Facultative Parasitism by the Bivalve Kurtiella pedroana in the Mole Crab Emerita analoga.

Evolutionary transitions to parasitism are rare. In this study, we documented a potential step toward parasitism in the commensal clam Kurtiella pedroana (Bivalvia: Galeommatoidea). Galeommatoideans are known commensals of various invertebrates, including crustaceans. Emerita analoga (Decapoda: Hippidae) is an abundant intertidal mole crab inhabiting Pacific coast beaches in North and South America. Mole crabs collected from Monterey Bay, California, were measured and examined externally and internally for associated molluscs. Out of the 520 mole crabs, 37 large female individuals harbored 49 clams (prevalence of 7.11% and mean intensity of 1.3). Forty-one ectocommensal clams were attached by their byssal threads to the inside of the gill chambers or to the lateroventral surfaces. However, our key finding was 8 clams that lacked byssal threads and were living in the hemocoel of 6 crabs. These internal clams were smaller than the ectocommensals. Because these internal clams lacked access to their normal food, we hypothesize they might have fed on hemolymph as would a parasite. Clam larvae have no obvious exit from the hemocoel, implying that endoparasitism is a dead-end for K. pedroana. Regardless, facultative parasitism in a free-living or an ectocommensal is uncommon and suggests a pathway to parasitism.

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).

Changing epidemiology of group B streptococcal colonization.

OBJECTIVES: To define factors influencing vertical transmission of and neonatal colonization with group B streptococci (GBS) in neonates representing ethnically and economically diverse populations, and to determine the serotype distribution of isolates, especially new types IV-VIII. STUDY DESIGN: Prospective, cross-sectional study of neonates born to women evaluated for GBS colonization at admission for delivery to one of four hospitals between January 1994 and February 1995. Cultures of throat, umbilicus, and rectum were obtained from 24- to 48-hour-old infants for isolation of GBS. Isolates were classified by capsular polysaccharide (I-VIII) and C protein (alpha and beta) antigen components. RESULTS: Colonization was detected in 28% of 546 mothers, was higher in blacks than whites (40.6% vs 20.3%) and Hispanics (26. 9%), and was not influenced by socioeconomic status. Overall, ethnic origin did not seem to be related to GBS serotype, but whites were more likely to carry the new type V strain than blacks (6 out of 24 [25%] vs 1 out of 43 [2%]). Vertical transmission of GBS to neonates was significantly diminished when their mothers had intrapartum antibiotics (0% vs 52%), rupture of membranes <12 hours before delivery (38.4% vs 73.3%), or delivery by cesarean section (25.9% vs 45.2%). Colonization with GBS was found in 13.8% of 549 neonates, was acquired vertically in 97%, and was less frequent in neonates at the private hospitals (4% vs 20%) where intrapartum antibiotics were given more frequently (34.7% vs 17.3%). Among isolates from neonates, serotype Ia predominated (31.6%) followed by types II (25%), III (22.4%), and V (11.8%); approximately 40% of strains contained C protein antigen. CONCLUSIONS: Changes in the epidemiology of GBS colonization included diminished rates in some populations associated with use of maternal intrapartum antibiotics, and a shift in serotype prevalence, with Ia as predominant and V, in addition to II and III, as common.

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

1. 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. 2. 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. 3. 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. 4. 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. 5. 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. 6. 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.

Calcium- and cyclic AMP-dependent chloride secretion in human colonic epithelia.

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.

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.

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.

Effects of D- and L- enantiomers of adenosine, AMP and ADP and their 2-chloro- and 2-azido- analogues on human platelets.

Aggregation of human platelets by ADP and the inhibition of this effect by adenosine are apparently mediated by different receptors. One of the criteria for receptors is that they show stereospecificity for their ligands. We have synthesized L-enantiomers of D-adenosine, AMP and ADP, together with their corresponding photolysable 2-azido analogues so that platelet receptors could be tested for stereospecificity. All of the L-enantiomers were completely inactive as aggregators or inhibitors of platelet function. None of the L-enantiomers changed levels of platelet cAMP. 2-Azido-L-adenosine, AMP and ADP are proposed as useful controls in photoaffinity experiments for non-specific labelling.

