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Background: Congenital stationary night-blindness (CSNB) is a recessive autosomal defect in low-light vision in Appaloosa and other horse breeds. This condition has been mapped by linkage analysis to a gene coding for the Transient Receptor Potential cation channel Member 1 (TRPM1). TRPM1 is normally expressed in the ON-bipolar cells of the inner nuclear layer of the retina. Down-regulation of TRPM1 expression in CSNB results from a transposon-like insertion in intron 1 of the TRPM1 gene. Stop transcription signals in this transposon significantly reduce TRPM1 primary transcript levels in CSNB horses. This study describes additional contributions by a second mutation of the TRPM1 gene, the ECA1 108,249,293 C > T SNP, to down-regulation of transcription of the TRPM1 gene in night-blind horses. This TRPM1 SNP introduces a consensus binding site for neuro-oncological ventral antigen 1 (Nova-1) protein in the primary transcript. Nova-1 binding disrupts normal splicing signals, producing unstable, non-functional mRNA transcripts., Results: Retinal bipolar cells express both TRPM1 and Nova-1 proteins. In vitro addition of Nova-1 protein retards electrophoretic migration of TRPM1 RNA containing the ECA1 108,249,293 C > T SNP. Up-regulating Nova-1 expression in primary cultures of choroidal melanocytes carrying the intron 11 SNP caused an average log 2-fold reduction of ~6 (64-fold) of TRPM1 mRNA expression., Conclusions: These finding suggest that the equine TRPM1 SNP can act independently to reduce survival of TRPM1 mRNA escaping the intron 1 transcriptional stop signals in CSNB horses. Coexistence and co-inheritance of two independent TRPM1 mutations across 1000 equine generations suggests a selective advantage for the apparently deleterious CSNB trait.

The objective of this study was to develop a murine retinal/choroidal/scleral explant culture system to facilitate the intravitreous delivery of vectors. Posterior segment explants from adult mice of 2 different age groups (4 wk and 15 wk) were cultured in serum-free medium for variable time periods. Tissue viability was assessed by gross morphology, cell survival quantification, activated caspase-3 expression, and immunohistochemistry. To model ocular gene therapy, explants were exposed to varying transducing units of a lentiviral vector expressing the gene for green fluorescent protein for 48 h. Explant retinal cells remained viable for approximately 1 wk, although the ganglion cell layer developed apoptosis between 4 and 7 d. Following vector infusion into the posterior segment cups, viral transduction was noted in multiple retinal layers in both age groups. An age of donor mouse influence was noted and older mice did not transduce as well as younger mice. This explant offers an easily managed posterior segment ocular culture with minimum disturbance of the tissue, and may be useful for investigating methods of enhancing retinal gene therapy under controlled conditions.

Mitochondrial transcription factor A (Tfam) has been implicated in the pathogenesis of retinal dysplasia in miniature schnauzer dogs and it has been proposed that affected dogs have altered mitochondrial numbers, size, and morphology. To test these hypotheses the Tfam gene of affected and normal miniature schnauzer dogs with retinal dysplasia was sequenced and lymphocyte mitochondria were quantified, measured, and the morphology was compared in normal and affected dogs using transmission electron microscopy. For Tfam sequencing, retina, retinal pigment epithelium (RPE), and whole blood samples were collected. Total RNA was isolated from the retina and RPE and reverse transcribed to make cDNA. Genomic DNA was extracted from white blood cell pellets obtained from the whole blood samples. The Tfam coding sequence, 5' promoter region, intron1 and the 3' non-coding sequence of normal and affected dogs were amplified using polymerase chain reaction (PCR), cloned and sequenced. For electron microscopy, lymphocytes from affected and normal dogs were photographed and the mitochondria within each cross-section were identified, quantified, and the mitochondrial area (μm²) per lymphocyte cross-section was calculated. Lastly, using a masked technique, mitochondrial morphology was compared between the 2 groups. Sequencing of the miniature schnauzer Tfam gene revealed no functional sequence variation between affected and normal dogs. Lymphocyte and mitochondrial area, mitochondrial quantification, and morphology assessment also revealed no significant difference between the 2 groups. Further investigation into other candidate genes or factors causing retinal dysplasia in the miniature schnauzer is warranted.

