Transporting epithelia as targets for kinin effects.
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Animals↗
Biomedical subjects
A W Cuthbert
Publications and source records attributed to A W Cuthbert.
Electrogenic anion secretion in cultured rat epididymal epithelium.
Primary monolayer cultures enriched with principal cells from rat cauda epididymis were grown on pervious supports until confluence was reached. The epithelia so formed were used for short-circuit current recording. Epididymal monolayers had a transepithelial potential of 0.2 mV (apical side negative), a basal short-circuit current of 2 microA/cm2 and a transepithelial resistance of 60-90 omega cm2 when bathed on both sides with Krebs-Henseleit solution at 37 degrees C and gassed with 95% O2-5% CO2. 8-bromo-adenosine 3',5'-phosphate (1 mM) and forskolin (10 microM) caused an inward-flowing current in short-circuited monolayers. The current was partially sensitive to acetazolamide and to frusemide, suggesting that anion secretion was responsible for the responses. In the absence of chloride in the bathing fluid the response to forskolin was sensitive to acetazolamide but not to frusemide. The peptide lysylbradykinin (LBK) and prostaglandin E2 (PGE2) also produced inward-flowing currents which again appeared to be due to anion secretion. The actions of PGE2 were seen only when this agent was added to the basolateral side of the tissue, while LBK had effects from both sides. The responses to kinin, but not to PGE2, were inhibited by cyclo-oxygenase inhibitors such as piroxicam and indomethacin. It seems the kinin effects are dependent, at least in part, upon eicosanoid formation. Responses to kinin and PGE2 were severely attenuated in chloride-free bathing solution. Readdition of chloride-containing solution restored the responses to both agents. Assuming electrogenic ion secretion in the epididymis is accompanied by osmotic fluid flow it is evident that the transit time of sperm in the cauda epididymis will be reduced. Possible consequences for the maturation of spermatozoa are discussed.
8-Bromo Cyclic Adenosine Monophosphate↗
Regulation of transepithelial ion transport by intracellular calcium ions.
A photodynamic effect of erythrosine B on the basolateral surface of rat colon epithelium under short circuit conditions is described. The resulting irreversible increase in short circuit current was the result of electrogenic chloride secretion. The effect was dependent upon oxygen and calcium ions, and is probably due to the generation of singlet oxygen which then permeabilises the membranes to calcium. Half maximal activation of secretion in permeabilised preparations occurred at an external calcium concentration of 1 microM. In tight sodium transporting epithelia increased Cai reduces SCC, possibly by a direct effect on apical sodium permeability. In toad urinary bladder SCC fell in response to conditions outlined above for rat colon.
Amiloride↗
Indirect effects of adenosine triphosphate on chloride secretion in mammalian colon.
The effects of adenosine triphosphate (ATP) on short-circuit current (SCC) in rat colonic epithelium are described. ATP caused a large increase in inward-going current and was considerably more potent in this respect than ADP, AMP or adenosine. The response to ATP was sided, there being only minor effects when the nucleotide was added to the apical side of the tissue. The effects of ATP were not modified by the cyclooxygenase inhibitor, indomethacin, eliminating eicosanoid formation as a mechanism. The effects of ATP were potentiated by theophylline and not blocked by alpha, beta-methylene ATP. The data are consistent with the effect being dependent on the activation of adenylate cyclase, but it has not been possible to classify the receptors into P1 or P2 categories. Using inhibitors of NaCl cotransport (piretanide), carbonic anhydrase (acetazolamide), and chloride channels (diphenylamine-2-carboxylate), it was concluded that the SCC response to ATP was due to chloride secretion with, perhaps, a minor contribution from bicarbonate. Flux measurements with 22Na and 36Cl confirmed this view, there being approximate equivalence of chloride secretion with the SCC responses. Additionally, flux measurements revealed an inhibition of electroneutral NaCl absorption in response to ATP. The effects of ATP were antagonized by tetrodotoxin (TTX), greater than 50% inhibition being achieved with 10 nM TTX. This result suggests that ATP does not act directly on receptors in the epithelial cells but rather on neuronal elements in the lamina propria. It will be necessary to re-examine other secretagogues for indirect effects of this kind and to search for the final effector neurotransmitter which evokes secretion.
