Bovine and porcine large intestine as model epithelia in a student lab course.

1993 ◽  
Vol 265 (6) ◽  
pp. S10 ◽  
Author(s):  
U Hegel ◽  
M Fromm ◽  
K M Kreusel ◽  
M Wiederholt
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Secretory Diarrhea ◽  
Tissue Preparation ◽  
Cl Secretion ◽  
Animal Physiology ◽  
Daily Schedule ◽  
K Secretion ◽  
Transepithelial Voltage

A short-circuit current experiment on epithelial ion transport is described that is suitable for student classes in human and animal physiology. Segments of late distal colon from either pig or cow are obtained from the slaughterhouse depending on the animals' daily schedule. Initial tissue preparation already in the slaughterhouse, cold storage, and proper choice of bath solutions are essential prerequisites for success. Students monitor spontaneous transepithelial voltage and short-circuit current (Isc) by use of manually operated voltage clamp units. Two main transport mechanisms are studied, electrogenic Na+ absorption and Cl- secretion. Electrogenic Na+ absorption is studied by measuring the Isc drop after amiloride. Then Cl- secretion is stimulated by theophylline and subsequently inhibited by furosemide. In some experiments K+ secretion can be detected by the blocking effect of mucosal Ba2+. Response of tissues from pig and cow is qualitatively similar but quantitatively different. The equipment is sturdy and inexpensive, can be provided by most departmental workshops, and has been tested for 3 yr in regular lab courses. Observations made during these experiments are closely related to clinical states, such as secretory diarrhea, cystic fibrosis, and hyperaldosteronism, as well as to the mechanisms of clinically used diuretics.

Active K transport across rabbit distal colon: relation to Na absorption and Cl secretion

1986 ◽  
Vol 251 (2) ◽  
pp. C252-C267 ◽  
Author(s):  
D. R. Halm ◽  
R. A. Frizzell
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Secretory Process ◽  
Cl Secretion ◽  
K Secretion ◽  
Transport Inhibitors ◽  
Unidirectional Fluxes ◽  
K Transport

We measured isotopic unidirectional fluxes of K to elucidate the mechanisms of active K transport across the distal colon of the rabbit. Separate pathways for active K absorption and active K secretion were detected using various transport inhibitors and stimulators. The rate and direction of net K transport depend on the activities of these two pathways. K absorption was reduced by orthovanadate (both solutions) or serosal Ba, consistent with ATPase-dependent uptake of K across the apical membrane and exit via a Ba-sensitive basolateral K conductance. K secretion was inhibited by serosal ouabain or mucosal Ba, indicating that K secretion involves basolateral uptake via the Na-K pump and apical exit via a Ba-sensitive K conductance. Active K secretion appears to be electrogenic, since inhibition by ouabain produced equivalent changes in the net K flux and short-circuit current. Addition of bumetanide to the serosal solution or the removal of either Na or Cl from the serosal solution inhibited K secretion; mucosal solution amiloride was without effect. These results indicate that this K secretory process is independent of electrogenic Na absorption but is mechanistically similar to Cl secretory processes. Both epinephrine and prostaglandin E2 (PGE2) stimulate K secretion, but only PGE2 also stimulates Cl secretion. The response to these secretogogues suggests that the mechanisms underlying K and Cl secretion are closely linked but can be regulated independently.

cGMP and Ca2+ regulation of ion transport across the isolated porcine distal colon epithelium

1994 ◽  
Vol 267 (4) ◽  
pp. R1026-R1033 ◽  
Author(s):  
M. D. DuVall ◽  
S. M. O'Grady
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Nacl Absorption ◽  
Flux Measurements ◽  
K Secretion ◽  
Baseline Values ◽  
Distal Colon Epithelium

