Effects of tetrodotoxin on chloride secretion in rabbit distal colon: tissue and cellular studies

1990 ◽  
Vol 258 (2) ◽  
pp. G223-G230 ◽  
Author(s):  
B. Biagi ◽  
Y. Z. Wang ◽  
H. J. Cooke
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Electrical Field Stimulation ◽  
Distal Colon ◽  
Chloride Secretion ◽  
Resting Potential ◽  
Colon Tissue ◽  
Colonic Crypts ◽  
Electrophysiological Studies ◽  
Release Of Acetylcholine

The effects of tetrodotoxin (TTX) were examined in muscle-stripped segments of rabbit distal colon and in cells of isolated colonic crypts. Electrical field stimulation (EFS) of the submucosa/mucosa evoked an increase in short-circuit current (ISC) that was due to an increase in chloride secretion. The EFS-evoked response was reduced 81% by 10(-7) M TTX and 30% by 5 X 10(-6) M atropine. Vasoactive intestinal peptide (VIP), carbachol, aminophylline, and 1,1-dimethyl-4-phenylpiperazinium increased ISC. Bumeta nide reduced the responses to neural stimulation, aminophylline, and VIP. To determine whether TTX had direct effects on crypt epithelial cells, crypts were isolated and cells were impaled with microelectrodes. Mean resting potential (Vbl) was -67 +/- 1.1 mV (n = 63). VIP and aminophylline depolarized Vbl by 34 +/- 4.6 (n = 13) and 34 +/- 3.5 mV (n = 18), respectively. TTX had no significant effect on resting Vbl or on the responses to VIP or aminophylline. We conclude that stimulation of submucosal neurons in the rabbit distal colon evokes a TTX- and bumetanide-sensitive increase in net chloride secretion that is dependent on the release of acetylcholine and other secretory neurotransmitters. Electrophysiological studies rule out a direct effect of TTX on colonic crypt cells.

Neural 5-hydroxytryptamine receptors regulate chloride secretion in guinea pig distal colon

1991 ◽  
Vol 261 (5) ◽  
pp. G833-G840 ◽  
Author(s):  
H. J. Cooke ◽  
Y. Z. Wang ◽  
T. Frieling ◽  
J. D. Wood
Keyword(s):  
Guinea Pig ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Electrical Field Stimulation ◽  
Distal Colon ◽  
Chloride Secretion ◽  
Cl Secretion ◽  
Release Of Acetylcholine ◽  
Ics 205 930 ◽  
Stimulation Of

The effects of 5-hydroxytryptamine (5-HT) on epithelial short-circuit current (Isc) were determined and related to the 5-HT effects on electrical and synaptic behavior of neurons in the submucosal plexus of the guinea pig colon. 5-HT evoked a biphasic increase in Isc that was reduced by bumetanide, Cl(-)-free solutions, atropine, and mecamylamine and abolished by tetrodotoxin. The 5-HT response was mimicked by 2-methyl-5-HT, but not by 5-hydroxyindalpine, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, and 5-methoxytryptamine (5-MeOT). ICS 205-930 suppressed the 5-HT response. Electrical field stimulation of submucosal neurons evoked an increase in Isc indicative of Cl- secretion that was reduced by 5-MeOT and enhanced by 2-methyl-5-HT. Application of 5-HT to submucosal neurons by micropressure ejection resulted in membrane depolarization, augmented excitability, and repetitive spike discharge. The depolarization was biphasic, consisting of rapidly and slowly activating components. The rapidly activating component was suppressed by ICS 205-930. Fast excitatory postsynaptic potentials evoked by electrical stimulation of interganglionic connectives were suppressed by 5-HT and 5-MeOT. These results suggest that 5-HT activates 5-HT3 receptors, which mediate fast excitatory responses in submucosal neurons, leading to release of acetylcholine at nicotinic and muscarinic synapses and stimulation of Cl- secretion. Presynaptic inhibition suppresses acetylcholine release and results in attenuation of neurally evoked Cl- secretion.

