To investigate the mechanism for bradykinin (BK) to stimulate intestinal secretomotor neurons and intestinal chloride secretion.

Muscle-stripped guinea pig ileal preparations were mounted in Ussing flux chambers for the recording of short-circuit current (Isc). Basal Isc and Isc stimulated by BK when preincubated with the BK receptors antagonist and other chemicals were recorded using the Ussing chamber system. Prostaglandin E2 (PGE2) production in the intestine was determined by enzyme immunologic assay (EIA).

Application of BK or B2 receptor (B2R) agonist significantly increased the baseline Isc compared to the control. B2R antagonist, tetrodotoxin and scopolamine (blockade of muscarinic receptors) significantly suppressed the increase in Isc evoked by BK. The BK-evoked Isc was suppressed by cyclooxygenase (COX)-1 or COX-2 specific inhibitor as well as nonselective COX inhibitors. Preincubation of submucosa/mucosa preparations with BK for 10 min significantly increased PGE2 production and this was abolished by the COX-1 and COX-2 inhibitors. The BK-evoked Isc was suppressed by nonselective EP receptors and EP4 receptor antagonists, but selective EP1 receptor antagonist did not have a significant effect on the BK-evoked Isc. Inhibitors of PLC, PKC, calmodulin or CaMKII failed to suppress BK-induced PGE2 production.

The results suggest that BK stimulates neurogenic chloride secretion in the guinea pig ileum by activating B2R, through COX increasing PGE2 production. The post-receptor transduction cascade includes activation of PLC, PKC, CaMK, IP3 and MAPK.

Kinins are acknowledged as important regulators of intestinal function during inflammation; however, their effects on human intestinal ion transport have not been reported. Here, we used muscle-stripped human colonic tissue and cultured T(84)-cell monolayers to study bradykinin (BK) actions on human intestinal ion transport.

Ion transport was measured as changes in short-circuit current (I(sc)) across colonic epithelia mounted in Ussing chambers.

In intact tissue, there was a distinct polarity to BK-elicited I(sc) responses. Whereas basolateral BK stimulated sustained responses (EC(50)=0.5+/-0.1 microM), those to apical BK were more rapid and transient (EC(50)=4.1+/-1.2 nM). In T(84) cells, responses to both apical and basolateral BK were similar to those seen upon apical addition to intact tissues. Cross-desensitization between apical and basolateral domains was not observed. BK-induced responses were largely due to Cl(-) secretion as shown by their sensitivity to bumetanide and removal of Cl(-) from the bathing solution. Studies using selective agonists and antagonists indicate responses to BK are mediated by B(2) receptors. Finally, responses to basolateral BK in intact tissues were inhibited by tetrodotoxin (1 microM), atropine (1 microM), capsaicin (100 microM) and piroxicam (10 microM). BK-stimulated prostaglandin (PG)E(2) release from colonic tissue.

BK stimulates human colonic Cl(-) secretion by activation of apical and basolateral B(2) receptors. Responses to apical BK reflect a direct action on epithelial cells, whereas those to basolateral BK are amplified by stimulation of enteric nerves and PG synthesis.

The distribution of tissue kallikrein (TK) and its plasma inhibitor, kallistatin in plasma and intestinal tissue, was studied in patients with active ulcerative colitis (UC) and Crohn's disease (CD). TK was localized to goblet cells and kallistatin to epithelial cells of normal human intestine. Both proteins are visualized in macrophages inside granulomas in CD as well as in plasmocytes in both CD and UC. Intestinal tissue kallikrein (ITK) and kallistatin are significantly decreased in inflamed intestine compared to noninflammatory controls. TK mRNA is significantly decreased in intestinal biopsy samples from active UC patients compared with inactive patients or controls. Immunoreactive TK is present in plasma in very low concentrations in patients and did not differ in normal subjects. Plasma kallistatin was significantly decreased in patients with active disease compared to normal controls. Our data suggest that release of TK during inflammation plays a role in inflammatory bowel disease.