Intact epithelium from the porcine distal colon was stripped of serosal muscle and mounted in Ussing chambers to investigate the regulation of Na, Cl, and K transport by guanosine 3',5'-cyclic monophosphate (cGMP) and elevations in intracellular [Ca2+]. Under voltage-clamped conditions cGMP (250 microM) produced an increase in tissue short-circuit current (Isc) that reached a maximal value within 10-20 min and remained elevated > 40 min. This response was associated with an inhibition of NaCl absorption and stimulation of Cl and K secretion. In the absence of Cl the Isc also slowly increased but returned to baseline values within 20 min. Bicarbonate removal from both serosal and mucosal solutions or serosal bumetanide (20 microM) reduced the effect of cGMP on Isc by approximately 40%. When performed simultaneously, these conditions reduced the cGMP response by approximately 60%. Transepithelial Na and Cl flux measurements indicated that serosal bumetanide blocked increased Cl secretion without effecting changes in NaCl absorption. In contrast, mucosal amiloride blocked the effects of cGMP on NaCl absorption but not Cl secretion. The cGMP Isc response was potentiated in the presence of 1 mM, but not 10 microM, amiloride. Moreover, 1 mM amiloride inhibited Isc under control conditions but was ineffective in the presence of cGMP. The Ca2+ ionophore ionomycin (3 microM) produced a transient increase in the Isc that was also associated with a decrease in transepithelial NaCl absorption and an increase in Cl and K secretion. In contrast to cGMP, the ionomycin Isc response was eliminated after Cl removal from the bath.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Cyclic AMP-induced K+secretion occurs independently of Cl−secretion in rat distal colon

2012 ◽  
Vol 303 (3) ◽  
pp. C328-C333 ◽  
Author(s):  
Geoffrey I. Sandle ◽  
Vazhaikkurichi M. Rajendran
Keyword(s):  
Channel Blocker ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Secretory Diarrhea ◽  
Atpase Inhibitor ◽  
Rat Distal Colon ◽  
Electrogenic Ion Transport ◽  
K Secretion

cAMP induces both active Cl−and active K+secretion in mammalian colon. It is generally assumed that a mechanism for K+exit is essential to maintain cells in the hyperpolarized state, thus favoring a sustained Cl−secretion. Both Kcnn4c and Kcnma1 channels are located in colon, and this study addressed the questions of whether Kcnn4c and/or Kcnma1 channels mediate cAMP-induced K+secretion and whether cAMP-induced K+secretion provides the driving force for Cl−secretion. Forskolin (FSK)-enhanced short-circuit current (indicator of net electrogenic ion transport) and K+fluxes were measured simultaneously in colonic mucosa under voltage-clamp conditions. Mucosal Na+orthovanadate (P-type ATPase inhibitor) inhibited active K+absorption normally present in rat distal colon. In the presence of mucosal Na+orthovanadate, serosal FSK induced both K+and Cl−secretion. FSK-induced K+secretion was 1) not inhibited by either mucosal or serosal 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34; a Kcnn4 channel blocker), 2) inhibited (92%) by mucosal iberiotoxin (Kcnma1 channel blocker), and 3) not affected by mucosal cystic fibrosis transmembrane conductance regulator inhibitor (CFTRinh-172). By contrast, FSK-induced Cl−secretion was 1) completely inhibited by serosal TRAM-34, 2) not inhibited by either mucosal or serosal iberiotoxin, and 3) completely inhibited by mucosal CFTRinh-172. These results indicate that cAMP-induced colonic K+secretion is mediated via Kcnma1 channels located in the apical membrane and most likely contributes to stool K+losses in secretory diarrhea. On the other hand, cAMP-induced colonic Cl−secretion requires the activity of Kcnn4b channels located in the basolateral membrane and is not dependent on the concurrent activation of apical Kcnma1 channels.

scholarly journals Prostanoids stimulate K secretion and Cl secretion in guinea pig distal colon via distinct pathways

2001 ◽  
Vol 281 (4) ◽  
pp. G984-G996 ◽  
Author(s):  
Dan R. Halm ◽  
Susan Troutman Halm
Keyword(s):  
Guinea Pig ◽  
Rank Order ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Prostanoid Receptors ◽  
Cl Secretion ◽  
Inflammatory Conditions ◽  
K Secretion

