Biomedical subjects

G W Forsyth

Publications and source records attributed to G W Forsyth.

At least 19 recordsLinked to original sources

PubMed2005

Chloride concentration affects Kv channel voltage-gating kinetics: Importance of experimental anion concentrations.

Chloride concentration has been shown to have a dramatic impact on protein folding and subsequent tertiary conformation [K.D. Collins, Ions from the Hofmeister series and osmolytes: effects on proteins in solution and in the crystallization process, Methods 34 (2004) 300-311; I. Jelesarov, E. Durr, R.M. Thomas, H.R. Bosshard, Salt effects on hydrophobic interaction and charge screening in the folding of a negatively charged peptide to a coiled coil (leucine zipper), Biochemistry 37 (1998) 7539-7550]. As it is known that Kv channel gating is linked to the stability of the cytoplasmic T1 multimerization domain conformation [D.L. Minor, Y.F. Lin, B.C. Mobley, A. Avelar, Y.N. Jan, L.Y. Jan, J.M. Berger, The polar T1 interface is linked to conformational changes that open the voltage-gated potassium channel, Cell 102 (2000) 657-670; B.A. Yi, D.L. Minor Jr., Y.F. Lin, Y.N. Jan, L.Y. Jan, Controlling potassium channel activities: interplay between the membrane and intracellular factors, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 11016-11023] and that intracellular chloride concentration has been linked to Kv channel kinetics [L.K. Bekar, W. Walz, Intracellular chloride modulates A-type potassium currents in astrocytes, Glia 39 (2002) 207-216; W.B. Thoreson, S.L. Stella, Anion modulation of calcium current voltage dependence and amplitude in salamander rods, Biochim. Biophys. Acta 1464 (2000) 142-150], the objective of the present study was to address how chloride concentration changes affect Kv channel kinetics more closely in an isolated expression system. Initially, no significant chloride concentration-dependent effects on channel steady-state gating kinetics were observed. Only after disruption of the cytoskeleton with cytochalasin-D did we see significant chloride concentration-dependent shifts in gating kinetics. This suggests that the shift in gating kinetics is mediated through effects of intracellular chloride concentration on cytoplasmic domain tertiary conformation as cytoskeletal interaction appears to mask the effect. Furthermore, as cytoskeletal disruption only impacts channel gating kinetics at low physiological intracellular chloride concentrations, these studies highlight the importance of paying close attention to anion concentrations used under experimental conditions.

Cell Line↗

PubMed2000

Isoform-specific exon skipping in a variant form of ClC-2.

A new form of the widely expressed, volume-regulated chloride channel, ClC-2, has been cloned from a pig ileal cDNA library. This ClC-2 homologue, called ClC-2i, has interesting variability within its cDNA sequence, including the deletion of bases that correspond to positions 1326 through 1401 in rat ClC-2 cDNA sequence. This 75 bp deletion corresponds to the complete loss of exon 13 plus the first four bases of exon 14, and involves an atypical intron-exon splice site. Tissue-specific mRNA expression patterns in the pig show variable degrees of exon 13 skipping in ClC-2i. Exon 13 skipping was also observed in rat ClC-2i, albeit at a higher frequency than in the pig in tissues that were examined. A relatively purine-rich 76 bp insertion in the pig genomic sequence of ClC-2i, close to the 3' end of intron 12, may be responsible for the relatively high frequency of exon 13 skipping during the processing of this mRNA.

Animals↗

PubMed2000

Cloning a chloride conductance mediator from the apical membrane of porcine ileal enterocytes.

Attempts to attribute ileal brush-border chloride conductance to specific proteins were pursued by screening a porcine intestinal cDNA library. A 0.94-kb clone was identified on expression screening with a monoclonal antibody that inhibited enterocyte brush-border chloride conductance. Further screening approaches led to the isolation of a 3.1-kb full-length sequence called pCLCA1, consistent with the identification of a 2.9-kb transcript through Northern analysis. This sequence had significant homology to the CLCA gene family of calcium-regulated chloride channels, especially to hCLCA1. However, a strong A-kinase consensus phosphorylation site in a predicted cytoplasmic loop of the protein was a notable difference from the hCLCA1 gene product. Several porcine exocrine epithelial tissues, including ileum, trachea, and the major salivary glands express pCLCA1 mRNA. In situ hybridization studies localized the expression of pCLCA1 mRNA to the crypt and villus epithelia of porcine ileum, whereas tracheal expression was observed in both surface epithelium and submucosal glands. In situ expression of pCLCA1 in mouse 3T3 cells induces an ionomycin-dependent chloride conductance activity in these cells.