1-Methyl-3-isobutylxanthine↗
Kinin effects on ion transport in monolayers of HCA-7 cells, a line from a human colonic adenocarcinoma.
Using epithelial monolayers of HCA-7 cells, derived from a primary human colonic adenocarcinoma and grown on pervious supports, it is shown that responses to lysylbradykinin can be elicited from either side. It is proposed that kinin receptors are inserted into both apical and basolateral membrane domains.
Adenocarcinoma↗
Immediate hypersensitivity reactions in epithelia from rats infected with Nippostrongylus brasiliensis.
Colonic epithelia from rats infected with the nematode Nippostrongylus brasiliensis have been studied under short circuit conditions and in response to challenge with worm antigen. Challenge from the serosal but not the mucosal side with antigen caused a transient increase in inwardly directed short circuit current. No effects were observed in comparable tissues from noninfected animals. Simultaneous measurements of short circuit current and of the fluxes of sodium or chloride ions showed there was an increase in electrogenic chloride secretion and an inhibition of electroneutral sodium chloride absorption, associated with antigen challenge. This result, together with the inhibitory effects of piretanide on the response to antigen challenge, indicate that chloride ions are a major carrier of the short circuit current response. However, the equivalence of the biophysical response to ion fluxes was not established, there being an excess of chloride secretion. The mast cell stabilizing agent, FPL 52694, significantly inhibited the current responses to antigen, while cromoglycate and doxantrazole were ineffective. Mepyramine, an H1-receptor antagonist, and indomethacin, an inhibitor of fatty acid cyclo-oxygenase, were without effect on the responses to antigen challenge. Anti-rat IgE produced qualitatively similar responses to antigen in both normal and sensitized colonic epithelia. However, the responses were significantly greater in tissues derived from infected animals. Maximally effective antigen concentrations prevented subsequent responses to anti-rat IgE in sensitized tissues, while anti-rat IgE only attenuated the responses to antigen. The ways in which antigen challenge modifies epithelial function is discussed, particularly in relation to its possible role in promoting rejection of the nematodes during secondary infection.
Animals↗
Changed sensitivity to antigen in a gut epithelium treated with bile salts.
Colonic epithelia from guinea-pigs, sensitized by feeding with cow milk, responded to antigen (beta-lactoglobulin) challenge when applied to the serosal, but not the mucosal, side of the tissue. The response, under short circuit conditions, was an inwardly directed current due to chloride secretion. Two detergents, deoxycholate and Triton X-100, caused the basal short circuit current to decrease and transepithelial conductance to increase when applied to the mucosal surface. After removing detergents from the bathing solution tissues now responded to antigen challenge from the mucosal side, without impairment of the overall response. There was a correlation between the conductance change induced by detergents and the fraction of the total response which could be elicited form the mucosal side of the tissue. It was concluded that models of local hypersensitivity reactions to ingested foodstuffs require both development of immunological sensitivity plus increased permeability to antigen. The role of bile salts in inducing the latter is discussed.
Animals↗
Calcium-dependent chloride secretion in rat colon epithelium.
