scholarly journals P2Y receptors mediate Ca2+ signaling in duodenocytes and contribute to duodenal mucosal bicarbonate secretion

2009 ◽  
Vol 296 (2) ◽  
pp. G424-G432 ◽  
Author(s):  
Xiao Dong ◽  
Eric James Smoll ◽  
Kwang Hyun Ko ◽  
Jonathan Lee ◽  
Jimmy Yip Chow ◽  
...  
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
P2y Receptors ◽  
Epithelial Cell Line ◽  
Initial Increase ◽  
Bicarbonate Secretion ◽  
Duodenal Epithelium ◽  
And Function

Since little is known about the role of P2Y receptors (purinoceptors) in duodenal mucosal bicarbonate secretion (DMBS), we sought to investigate the expression and function of these receptors in duodenal epithelium. Expression of P2Y2 receptors was detected by RT-PCR in mouse duodenal epithelium and SCBN cells, a duodenal epithelial cell line. UTP, a P2Y2-receptor agonist, but not ADP (10 μM), significantly induced murine duodenal short-circuit current and DMBS in vitro; these responses were abolished by suramin (300 μM), a P2Y-receptor antagonist, or 2-aminoethoxydiphenyl borate (2-APB; 100 μM), a store-operated channel blocker. Mucosal or serosal addition of UTP induced a comparable DMBS in wild-type mice, but markedly impaired response occurred in P2Y2 knockout mice. Acid-stimulated DMBS in vivo was significantly inhibited by suramin (1 mM) or PPADS (30 μM). Both ATP and UTP, but not ADP (1 μM), raised cytoplasmic-free Ca2+ concentrations ([Ca2+]cyt) with similar potencies in SCBN cells. ATP-induced [Ca2+]cyt was attenuated by U-73122 (10 μM), La3+ (30 μM), or 2-APB (10 μM), but was not significantly affected by nifedipine (10 μM). UTP (1 μM) induced a [Ca2+]cyt transient in Ca2+-free solutions, and restoration of external Ca2+ (2 mM) raised [Ca2+]cyt due to capacitative Ca2+ entry. La3+ (30 μM), SK&F96365 (30 μM), and 2-APB (10 μM) inhibited UTP-induced Ca2+ entry by 92, 87, and 94%, respectively. Taken together, our results imply that activation of P2Y2 receptors enhances DMBS via elevation of [Ca2+]cyt that likely results from an initial increase in intracellular Ca2+ release followed by extracellular Ca2+ entry via store-operated channel.

Role of Ca2+-activated K+ channels in duodenal mucosal ion transport and bicarbonate secretion

2006 ◽  
Vol 291 (6) ◽  
pp. G1120-G1128 ◽  
Author(s):  
Hui Dong ◽  
Anders Smith ◽  
Marjan Hovaida ◽  
Jimmy Y. Chow
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Molecular Targets ◽  
Short Circuit Current ◽  
Duodenal Mucosa ◽  
Biological Action ◽  
Bicarbonate Secretion ◽  
Epithelial Ion Transport ◽  
And Function

Stimulation of muscarinic receptors in the duodenal mucosa raises cytosolic free Ca2+ concentration ([Ca2+]cyt), thereby regulating duodenal epithelial ion transport. However, little is known about the downstream molecular targets that account for this Ca2+-mediated biological action. Ca2+-activated K+ (KCa) channels are candidates, but the expression and function of duodenal KCa channels are poorly understood. Therefore, we determined whether KCa channels are expressed in the duodenal mucosa and investigated their involvement in Ca2+-mediated duodenal epithelial ion transport. Two selective blockers of intermediate-conductance Ca2+-activated K+ (IKCa) channels, clotrimazole (30 μM) and 1-[(2-chlorophenyl)diphenylmethyl]-1 H-pyrazole (TRAM-34; 10 μM), significantly inhibited carbachol (CCh)-induced duodenal short-circuit current ( Isc) and duodenal mucosal bicarbonate secretion (DMBS) in mice but did not affect responses to forskolin and heat-stable enterotoxin of Escherichia coli. Tetraethylammonium, 4-aminopyridine, and BaCl2 failed to inhibit CCh-induced Isc and DMBS. A-23187 (10 μM), a Ca2+ ionophore, and 1-ethyl-2-benzimidazolinone (1-EBIO; 1 mM), a selective opener of KCa channels, increased both Isc and DMBS. The effect of 1-EBIO was more pronounced with serosal than mucosal addition. Again, both clotrimazole and TRAM-34 significantly reduced A23187- or 1-EBIO-induced Isc and DMBS. Moreover, clotrimazole (20 mg/kg ip) significantly attenuated acid-stimulated DMBS of mice in vivo. Finally, the molecular identity of IKCa channels was verified as KCNN4 (SK4) in freshly isolated murine duodenal mucosae by RT-PCR and Western blotting. Together, our results suggest that the IKCa channel is one of the downstream molecular targets for [Ca2+]cyt to mediate duodenal epithelial ion transport.