1. In the present study, we used a microphysiometer to measure bradykinin-induced acidification responses in IMR-90, a human lung fibroblast cell line, and INT-407, a human colonic epithelial cell line. Furthermore, we investigated the effect of 24 h exposure of transforming growth factor (TGF)-alpha on the bradykinin response in INT-407 cells. 2. Bradykinin (0.1-100 nmol/L) was potent in producing acidification responses in IMR-90 cells (pEC50 8.79+/-0.13; Hill slope 0.96+/-0.04) and INT-407 cells (pEC50 8.90+/-0.04; Hill slope 1.00+/-0.07). These responses were competitively antagonized by the bradykinin B2 receptor antagonist icatibant in both IMR-90 cells (apparent pKB = 8.54+/-0.15; Hill slope = 1.09+/-0.13 and 1.66+/-0.26 in the absence and presence of 10 nmol/L icatibant, respectively) and INT-407 cells (pKB = 8.12+/-0.07 (3, 10 and 30 nmol/L icatibant); Hill slope = 1.06+/-0.04). However, the bradykinin B1 receptor antagonist des-Arg9Leu8-bradykinin (3 micromol/L) had no effect on the bradykinin responses. 3. The non-peptide bradykinin B2 receptor antagonist FR173657 selectively antagonized bradykinin-induced acidification responses in INT-407 cells in a competitive manner (pKB = 8.76+/-0.10; Hill slope = 0.92+/-0.05) at lower concentrations (1 and 3 nmol/L) but in an insurmountable manner at higher concentrations (10 nmol/L; Hill slope = 1.04+/-0.09). This compound, at concentrations of 10 and 100 nmol/L (Hill slope = 1.38+/-0.15), also proved to be an insurmountable antagonist in IMR-90 cells. 4. The bradykinin B1 receptor selective agonist Lys0des-Arg10-bradykinin (0.1 nmol/L to 0.1 micromol/L) failed to produce acidification responses in IMR-90 cells, even after 24 h pre-incubation with bacterial lipopolysaccharide (0.1 microg/mL). 5. A 24 h pre-incubation of INT-407 cells with TGF-alpha (1, 10 and 100 ng/mL) caused a significant concentration-dependent decrease in maximal bradykinin response without affecting the pEC50. 6. In addition to this study being the first to use a microphysiometer to characterize bradykinin B2 receptors in cultured IMR-90 human lung fibroblast cells and INT-407 human colonic epithelial cells, we also showed that pre-incubation of INT-407 cells with TGF-alpha caused a significant decrease in maximal acidification response mediated by bradykinin B2 receptors.

Kinin effects on epithelial electrogenic ion transport are reviewed, with reference to the alimentary tract. The transported ion is usually chloride, but some epithelia also transport bicarbonate. The key components of the transport system are the sodium-potassium-chloride cotransporter, Na+-K+ ATPase (both located basolaterally) and the CFTR chloride channel (located apically). Activation of K+-channels in both membranes may secondarily affect the anion transport mechanism. The types of kinin receptors that cause chloride secretion, the second messengers involved and the possible functional responsibilities of the kinin-activated secretory mechanism are discussed.

The effect of chronic exposure to transforming growth factor-alpha (TGF-alpha) on bradykinin-stimulated acute prostanoid production and ion secretion in monolayers of HCA-7 colony 29 colonic epithelial cells has been studied. Monolayers synthesized prostaglandin E2 (PGE2) at a basal rate of 2.10 +/- 0.31 pg. monolayer-1. min-1 over 24 h. Bradykinin (10(-8)-10(-5) M) dose dependently increased acute PGE2 release by three orders of magnitude. This was associated with a rise in cAMP from 1.60 +/- 0.14 to 2.90 +/- 0.1 pmol/monolayer (P < 0.02) and a dose-dependent increase in short-circuit current (SCC). When monolayers were primed by a 24-h exposure to TGF-alpha, basal PGE2 release rose to 6.31 +/- 0.38 pg. monolayer-1. min-1 (TGF-alpha concn 10 ng/ml; P = 0.001). However, the stimulation of acute prostaglandin release, intracellular cAMP, and increased SCC by bradykinin was significantly reduced by preincubation with TGF-alpha. Priming with PGE2 (10(-8)-10(-6) M) over 24 h mimicked the effect of TGF-alpha on bradykinin-induced changes in cAMP and SCC. These data suggest that enhanced chronic release of prostaglandins in response to stimulation with TGF-alpha may downregulate acute responses to bradykinin. In vivo, TGF-alpha could have an important modulatory function in regulating secretion under inflammatory conditions.