Acetylcholine release from colonic submucous neurons associated with chloride secretion in the guinea pig

1992 ◽  
Vol 262 (1) ◽  
pp. G131-G136 ◽  
Author(s):  
N. H. Javed ◽  
H. J. Cooke
Keyword(s):  
Guinea Pig ◽  
Ion Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Electrical Field Stimulation ◽  
Distal Colon ◽  
Chloride Secretion ◽  
Ach Release ◽  
Response Characteristics ◽  
Flux Chambers

Electrical field stimulation of submucous neurons in the guinea pig distal colon evokes an increase in chloride secretion sensitive to cholinergic blockade. This study was undertaken in the guinea pig to determine the feasibility of measuring acetylcholine (ACh) release simultaneously with ion transport in sheets of colonic submucosa/mucosa set up in flux chambers modified for perfusion of the submucosal surface. Release of [3H]ACh was determined in the absence of cholinesterase inhibitors as the stimulus-evoked outflow of 3H from preparations preloaded with [3H]choline. [3H]ACh released in response to electrical stimulation correlated with short-circuit current at frequencies from 0.5 to 10 Hz. At 5 and 10 Hz, the stimulus-evoked release of [3H]ACh decreased during subsequent stimulation periods. The stimulus-evoked increase in [3H]ACh was attenuated by tetrodotoxin. [3H]ACh release evoked at stimulus frequencies of 0.5-10 Hz was not altered by atropine despite a reduction in short-circuit current. This study illustrates the feasibility of measuring ACh release simultaneously with ion transport in flux chambers. The results provide new information on the response characteristics of colonic submucous neurons and provide direct evidence for regulation of chloride secretion by ACh.

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)

Chymotrypsin, ileal chloride transport, and neurotransmitters

1984 ◽  
Vol 247 (3) ◽  
pp. G253-G260 ◽  
Author(s):  
K. A. Hubel
Keyword(s):  
Cell Function ◽  
Chloride Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Electrical Field Stimulation ◽  
Chloride Secretion ◽  
Target Cells ◽  
Flux Chamber ◽  
Rabbit Ileum ◽  
Electrical Field

Electrical field stimulation (EFS) depolarizes nerves and causes chloride secretion by mucosa of rabbit ileum mounted in a flux chamber. To test the hypothesis that the transmitter is a peptide, we determined whether the EFS response was prevented by the endopeptidase chymotrypsin (CT). Serosal, but not mucosal, addition of CT (200 micrograms/ml) reduced the short-circuit current (Isc) response to EFS by 90% or more. CT also reduced Cl absorption by decreasing the mucosal-to-serosal flux, but it did not affect net Na absorption. CT prevented the response to vasoactive intestinal polypeptides, but the response returned when CT activity was eliminated. The response to EFS did not return, however, implying that CT damaged cells that released transmitter or epithelial target cells. CT reduced the Isc response to serotonin by 69% and to A23187 by 10% and did not affect the theophylline response. We conclude that 1) the effects of CT on cell function limit its usefulness in identifying peptide neurotransmitters in epithelium, 2) CT irreversibly inhibits ion transport responses to EFS and to serotonin, and 3) CT reduces absorption of Cl probably by affecting a calcium pathway that modifies Cl transport.

Histamine action on guinea pig ileal mucosa

1984 ◽  
Vol 246 (4) ◽  
pp. G372-G377 ◽  
Author(s):  
H. J. Cooke ◽  
P. R. Nemeth ◽  
J. D. Wood
Keyword(s):  
Receptor Agonist ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Electrical Field Stimulation ◽  
Chloride Secretion ◽  
Evoked Response ◽  
Field Stimulation ◽  
H1 Receptor ◽  
Electrical Field ◽  
Mucosal Response