Epithelia, dissected from the descending rat colon, were studied under short-circuit conditions in Ussing chambers. The latter were modified to accept flexible light guides, so that the tissue could be irradiated, with white light, normal to its surface. Irradiation alone had no effect on short-circuit current (s.c.c.). In the presence of erythrosine B (which by itself had no effect on the s.c.c.) on the basolateral side of the tissue, irradiation produced a substantial increase in s.c.c.; this increase was sustained after irradiation had ceased and the dye had been washed away. The photodynamic effect of erythrosine B required the presence of oxygen in the bathing solution. Also calcium was essential for the s.c.c. response to occur. Thus, irradiation in the presence of the dye in the absence of calcium had no effect on s.c.c., but a s.c.c. increase could be revealed by subsequent addition of calcium after irradiation had ceased. Cobalt and magnesium ions antagonized the effect of calcium in the conditions described above. Ion flux measurements with 36Cl and 22Na showed that the photodynamic effect of erythrosine B abolished net sodium absorption and reversed net chloride absorption to secretion. The data are consistent with abolition of electroneutral sodium chloride absorption and the stimulation of electrogenic chloride secretion to an extent equivalent to the s.c.c. responses. Using calcium-containing buffers it was possible to compare the s.c.c. responses at low, known ionized calcium concentrations with the maximal chloride secretory effect following photodynamic activation. Chloride secretion was half-maximally activated when the basolateral bathing fluid contained 1 microM-ionized calcium and after the basolateral face of the tissue had been permeabilized by the photodynamic action of erythrosine B. The relation between ionized calcium concentration in the basolateral fluid and the chloride secretory response was steep.
Action Potentials↗
Kinin effects on electrogenic ion transport in primary cultures of pig renal papillary collecting tubule cells.
Confluent monolayers of pig renal papillary collecting tubule (RPCT) cells were formed on Millipore filters coated with collagen. They were clamped in Ussing-type chambers and used to measure short-circuit current (SCC). The monolayers had low potentials (0.1 mV) with the basolateral side positive. Small inward currents flowed under short-circuit conditions. Increases in SCC were obtained following addition of a number of agents. Receptors associated with SCC changes were disposed as follows: for kinins (e.g., lysyl-bradykinin) they were present on both sides of the tissue, while those for arginine vasopressin and norepinephrine were present on the basolateral side only. Epithelia responded to PGE2 added to the apical or basolateral face of the tissue; application to one side prevented the response from the contralateral side. The tissues also responded to forskolin, an activator of adenylate cyclase, with a sustained inward current that was sensitive to furosemide. Similar sustained inward currents were recorded following exposure to 8-bromoadenosine-3',5'-cyclic monophosphate (BrcAMP). Responses to kinins were attenuated by inhibition of fatty acid cyclooxygenase with either indomethacin or piroxicam or by replacing chloride with impermeant ions. If the SCC was first increased with forskolin, BrcAMP, or norepinephrine, the kinin effects on SCC were either abolished or reversed. It is concluded that kinin can cause chloride secretion in RPCT monolayers, possibly via a prostaglandin or a prostaglandin-adenylate cyclase mechanism. Secondary effects of kinin, exposed by first raising tissue cAMP levels, are not precluded.
8-Bromo Cyclic Adenosine Monophosphate↗
Studies of the kallikrein-kinin system and prostaglandins in epithelial ion transport.
Tissue kallikrein of colon mucosa is synthesized rapidly, and this synthetic process can now be examined in relation to hormonal or dietary manipulations or pathological circumstances that affect intestinal ion transport. Although the identical renal tissue enzyme is known to be enriched in membranes of distal convoluted tubular epithelial cells, the precise localization of the intestinal enzyme is uncertain. An understanding of the intestinal cellular locale of kallikrein will help in defining its local role. That tissue kallikreins can be inhibited by monovalent cations and some drugs (e.g., amiloride) and that kallikrein inhibitors affect cation transport across epithelial surfaces containing such enzymes must be reconciled with the new observations of kinin-induced chloride secretion. Extracellular calcium, eicosanoid synthesis, and cyclic nucleotide production are involved in the secretory response to kinins, although an absolute requirement for intact eicosanoid synthesis may not exist.
Amiloride↗
Role of calcium ions in kinin-induced chloride secretion.