Objective: To compare relative amounts of WBC mitochondrial DNA (mtDNA; assessed via real-time PCR assay) and morphology of lymphocyte mitochondria (assessed via transmission electron microscopy [TEM]) in blood samples collected from English Springer Spaniels with and without retinal dysplasia., Animals: 7 and 5 client-owned English Springer Spaniels (1 to 11 years old) with and without retinal dysplasia, respectively., Procedures: Blood samples were obtained from affected and unaffected dogs via venipuncture. Genomic DNA was extracted from WBCs of the 7 affected and 5 unaffected dogs, and relative quantification of the cytochrome c oxidase subunit 1 gene (COX1) was determined via analysis of real-time PCR results. White blood cells from 3 affected and 4 unaffected dogs were embedded in epoxide resin for TEM; cross sections were examined for lymphocytes, which were measured. The mitochondria within lymphocytes were quantified, and the mitochondrial surface area per lymphocyte cross section was calculated. A masked technique was used to compare mitochondrial morphology between the 2 groups., Results: Compared with the smallest measured quantity of mtDNA among unaffected dogs, mtDNA amounts varied among unaffected (1.08- to 4.76-fold differences) and affected dogs (1- to 2.68-fold differences). Analysis of lymphocyte measurements and mitochondrial surface area, morphology, and quantity revealed no significant differences between affected and unaffected dogs., Conclusions and Clinical Relevance: No significant differences were detected in relative amounts of WBC mtDNA or the size, number, or morphology of lymphocyte mitochondria in English Springer Spaniels affected with retinal dysplasia, compared with results for unaffected control dogs.

Purpose: To investigate the molecular basis of inherited retinal dysplasia in miniature Schnauzers., Methods: Retina and retinal pigment epithelial tissues were collected from canine subjects at the age of 3 weeks. Total RNA isolated from these tissues was reverse transcribed to make representative cDNA pools that were compared for differences in gene expression by using a subtractive hybridization technique referred to as representational difference analysis (RDA). Expression differences identified by RDA were confirmed and quantified by real-time reverse-transcription PCR. Mitochondrial morphology from leukocytes and skeletal muscle of normal and affected miniature Schnauzers was examined by transmission electron microscopy., Results: RDA screening of retinal pigment epithelial cDNA identified differences in mRNA transcript coding for two mitochondrial (mt) proteins--cytochrome oxidase subunit 1 and NADH dehydrogenase subunit 6--in affected dogs. Contrary to expectations, these identified sequences did not contain mutations. Based on the implication of mt-DNA-encoded proteins by the RDA experiments we used real-time PCR to compare the relative amounts of mt-DNA template in white blood cells from normal and affected dogs. White blood cells of affected dogs contained less than 30% of the normal amount of two specific mtDNA sequences, compared with the content of the nuclear-encoded glyceraldehyde-3-phosphate dehydrogenase (GA-3-PDH) reference gene. Retina and RPE tissue from affected dogs had reduced mRNA transcript levels for the two mitochondrial genes detected in the RDA experiment. Transcript levels for another mtDNA-encoded gene as well as the nuclear-encoded mitochondrial Tfam transcription factor were reduced in these tissues in affected dogs. Mitochondria from affected dogs were reduced in number and size and were unusually electron dense., Conclusions: Reduced levels of nuclear and mitochondrial transcripts in the retina and RPE of miniature Schnauzers affected with retinal dysplasia suggest that the pathogenesis of the disorder may arise from a lowered energy supply to the retina and RPE.