The plasma kallikrein-kinin system is a mediator of intestinal inflammation induced by peptidoglycan-polysaccharide from group A streptococci (PG-APS) in rats. In this study we investigated the participation of intestinal tissue kallikrein (ITK). Lewis rats were injected intramurally with PG-APS. ITK was visualized by immunohistochemical staining. Cecal ITK concentration was measured by radioimmunoassay, and gene expression was evaluated by RNase protection assay. Kallikrein-binding protein (KBP) was evaluated in plasma by ELISA. Tissue kallikrein was identified in cecal goblet cells in both control and PG-APS-injected rats and in macrophages forming granulomas in inflamed tissues. Cecal ITK was significantly lower in acute and chronic phases of inflammation and in supernatant from in vitro cultures of inflamed cecum. ITK mRNA levels were not significantly different. Plasma KBP levels were significantly reduced in inflamed rats. The presence of tissue kallikrein in macrophages suggests participation in experimental colitis. The decrease of ITK in the inflamed intestine associated with unchanged mRNA levels suggests ITK release during intestinal inflammation.

The effect of the inflammatory mediator bradykinin on glycoprotein synthesis and mucin secretion in the human colonic adenocarcinoma cell line HT29-18N2 was examined. Bradykinin, at a threshold of 0.01 microM, accelerated the rate of mucin discharge as assessed by a mucin-specific ELISA. Using immunofluorescence microscopy, a thick meshwork of extracellular mucus was observed over bradykinin-treated monolayers but not mock-treated controls. Morphometric analysis of bradykinin-treated monolayers revealed no decreases in intracellular mucin stores or any other easily discernable morphological alteration. The ability of the cyclooxygenase inhibitors indomethacin and naproxen to decrease the response to bradykinin by approximately 68% indicates the effect is mediated, at least partially, through the generation of prostaglandins. Bradykinin did not alter the rate of incorporation of 3H-glucosamine into newly synthesized glycoproteins. Bradykinin-accelerated mucin secretion may be linked to the depletion of intracellular mucin stores in the inflammatory bowel disease ulcerative colitis.

The canine proximal colon set up in Ussing chambers responded to the serosal addition of bradykinin (BK) with changes in short-circuit current (Isc). Two preparations were used to analyze these effects - an innervated mucosal preparation and a 'functionally nerve-free' epithelial preparation. The specific questions that this study sought to answer were (1) is there a significant neural component to the effects noted?, and (2) what is the receptor subtype involved? BK produced dose-dependent increases across both the mucosa and the epithelial preparations. A secondary decrease in Isc was noted in the mucosal but not the epithelial preparation. Tetrodotoxin (TTX) significantly inhibited the magnitude of mucosal responses and delayed their onset as well, indicating the presence of a significant neural component. Addition of the B2 antagonist, D-Arg0[Hyp3,Thi5,8, D-Phe7]BK produced a surmountable inhibition of the responses to the agonist. The B1 selective agonist, des-Arg9BK produced increases in Isc across both preparations, though TTX had no significant effects on these responses. Cross-desensitization was seen between BK and des-Arg9 BK. However, since the B1 selective antagonist, des-Arg9[Leu8]BK acted as a partial agonist in our preparation, these effects could not be defined further. Clearly, B2 receptors are involved in mediating canine colonic BK responses, however the role of B1 receptors in this tissue requires further definition.

Carbonic anhydrase 1 (CA1) catalyses the reversible hydration of CO2 and is important for cellular diffusion of CO2, ion transport and pH regulation. The gene encoding CA1 (CA1) has two promoters. In adult colon epithelia the proximal promoter determines high levels of expression and the distal erythroid promoter is repressed. RNA in situ hybridisation shows that CA1 mRNA is abundant in differentiating cells of the colonic crypt as they migrate to the luminal surface, but is not present at the base of the crypts and levels are low on the luminal surface. It is likely that CA1 gene expression in these cells is regulated by differential transcription and/or mRNA stability. In contrast CA1 protein is localised predominantly on the luminal surface. Since CA1 mRNA and protein do not exactly co-localise it can be inferred that CA1 expression is also subject to post-transcriptional control. CA1 mRNA is significantly reduced in colon carcinoma and in adenomas from familial adenomatous polyposis patients. Loss of CA1 expression is associated with the disappearance of differentiated epithelial cells. Out of twelve colon carcinoma cell lines three, LIM1215, LIM1899 and HT115, expressed CA1 and nine did not. This variation in expression may also be associated with cell type differentiation.