3T3 Cells↗

PubMed1996

Monoclonal antibody against conductive chloride transport in pig ileal apical membrane vesicles.

Conductive chloride transport in the small intestine is an important factor controlling fluid movement from the blood to the lumen of the gut. Several proteins with potential conductive chloride ion channel activity are expressed in the enterocyte cell population. However, it is not clear whether one or more than one protein species is normally responsible for mediating conductive chloride transport. We have raised monoclonal antibodies that inhibit conductive chloride transport in apical membrane vesicles prepared from porcine ileal enterocytes. These monoclonal antibodies have been used to identify a unique protein involved with this conductive chloride transport. Here, we report that anti-chloride conductance monoclonal antibodies did not detect any antigen in Western blots of enterocyte apical membrane protein. Dot blotting and immunoprecipitation experiments indicated that the antigen recognized by these monoclonal antibodies was not the cystic fibrosis transmembrane conductance regulator. The antigen was localized to both villus and crypt regions of ileum on immunohistochemistry. A 90-kDa protein species was immunoprecipitated from a primary enterocyte cell line by these monoclonal antibodies. This 90-kDa protein may be a chloride ion channel or may play some regulatory role in conductive chloride transport in enterocyte apical membrane vesicles.

Animals↗

PubMed1993

Variable expression of leukocyte cytosolic broad-specificity beta-glucosidase activity.

The cytosolic beta-glucosidase activity that is found in a variety of mammalian tissues has no clearly defined function. In vitro assay conditions under which the broad-specificity beta-glucosidase hydrolyzes glucocerebroside at a significant rate have not been described. Nonetheless, it has been suggested that this enzyme might play an accessory role with lysosomal glucocerebrosidase in catalyzing the hydrolysis of glucosylceramide. However, this hypothesis would require that activity of both enzymes be low in severe cases of Gaucher disease in which there are pathological accumulations of glucosylceramide in one or more of the affected organs, i.e. spleen, liver and bone marrow. Information is lacking regarding the normal range of cytosolic beta-glucosidase activity in humans. p-Nitrophenyl-beta-D-mannoside was found to be a potent inhibitor (Ki = 0.068 mM) of cytosolic beta-glucosidase. In parallel studies, the activity of glucocerebrosidase was found to be minimally affected by p-nitrophenyl-beta-D-mannoside at concentrations as high as 2.5 mM. This information was used to design an assay system that would allow us to estimate glucocerebrosidase and cytosolic beta-glucosidase activities in extracts of human leukocytes. Average cytosolic beta-glucosidase activity with 4-heptyl-umbelliferyl-beta-D-glucoside as a substrate was 8.8 nmol/h/mg protein in leukocytes from 356 subjects (range, 0.2-28). Average leukocyte glucocerebrosidase specific activity was 16 nmol/h/mg protein (range 5.3-45.7). No correlation was observed between cytosolic beta-glucosidase and glucocerebrosidase activity for control and Gaucher heterozygote populations (r = 0.19 and 0.25, respectively). The wide range of leukocyte cytosolic beta-glucosidase activity in individuals tested in this study indicates that a substantial proportion of the population may lack sufficient cytosolic beta-glucosidase activity to assist a defective lysosomal glucocerebrosidase in hydrolyzing glucosylceramide.

Chromatography, High Pressure Liquid↗

PubMed1992

Effect of covalent modification on the binding of cholera toxin B subunit to ileal brush border surfaces.

A competitive binding assay has been developed to determine how modifications to the B subunit of cholera toxin affect the binding affinity of the subunit for an ileal brush border membrane surface. The Ricinus communis120 agglutinin (RCA120) specifically binds to terminal beta-D-galactosyl residues such as those found in oligosaccharide side chains of glycoproteins and ganglioside GM1. Conditions were designed to produce binding competition between the B subunit of cholera toxin and the RCA120 agglutinin. Displacement of RCA120 from brush border surfaces was proportional to the concentration of B subunit added. This assay was used to study the effect of modification of B subunit on competitive binding affinity for the ileal brush border surface. The B subunit of cholera toxin was modified by coupling an average of five sulfhydryl groups to each B subunit molecule and by reaction of the SH-modified B subunit with liposomes containing a surface maleimide group attached to phosphatidylethanolamine. SH-modified B subunit was approximately 200-fold more effective than native B subunit in displacing lectin from brush border surfaces in the competitive binding assay. The enhanced binding activity was retained on covalent attachment of the modified B subunit to the liposome surface. We conclude that the B subunit of cholera toxin may be a useful targeting agent for directing liposomes to cell surfaces that contain a ganglioside GM1 ligand.