CLCA proteins were discovered in bovine trachea and named for a calcium-dependent chloride conductance found in trachea and in other secretory epithelial tissues. At least four closely located gene loci in the mouse and the human code for independent isoforms of CLCA proteins. Full-length CLCA proteins have an unprocessed mass ratio of approximately 100 kDa. Three of the four human loci code for the synthesis of membrane-associated proteins. CLCA proteins affect chloride conductance, epithelial secretion, cell-cell adhesion, apoptosis, cell cycle control, mucus production in asthma, and blood pressure. There is a structural and probable functional divergence between CLCA isoforms containing or not containing beta4-integrin binding domains. Cell cycle control and tumor metastasis are affected by isoforms with the binding domains. These isoforms are expressed prominently in smooth muscle, in some endothelial cells, in the central nervous system, and also in secretory epithelial cells. The isoform with disrupted beta4-integrin binding (hCLCA1, pCLCA1, mCLCA3) alters epithelial mucus secretion and ion transport processes. It is preferentially expressed in secretory epithelial tissues including trachea and small intestine. Chloride conductance is affected by the expression of several CLCA proteins. However, the dependence of the resulting electrical signature on the expression system rather than the CLCA protein suggests that these proteins are not independent Ca2+-dependent chloride channels, but may contribute to the activity of chloride channels formed by, or in conjunction with, other proteins.

Voltage-gated potassium channels are well established as critical for setting action potential frequency, membrane potential, and neurotransmitter release in neurons. However, their role in the "nonexcitable" glial cell type is yet to be fully understood. We used whole cell current kinetics, pharmacology, immunocytochemistry, and RT-PCR to characterize A-type current in hippocampal astrocyte cultures to better understand its function. Pharmacological analysis suggests that approximately 70, 10, and <5% of total A current is associated with Kv4, Kv3, and Kv1 channels, respectively. In addition, pharmacology and kinetics provide evidence for a significant contribution of KChIP accessory proteins to astrocytic A-channel composition. Localization of the Shaw Kv3.4 channel to astrocytic processes and the Shal Kv4.3 channel to soma suggest that these channels serve a specific function. Given this complex A-type channel expression pattern, we assessed the role of A currents in membrane voltage oscillations in response to current injections. Although TEA-sensitive delayed-rectifying currents are involved in the extent of repolarization, 4-AP-sensitive A currents serve to increase the rate. As in neurons, this effect may enable astrocytes to respond rapidly to high-frequency synaptic events. Our results indicate that hippocampal astrocytes in vitro express multiple A-type Kv channel alpha-subunits with accessory, possibly Ca(2+)-sensitive, cytoplasmic subunits that appear to be specifically localized to subcellular membrane compartments. Function of these channels remains to be determined in a physiological setting. However, this study suggests that they enable astrocytes to respond rapidly with membrane voltage oscillations to high-frequency incoming signals, possibly synchronizing astrocyte function to neuronal activity.

A reference database of differences in mRNA expression in normal healthy canine retinal pigment epithelium (RPE) has been established. This database identifies non-informative differences in mRNA expression that can be used in screening canine RPE for mutations associated with clinical effects on vision. Complementary DNA (cDNA) pools were prepared from mRNA harvested from RPE, amplified by PCR, and used in a subtractive hybridization protocol (representational differential analysis) to identify differences in RPE mRNA expression between canines. The effect of relatedness of the test canines on the frequency of occurrence of differences was evaluated by using 2 unrelated canines for comparison with 2 female sibling canines of blue heeler/bull terrier lineage. Differentially expressed cDNA species were cloned, sequenced, and identified by comparison to public database entries. The most frequently observed differentially expressed sequence from the unrelated canine comparison was cDNA with 21 base pairs (bp) identical to the human epithelial membrane protein 1 gene (present in 8 of 20 clones). Different clones from the same-sex sibling RPE contained repetitions of several short sequence motifs including the human epithelial membrane protein 1 (4 of 25 clones). Other prevalent differences between sibling RPE included sequences similar to a chicken genetic marker sequence motif (5 of 25), and 6 clones with homology to porcine major histocompatibility loci. In addition to identifying several repetitively occurring, noninformative, differentially expressed RPE mRNA species, the findings confirm that fewer differences occurred between siblings, highlighting the importance of using closely related subjects in representational difference analysis studies.