1. The patch-clamp technique, both cell attached and inside-out patches, was used to examine the effects of lysylbradykinin (LBK) and A23187 on ion channels in cultured Colony 29 epithelial cells derived from a human adenocarcinoma. 2. LBK and A23187 applied directly to the intact cell stimulated the opening of a number of types of ion channel including Ca(2+)-activated K+ channels. 3. By use of inside-out patches, anion channels could be stimulated to open by application of protein kinase A and ATP to the cytosolic surface. Ca(2+)-activated K+ channels were also identified in isolated membrane patches. 4. The results suggest that the anion secretion which is stimulated by LBK is a complex event, involving the activation of a number of different types of ion channel, and that part of the response is the result of hyperpolarization of the cell by activation of Ca(2+)-activated K+ channels. From the data presented in this and the accompanying papers it appears that the Ca(2+)-sensitive K+ channels would be equally effective in either the apical or basolateral membranes.

Primary cultures of glandular endometrial epithelial cells grown on permeable supports formed monolayers with a high transepithelial electrical resistance [1096 +/- 83 omega.cm2 (n = 34)] and displayed electrogenic ion transport as demonstrated by an inward short circuit current (Isc; 20 +/- 2 microA/cm2). Bradykinin, 10(-8)-10(-6) M, added to either the basolateral or apical solutions enhanced the inward ISC. The concentration-response curves for bradykinin were bell-shaped in nature. The ISC response was more sensitive to apical addition of bradykinin and the maximum response was also greater with apical bradykinin. The increases in ISC were accompanied by two- to three-fold increases in transepithelial conductance. Apical addition of amiloride, 10(-4) M, reduced the unstimulated ISC by 80%. In the presence of amiloride, the response to both apical and basolateral bradykinin was reduced by > 50% in 8 out of 18 layers, and the mean response was reduced by approximately 25%. The data are consistent with a physiological role for bradykinin in the control of the intrauterine electrolyte environment, mediated in part by enhanced Na+ absorption.

1. Hoe-140, a potent kinin receptor antagonist, was investigated for its ability to inhibit the effects of lysylbradykinin (kallidin) on a cultured colonic epithelium, HCA-7 Colony 29, derived from a human adenocarcinoma. 2. Measurements of electrogenic chloride secretion (as short circuit current), and of intracellular Ca2+ (from Fura-2 fluorescence) were used to assess the action of lysylbradykinin in the absence and presence of Hoe 140. 3. From short circuit current data, Hoe 140 appeared to be a competitive antagonist with a Ki value of 5 nM. However, with measurements of intracellular Ca2+ Hoe 140 was apparently a non-competitive antagonist with a Ki of between 4-6 nM. 4. Because of the unexpected finding of non-competitive antagonism, measurements were made with a second antagonist pair, histamine and mepyramine. Mepyramine behaved as a competitive antagonist against responses to histamine with a Ki value of approximately 5 nM when short circuit current measurements were evaluated. However, when intracellular Ca2+ concentration was used as a measure mepyramine, 30 nM, produced a near parallel shift in the response curve, but at 100 nM the maximal response was depressed. 5. The reasons why the apparent type of antagonism depends upon the method of measurement is discussed, bearing in mind that the increase in intracellular Ca2+ is a signal which precedes the increase in short circuit current.

The formation of ion channels by the nonadecapeptide antibiotic duramycin was examined using black lipid membranes and using the patch-clamp technique. In black lipid membranes made from glyceryl monooleate or a phosphatidylcholine/phosphatidylethanolamine mixture, duramycin induced complex fluctuations in membrane conductance, some step-like and some which were incapable of being resolved into discrete conductance states. Both conductance and largest step size increased with time. A similar time-dependent increase in conductance was seen in patch-clamp experiments with HCA-7 Colony 29 human colonic epithelial cell. The channels displayed weak anion selectivity and the smaller channels formed in patches from epithelial cells showed weak inward-rectification. Channel formation by duramycin was achieved at lower concentrations when the black lipid membrane was made with phospholipid rather than with glyceryl monooleate. Lower concentrations were effective in generating conductances in epithelial cells than in bilayers. It is concluded that duramycin forms ion channels in both artificial and biological membranes. Accumulation of duramycin and coalescence of initially small channels into larger ones is considered to be responsible for the recorded behaviour and to final disruption of membranes.