Animals↗

PubMed1992

Inhibition of ileal brush-border chloride conductance by specific antibody.

Antibody raised in mice was used in attempting to identify proteins responsible for the conductive chloride transport that can be measured in porcine ileal brush border membrane vesicles. Ileal brush-border membrane vesicle protein from pig was separated into five different molecular mass fractions by preparative SDS polyacrylamide disc gel electrophoresis. Separated protein fractions were used to immunize mice. Antibody was screened for reactivity with antigen by Western blotting, and for effects on conductive chloride transport in ileal brush border membrane vesicles. Immunization with brush-border protein from fraction I proteins (> 110 kDa) produced polyclonal antisera which specifically inhibited the conductive component of chloride uptake by ileal brush border vesicle preparations. Western blotting of the antigen showed the presence of several protein species of molecular mass > 100 kDa that were recognized by immune serum. Spleen cells from a mouse producing antiserum that inhibited conductive chloride transport were fused with a myeloma cell line. The resulting hybridoma colonies produced antibody that reacted with at least seven distinct protein bands by Western blot assay and inhibited chloride conductance in brush-border membrane vesicles.

Animals↗

PubMed1992

Liposomes targeted to deliver antisecretory agents to jejunal mucosa.

The B subunit of cholera toxin has been covalently attached to the surface of liposomes made from a mixture of phosphatidylethanolamine, phosphatidylcholine and cholesterol. Adenylate cyclase inhibitors and chloride conductance inhibitors were encapsulated within the liposomes. These "targeted" liposomes were used to study the combined effects of this novel delivery system, and a limited number of possible antisecretory agents, on net fluid flux into the pig jejunum. A state of net secretory fluid flux was induced in isolated jejunal loops in weanling pigs by adding theophylline or cholera toxin to the lumen of the isolated loops. There was no reduction in net fluid secretion when liposome suspensions without encapsulated secretory inhibitors were added to fluid in the lumen of loops treated with theophylline. There was also no reduction in net fluid secretion when miconazole, alpha-phenylcinnamate or 5 nitro-2-(3-phenethylamino)benzoate were encapsulated within targeted liposomes added to isolated jejunal loops. The net fluid flux induced by exposure of jejunal loops to theophylline was significantly reduced by adding targeted liposomes containing 2'-deoxy-3'-AMP. The reduction involved a reversal of net secretory fluid flux to an absorptive value. The net fluid secretory response to treatment of loops with cholera toxin was also inhibited by treating loops with targeted liposomes containing 2'-deoxy-3'-AMP. However, the reversal of secretion was less complete for secretion induced by cholera toxin than for secretion induced by theophylline. The reduced antisecretory efficacy versus cholera toxin was not improved by encapsulating higher concentrations of 2'-deoxy-3'-AMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Candidate proteins for conductive chloride transport in porcine ileal brush-border membrane.

Conductive transport of chloride ion is important in controlling ion and fluid secretion by exocrine tissues. The current study was directed at identifying proteins in the intestinal brush-border membrane that may be involved with conductive chloride transport. Reaction of total brush-border membrane protein with phenyl-isothiocyanate inhibited conductive chloride transport into brush-border membrane vesicles. The conductive transport process was protected from this inhibition by including the ligands Cl- and alpha-phenylcinnamate in the reaction mixture. Brush-border membrane protein protected by this procedure and labeled with fluorescein had an apparent molecular mass in the region of 130 and 23 kDa on separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphorylation of brush-border membrane protein with [gamma-32P] ATP and endogenous protein kinase under conditions causing activation of chloride conductance in membrane vesicles caused the transfer of 32P to several proteins, including ones in the same molecular size range (130 and 23 kDa) as those identified by the fluorescein labeling procedure. Conductive chloride transport in porcine intestinal brush-border vesicles may occur via proteins identified by this differential labeling procedure.

Animals↗

PubMed1990

Effects of chloride conductance inhibitors on fluid secretion into ligated ileal and jejunal loops in pigs.

