Margaret E. Hickman

Chloride secretion in response to guanylin in colonic epithelial from normal and transgenic cystic fibrosis mice.

1 Guanylin, a 15 amino acid endogenous gut peptide, increased the short circuit current (SCC) in the epithelium of the mouse colon, but only when applied to the apical and not the basolateral surface. 2 By use of selective blockers of epithelial ion transport and modification of the bathing solution, it was concluded that guanylin increased electrogenic chloride secretion but also had a minor effect on electrogenic sodium absorption. In addition there were small residual currents which remained unresolved. 3 The threshold concentration of guanylin causing a SCC increase was less than 50 nm, but at concentrations 40 times greater no indication of a maximally effective concentration was found. 4 Two guanylin isomers with the same amino acid sequence but with the disulphide bridges joined in an alternate fashion showed no activity. Thus only guanylin with the greatest structural homology to heat stable enterotoxin (STa) showed biological activity. 5 The action of guanylin was virtually eliminated in colonic epithelia from transgenic cystic fibrosis (CF) mice. As these animals lack the chloride channel coded by the CF gene sequence, it is likely that the final effector process in murine colonic epithelia involves the CFTR (cystic fibrosis transmembrane conductance regulator) chloride channel. 6 Opportunistic infections of the gut generating STa lead to diarrhoeal conditions via an action of the toxin on apical guanylin receptors. Thus, as discussed, the CF heterozygote may have a genetic advantage in this circumstance.

Importance of basolateral K+ conductance in maintaining Cl− secretion in murine nasal and colonic epithelia

1 Epithelia lining the nasal passages and descending colon of wild‐type and cystic fibrosis (CF) mice were examined by the short‐circuit current technique. Additionally, intracellular Ca2+ ion determinations were made in nasal epithelial cells. Forskolin produced anion secretory currents in wild‐type and CF nasal epithelia. It produced similar effects in wild‐type colonic epithelia, but not in colonic epithelia from CF mice. 2 After electrogenic Na+ transport was blocked with amiloride and electrogenic Cl− secretion was stimulated with forskolin, the ability of K+ channel blockers to inhibit the forskolin‐induced Cl− current was determined. The order of efficiency for nasal epithelium was: Ba2+ > clofilium >>> TEA = azimilide >>> trans‐6‐cyano‐4‐(N‐ethylsulphonyl‐N‐methylamino)‐3‐hydroxy‐2,2‐dimethyl‐chromane (293B) = charybdotoxin, whereas for the colonic epithelium the order was: Ba2+= 293B >>> azimilide = TEA >>> clofilium = charybdotoxin. 3 1‐Ethyl‐2‐benzimdazolinone (1‐EBIO) was able to generate large Cl−‐secretory currents in colonic epithelia which were partially sensitive to charybdotoxin, with the remaining current being inhibited by 293B. In nasal epithelia 1‐EBIO produced only a small transient effect on current. 4 Forskolin released intracellular Ca2+ in nasal epithelial cells; this activity was attenuated when more powerful Ca2+‐releasing agents were applied first. 5 It is concluded that an action on basolateral cAMP‐sensitive K+ channels is an important determinant of the maintained responses to forskolin in nasal and colonic epithelia, in addition to the effects on the cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane. In CF nasal epithelia the activation of calcium‐activated chloride channels (CACs) substitutes for the effect on CFTR. On the basis of the different orders of potency of the blocking agents and the differential response to 1‐EBIO it is concluded that the cAMP‐sensitive K+ channels are different in the airways and the gut.

Ion-transporting activity in the murine colonic epithelium of normal animals and animals with cystic fibrosis.