Nerve-mediated and direct actions of histamine on mucosal transport function in the guinea pig ileum were investigated. Addition of histamine to the serosal side of flat sheet preparations in Ussing chambers evoked a transient increase in base-line short-circuit current that was due primarily to an increase in active chloride secretion. The mucosal response to histamine was mimicked by the H1-receptor agonist 2-methylhistamine, but not by the H2-receptor agonist dimaprit. The histamine-evoked response was prevented by the H1-receptor blocker pyrilamine, but not by the H2-receptor antagonist cimetidine. Thirty percent of the mucosal response to histamine was inhibited by tetrodotoxin. Intracellular electrical recording showed that histamine activated AH/type 2 myenteric neurons, and this response was abolished in the presence of pyrilamine. Local anesthetic action of pyrilamine was ruled out by direct electrical recording from myenteric neurons in the presence and absence of pyrilamine. Electrical field stimulation evoked a biphasic increase in short-circuit current. Histamine and 2-methylhistamine did not alter the sustained phase of the short-circuit current response to electrical field stimulation, although pyrilamine reduced the electrically evoked response by 22%. Muscarinic blockade with atropine reduced the stimulus-evoked response by 55%. When muscarinic receptors were blocked and electrical field stimulation applied, histamine increased the stimulus-evoked mucosal response by 22.3%. These results suggest that histamine increases short-circuit current and chloride secretion by acting at H1-receptor sites on both the enteric innervation of the mucosa and on the enterocytes.

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.

Substance P as a mediator of colonic secretory reflexes

1997 ◽  
Vol 272 (2) ◽  
pp. G238-G245 ◽  
Author(s):  
H. J. Cooke ◽  
M. Sidhu ◽  
P. Fox ◽  
Y. Z. Wang ◽  
E. M. Zimmermann
Keyword(s):  
Substance P ◽  
Messenger Rna ◽  
Short Circuit ◽  
Chronic Administration ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Chloride Secretion ◽  
Intestinal Epithelial ◽  
Axon Reflex ◽  
Reflex Pathways

The role of substance P in neural reflex pathways activated by stroking was investigated in muscle-stripped segments of distal colon from guinea pigs. Stroking the mucosal surface with a brush at 1 stroke/s evoked an increase in short-circuit current (Isc) indicative of chloride secretion. The response to mucosal stroking was maximally reduced by 69-75% by the antagonist GR-82334. The agonist [Sar9,Met(O2)11] substance P caused a bumetanide-sensitive increase in Isc when added to the mucosal or serosal bath. Ablation of extrinsic afferents with acute or chronic administration of capsaicin did not alter the mucosal stroking response. Reverse transcription-polymerase chain reaction and in situ hybridization revealed the presence of neurokinin1 (NK1) receptor messenger RNA in isolated colonocytes or crypt glands. Ligand binding of 125I-Bolton-Hunter-labeled substance P was inhibited by GR-82334. The 50% inhibitory concentration was 0.84 nM. The results demonstrate a role for substance P released from capsaicin-insensitive submucosal neurons and in mucosal stroking reflexes. The presence of NK1 receptors on isolated colonocytes suggests that appropriate elements are present for axon reflex activation of intestinal epithelial cells.

5-HT activates nitric oxide-generating neurons to stimulate chloride secretion in guinea pig distal colon

1998 ◽  
Vol 275 (4) ◽  
pp. G829-G834 ◽  
Author(s):  
Atsukazu Kuwahara ◽  
Hirofumi Kuramoto ◽  
Makoto Kadowaki
Keyword(s):  
Nitric Oxide ◽  
Guinea Pig ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Chloride Secretion ◽  
Evoked Response ◽  
Evoked Responses ◽  
No Donor ◽  
Simultaneous Application

The participation of nitric oxide (NO) in serotonin (5-hydroxytryptamine; 5-HT)-evoked chloride secretion in guinea pig distal colon was examined. Submucosal/mucosal segments were mounted in Ussing flux chambers, and an increase in short-circuit current ( I sc) was used as an index of secretion. Addition of 5-HT to the serosal side produced a concentration-dependent (10−7–10−5M) increase in I sc caused by chloride secretion. N G-nitro-l-arginine (l-NNA) significantly reduced the 5-HT-evoked early (P-1) and late (P-2) responses to 61.1 and 70.6% of control, respectively. Neurally evoked response was also inhibited by l-NNA. The NO donor sodium nitroprusside (SNP, 10−4 M) increased basal I sc mainly because of chloride secretion. The SNP-evoked response was significantly reduced by tetrodotoxin but was unchanged by atropine or indomethacin. These results suggest that the 5-HT-evoked increase in I sc is associated with an NO-generating mechanism. Atropine significantly reduced the 5-HT (10−5 M)-evoked P-1 and P-2 responses to 71.8 and 19.7% of control, respectively. Simultaneous application of atropine andl-NNA further decreased the 5-HT-evoked responses more than either drug alone; application ofl-NNA and atropine decreased the 5-HT-evoked P-1 and P-2 responses to 68.5 and 39.2% of atropine-treated tissues, respectively. These results suggest that noncholinergic components of P-1 and P-2 responses are 71.8 and 19.7% of control, respectively, and that NO components of P-1 and P-2 responses are 32 and 61%, respectively, of the noncholinergic component of the 5-HT-evoked responses. The results provide evidence that NO may participate as a noncholinergic mediator of 5-HT-evoked chloride secretion in guinea pig distal colon.