Short-circuit current ( I sc) and transepithelial conductance ( G t) were measured in guinea pig distal colonic mucosa isolated from submucosa and underlying muscle layers. Indomethacin (2 μM) and NS-398 (2 μM) were added to suppress endogenous production of prostanoids. Serosal addition of PGE2 (10 nM) stimulated negative I scconsistent with K secretion, and concentrations >30 nM stimulated positive I sc consistent with Cl secretion. PGE2 also stimulated G t at low and high concentrations. Dose responses to prostanoids specific for EP prostanoid receptors were consistent with stimulating K secretion through EP2 receptors, based on a rank order potency (from EC50 values) of PGE2 (1.9 nM) > 11-deoxy-PGE1 (8.3 nM) > 19( R)-hydroxy-PGE2 (13.9 nM) > butaprost (67 nM) > 17-phenyl-trinor-PGE2 (307 nM) ≫ sulprostone (>10 μM). An isoprostane, 8-iso-PGE2, stimulated K secretion with an EC50 of 33 nM. Cl secretory response was stimulated by PGD2 and BW-245C, a DP prostanoid receptor-specific agonist: BW-245C (15 nM) > PGD2 (30 nM) > PGE2 (203 nM). Agonists specific for FP, IP, and TP prostanoid receptors were ineffective in stimulating I sc and G t at concentrations <1 μM. These results indicate that PGE2stimulated electrogenic K secretion through activation of EP2 receptors and electrogenic KCl secretion through activation of DP receptors. Thus stimulation of Cl secretion in vivo would occur either via physiological concentrations of PGD2(<100 nM) or pathophysiological concentrations of PGE2(>100 nM) that could occur during inflammatory conditions.

Defective cholinergic Cl− secretion and detection of K+ secretion in rectal biopsies from cystic fibrosis patients

2000 ◽  
Vol 278 (4) ◽  
pp. G617-G624 ◽  
Author(s):  
M. Mall ◽  
A. Wissner ◽  
H. H. Seydewitz ◽  
J. Kuehr ◽  
M. Brandis ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Channel Blockers ◽  
K Secretion ◽  
Transepithelial Voltage ◽  
Human Airways ◽  
Rectal Biopsies

Rectal biopsies from cystic fibrosis (CF) patients show defective cAMP-activated Cl− secretion and an inverse response of the short-circuit current ( I sc) toward stimulation with carbachol (CCh). Alternative Cl− channels are found in airway epithelia and have been attributed to residual Cl− secretion in CF colon. The aim of the present study was to investigate ion conductances causing reversed I sc upon cholinergic stimulation. Furthermore, the putative role of an alternative Ca2+-dependent Cl− conductance in human distal colon was examined. Cholinergic ion secretion was assessed in the absence and presence of cAMP-dependent stimulation. Transepithelial voltage and I sc were measured in rectal biopsies from non-CF and CF individuals by means of a perfused micro-Ussing chamber. Under baseline conditions, CCh induced a positive I sc in CF rectal biopsies but caused a negative I sc in non-CF subjects. The CCh-induced negative I sc in non-CF biopsies was gradually reversed to a positive response by incubating the biopsies in indomethacin. The positive I sc was significantly enhanced in CF and was caused by activation of a luminal K+ conductance, as shown by the use of the K+ channel blockers Ba2+ and tetraethylammonium. Moreover, a cAMP-dependent luminal K+conductance was detected in CF individuals. We conclude that the cystic fibrosis transmembrane conductance regulator is the predominant Cl− channel in human distal colon. Unlike human airways, no evidence was found for an alternative Cl−conductance in native tissues from CF patients. Furthermore, we demonstrated that both Ca2+- and cAMP-dependent K+ secretion are present in human distal colon, which are unmasked in rectal biopsies from CF patients.

Role of calcium in chloride secretion mediated by cAMP pathway activation in rabbit distal colon mucosa

1993 ◽  
Vol 264 (2) ◽  
pp. G252-G260 ◽  
Author(s):  
V. Calderaro ◽  
E. Chiosi ◽  
R. Greco ◽  
A. M. Spina ◽  
A. Giovane ◽  
...  
Keyword(s):  
Short Circuit ◽  
Fold Increase ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Chloride Secretion ◽  
Free Medium ◽  
Cl Secretion ◽  
Pathway Activation ◽  
Flux Measurements ◽  
Pg E2