Electrogenic ion transport in the isolated co-Ionic epithelium from normal and transgenic mice with cystic fibrosis (CF mice) has been investigated under short-circuit current (Isc) conditions. Normal tissues showed chloride secretion in response to carbachol or forskolin, which was sensitive to the Na-K-2Cl cotransport inhibitor, frusemide. Responses to both agents were maintained for at least 12 h in vitro, but the responses to carbachol changed in format throughout this period. By contrast CF colons failed to show the normal secretory responses to carbachol and forskolin, most preparations showing a decrease in Isc that was immediately reversed by frusemide. In CF colons addition of Ba2+ ions or tetraethylammonium (TEA+) to the apical bathing solution antagonised the reduction in Isc caused by the secretagogues. It is concluded that the reduction in Isc in CF colons is due to electrogenic K+ secretion and this was confirmed by flux studies using rubidium-86. In normal colons exposed to TEA+ the responses to for-skolin were greater, but not significantly so, presumably because the minor K+-secretory responses are dominated by major chloride-secretory responses. Again rubidium-86 fluxes showed an increase of K+ secretion in normal colons receiving forskolin. Since the amiloride-sensitive current was not different in CF and normal colons there was no evidence that the CF mice were stressed in a way that increased mineralocorticoid levels and hence K+ secretion. Knowledge of the phenotype of the colonic epithelium of the CF mouse sets the baseline from which attempts at gene therapy for the gut must be judged.

Activation of Ca2+- and cAMP-sensitive K+ channels in murine colonic epithelia by 1-ethyl-2-benzimidazolone

1-Ethyl-2-benzimidazolone (EBIO) caused a sustained increase in electrogenic Cl- secretion in isolated mouse colon mucosae, an effect reduced by blocking basolateral K+ channels. The Ca2+-sensitive K+ channel blocker charybdotoxin (ChTX) and the cAMP-sensitive K+channel blocker 293B were more effective when the other had been added first, suggesting that both types of K+ channel were activated. EBIO did not cause Cl- secretion in cystic fibrosis (CF) colonic epithelia. In apically permeabilized colonic mucosae, EBIO increased the K+ current when a concentration gradient was imposed, an effect that was completely sensitive to ChTX. No current sensitive to trans-6-cyano-4-( N-ethylsulfonyl- N-methylamino)-3-hydroxy-2,2-dimethylchromane (293B) was found in this condition. However, the presence of basolateral cAMP-sensitive K+channels was demonstrated by the development of a 293B-sensitive K+ current after cAMP application in permeabilized mucosae. In isolated colonic crypts EBIO increased cAMP content but had no effect on intracellular Ca2+. It is concluded that EBIO stimulates Cl-secretion by activating Ca2+-sensitive and cAMP-sensitive K+ channels, thereby hyperpolarizing the apical membrane, which increases the electrical gradient for Cl- efflux through the CF transmembrane conductance regulator (CFTR). CFTR is also activated by the accumulation of cAMP as well as by direct activation.

Calcium‐and cyclic AMP‐dependent chloride secretion in human colonic epithelia

1 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 a variety of secretagogues. Similar monolayers, but grown on plastic dishes, were used for measurements of tissue cyclic AMP. 2 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°C. Responses to kinin were not significantly attenuated by piroxicam, an inhibitor of cyclo‐oxygenase. 3 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. 4 The effects of the secretagogues were inhibited by piretanide, a loop diuretic, applied basolaterally. It is presumed that SCC responses represent electrogenic chloride secretion. 5 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. 6 A23187 increased SCC when applied to HCA‐7 and HCA‐7‐Col 3 monolayers with little effect on HCA‐7‐Col 1. 7 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.