Galanin-induced alteration of electrolyte transport in the rat intestine

1992 ◽  
Vol 263 (4) ◽  
pp. G502-G507
Author(s):  
T. Kiyohara ◽  
M. Okuno ◽  
H. Ishikawa ◽  
T. Nakanishi ◽  
Y. Shinomura ◽  
...  
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Electrical Field Stimulation ◽  
Distal Colon ◽  
Rat Colon ◽  
Chloride Absorption ◽  
Neural Transmission ◽  
36Cl Fluxes ◽  
Intestinal Ion Transport

Effects of rat and porcine galanin on rat intestinal ion transport were examined in vitro. In the rat distal colon, a sustained increase in short-circuit current (Isc) was produced by the serosal addition of rat galanin at a concentration as low as 10(-9) M, and a maximal increment was observed at 10(-7) M. Porcine galanin was approximately 100 times less potent than rat galanin. In the rat jejunum, rat galanin produced only a slight and transient decrease in basal Isc. The response to rat galanin was not influenced by atropine, hexamethonium, or amiloride, but was virtually abolished by tetrodotoxin or furosemide. Rat galanin did not significantly influence the increase in Isc elicited by electrical field stimulation in the rat colon and jejunum. Transmural unidirectional 22Na and 36Cl fluxes in the rat colonic mucosa were measured under short-circuited conditions, and rat galanin significantly decreased net sodium and net chloride absorption. These findings suggest that galanin acts as a secretory modulator in the rat colon via noncholinergic neural transmission.

Defective modulation of colonic secretomotor neurons in a rabbit model of colitis

1993 ◽  
Vol 264 (4) ◽  
pp. G671-G677 ◽  
Author(s):  
J. M. Goldhill ◽  
R. Burakoff ◽  
V. Donovan ◽  
K. Rose ◽  
W. H. Percy
Keyword(s):  
Short Circuit ◽  
Rabbit Model ◽  
In Vitro Study ◽  
Short Circuit Current ◽  
Electrical Field Stimulation ◽  
Distal Colon ◽  
Trinitrobenzenesulfonic Acid ◽  
Cyclic Monophosphate ◽  
Secretomotor Neurons

The present in vitro study was conducted to investigate possible alterations in the control of colonic electrolyte transport in an experimental model of colitis. Intrarectal administration of trinitrobenzenesulfonic acid induced a colitis-like inflammation in the rabbit distal colon. Responses to amiloride and residual short-circuit current after this treatment were unchanged, suggesting that the absorptive and secretory mechanisms remained intact. Electrical field stimulation and vasoactive intestinal polypeptide, a candidate secretomotor neurotransmitter, both elicited similar responses in control and colitic tissue. This suggests that communication at the neuroepithelial junction was unimpaired. In untreated tissue, the effects of prostaglandin E2 (PGE2) and of acetylcholine were attenuated by tetrodotoxin, suggesting, therefore, that both play a role in the modulation of secretomotor neurons. In addition, PGE2 had an appreciable direct epithelial effect. Responses to both of these agonists were absent in colitis. The effects of N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate were unchanged in colitis, suggesting that altered PGE2 responsiveness may involve changes in epithelial receptor number, affinity, or in their ability to mediate an increase in adenosine 3',5'-cyclic monophosphate levels. It is concluded that this rabbit model of colitis exhibits 1) defects in the modulation of secretomotor neurons by acetylcholine and PGE2 and 2) an attenuated epithelial response to PGE2.

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