1. Colonic epithelial cells, derived from a human adenocarcinoma (HCA-7), were examined by the patch clamp technique. 2. Outwardly rectifying anion (Cl-) channels were identified in the apical membrane. The conductance was g(in) approximately 26 pS, g(out) approximately 40 pS. The open state probability of the channels increased with depolarization and the selectivity for Cl- over K+ (PCl/PK) was approximately 7.5. 3. The channels were sensitive to intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP, 0.1 mM), but not to Ca2+ (at concentrations up to 1 mM). At depolarized potentials the channels were blocked by pirentanide (1-5 microM) applied intracellularly. 4. HCA-7 monolayers loaded with 125I- (as a marker for Cl-) were used to measure I- efflux and converted to instantaneous rate constants. 5. The rate constant for I- efflux was increased by forskolin and lysylbradykinin (LBK). The effects of forskolin were not effected by BAPTA (an intracellular calcium chelator). The effects of LBK were inhibited by BAPTA and by Ba2+, indicating that LBK raised intracellular Ca2+ (Cai) which activates Ca(2+)-sensitive K-channels, the latter being blocked by Ba2+. 6. Although it cannot be conclusively proved that the outwardly rectifying chloride channels described here are solely or partially responsible for the increased anion efflux or transepithelial chloride secretion, the channels are likely to be more relevant for cyclic AMP-requiring rather than Ca(2+)-requiring secretagogues.

The production of tissue kallikrein and the short-circuit current (SCC) response in the human colonic epithelial cell line T84 to bradykinin have been studied. These cells produce true tissue kallikrein and secrete it into the growth medium, and addition of bradykinin results in a small increase in SCC. After growth of T84 cells as a xenograft in athymic (nude) mice (NuT84), however, the cellular concentration of the enzyme increases 38-fold, and the maximal change in SCC (delta SCC) induced by bradykinin increases 35-fold. The SCC response to prostaglandin E1 is not changed, and the response to forskolin increases eightfold. The bradykinin-induced delta SCC was inhibited by piretanide, suggesting that a secretory movement of chloride was responsible for the change, and the effect was attenuated by an antagonist to kinin receptors. We conclude that both the production of true tissue kallikrein and the chloride secretory response to bradykinin can be regulated in T84 cells by changes in the cell environment.

A cultured human epithelial cell line, Colony 29, has been used to investigate the relation between anion secretion and intracellular Ca2+ concentration (Cai) in response to the secretagogues, lysylbradykinin (LBk) and histamine. Anion secretion was measured as short-circuit current (SCC) responses in epithelia cultured on previous supports. Cai was measured both in cell suspensions and epithelial monolayers using Fura-2 fluorescence. While it is concluded that raised Cai is responsible for anion secretion the relationship is complex. For both secretagogues there is a receptor reserve, that is the maximal Cai increase is greater than that required to cause a maximal secretory response. By examining the interactions between maximally effective concentrations of LBk and histamine it was shown that neither the SCC nor Cai responses behaved additively. From observations in the absence of external Ca2+ it was concluded that both secretagogues cause Ca2+ release from the same intracellular source, but that in normal conditions Ca2+ derived from intracellular and extracellular sources is responsible for the full effect.

Distension of the rat colon descendens in vitro by a hydrostatic gradient induced an increase in short-circuit current (Isc). In a mucosa-submucosa preparation containing the plexus submucosus, the increase in Isc was biphasic with a half-time of about 200 s for the spontaneous returning to the baseline. The time course was monophasic in a mucosa preparation without the plexus submucosus. The increase in Isc in the mucosa-submucosa preparation was inhibited by an inhibitor of phospholipase A2, quinacrine, and by indomethacin, tetrodotoxin or atropine; each of these compounds also abolished the second phase of the response. In contrast, only indomethacin was effective in reducing the increase in Isc in the mucosa preparation. In both preparations the response to distension was inhibited by scilliroside, by replacement of Cl- with gluconate, and by administration of frusemide or the chloride channel blocker, anthracene-9-carboxylic acid. The results indicate that distension induces chloride secretion by causing the release of prostaglandins, which act indirectly, i.e. mediated by the submucosal plexus, and directly at the epithelium.