Antimicrobial susceptibilities of group B streptococci isolated between 1992 and 1996 from patients with bacteremia or meningitis.

In vitro testing of 229 group B streptococcal isolates from a variety of patients with invasive infections indicated uniform penicillin G susceptibility. However, 17 (7.4%) isolates were resistant to erythromycin and 8 (3.4%) were resistant to clindamycin. These results support the continued use of penicillin G as the drug of choice for the treatment and prevention of group B streptococcal disease.

Complement and antibody in neutrophil-mediated killing of type V group B streptococcus.

Serious infections caused by type V group B streptococci (GBS) are increasing. The requirements for antibody, complement, and neutrophil receptors in the killing of 12 clinical type V GBS isolates were investigated. When tested at concentrations of 33%, 5% and 1%, a human serum pool promoted neutrophil-mediated killing of the 12 isolates at a mean of 83% +/- 9%, 77% +/- 17%, and 14% +/- 28%, respectively. Addition of heated immune rabbit serum to the 5% or 1% pool increased killing significantly (97% +/- 2% or 80% +/- 15%, respectively, P < .01). With hypogammaglobulinemic serum, complement-mediated killing ranged from 74% +/- 2% for a strain designated resistant to 98% +/- 1% for a strain designated sensitive. Neutrophil-mediated killing was not altered by use of human sera deficient in C4 or C7 but was reduced significantly with C3-deficient serum (P < .05). Maximal inhibition of neutrophil-mediated killing was observed by monoclonal antibody blockade of complement receptor (CR) 3 alone or in combination with CR1 or Fc receptor III. Thus, C3 is required and specific antibody promotes neutrophil-mediated killing of type V GBS. Neutrophil CR3 and either CR1 or Fc receptor III optimize phagocytosis. A number of host responses function in concert to effect optimal neutrophil-mediated killing of clinical isolates of type V GBS.

A 5-year review of recurrent group B streptococcal disease: lessons from twin infants.

Recurrent invasive disease due to group B Streptococcus (GBS) in twin infants has not been reported. We report 2 cases of recurrent GBS afflicting both siblings of a set of dichorionic twin infants. The maternal and infant colonizing and invasive strains were identical by serotyping and pulsed-field gel electrophoresis (PFGE). Despite attempts at eradication with different antibiotic regimens, the infants remained colonized after treatment of the second episode. A 5-year review of recurrent invasive GBS disease in infants in our affiliated hospitals was undertaken, and 6 further cases were identified. Serotyping and PFGE of isolates from initial and second episodes were genotypically identical for each case. Three infants each had GBS serotype Ia or V disease and 2 had GBS serotype III disease. The exact pathogenesis of recurrent GBS disease remains unclear, but our data support the hypothesis that persistent mucosal colonization with the original GBS strain rather than new acquisition is a pivotal factor in disease recurrence.

Activation of Ca(2+)- 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 Ca(2+)-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 Ca(2+). It is concluded that EBIO stimulates Cl(-) secretion by activating Ca(2+)-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.

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

Electrogenic ion transport in the isolated colonic 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 forskolin 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.

Electrogenic bicarbonate secretion in mouse gallbladder.

Mouse gallbladders (4 mm2) were investigated using the short-circuit current (Isc) technique. Responses of 50 microA/cm2 were obtained in response to forskolin and agents that stimulated the adenylate cyclase system (IBMX and dibutyryl-cAMP). The calcium ionophore ionomycin increased Isc to 30% of the forskolin-stimulated increase. The forskolin-dependent current was inhibited 40% by acetazolamide but was insensitive to furosemide. Forskolin responses were dependent on the presence of bicarbonate ions; removal from both sides of the membrane or the basolateral side alone caused a significant reduction in responses. Removal of chloride ions from the basolateral side had no effect, while removal from the apical side caused a significant reduction in the forskolin responses, but only by 30%. It is argued that the remaining current (70%) cannot result from a parallel arrangement of a chloride channel and a chloride-bicarbonate exchanger and that bicarbonate is secreted through the apical membrane by a predominantly conductive mechanism. Apparently, forskolin converts a near electrically silent epithelium to an electrogenically secreting tissue.