The effects of CLCA protein expression on the regulation of Cl(-) conductance by intracellular Ca(2+) and cAMP have been studied previously in nonepithelial cell lines chosen for low backgrounds of endogenous Cl(-) conductance. However, CLCA proteins have been cloned from, and normally function in, differentiated epithelial cells. In this study, we examine the effects of differentiation of the Caco-2 epithelial colon carcinoma cell line on modulation of Cl(-) conductance by pCLCA1 protein expression. Cl(-) transport was measured as (36)Cl(-) efflux, as transepithelial short-circuit currents, and as whole cell patch-clamp current-voltage relations. The rate of (36)Cl(-) efflux and amplitude of currents in patch-clamp studies after the addition of the Ca(2+) ionophore A-23187 were increased significantly by pCLCA1 expression in freshly passaged Caco-2 cells. However, neither endogenous nor pCLCA1-dependent Ca(2+)-sensitive Cl(-) conductance could be detected in 14-day-postpassage cells. In contrast to Ca(2+)-sensitive Cl(-) conductance, endogenous cAMP-dependent Cl(-) conductance does not disappear on Caco-2 differentiation. cAMP-dependent Cl(-) conductance was modulated by pCLCA1 expression in Caco-2 cells, and this modulation was observed in freshly passaged and in mature 14-day-postpassage Caco-2 cultures. pCLCA1 mRNA expression, antigenic pCLCA1 protein epitope expression, and pCLCA1 function as a modulator of cAMP-dependent Cl(-) conductance were retained through differentiation in Caco-2 cells, whereas Ca(2+)-dependent Cl(-) conductance disappeared. We conclude that pCLCA1 expression may increase the sensitivity of preexisting endogenous Cl(-) channels to Ca(2+) and cAMP agonists but apparently lacks inherent Cl(-) channel activity under growth conditions where endogenous channels are not expressed.

Problems in ion and fluid transfer across the retinal pigment epithelium (RPE) are a probable cause of inappropriate accumulations of fluid between the photoreceptors of the retina and the RPE. The activities of Cl- transporters involved in basal fluid transfer across the RPE have been compared to determine whether Ca2+- or cAMP-dependent channels may be responsible for basal housekeeping levels of secretory activity in this tissue. The role of a candidate Ca2+-dependent CLCA protein in the basal RPE transport of Cl- has been investigated. Low concentrations of the Cl- conductance inhibitors glibenclamide and 5-nitro-2-(3-phenylpropylamino)benzoate reduced the short-circuit current in dog RPE preparations mounted in Ussing chambers and decreased the Ca2+-dependent Cl- efflux from fibroblasts expressing the pCLCA1 Cl- conductance regulator. However, these same agents did not inhibit the rate of Cl- release from cultured fibroblasts expressing the cystic fibrosis transmembrane regulator (CFTR) conductive Cl- channel. Addition of ionomycin to primary cultures of canine RPE cells or to fibroblasts expressing the pCLCA1 channel regulator increased the rate of release of Cl- from both types of cultured cells. However, the presence of pCLCA1 also increased cAMP-dependent Cl- release from fibroblasts expressing CFTR. We conclude that Ca2+-dependent Cl- transport may be more important than cAMP-dependent Cl- transport for normal fluid secretion across the RPE. Furthermore, CLCA proteins expressed in the RPE appear to regulate the activity of other Cl- transporters, rather than functioning as primary ion transport proteins.

The regulatory behavior, inhibitor sensitivity, and properties of the whole cell chloride conductance observed in cells expressing the cDNA coding for a chloride conductance mediator isoform of the CLCA gene family, pCLCA1, have been studied. Common C-kinase consensus phosphorylation sites between pCLCA1 and the closely related human isoform hCLCA1 are consistent with a role for calcium in channel activation. Both channels are activated rapidly on exposure to the calcium ionophore ionomycin. Direct involvement of calcium in the activation of pCLCA1 was supported by the finding that treatment with the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM reduced the rate of chloride efflux from NIH/3T3 cells expressing the pCLCA1 channel. No combination of A-kinase activators used was effective in activating chloride efflux via this channel despite the presence of a unique strong A-kinase consensus site in pCLCA1. Notable differences of pCLCA1 from the reported properties of CLCA family members include the failure of phorbol 12-myristate 13-acetate to activate chloride efflux in cells expressing pCLCA1 and a lack of inhibition of chloride efflux from these cells after treatment with DIDS or dithiothreitol. However, selected inhibitors of anionic conductance inhibited pCLCA1-dependent anion efflux. The electrogenic nature of the ionomycin-dependent efflux of chloride from cells expressing pCLCA1 was confirmed by detection of outwardly rectifying chloride current and inhibition of this current by chloride conductance inhibitors in a whole cell patch-clamp study.