1. Thapsigargin, a sesquiterpene lactone, was shown to cause electrogenic anion secretion in monolayers of human colonic epithelial cells, an effect which was crucially dependent upon calcium and did not involve eicosanoid formation. 2. To measure the secretory effect calcium needed to be present in the external bathing solution. By means of Fura-2 fluorescence measurements thapsigargin was shown to raise Cai by around 250 nM when the bathing solution contained calcium. In the nominal absence of external calcium thapsigargin raised Cai by only 60 nM, but from a lower basal value. This was insufficient to cause secretion. 3. Effects of other calcium-dependent secretagogues (e.g. lysylbradykinin) were inhibited in the presence of thapsigargin, whereas kinin responses were potentiated if the peptide was added following a stimulus which increases cyclic AMP. 4. From the data given here and the known behaviour of colonic epithelia it is concluded that thapsigargin increases Cai by a non-ionophoric mechanism by release from internal stores. Calcium-stimulated calcium influx then follows resulting in the opening of basolateral K channels, increasing the electrochemical gradient for chloride efflux, or alternatively by activating anion channels in the apical membrane. It is concluded that thapsigargin is a potentially important tool for examining epithelial mechanisms.

1. Sheets of muscle-stripped rat and rabbit colon with epithelium intact or removed were mounted in Ussing-type chambers for recording of transepithelial p.d., resistance and short circuit current (Isc), and measurement by radioimmunoassay (RIA) of the release of prostaglandins into serosal and mucosal bathing solutions. 2. In epithelial-intact preparations prostaglandin E2 (PGE2), PGE1, PGF2 alpha, U46619 and prostacyclin (10(-7)-10(-6) M) caused increases in Isc and transepithelial p.d., in (approximate) descending order of potency. Epithelial-removed preparations did not exhibit any transepithelial p.d. 3. In epithelial-intact preparations, lysyl-bradykinin (LBk) applied serosally but not mucosally caused increased p.d. and release of PGE2 (and to a lesser extent other prostaglandins) into serosal but not mucosal bathing solutions. In epithelial-removed tissues, responsiveness to LBk was maintained, but it did not exhibit 'sidedness', i.e. LBk was effective when applied on either side and PGE2 release occurred into both compartments. 4. Indomethacin and other non steroidal anti-inflammatory drugs (NSAIDs) abolished the LBk-induced p.d. and reduced PGE2 release if applied serosally but not mucosally in epithelial-intact preparations. In epithelial-removed tissues, indomethacin added to either side abolished prostaglandin release into both compartments. 5. Calcium removal from serosal but not mucosal bathing solution (Ca2+-free EGTA Krebs) abolished p.d. generation by LBk in epithelial-intact preparations, and reduced PGE2 release in rabbit but not rat colon. Similarly, in epithelial-removed preparations, calcium removal did not affect kinin-induced PGE2 generation in rat but strongly attenuated it in rabbit colon. 6. We conclude that (i) kinins activate the arachidonate cascade principally by interactions with cells in the subepithelial (lamina propria) layer, rather than with the epithelial cells themselves, (ii) PGE2 contributes substantially to the kinin-induced increase of transepithelial p.d. as a messenger released from kinin-responsive subepithelial cells and acting on the basolateral pole of the epithelial cells, (iii) the apparent sidedness of colonic epithelium in terms of responses to kinins, NSAIDs and calcium removal is due to the barrier properties of the epithelial cell layer, and (iv) there are differences in calcium sequestration and apparent calcium dependence of prostaglandin biosynthesis between rat and rabbit colonic subepithelial cells.

We study bradykinin-stimulated K+ efflux in Madin-Darby canine kidney (MDCK) cells using 86Rb as an isotopic tracer. Bradykinin brings about a rapid increase in the permeability of MDCK cells to K+, the effect is dose-dependent with a plateau at 10(-6) M. The effect seems to be mediated by Ca2+-activated K+ channels, localised at the basolateral aspect of the epithelium. Unlike alpha-receptors, which mediate a similar effect of adrenalin in these cells, bradykinin receptors seem to be present at both sides of the epithelium. Bradykinin increases the labelling of IP3, and bradykinin-stimulated K+ efflux persists even in cells which are bathed in Ca2+-free medium, suggesting that the effects seen in the present work are probably due to Ca2+ release from intracellular stores. Some extracellular Ca2+ also might be involved in the bradykinin effect, consistent with the kinin-increasing membrane permeability to Ca2+.