Formal analysis of electrogenic sodium, potassium, chloride and bicarbonate transport in mouse colon epithelium

The mammalian colonic epithelium carries out a number of different transporting activities simultaneously, of which more than one is increased following activation with a single agonist. These separate activities can be quantified by solving a set of equations describing these activities, provided some of the dependent variables can be eliminated. Using variations in the experimental conditions, blocking drugs and comparing wild type tissues with those from transgenic animals this has been achieved for electrogenic ion transporting activity of the mouse colon. Basal activity and that following activation with forskolin was measured by short circuit current in isolated mouse colonic epithelia from normal and cystic fibrosis (CF) mice. Using amiloride it is shown that CF colons show increased electrogenic sodium absorption compared to wild type tissues. CF mice had elevated plasma aldosterone, which may be responsible for part or all of the increased sodium absorbtion in CF colons. The derived values for electrogenic chloride secretion and for electrogenic potassium secretion were increased by 13 and 3 fold respectively by forskolin, compared to basal state values for these processes. The loop diuretic, frusemide, completely inhibited electrogenic potassium secretion, but apparently only partially inhibited electrogenic chloride secretion. However, use of bicarbonate‐free solutions and acetazolamide reduced the frusemide‐resistant current, suggesting that electrogenic bicarbonate secretion accounts for the frusemide‐resistant current. It is argued that the use of tissues from transgenic animals is an important adjunct to pharmacological analysis, especially where effects in tissues result in the activation of more than one sort of response.

Restoration by intratracheal gene transfer of bicarbonate secretion in cystic fibrosis mouse gallbladder

Gallbladders from cystic fibrosis (CF) mice ( Cftrtm1Cam and Cftrtm2Cam ) were examined with the short-circuit current technique. The tissues failed to show any electrogenic anion transport in response to forskolin (cAMP stimulus) but responded to the Ca2+ ionophore ionomycin. Administration of the plasmid pTrial10-CFTR2 complexed with cationic liposomes {3β-[ N-(dimethylaminoethane)-carbamoyl]cholesterol andl-α-phosphatidylethanolamine dioleolyl} to the airways restored the phenotype of CF gallbladders to that of the wild type, but did not do so when given orally. Formation of human CFTR mRNA in gallbladders of transfected CF null mice was demonstrated. Using the reporter genes pCMV- luc and pCMV- LacZ, we showed that 1) the intratracheal route was more effective than the oral, intravenous, intramuscular, subcutaneous, or intraperitoneal routes in expressing luciferase activity in the gallbladder and 2) β-galactosidase staining after pCMV- LacZ was confined to the columnar epithelium lining the gallbladder without any discernible activity in its smooth muscle. The discovery of an unusual route for gene transfer to the biliary system may give useful insight into counteracting the consequences of biliary fibrosis in human CF patients.Gallbladders from cystic fibrosis (CF) mice (Cftrtm1Cam and Cftrtm2Cam) were examined with the short-circuit current technique. The tissues failed to show any electrogenic anion transport in response to forskolin (cAMP stimulus) but responded to the Ca2+ ionophore ionomycin. Administration of the plasmid pTrial10-CFTR2 complexed with cationic liposomes (3beta-[N-(dimethylaminoethane)-carbamoyl]cholesterol and L-alpha-phosphatidylethanolamine dioleolyl) to the airways restored the phenotype of CF gallbladders to that of the wild type, but did not do so when given orally. Formation of human CFTR mRNA in gallbladders of transfected CF null mice was demonstrated. Using the reporter genes pCMV-luc and pCMV-LacZ, we showed that 1) the intratracheal route was more effective than the oral,intravenous, intramuscular, subcutaneous, or intraperitoneal routes in expressing luciferase activity in the gallbladder and 2) beta-galactosidase staining after pCMV-LacZ was confined to the columnar epithelium lining the gallbladder without any discernible activity in it smooth muscle. The discovery of an unusual route for gene transfer to the biliary system may give useful insight into counteracting the consequences of biliary fibrosis in human CF patients.

Hypotonicity-induced anion fluxes in cells expressing the multidrug-resistance-associated protein, MRP.