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.

Use of capsular polysaccharide-tetanus toxoid conjugate vaccine for type II group B Streptococcus in healthy women.

An estimated 15% of invasive group B streptococcal (GBS) disease is caused by type II capsular polysaccharide (II CPS). In developing a pentavalent vaccine for the prevention of GBS infections, individual GBS CPSs have been coupled to tetanus toxoid (TT) to prepare vaccines with enhanced immunogenicity. Type II GBS (GBS II) vaccine was created by direct, covalent coupling of II CPS to TT by reductive amination. In 2 clinical trials, 75 healthy nonpregnant women 18-45 years old were randomized to receive II CPS-TT (II-TT) conjugate (dose range, 3.6-57 microg of CPS component) or uncoupled II CPS vaccine. Both vaccines were well tolerated. II CPS-specific IgG serum concentrations (as well as IgM and IgA) peaked 2 weeks after immunization, being significantly higher in recipients of conjugated vaccine than in recipients of uncoupled CPS. Immunological responses to conjugate were dose dependent and correlated with opsonophagocytosis in vitro. These results support inclusion of II-TT conjugate when preparing a multivalent GBS vaccine.

Selective in vivo transfection of murine biliary epithelia using polycation-enhanced adenovirus.

We have investigated the use of polycations to increase adenovirus-mediated expression of transgenic protein to the biliary epithelia with a view to gene therapy for hepatobiliary disease in CF. We have shown that adenovirus carrying the beta-galactosidase transgene transfect both human and mouse biliary epithelia in primary culture and that in both instances adenovirus transfection can be significantly increased by co-complexing with polycation. In vivo administration of 1 x 109 p.f.u. adenovirus co-complexed with the polyamine polyethyenimine (PEI) into the mouse biliary duct leads to >80% positively stained biliary epithelia while adenovirus alone at the same titre infected <5% biliary epithelia. We suggest that the use of low titre polycation enhanced adenoviral delivery to the biliary tree of CF patients could be of therapeutic significance. As a prelude to an extensive in vivo functional investigation in CF null mice we have shown that Ad5/polycation complexes deliver functional CFTR to non-CFTR expressing cells in vitro more efficiently than Ad5 alone.

Safety and immunogenicity of capsular polysaccharide-tetanus toxoid conjugate vaccines for group B streptococcal types Ia and Ib.

About 40% of invasive group B streptococcal (GBS) isolates are capsular polysaccharide (CPS) types Ia or Ib. Because infant and maternal GBS infections may be preventable by maternal vaccination, individual GBS CPS have been coupled to tetanus toxoid (TT) to prepare vaccines with enhanced immunogenicity. Immunogenicity in rabbits and protective capacity in mice of a series of type Ia- and Ib-TT conjugates increased with the degree of polysaccharide-to-protein cross-linking. In total, 190 healthy, nonpregnant women aged 18-40 years were randomized in four trials to receive Ia- or Ib-TT conjugate (dose range, 3.75-63 microg of CPS component), uncoupled Ia or Ib CPS, or saline. All vaccines were well-tolerated. CPS-specific IgG serum concentrations peaked 4-8 weeks after vaccination and were significantly higher in recipients of conjugated than of uncoupled CPS. Immune responses to the conjugates were dose-dependent and correlated in vitro with opsonophagocytosis. These results support inclusion of Ia- and Ib-TT conjugates when formulating a multivalent GBS vaccine.

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 (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-resistant 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 hypotonic 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 gluconate 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.