establishing a transmucosal osmotic gradient.

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