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.

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)

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.

Two dogs were diagnosed as malignant hyperthermia susceptible based on increased susceptibility (P less than 0.001) of biopsied muscle to caffeine-induced contracture. Erythrocytes from malignant hyperthermia and normal dogs were then examined for an antioxidant system deficiency. Values for serum muscle enzymes, reticulocytes and corpuscular hemoglobin were mildly elevated. Osmotic fragility was increased: hemolysis occurred at a NaCl concentration 10 mM higher than for normal dogs (P less than 0.001). A 35% glucose-6-phosphate dehydrogenase deficiency (P less than 0.001) with a 40% compensatory increase (P less than 0.01) in 6-phosphogluconate dehydrogenase activity was found. The membrane Ca2+-activated ATPase activity was abnormal: 100% increased with a 40% decreased Arrhenius activation energy (P less than 0.005) and increased thermostability. A 40% increased intracellular accumulation of total Ca2+ occurred in response to in vitro energy depletion in erythrocytes from one malignant hyperthermia dog (P less than 0.01). The multifactorial pattern of inheritance and the broad spectrum of malignant hyperthermia susceptibility are proposed to result from an antioxidant system deficit unmasking or aggravating an intrinsic muscle membrane anomaly. An individual from a family with a history of malignant hyperthermia or unexplained anesthetic death should be considered malignant hyperthermia susceptible if erythrocyte osmotic fragility is abnormal and there is a mild, unexplained elevation in serum creatine kinase.

The use of nicotinic acid for preventing intestinal secretion caused by cholera toxin and by the heat-stable enterotoxin of Escherichia coli has been investigated in the weanling pig. Secretory effects were measured in ligated jejunal loops of halothane-anesthetized pigs by dilution of a nonabsorbable marker added to the loop fluid. Different routes of administration and different initial pH values for nicotinate solutions were studied to determine optimal conditions for secretory inhibition. The neutral sodium salt of nicotinic acid had no significant antisecretory activity under any conditions used in these trials. Inhibition of secretion was most effective with partly neutralized nicotinic acid at pH 4.5 added directly to loops containing enterotoxin. Net fluid secretion induced by cholera toxin or heat-stable enterotoxin of E. coli was prevented by this treatment. Reversal of secretion was not accompanied by any measurable changes in cyclic nucleotide concentration in intestinal mucosa. Nicotinic acid antagonism of a secretory step common to cholera toxin and heat-stable enterotoxin of E. coli but subsequent to cyclic nucleotide involvement is indicated by these data.

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.

The effect of heat stable and heat labile Escherichia coli enterotoxins or cholera toxin in combination with theophylline on net water, sodium and chloride and unidirectional sodium and chloride fluxes was examined in acute isolated loops of jejunum of weanling swine. The effect of heat stable enterotoxin in combination with theophylline was determined in loops located in the proximal jejunum, while combinations of theophylline and either heat labile enterotoxin or cholera toxin were studied in the distal jejunum. In each situation the addition of theophylline resulted in an additive rather than a synergistic increment of intestinal secretory activity. This study implies that the intestinal adenyl cyclase system and enterotoxin induced intestinal secretion may not be directly related in the swine small intestine.

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.

Changes in serum enzyme levels, liver histology and liver function tests have been correlated to determine the usefulness of these tests in assessing liver status. The effects of carbon tetrachloride administration on these parameters has been determined in a group of 20 sheep. Normal levels, elevated levels after injury and the effect of elapsed time after injury are reported for serum glutamic dehydrogenase, sorbitol dehydrogenase, glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, lactate dehydrogenase, fructose-1-phosphate adlolase, alkaline phosphatase, cholesterol and proteins. Variation in the time of elevation of enzyme activities may be useful in determining the elapsed time between acute injury and serum sampling. In comparison to sheep fed an adequate diet, a diet with a restricted protein intake was associated with increased severity of histological lesions and decreased liver function.