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.

Prolactin effects on ion transport across cultured mouse mammary epithelium

1981 ◽  
Vol 240 (3) ◽  
pp. C110-C115 ◽  
Author(s):  
C. A. Bisbee
Keyword(s):  
Cell Cultures ◽  
Short Circuit ◽  
Collagen Gel ◽  
Mammary Epithelium ◽  
Short Circuit Current ◽  
Sodium Absorption ◽  
Collagen Gels ◽  
Mouse Mammary Epithelium

Prolactin is a known osmoregulatory hormone in lower vertebrates, and recent evidence indicates that this hormone modulates ionic concentrations in milk. In an ultrastructurally and biochemically differentiated primary cell culture system in which mouse mammary epithelium is maintained on floating collagen gels, prolactin causes an increase in short-circuit current (Isc) of monolayers of cells derived from midpregnant (24.6 to 48.0 microA . cm-2) and lactating (10.4 to 16.1 microA . cm-2) glands. Transepithelial potential differences (basal side ground) average about -12 mV and are similar to those seen in vivo. Prelactating mammary epithelial cell cultures have transepithelial resistances ranging from 374 omega . cm2 (prolactin present) to 507 omega . cm2 (prolactin absent), and lactating cell cultures have resistances averaging almost 1,000 omega . cm2. Prolactin effects require at most one day of culture maintenance in prolactin-containing medium, and the effects are not due to known contamination of prolactin preparations with arginine vasopressin or growth hormone. Medium concentrations of prolactin as low as 1 ng/ml can elicit these effects. In prelactating cell cultures not treated with prolactin, the Isc is equal to the rate of sodium absorption. Prolactin increases sodium absorption fourfold but increases Isc only twofold. Clearly, prolactin induces other active transport; neither potassium nor chloride movements can account for this additional transport. Resistance values, current-voltage plots, and permeability coefficients indicate that in vitro mammary epithelium is a moderately “tight” tissue. Comparisons with intact glands indicate that in vitro mammary epithelium closely resembles its in vivo counterpart. Floating collagen gel cultures appear suitable for elucidating transport properties in cellularly heterogeneous and structurally complex mammalian tissues.

Water and electrolyte transport by rabbit esophagus

1975 ◽  
Vol 229 (2) ◽  
pp. 438-443 ◽  
Author(s):  
DW Powell ◽  
SM Morris ◽  
DD Boyd
Keyword(s):  
Sodium Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Electrolyte Transport ◽  
Potential Difference ◽  
Sodium Absorption ◽  
Bathing Solution ◽  
Electrical Potential Difference

The nature of the transmural electrical potential difference and the characteristics of water and electrolyte transport by rabbit esophagus were determined with in vivo and in vitro studies. The potential difference of the perfused esophagus in vivo was -28 +/- 3 mV (lumen negative). In vitro the potential difference was -17.9 +/- 0.6 mV, the short-circuit current 12.9 +/- 0.6 muA/cm2, and the resistance 1,466 +/- 43 ohm-cm2. Net mucosal-to-serosal sodium transport from Ringer solution in the short-circuited esophagus in vitro accounted for 77% of the simultaneously measured short-circuit current and net serosal-to-mucosal chloride transport for 14%. Studies with bicarbonate-free, chloride-free, and bicarbonate-chloride-free solutions suggested that the net serosal-to mucosal transport of these two anions accounts for the short-circuit current not due to sodium absorption. The potential difference and short-circuit current were saturating functions of bathing solution sodium concentration and were inhibited by serosal ouabain and by amiloride. Thus active mucosal-to-serosal sodium transport is the major determinant of the potential difference and short-circuit current in this epithelium.

An Investigation of the Role of Possible Neural Mechanisms in Cholera Toxin-Induced Secretion in Rabbit Ileal Mucosa in Vitro

1989 ◽  
Vol 77 (2) ◽  
pp. 161-166 ◽  
Author(s):  
K. J. Moriarty ◽  
N. B. Higgs ◽  
M. Woodford ◽  
L. A. Turnberg
Keyword(s):  
Cholera Toxin ◽  
Fluid Transport ◽  
Short Circuit ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Electrical Potential Difference ◽  
Rabbit Ileum ◽  
Ileal Mucosa