Compounds that prevent chloride transport in membrane vesicles have been tested for in vivo activity against the effects of intestinal secretory agents. Chloride channel blockers including diphenylamine-2-carboxylate, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate, 5-nitro-2-(2-phenylethylamino)benzoic acid, and alpha-phenylcinnamic acid were tested for effects on jejunal or ileal secretion in weanling pigs. Secretion was studied in ligated intestinal loops in a control state, during exposure to secretory concentrations of theophylline, and after prior treatment with cholera toxin. Increases in net fluid flux induced by either theophylline or cholera toxin were not prevented by adding chloride channel blockers into the intestinal lumen. Channel blocker concentrations that reduced chloride transport by greater than 50% in pig jejunal brush border vesicles did not cause significant changes in unidirectional blood to lumen chloride flux measured in situ. Several routes of administration of the specific chloride channel blocker alpha-phenylcinnamate failed to reduce fluid secretion induced by theophylline. Chloride channel blocker effectiveness appears to be significantly different between in vitro and in vivo experimental models. In contrast to the chloride channel blockers, loperamide significantly reduced net fluid and chloride flux in ileal loops secreting fluid in response to theophylline. Antagonism of the production or actions of second messenger by loperamide was more effective than the chloride channel blockers in reducing conductive chloride transport associated with intestinal secretion.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed1989

Inhibiting conductive Cl uptake in membrane vesicles: specificity of alpha-phenylcinnamate.

alpha-Phenylcinnamate has been investigated in comparison to other inhibitors of chloride ion transport into porcine jejunal brush-border membrane vesicles. The transport modes studied included uptake driven only by a chemical Cl gradient, Cl uptake dependent on a transmembrane potential, self-exchange of Cl with no chemical or potential gradient, and Cl uptake dependent on a chemical gradient for bicarbonate. Uptake driven by the chemical gradient for Cl was strongly inhibited by millimolar concentrations of diphenylamine-2-carboxylate, 5-nitro-2-(2-phenylethylamino)benzoate (NPEB), and to a lesser extent by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS). Similar concentrations of alpha-phenylcinnamate did not reduce this mode of Cl uptake. Conductive Cl uptake driven by a potassium gradient was inhibited by approx. 50% at 2.5 mM alpha-phenylcinnamate. alpha-Phenylcinnamate was equally effective in reducing the initial rate of conductive chloride accumulation in vesicles with naturally opened Cl channels (conductance activation by cyclic AMP and Ca2+), or with Cl channels opened by exposure to tetramethylammonium (TMA) buffer. In comparison with diphenylamine-2-carboxylate, NPEB and SITS, alpha-phenylcinnamate had the least effect on Cl-HCO3 exchange at inhibitor concentrations which reduced conductance activity. Self-exchange rates of physiological concentrations of Cl were also relatively unaffected by low mM concentrations of alpha-phenylcinnamate. Kinetic analysis indicated that alpha-phenylcinnamate was an uncompetitive inhibitor, requiring the presence of the normal Cl ligand for binding to, and inhibition of, conductive Cl transport by pig intestinal brush-border vesicles.

Animals↗

PubMed1989

Activation of chloride conductance in pig jejunal brush border vesicles.

Requirements for the activation of Cl- conductance have been investigated in pig jejunal brush border vesicles. The stability of ATP as a substrate for protein kinase activity, the stability of the phosphoprotein product of protein kinase action, and the choice of buffer system used for vesicle preparation were studied as variables which affected the outcome of in vitro activation attempts. Arsenate was selected as the most effective agent in protecting ATP from hydrolysis by the phosphatase activity in this vesicle system. Brush border vesicle protein appeared to prevent the accumulation of phosphoprotein in a cAMP-dependent protein kinase reaction, and vesicle protein only had phosphate acceptor activity when KF was added as a presumptive inhibitor of phosphoprotein phosphatase. A Cl- conductance response to a potassium gradient and valinomycin was present in vesicles prepared in buffers containing tetramethylammonium. Cl- conductance activity was not increased in this system by the addition of ATP, dibutyryl cyclic AMP, and cyclic AMP-dependent protein kinase. There was no Cl conductance response to a potassium gradient in vesicles buffered with imidazolium-acetate. Incorporation of ATP, AsO4(3-), and F- into these nonconductive vesicles by homogenization, followed by addition of dibutyryl cAMP, produced substantial conductance activity. Maximal activation of Cl- conductance was obtained with vesicles prepared in imidazolium-acetate buffering, using precautions to stabilize ATP and phosphoprotein prior to conductance measurements.