Anion transport in human multidrug-resis-tant large cell lung tumour cells (COR-L23/R) which overexpress the multidrug-resistance-associated protein (MRP) has been compared with that in cells of the parent line (COR-L23/P). Whole-cell patch-clamp recordings reveal variability between individual cells in basal anion conductance and in anion conductance increases following exposure to hypotonic media. The increase of stimulated over basal conductance is significantly larger for resistant cells than for parent cells. The chloride channel blocker, diisothiocyanatostilbene-2-2′-disulphonic acid (DIDS), rapidly and reversibly inhibits the increase in outward but not inward conductance when applied externally at 10−4 M during recording, but it is without effect when introduced into the cells via the patch pipette. Preincubation with DIDS greatly reduces both inward and outward conductance. 125I− efflux has been used to measure anion movement in cell populations. Basal efflux is similar in the two cell lines, but following a hypotonie challenge, the increase in rate constant for efflux from COR-L23/R cells is at least double that from COR-L23/P cells. This increase in efflux is greatly reduced by incubation with DIDS at 10−4 M. Replacement of external chloride by glu-conate does not affect efflux, thus excluding the possible involvement of DIDS-sensitive chloride exchange. Results from both techniques suggest that DIDS-sensitive, hypotonicity-induced anion channel activity is augmented in COR-L23/R multidrug-resistant variant cells which overexpress MRP. This augmentation may be caused by MRP itself or by other genes coexpressed with MRP.

Hypotonicity-induced changes in anion permeability of cultured rat brain endothelial cells

Iodide efflux, an index of anion permeability, has been monitored in cultured rat brain endothelial cells. Following hypotonicity-induced swelling, large, rapid increases in permeability occur, the extent of these increases depending on the degree of hypotonicity. Such large responses are not observed with rat aortic endothelial cells. Results of anion substitution experiments suggest that iodide efflux is via a chloride channel rather than an exchanger. The efflux increase is blocked by NPPB (100 microM) but not by DIDS or DPC at 100 microM. It is dependent on intracellular ATP but unaffected by removal of external calcium. Increasing internal calcium using A23187 does not produce a change in efflux, but depletion of calcium reduces or eliminates the response to hypotonicity. The response is reduced by pimozide (2-50 microM) that inhibits the actions of calmodulin and by pBPB (10 microM) that affects phospholipase A2 activity. It is eliminated by 5-lipoxygenase inhibitors (L-656,224 and ETH615, 10 microM) but is unaffected by cyclo-oxygenase inhibitors (indomethacin and piroxicam, 1-100 microM). It is blocked by some modulators of P-glycoprotein activity, e.g., verapamil (100 microM), tamoxifen (50 microM), and progesterone (100 microM) but not by others, e,g., forskolin (40 microM), dideoxyforskolin (40 microM), quinidine (100 microM) and cyclosporin A (10 microM).

Increases in aggregation by and uptake of 5-hydroxytryptamine with platelets from rabbits treated with chlorpromazine.

1 Citrated platelet‐rich plasma was prepared from New Zealand white rabbits before, during and after administration of chlorpromazine (2 mg/kg) intramuscularly once daily for 3 to 4 weeks. 2 In these plasmas, the velocity of platelet aggregation by 5‐hydroxytryptamine (5‐HT) added at 1, 3 and 10 μm increased greatly, beginning 3 to 4 days after the start of chlorpromazine injections and lasting for a similar period after they were terminated The increase had two maxima, the first after 6 to 10 days and the second after 17 to 24 days. Chlorpromazine treatment did not affect aggregation by adenosine diphosphate (ADP). 3 The uptake of 5‐HT by rabbit platelets was very fast and linear for less than 10 s. In platelets from untreated rabbits the uptake had a Km of 0.35 ± 0.08 μm and a Vmax of 39.8 ± 6.1 pmol 10−8 platelets 10−1 (n = 5). 4 In platelets from rabbits injected with chlorpromazine (see (2) above) both kinetic constants increased significantly, the Km to 0.88 ± 0.08 μm and the Vmax to 67.8 ± 5.5 pmol. 10−8 platelets. 10−1 s (n = 9).