1. Cholera toxin stimulates intestinal secretion in vitro by activation of mucosal adenylate cyclase. However, it has been proposed that cholera toxin promotes secretion in vivo mainly through an indirect mechanism involving enteric neural reflexes. 2. We examined this hypothesis further by studying the influence of neuronal blockade on cholera toxin-induced changes in fluid transport across rabbit ileum in vitro. Mucosa, stripped of muscle layers, was mounted in flux chambers and luminal application of crude cholera toxin (2 μg/ml) caused a delayed but sustained rise in the short-circuit current, electrical potential difference and Cl− secretion. Pretreatment with the nerve-blocking drug, tetrodotoxin (5 × 10−6 mol/l serosal side), failed to influence the secretory response to cholera toxin, and addition of tetrodotoxin at the peak response to cholera toxin also had no effect. 3. That tetrodotoxin could block neurally mediated secretagogues was confirmed by the demonstration that the electrical responses to neurotensin (10−7 mol/l and 10−8 mol/l) were blocked by tetrodotoxin (5 × 10−6 mol/l). Furthermore, the response to cholera toxin of segments of ileum, which included the myenteric, submucosal and mucosal nerve plexuses, was not inhibited by tetrodotoxin. 4. We conclude that cholera toxin-induced secretion in rabbit ileum in vitro is not mediated via a neurological mechanism.

NaCl transport across equine proximal colon and the effect of endogenous prostanoids

1990 ◽  
Vol 259 (1) ◽  
pp. G62-G69 ◽  
Author(s):  
L. L. Clarke ◽  
R. A. Argenzio
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Proximal Colon ◽  
Nacl Transport ◽  
Apparent Lack ◽  
Ussing Chambers ◽  
Cl Secretion

In contrast to in vivo findings, the equine proximal colon fails to demonstrate significant net absorption of Na+ and Cl- under in vitro conditions. The present study was undertaken to determine if endogenous prostanoids are responsible for this apparent lack of ion transport. Proximal colonic tissues from ponies were preincubated in either normal Ringer solution or in Ringer containing 1 microM indomethacin and studied in Ussing chambers containing these solutions. Untreated colonic mucosa demonstrated negligible Na(+)-Cl- absorption in the basal state. In contrast, indomethacin-treated colon significantly absorbed Na+ and Cl-, primarily as the result of an equivalent increase in the mucosal-to-serosal flux of these ions. Preincubation of proximal colon in 0.1 mM ibuprofen-treated Ringer yielded similar results. Treatment of indomethacin colon with 1 mM mucosal amiloride eliminated net Na(+)-Cl- absorption without affecting the short-circuit current (Isc). The Isc in control tissue was significantly greater than in indomethacin-treated tissue and was reduced by 0.1 mM serosal furosemide. Serosal addition of 0.1 microM prostaglandin E2 or 10 mM serosal plus mucosal theophylline to indomethacin-treated tissues abolished net Na(+)-Cl- absorption and increased the Isc to levels indistinguishable from control. In contrast, control tissues were essentially unaffected by these secretagogues. These findings indicated that Na(+)-Cl- absorption in equine proximal colon was electroneutral (possibly involving Na(+)-H+ exchange) and that the tissue was capable of electrogenic Cl- secretion. However, under the in vitro conditions, basal ion transport was dominated by endogenous prostanoids that abolished Na(+)-Cl- absorption and elicited near-maximal electrogenic Cl- secretion.

Chronic regulation of transepithelial Na+ transport by the rate of apical Na+ entry

1996 ◽  
Vol 270 (2) ◽  
pp. C600-C607 ◽  
Author(s):  
M. D. Rokaw ◽  
E. Sarac ◽  
E. Lechman ◽  
M. West ◽  
J. Angeski ◽  
...  
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Maximal Activity ◽  
Na Channels ◽  
Na Transport ◽  
Bottom Structures ◽  
Stimulation Of

In several settings in vivo, prolonged inhibition of apical Na+ entry reduces and prolonged stimulation of apical entry enhances the ability of renal epithelial cells to reabsorb Na+, an important feature of the load-dependent regulation of renal tubular Na+ transport. To model this load dependency, apical Na+ entry was inhibited or stimulated for 18 h in A6 cells and vectorial transport was measured as short-circuit current (Isc) across monolayers on filter-bottom structures. Basal amiloride-sensitive Isc represents the activity of apical Na+ channels, whereas Isc after permeabilization of the apical membrane to cations with nystatin represents maximal activity of the basolateral Na(+)-K(+)-ATPase. Chronic inhibition of apical Na+ entry by 18-h apical exposure to amiloride or replacement of apical Na+ with tetramethylammonium (TMA+), followed by washing and restoration of normal apical medium, revealed a persistent decrease in Isc that remained despite exposure to nystatin. Both basal and nystatin-stimulated Isc recovered progressively after restoration of normal apical medium. In contrast, chronic stimulation of apical Na+ entry by short circuiting the epithelium increased Isc in the absence and presence of nystatin, indicating upregulation of both apical Na+ channels and basolateral Na(+)-K(+)-ATPase. Basolateral equilibrium [3H]ouabain binding was reduced to 67 +/- 5% in TMA+ vs. control cells, whereas values in 18-h short-circuited cells increased by 42 +/- 19%. The results demonstrate that load dependency of tubular Na+ transport can be modeled in vitro and indicate that the regulation of Na(+)-K(+)-ATPase observed in these studies occurs in part by changes in the density of functional transporter proteins within the basolateral membrane.