Adenosine Triphosphate↗

PubMed1988

Chloride ion transport into pig jejunal brush-border membrane vesicles.

1. This study was carried out to determine the types and activities of carrier proteins which transport the chloride ion in pig jejunal brush-border membranes, with an emphasis on studying the properties of chloride conductance activity in vesicles prepared from these membranes. 2. Sodium-chloride co-transport activity was not detected in this tissue. A sodium-proton antiport with typical amiloride sensitivity was present. An anion exchanger linking chloride to hydroxyl or bicarbonate ions was also found in the pig jejunal brush-border membrane vesicles. 3. Chloride conductance activity in this system was specifically dependent on the buffering agents used for vesicle preparation. Conductance activity could not be demonstrated in vesicles prepared in imidazolium acetate or in HEPES-Tris buffers. HEPES-tetramethylammonium buffering of vesicles in the chloride uptake system produced a significant conductance response to a potassium gradient plus valinomycin. 4. Chloride conductance showed saturable kinetics with respect to substrate concentration, with a Michaelis-Menten constant (Km) of approximately 116 mM and a maximum velocity (Vmax) of 132 nmol (mg protein)-1 min-1. 5. Preliminary screening of potential inhibitors of chloride conductance showed only minimal inhibitor effects of SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-sulphonic acid), anthracene-9-carboxylate, N-phenylanthranilate and piretanide. 6. The conductance activity in pig jejunal vesicles appears to have stringent buffer requirements, and to be relatively insensitive to the effects of reported conductance inhibitors.

Animals↗

PubMed1987

Calcium transport affinity, ion competition and cholera toxin effects on cytosolic Ca concentration.

The physiological relevance of an apparent ionophore activity of cholera toxin towards Ca2+ has been examined in several different systems designed to measure affinity, specificity, rates of ion transfer, and effects on intracellular ion concentrations. Half-maximal transfer rates across porcine jejunal brush-border vesicles were obtained at a concentration of 0.20 microM Ca2+. When examined in the presence of competing ions the transfer process was blocked by very low concentrations of La3+ or Cd2+, Sr2+, Ba2+ and Mg2+ were relatively inefficient competitors for Ca2+ transport mediated by cholera toxin. The relative affinities observed would be compatible with a selectivity for Ca2+ transfer at physiological ion concentrations, as well as an inhibition of this ionophore activity by recognized antagonists of cholera toxin such as lanthanum ions. Entry rates of Ca2+ into brush-border vesicles exposed to cholera toxin were large enough to accelerate the collapse of a Ca2+ gradient generated by endogenous Ca, Mg-ATPase activity. The treatment of isolated jejunal enterocytes with cholera toxin caused a significant elevation in cytosolic Ca2+ concentrations as measured by Quin-2 fluorescence. This effect was specifically prevented by prior exposure of the cholera toxin to excess ganglioside GM1. We conclude that cholera toxin has many of the properties required for promoting transmembranes Ca2+ movement in membrane vesicles and appears to be an effective Ca2+ ionophore in isolated mammalian cells.

Animals↗

PubMed1987

Use of calcium depletion and chlorpromazine to study calcium dependence of secretory detergent action in the colon.

The role of Ca2+ in the in situ secretory response of rat colon and pig ileum was studied by chelation depletion of Ca2+ and by treatment with chlorpromazine. The effect of depleting lumenal Ca2+ by chelation and the effect of intraperitoneal administration of chlorpromazine were determined relative to colonic permeability and net fluid flux measured across the rat colon or pig ileum. Replacement of Ca2+ in the perfusate by 1.0 mM ethyleneglycol-(bis-beta-ethylaminoether) (EGTA) did not produce significant changes in the net absorptive fluid flux measured in the control state or in the net secretory fluid flux caused by secretory detergent agents. The concentration of EGTA used in the perfusate did not alter mucosal permeability. Nonsecretory bile acids or A23187 had no effect on net colonic fluid flux or on colonic permeability to mannitol in the rat. The known correlation between net fluid flux and increased colonic permeability to polar molecules has been confirmed for the secretory detergent compounds. Chlorpromazine pretreatment caused a partial reversal of net secretory fluid fluxes induced by deoxycholate and high concentrations (6.0 mM) of ricinolate and dioctyl sulfosuccinate without significantly altering mucosal permeability to mannitol. We conclude that depletion of lumenal Ca2+ is not an effective method for determining the possible Ca2+ dependence of these intestinal secretory events. The antisecretory actions of chlorpromazine may provide some indirect evidence for Ca2+ involvement in the secretory effects of the detergent class of laxative compounds. Permeability may be essential for secretion caused by these agents, but the driving force would appear to be provided by the active transfer of electrolytes from the blood to the lumen of the colon.