Effects of Ca2+ on adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion were investigated in intact mucosa and isolated crypt cells of rabbit descending colon. Addition of 10 microM prostaglandin (PG)E2 or forskolin to tissues incubated in Ca(2+)-free medium increased the size of short-circuit current (Isc) and Cl- secretion as estimated by unidirectional 36Cl flux measurements (net flux = -2.31 +/- 0.24 vs. -1.22 +/- 0.10 mueq.h-1.cm-2, n = 4, P < 0.001). Addition of 10 microM PGE2 to tissues incubated in 1.2 mM Ca2+ Ringer induced a 7-fold increase in mean cAMP level, whereas it produced an 11-fold increase in tissues exposed to Ca(2+)-free medium. Membrane preparations from whole mucosa incubated in Ca(2+)-free medium displayed a cyclic nucleotide phosphodiesterase activity significantly lower than controls (18.76 +/- 0.54 vs. 31.20 +/- 0.39 pmol cAMP. mg protein-1.min-1, means +/- SE, n = 4, P < 0.001). Ca2+ removal also affected adenylate cyclase (AC) responsiveness to agonists; AC activity increased in controls by 54 and 226% after stimulation with 10 microM PGE2 and forskolin, respectively, but it increased more (77 and 325%, respectively) after incubation in Ca(2+)-free solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

cAMP-dependent sulfate secretion by the rabbit distal colon: a comparison with electrogenic chloride secretion

1997 ◽  
Vol 273 (1) ◽  
pp. C148-C160 ◽  
Author(s):  
R. W. Freel ◽  
M. Hatch ◽  
N. D. Vaziri
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Chloride Secretion ◽  
Disulfonic Acid ◽  
Cl Secretion ◽  
Cyclic Monophosphate ◽  
Anion Conductance ◽  
Flux Measurements

The ability of a Cl-secreting epithelium to support net secretion of an anion other than a halide was investigated with 35SO4 flux measurements across the isolated, short-circuited rabbit distal colon. In most experiments, 36Cl fluxes were simultaneously measured to validate the secretory capacity of the tissues. Serosal addition of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP, 0.5 mM) stimulated a sustained net secretion of SO4 (about -3.0 nmol.cm-2.h-1 from a 0.20 mM solution) via an increase in the serosal-to-mucosal unidirectional flux, whereas Ca ionophore A-23187 (1 microM, serosal) produced a more transient stimulation of SO4 and Cl secretion. Net adenosine 3',5'-cyclic monophosphate (cAMP)-dependent SO4 and Cl secretion were strongly voltage sensitive, principally through the potential dependence of the serosal-to-mucosal fluxes, indicating an electrogenic transport process. Symmetrical replacement of either Na, K, or Cl inhibited cAMP-dependent SO4 secretion, whereas HCO3-free buffers had no effect on SO4 secretion. Serosal bumetanide (50 microM) or furosemide (100 microM) reduced DBcAMP-stimulated SO4 and Cl secretion, whereas serosal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (50 microM) blocked DBcAMP-induced SO4 secretion while enhancing net Cl secretion and short-circuit current. Mucosal 5-nitro-2-(3-phenylpropylamino)benzoic acid partially inhibited SO4 secretion and completely inhibited Cl secretion. It is concluded that secretagogue-stimulated SO4 secretion, like Cl secretion, may be an electrogenic process mediated by diffusive efflux through an apical anion conductance. Cellular accumulation of SO4 across the basolateral membrane appears to be achieved by a mechanism that is distinct from that employed by Cl.

Neuromodulation of ion transport in porcine distal colon: NPY reduces secretory actions of leukotrienes

1995 ◽  
Vol 269 (2) ◽  
pp. R426-R431 ◽  
Author(s):  
T. R. Traynor ◽  
D. R. Brown ◽  
S. M. O'Grady
Keyword(s):  
Ion Transport ◽  
Colonic Mucosa ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Effective Concentration ◽  
Leukotriene C4 ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Half Maximal Effective Concentration