Restitution of barrier and transport function of porcine colon after acute mucosal injury

1988 ◽  
Vol 255 (1) ◽  
pp. G62-G71 ◽  
Author(s):  
R. A. Argenzio ◽  
C. K. Henrikson ◽  
J. A. Liacos
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Mucosal Injury ◽  
Acute Injury ◽  
Surface Epithelium ◽  
Mucosal Permeability ◽  
Nacl Transport ◽  
Crypt Epithelium

Acute injury of the porcine colonic epithelium was induced in vivo with the bile salt, deoxycholate. A concentration of 15 mM for 30 min completely destroyed the surface epithelium and induced a marked increase in mucosal permeability to mannitol. The crypt epithelium however was not significantly affected. Within 8 min of recovery, the colonic surface was reepithelialized with flattened, migrating cells, and within 40 min, mucosal permeability to mannitol was normalized. In vitro studies showed that in these early stages of recovery, NaCl transport, short-circuit current, and resistance were markedly impaired, whereas the theophylline-induced secretory response remained intact. Recovery of absorptive function paralleled the transition from flattened to columnar surface epithelium and was complete within 2 h. Results suggest that 1) active migratory events play an important role in rapid restitution of an epithelial barrier, 2) active absorption of ions is much slower to recover, and 3) active secretory events are intact and probably originate in the crypt epithelium.

Possible role of aldosterone and T3 in development of amiloride-blockable SCC across frog skin in vivo

1999 ◽  
Vol 277 (5) ◽  
pp. R1305-R1312 ◽  
Author(s):  
Makoto Takada ◽  
Michio Shiibashi ◽  
Miyoko Kasai
Keyword(s):  
Thyroid Hormone ◽  
Single Channel ◽  
Short Circuit ◽  
Noise Analysis ◽  
Short Circuit Current ◽  
Rate Coefficients ◽  
Adult Type ◽  
Apparent Dissociation Constant

There are inconsistencies between the in vitro and in vivo effects of thyroid hormone and aldosterone (Aldo) on the development of an amiloride-blockable short-circuit current (SCC) across bullfrog skin [Takada, M., H. Yai, and K. Takayama-Arita. Am. J. Physiol. 268 ( Cell Physiol. 37): C218–C226, 1995]. To address this issue, tadpoles were raised in Aldo + T3. An amiloride-blockable SCC developed across the skin before forelimbs appeared. Noise analysis of the characteristics (single-channel current, blocking and unblocking rate coefficients, and apparent dissociation constant) of this amiloride-blockable Na+ channel showed that it really was of the adult type. A similar SCC developed at stage XIX in the skin of tadpoles raised with Aldo alone. These results strongly support our hypothesis that the crucial hormone in the development of this SCC is Aldo but that a suppression mechanism attenuates its effect on SCC development until it is removed by the increase in the serum concentration of thyroid hormone (which starts at stages XVIII–XIX in vivo).

Role of active potassium transport by integumentary epithelium in secretion of larval-pupal moulting fluid during silkmoth development

1975 ◽  
Vol 62 (2) ◽  
pp. 357-366
Author(s):  
A. M. Jungreis ◽  
W. R. Harvey
Keyword(s):  
Short Circuit ◽  
Flux Ratio ◽  
Short Circuit Current ◽  
Potassium Transport ◽  
Open Circuit ◽  
Ratio Test ◽  
Potassium Flux

1. The exuvial side of the pharate pupal integument is usually positive to the haemolymph-side, both in vivo and in vitro, during the period when the moulting fluid is being secreted. 2. The ratio of potassium flux toward the exuvial space is higher than that toward the haemolymph, under both open-circuit conditions and short-circuit conditions, demonstrating by the Flux Ratio test that potassium is actively transported across the isolated integument during this secretion period. 3. Just prior to ecdysis, while moulting fluid is being reabsorbed, the potassium flux ratios become unity, suggesting that active potassium transport has ceased, but the short-circuit current that remains suggests that some other ion is actively transported at this time. 4. We argue that the potassium salt solution, formed in the exuvial space (as water presumably follows the actively transported potassium), has three functions (1) to accomplish the gel--sol transformation, (2) to activate the gel enzymes and (3) to buffer the enzyme solution at a pH favourable to the activity of the gel enzymes.

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