Animals↗

PubMed1986

Cholera toxin facilitates calcium transport in jejunal brush border vesicles.

Cholera toxin is very well characterized in terms of the activation of adenylate cyclase. In some systems, however, this cyclase activation does not seem to account for all of the physiological responses to the toxin. On the premise that cholera toxin may also exert effects through other second messenger compounds we have studied the effect of cholera toxin on the rate of Ca2+ movement across the membrane of intestinal brush border vesicles. Increasing concentrations of cholera toxin progressively accelerated the passive uptake of Ca2+ into, and the efflux of Ca2+ from, an osmotically active space in brush border membrane vesicles. This effect of cholera toxin was saturable by excess Ca2+ and was relatively specific, as the toxin did not affect vesicle permeability to an uncharged polar solute. The toxin had two high affinity Ca2+ binding sites on the A subunit as measured by equilibrium dialysis. Ca2+ transport facilitated by cholera toxin was temperature dependent, required the holotoxin, and could be inhibited by preincubation of the toxin with excess free ganglioside GM1. This increased rate of Ca2+ influx caused by the in vitro addition of cholera toxin to brush border membrane vesicles may have physiological significance as it was comparable to rates observed with the Ca ionophore A23187. Similar effects occurring in vivo could permit cholera toxin to increase cytoplasmic Ca2+ concentrations and to produce accompanying second messenger effects.

Animals↗

PubMed1986

The alkalinizing effects of metabolizable bases in the healthy calf.

The alkalinizing effect of citrate, acetate, propionate, gluconate, L and DL-lactate were compared in healthy neonatal calves. The calves were infused for a 3.5 hour period with 150 mmol/L solutions of the sodium salts of the various bases. Blood pH, base excess, and metabolite concentrations were measured and the responses compared with sodium bicarbonate and sodium chloride infusion. D-gluconate and D-lactate had poor alkalinizing abilities and accumulated in blood during infusion suggesting that they are poorly metabolized by the calf. Acetate, L-lactate and propionate had alkalinizing effects similar to bicarbonate, although those of acetate had a slightly better alkalinizing effect than L-lactate. Acetate was more effectively metabolized because blood acetate concentrations were lower than L-lactate concentrations. There was a tendency for a small improvement in metabolism of acetate and lactate with age. Sodium citrate infusion produced signs of hypocalcemia, presumably because it removed ionized calcium from the circulation. D-gluconate, D-lactate and citrate are unsuitable for use as alkalinizing agents in intravenous fluids. Propionate, acetate and L-lactate are all good alkalinizing agents in healthy calves but will not be as effective in situations where tissue metabolism is impaired.

Acetates↗

PubMed1985

Calcium mediation of the pig jejunal secretory response.

The involvement of Ca++ ions as secretory mediators in pig jejunal epithelia has been investigated with an in vitro system. Omission of Ca++ from the Ringer-HCO3 bathing media on both sides of the tissue had minor effects on the basal electrical activity of pig jejunal mucosa. There were only slight decreases in transepithelial potential difference and increases in conductance with Ca++ free media. Low EGTA concentrations which reversibly blocked potential difference responses to secretory agents also had minimal effects on basal electrical activity. The in vitro secretory responses to A23187, to theophylline, and to Escherichia coli heat-stable enterotoxin were all eliminated by Ca++ depletion and restored by replacing normal Ca++ concentrations in the bathing media. Dantrolene prevented the secretory response but not the potential difference increases caused by heat-stable enterotoxin and A23187, suggesting that intracellular Ca++ stores may be reservoirs of secretory signal agent. Verapamil only blocked the secretory response to heat-stable enterotoxin. Chlorpromazine had negligible effects on basal conditions, but totally blocked both the secretory response and the Ca++-dependent effects of A23187 and heat-stable enterotoxin on potential difference. The response to theophylline was only partially inhibited by chlorpromazine, implying some involvement of both cAMP and Ca++ as secretory signals for theophylline. Cytoplasmic Ca++ concentrations appear to be at least as important as cyclic nucleotides in regulating the secretory effects of pig jejunum.

Animals↗