Electrogenic ion transport across the epithelium lining the descending colon of male Sprague-Dawley rats has been measured under short-circuit conditions. Responses to kallidin (lysylbradykinin) were inhibited by 70% if calcium was removed from the solution bathing the basolateral aspect of the tissue. Under identical conditions responses to prostaglandin E1 and dibutyryl cyclic adenosine monophosphate were not changed. Forskolin, which directly activates the catalytic subunit of adenylate cyclase, was inhibited by 35% by calcium removal, whereas responses to the phosphodiesterase inhibitor isobutylmethylxanthine were inhibited by 45% by the same procedure. In the absence of calcium, strontium could substitute in promoting the chloride secretory events triggered by kallidin. Magnesium ions antagonized the effects of the kinin in the presence of calcium ions in the bathing solution. The effects of kallidin were partially antagonized by verapamil and trifluoperazine and were potentiated by isobutylmethylxanthine. These results, together with earlier evidence, suggest that kinin elicits a chloride secretory response in this epithelium by stimulating the formation of prostaglandins which then activate adenylate cyclase. Extracellular calcium ions appear to have an important role in the proximal part of this cascade for prostaglandin generation. However, biochemical correlates of these biophysical responses presented in the following paper indicate a more complex role for calcium in the genesis of the kinin response.
Animals↗
Mediators of the secretory response to kinins.
The output of immunoreactive (i) 6 keto prostaglandin F1 alpha (i6ketoPGF1 alpha), iPGE2 and ithromboxane B2 (iTXB2) from isolated colonic epithelium of the rat into the apical and basolateral bathing solution has been measured. In some instances tissues were also voltage clamped at 0 mV to measure short circuit current (SCC). Kallidin (lysylbradykinin) stimulated the output of all three eicosanoids, specifically from the basolateral face of the tissue. The output was similar whether or not the tissues were short circuited. Both the SCC response and eicosanoid output were dependent upon the concentration of kallidin, but not in a strictly proportional manner, there being relatively more eicosanoid output at submaximal kinin concentrations. Indomethacin, 5 microM, abolished the eicosanoid output, in response to kinin, while some part of the SCC response remained. Calcium removal from the basolateral bathing fluid severely attenuated the SCC response, reduced the output of i6ketoPGF1 alpha to half, but left the output of iPGE2 unchanged. In the presence or absence of calcium it is probable that sufficient PGE material is released to cause part of the SCC changes seen with kinin. Kinin and PGE1 increased the cyclic AMP content of intact epithelia, provided a phosphodiesterase inhibitor was added at the same time. It is proposed that kinin causes an increase in calcium influx at the basolateral pole of the tissue. This calcium is necessary for the production of some eicosanoids and the subsequent generation of cyclic AMP, which then increases apical chloride permeability. In addition, calcium may facilitate entry of chloride through the basolateral face of the cells by activating a cotransport mechanism.
6-Ketoprostaglandin F1 alpha↗
Kinin effects on chloride secretion do not require eicosanoid synthesis.
The actions of bradykinin on colonic epithelia from essential fatty acid-deficient (EFAD) rats has been examined. Electrogenic chloride secretion as short circuit current (SCC) and release of immunoreactive prostaglandin E2 (iPGE2) and i 6-keto PGF1 alpha have been measured. Resting release of prostanoids was significantly less in EFAD than in control tissues. Bradykinin, in a maximally effective concentration, produced no increase in prostanoid release in EFAD tissues in contrast to controls, while the SCC response was 55% of that in controls. In EFAD tissues the SCC response to bradykinin was the same whether or not the cyclooxygenase inhibitor piroxicam was present. EFAD tissues were not more sensitive to prostaglandins than control tissues. We conclude that while prostaglandin release contributes to the totality of the response to bradykinin, the latter's effect on electrogenic chloride secretion does not require the obligatory production of arachidonic acid metabolites.
6-Ketoprostaglandin F1 alpha↗
Sidedness of the reaction to beta-lactoglobulin in sensitised colonic epithelia.