Electrical transmural stimulation (ETS) was used to examine the neuroregulation of electrolyte transport in the porcine distal colon. ETS of the colonic mucosa-submucosa mounted in Ussing chambers produced rapid and transient increases in short-circuit current (Isc) that were inhibited 36% by serosal bumetanide, suggesting that a portion of the response may be attributed to Cl secretion. ETS actions were dependent upon stimulus intensity and frequency and were inhibited by tetrodotoxin and omega-conotoxin. Prazosin and pyrilamine had no effect on the mucosal responses to ETS, whereas atropine reduced the responses by 32%. Neuropeptide Y (NPY) also reduced the mucosal responses to ETS up to 60% (half-maximal effective concentration = 17 nM). In addition, the effects of leukotriene C4, previously shown to stimulate Cl secretion via a neuronal pathway, were also inhibited by NPY. These results indicate that cholinergic submucosal neurons play a role in the regulation of epithelial ion transport and that NPY acts as an inhibitory neuromodulator, particularly on leukotriene-sensitive neurons in the porcine distal colon.

Brain natriuretic peptide stimulates K and Cl secretion across porcine distal colon epithelium

1991 ◽  
Vol 260 (4) ◽  
pp. C750-C755 ◽  
Author(s):  
T. R. Traynor ◽  
S. M. O'Grady
Keyword(s):  
Brain Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Ringer Solution ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
The Difference ◽  
Distal Colon Epithelium

Porcine distal colon epithelium was mounted in Ussing chambers and bathed with porcine Ringer solution. The serosal addition of brain natriuretic peptide (BNP; 50 nM) or atriopeptin III (AP-III; 500 nM) produced significant increases (50-75 microA/cm2) in short-circuit current (Isc). These increases in Isc were not inhibited by pretreatment with tetrodotoxin (TTX) or 5,8,11,14-eicosatetraynoic acid (ETYA). Analysis of concentration-response relationships revealed that BNP was 5.8-fold more potent than AP-III in stimulating the Isc. BNP and AP-III significantly increased the serosal-to-mucosal (S----M) Cl flux and reduced net Cl absorption by 38 and 41%, respectively. The BNP-stimulated S----M Cl flux was abolished when HCO3 was removed. In contrast, the vasoactive intestinal peptide (VIP)-stimulated S----M Cl flux was not affected by HCO3 replacement. In addition to their effects on Cl transport, BNP and AP-III increased net Rb secretion by 79 and 58%, respectively. BNP-stimulated Rb secretion was reduced by 76% after HCO3 replacement. These results indicate that natriuretic peptides stimulate K- and HCO3-dependent Cl secretion which is not present under basal conditions or after VIP stimulation. The difference in potency between BNP and AP-III suggests that ANP-B receptors may mediate their effects on ion transport in the porcine colon.

Extracellular ATP stimulates K+ secretion across cultured human airway epithelium

1997 ◽  
Vol 272 (6) ◽  
pp. L1084-L1091 ◽  
Author(s):  
L. L. Clarke ◽  
T. Chinet ◽  
R. C. Boucher
Keyword(s):  
Airway Epithelium ◽  
Apical Membrane ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Extracellular Atp ◽  
Cl Secretion ◽  
Human Airway ◽  
Human Airway Epithelium ◽  
K Secretion

Extracellular ATP applied to the luminal side of human airway epithelium (HAE) activates an apical membrane Cl- conductance and transepithelial Cl- secretion. However, in some HAE preparations, we have found that luminal ATP induces a change in short-circuit current (Isc), consistent with K+ secretion. Using intracellular microelectrodes and radioisotopic flux studies, we investigated whether extracellular ATP regulates transepithelial K+ secretion in primary HAE cultures. In physiological Ringer solution, HAE had a negligible electrochemical driving force for Cl- secretion (DFCl), and luminal ATP induced a change in Isc opposite in polarity to Cl- secretion. Intracellular microelectrode measurements indicated that the "reversed" Isc was associated with activation of a hyperpolarizing (K+) conductance in the apical membrane. Radioisotope studies of HAE pretreated with amiloride to induce a favorable DFCl revealed that luminal ATP stimulates a small 42K secretory flux concurrently with Cl- secretion. In ion-substituted Ringer solution, luminal ATP stimulated both the outward (K+) current and the inward (Cl-) current with approximately equal potency (approximately 10(-6) M). We conclude that luminal ATP activates an apical membrane K+ conductance and transepithelial K+ secretion across HAE.

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