The active transport of ions by the intestinal epithelium is regulated by a number of enteric neurotransmitters, hormones and other substances. Our knowledge of the receptors mediating the actions of these substances is generally fragmentary. This review summarizes current knowledge on the location and functional characteristics of transmitter receptors regulating transport function in the small intestine, highlighting recent research on cholinergic and bradykinin receptors.

1. Type 1 hypersensitivity reactions in response to antigen challenge have been measured as short circuit current (SCC) responses in reconstructed tissues consisting of syngeneic cell types. 2. In all instances reconstructed tissues consisted of an epithelial monolayer grown on collagen-coated Millipore filters and a pad of peritoneal cells. Monolayers were formed of either HCA-7 or HCA-7-Col 1 cells derived from a human adenocarcinoma. Peritoneal cells were derived from rats or guinea-pigs sensitized to either ovalbumin or beta-lactoglobulin. 3. The SCC responses of the monolayers were dependent upon the 'concentration' of peritoneal cells in the reconstructed tissue. The threshold concentration was 0.4 X 10(6) cells when rat peritoneal cells are combined with an epithelial monolayer of 0.2 cm2. 4. The SCC responses in response to antigen challenge were selectively inhibited by the H1-receptor antagonist, mepyramine. Similarly the effects of exogenously applied histamine were antagonised by mepyramine. 5. The responses to antigen challenge were not inhibited by tetrodotoxin in reconstructed tissues. This result is in contrast to that with isolated intestinal epithelia from sensitized animals where tetrodotoxin inhibits the SCC responses to external field stimulation and to challenge with antigens. The consequences of these results for understanding the mechanisms of epithelial Type 1 hypersensitivity reactions are discussed. Suggestions are made to illustrate how the methods developed here may be employed to ask questions about the nature of mediators released and the types of cell responsible in human disease conditions.

Three stable epithelial cell lines (HCA-7, HCA-7-Col 1 and HCA-7-Col 3) all derived from the same human adenocarcinoma have been cultured on collagen-coated Millipore filters. These epithelial monolayers have been used to record short circuit current (SCC) in response to of secretagogues. Similar monolayers, but grown on plastic dishes, were used for measurements of tissue cyclic AMP. Lysylbradykinin, applied to either side of the monolayers, increased SCC in HCA-7 cells but had little effect on the other two lines. The responses showed rapid desensitization, which could be prevented by cooling to 4 degrees C. Responses to kinin were not significantly attenuated by piroxicam, an inhibitor of cyclo-oxygenase. Other secretagogues, vasoactive intestinal polypeptide (VIP) and carbachol also increased SCC in monolayers. The responses to VIP were greatest in HCA-7-Col 1 monolayers while responses were virtually absent in HCA-7-Col 3. A similar profile was seen with carbachol except that responses of HCA-7 and HCA-7-Col 1 monolayers were more equal. With one exception the responses to VIP and carbachol showed sidedness, acting only from the basolateral side. The effects of the secretagogues were inhibited by piretanide, a loop diuretic, applied basolaterally. It is presumed that SCC responses represent electrogenic chloride secretion. Treatment with forskolin increased SCC in HCA-7 and HCA-7-Col 1 monolayers with little effect in HCA-7-Col 3. Nevertheless cyclic AMP levels were elevated most in HCA-7-Col 3 and least in HCA-7-Col 1 monolayers, in reciprocal relationship to the functional response. A23187 increased SCC when applied to HCA-7 and HCA-7-Col 3 monolayers with little effect on HCA-7-Col 1. The differential responses of the three human cell lines provide unique opportunities to discover the functional responsibilities of entities involved in the chloride secretory process. HCA-7-Col 3 cells which generate high levels of cyclic AMP in response to forskolin but which fail to show a substantial chloride secretory response may be a useful model of some disease conditions.

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.

The establishment and characterisation (morphology, ultrastructure, tumourigenicity) of six cell lines from primary human colorectal adenocarcinomas is described. These lines were established from surgical specimens, from 49 unselected patients, without the use of 'feeder' cells, 'conditioned' medium or passage of cells in nude mice. The six cell lines exhibit considerable variation in morphology, CEA secretion and tumourigenicity in nude mice. At least two of the lines retain some of the differentiated characteristics of colorectal epithelium.