The short circuit current reaction to beta-lactoglobulin shown by the epithelial lining of the colon from guinea-pigs fed with cows' milk is elicited only when the challenge is applied to the basolateral side of the tissue. However if the apical surface of the epithelium is subjected to controlled lesioning with ultraviolet irradiation then apical challenge with beta-lactoglobulin becomes effective. A similar situation ensues when preparations are aged in vitro at room temperature in oxygenated Krebs-Henseleit solution. Although irradiation causes insignificant changes in the electrical resistance of the tissue, the evidence suggests that it allows beta-lactoglobulin to penetrate the epithelium from the apical side. If the model is applicable to clinical conditions of food allergic disease then it appears that concomitant changes in epithelial permeability as well as the development of sensitivity are required.
Animals↗
Immediate hypersensitivity reactions to cow milk proteins in isolated epithelium from ileum of milk-drinking guinea-pigs: comparisons with colonic epithelia.
The epithelium lining the ileum has been isolated and studied in vitro under short-circuit conditions. Tissues were obtained from guinea-pigs fed cow milk. When challenged with beta-lactoglobulin on either the apical or basolateral side, a transient, inward flowing current was recorded. This reaction did not occur with tissues from non-milk drinking animals. Casein and alpha-lactalbumin, in contrast to beta-lactoglobulin, produced only minor effects while bovine serum albumin and bovine gamma-globulin were without effect. The effect with beta-lactoglobulin was associated with overall fluid secretion in the basolateral to apical direction, although the ions responsible for carrying the current have not been identified. Possible mediators of the response to beta-lactoglobulin have been investigated. Throughout the responses of the ileum have been compared with those in the colon. The differences between the two tissues is striking. The relevance of the model to mechanisms of food intolerance is discussed.
Animals↗
Conversion of sodium channels to a form sensitive to cyclic AMP by component(s) from red cells.
Sodium transport has been measured in the isolated epithelium from colons of male Sprague-Dawley rats. Sodium transport in colons was induced by pretreating the animals with dexamethasone (6 mg kg-1) which caused the appearance of an amiloride-sensitive short circuit current within a few hours. Forskolin, a diterpene, which activates adenylate cyclase, was found to increase the cyclic adenosine monophosphate (cyclic AMP) content of rat colons and also to increase short circuit current at the same time. However, measurements of chloride and sodium fluxes across the epithelium indicated that forskolin activates chloride secretion but has no effect on sodium transport. In confirmation of (3) it was found that the amiloride-sensitive short circuit current was unchanged after the short circuit current had been increased by forskolin under a variety of conditions. The behaviour of the mammalian colon as indicated in (3) and (4) is unlike that of amphibian sodium transporting epithelia. It is shown that in toad urinary bladder forskolin increases amiloride-sensitive short circuit current. Procedures were investigated which might make sodium transport in the mammalian colon sensitive to cyclic AMP. Exposing the apical surface to sonicated suspensions of nucleated red cells (frog, toad and duck), followed by washing, gave preparations with amiloride-sensitive short circuit currents which were increased by forskolin or dibutyryl cyclic AMP. It would appear that the sodium channel in the mammalian colon, unlike that of amphibian tissues, has lost the ability to have its properties modified by cyclic AMP. Incubation of colons with sonicated suspensions of nucleated red cells apparently modifies the tissues such that sodium transport across the tissue becomes sensitive to the nucleotide.
Amiloride↗
Selective activity of cyclokinins.
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Animals↗
Immediate hypersensitivity reaction to beta-lactoglobulin in the epithelium lining the colon of guinea pigs fed cows' milk.
Isolated epithelia from the colons of guinea pigs fed cows' milk have been studied in vitro under short circuit current conditions. When challenged with beta-lactoglobulin (beta LG) from the basolateral side, a large, transient inward flowing current was recorded. Pharmacological and ion flux measurements indicated this was due to inappropriate chloride secretion. There was no effect of beta LG on normal epithelia. The effects of challenge with beta LG were abolished by pretreatment with indomethacin, a prostaglandin synthetase inhibitor. By exposing epithelia from water-drinking animals in vitro to serum from milk-drinking guinea pigs sensitivity to beta LG could be conferred.
Animals↗