The effects of cholera toxin on mucosal cyclic nucleotide concentrations and on net fluid secretion in the porcine small intestine are reported. Cholera toxin causes net secretion of fluid into the small intestine of weanling pigs, and secretory rates are dependent on the dose of the toxin placed in intestinal loops. Intestinal secretion due to cholera toxin exposure was not consistently accompanied by elevated concentrations of mucosal cyclic AMP or cyclic GMP. Net fluid fluxes in individual loops did not correlate with mucosal cyclic AMP concentration in the same loop. Jejunal adenylate cyclase was activated to a lesser extent in pigs, compared with rabbits, after in vivo treatment with cholera toxin. In vitro activation in cell-free homogenates was similar for both species. Papaverine was similar to cholera toxin in causing fluid secretion without cyclic AMP accumulations, but 3-isobutyl-1-methyl xanthine significantly increased cyclic AMP concentration and induced fluid secretion in pigs. Weanling pigs appeared to differ from rabbits in having a secretory response to cholera toxin which was independent of elevations in total mucosal cyclic AMP concentration.

The effect of cholera toxin, heat labile and heat stable Escherichia coli enterotoxin on mucosal cyclic AMP concentrations was determined on the proximal jejunum of weanling pigs and young rabbits. Ligated loops were injected with solutions containing no enterotoxin for control and either cholera toxin, heat labile or heat stable E. coli enterotoxin. The loops were drained after either two, four or six hours incubation at which time accumulated fluid was recorded and mucosal samples removed for determination of cyclic AMP concentration. In the rabbit, cholera toxin and heat labile, but not heat stable E. coli enterotoxin stimulated intestinal secretion while in the pig all three enterotoxins induced net fluid accumulation. Cholera toxin and heat labile, but not heat stable E. coli enterotoxin elevated rabbit mucosal cyclic AMP concentrations. In the pig these enterotoxins had no significant effect on mucosal cyclic AMP concentrations. The results are inconsistent with the hypothesis that the adenyl cyclase system is an essential step for enterotoxin induced intestinal secretion. The activation of intestinal adenyl cyclase by bacterial enterotoxins may only be an associated and not a necessary event for the stimulation of intestinal secretion.

Heat-stable enterotoxins from a bovine and porcine strain of Escherichia coli were isolated and showed significant differences in amino acid composition and heat stability.

Heat-stable enterotoxins (ST-124 and ST-1261) have been isolated from two different enterotoxigenic Escherichia coli of bovine (124) and porcine (1261) origin. The enterotoxin preparations were isolated by ultrafiltration and ion-exchange chromatography and were both active in the suckling mouse test and pig ligated loop test in the nanogram range. The bovine (ST-124) enterotoxin was not stable to heating in its isolated form, and significant differences in amino acid composition were observed between the two enterotoxins. Although both toxins were active at similar levels in the suckling mouse and pig ligated loop tests, ST-124 lacked the ability to cause the profound secretory responses seen with ST-1261 in the weanling pig ligated loop.

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.

Effect of the M (modifier) gene of Escherichia coli W on the expression of wild-type structural genes of four arginine biosynthetic enzymes was studied by examining enzyme activity in cell-free extracts of cultures grown in minimal medium and medium containing arginine. The mutant M gene was originally identified as causing arginine-induced synthesis of acetylornithine delta-transaminase in a strain deficient for the enzyme. The strains used in this study received the mutant M gene by recombination. Noncoordinate repression has been demonstrated for two more enzymes of the arginine regulon of E. coli W and the M(-) gene increases the degree of noncoordinate repression for the regulon. Mutation of the M gene results in altered regulation of acetylornithine delta-transaminase, ornithine transcarbamylase, and acetylornithinase. In addition, a decreased growth rate is observed. It is proposed that the M gene is a regulatory gene. A model is presented to explain the data which involves changes in operator-repressor affinity for the structural genes and possibly for the gene controlling arginyl transfer ribonucleic acid synthetase.