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C M Fuller

Publications and source records attributed to C M Fuller.

13 recordsLinked to original sources
PubMed1992

Functional reconstitution of a chloride channel protein from bovine trachea.

We characterized the electrophysiological properties of a chloride channel protein isolated from bovine trachea after incorporation into planar lipid bilayers, and studied the effects of thiol-modulating agents on channel regulation both in bilayers and vesicular iodide uptake studies. Our experiments showed that this protein formed perfectly anion-selective channels in the bilayer, with an anion permeability sequence of I- (2.1) > NO3- (1.7) > Br- (1.2) > Cl- (1.0). The conductance of this channel was 25-30 picosiemens in 150 mM Cl-, and saturated with increasing chloride concentration. This channel could be completely inhibited by 4,4'-bis(isothiocyano)-2,2'-stilbenedisulfonate. Immunoblot analysis, using polyclonal antibodies (anti-p38), revealed one major band at 140 kDa. Upon reduction with dithiothreitol, 64- and 38-kDa polypeptides were observed. Functional experiments showed that reduction was accompanied by loss of 125I- uptake and single-channel activity. In the presence of dithiothreitol, only the low molecular mass protein forms (64 and 38 kDa) were detected by anti-p38 antibodies on Western blots. Cross-linking of S-S bonds with Cu(2+)-o-phenanthroline led to activation of chloride channels in vesicles and bilayers. Over-aggregation of chloride channels by this S-S cross-linking reagent caused inhibition of 125I- uptake by 80-100% and the abolishment of single-channel activity. We propose that the native chloride channel from bovine trachea can exist in vivo in different structural and functional forms depending upon its thiol-disulfide oxidation reduction status. The oxidized form has a molecular mass of 140 kDa and represents a fully active chloride channel. Inactivation of this channel might occur by over-aggregation of protein subunits, or by dissociation of the 140-kDa subunit by disulfide bond reduction.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Peroxynitrite inhibits sodium uptake in rat colonic membrane vesicles.

Peroxynitrite (ONOO-) is a potent oxidizing agent that initiates lipid peroxidation and sulfhydryl oxidation and may be responsible for a portion of the cytotoxicity attributed to superoxide anion (.O2-). We quantified the extent to which ONOO-, xanthine plus xanthine oxidase (XO) and hydrogen peroxide (H2O2), decreased sodium (Na+) uptake into membrane vesicles derived from colonic cells of dexamethasone-treated rats. Carrier-free 22Na+ uptake into vesicles was measured in the presence of an inside-negative membrane potential, produced by the addition of the potassium ionophore valinomycin (10 microM) after removal of all external potassium by cation exchange chromatography. Preincubation of vesicles with either 100 microM or 1 mM ONOO- for 30 s decreased the amiloride-blockable fraction of Na+ uptake by 27 +/- 7% and 65 +/- 2%, respectively (means +/- S.E.; n greater than or equal to 5; P less than 0.05 from control). However, the amiloride-insensitive part of Na+ uptake was not affected, indicating that there was no overt destruction of these vesicles by these ONOO- concentrations. Decomposed ONOO-, hydrogen peroxide (1 microM-10 mM), or xanthine (500 microM) plus XO (10-30 mU/ml), either in the absence or in the presence of 100 microM FeEDTA, did not decrease Na+ uptake. These data suggest that ONOO- is a potent injurious agent that can compromise Na+ uptake across epithelial cells, possibly by damaging Na+ channels.

Amiloride↗
PubMed1992

Identification of a membrane protein from T84 cells using antibodies made against a DIDS-binding peptide.

The outwardly rectified chloride channel of secretory epithelial cells is inhibited by disulfonic stilbene (DS) compounds such as 4,4'-diisothiostilbene-2,2'-disulfonic acid (DIDS) [R. J. Bridges, R. T. Worrell, R. A. Frizzell, and D. J. Benos, Am. J. Physiol. 256 (Cell Physiol. 25): C902-C912, 1989]. A 13-amino acid peptide (P49) corresponding to the putative DS binding site region of the murine anion exchange protein was synthesized, and polyclonal antibodies were generated against it and then purified over a P49 affinity column. The resulting monospecific antibodies reacted on Western blots with a 95- to 100-kDa protein from human erythrocytes and a 55- to 60-kDa protein from the human colonic tumor cell line, T84. The reaction with T84 protein did not appear to represent recognition of an anion exchanger because anion efflux from T84 cells was independent of external Cl-. In addition, monoclonal antibodies raised against human band 3 recognized the band 3 protein in human red cell ghost preparations but recognized nothing in T84 cell membrane preparations. In T84 cells, DIDS protected the 60-kDa protein from antibody binding. The anti-P49 antibody blocked outwardly rectified Cl- channels incorporated into planar lipid bilayer membranes from rat colon. Immunocytochemical data reveal specific binding of the anti-P49 antibody to perinuclear cytoplasmic vesicles. Forskolin caused these antibody-labeled vesicles to migrate from the perinuclear region to the plasma membrane under conditions and with a time course identical to that seen for stimulation of Cl- transport in these cells. Our results suggest that the protein may be a part of a chloride channel complex of secretory epithelial cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Antibodies against the cystic fibrosis transmembrane regulator.

Rabbit polyclonal antibodies have been raised against high-performance liquid chromatography purified synthetic peptides corresponding to two discrete regions of the cystic fibrosis transmembrane regulator (CFTR) protein: the R-domain (residues 785-796) and the extreme COOH-terminus (residues 1467-1480). Antibodies (Ab) to each of these peptides were affinity purified either by passage over a peptide-derivatized agarose matrix (Ab 785) to produce monospecific polyclonal antibodies or by protein A affinity chromatography to purify the immunoglobulin G1 fraction free of other serum proteins (Ab 1467). These antibodies recognize a candidate CFTR protein in the colonic cell line T84, as determined by Western blot analysis and by immunoprecipitation and labeling of the precipitate with [gamma-32P]ATP in the presence of protein kinase A. Both antibodies precipitated CFTR-related polypeptides from four mammalian cell types (HeLa, Bsc-40, HEp-2, and Chinese hamster ovary cells) transfected with the full-length CFTR cDNA clone using a vaccinia T7 protein expression system. Similar results were observed using a yeast CFTR expression system. In each case the Mr values of the bands observed were consistent with that expected for the CFTR protein. These antibodies should be useful probes for the immunocytochemical localization, immunoaffinity purification, and ultimately the functional characterization of the CFTR protein.

Animals↗
PubMed1992

CFTR!

Cystic fibrosis (CF) is a fatal genetic disease primarily affecting Caucasians, although cases have been reported from other ethnic groups. CF has a complex etiology, but it is chiefly a disease of electrolyte transport and is characterized by defects in fluid secretion by several epithelia, including the sweat duct, exocrine pancreas, and the pulmonary airways. The link between CF and a defect in cAMP-mediated Cl- transport in secretory epithelia was established in the early 1980s. Since then, numerous electrophysiological studies have focused on the characterization and regulation of individual Cl- channels underlying the macroscopic Cl- currents of secretory epithelia in the airways, sweat ducts, and gut. In this review the results of these studies in the light of current knowledge of the function of the CF gene product, the CF transmembrane conductance regulator (CFTR) protein, will be analyzed. The CFTR protein is a member of a family of ATP-binding proteins that act as unidirectional solute pumps. These proteins are membrane spanning, are found in both prokaryotic and eukaryotic cells, and have two ATP-binding domains. The family includes the p-glycoproteins that are involved with the expression of multidrug resistance in certain tumor cells. The majority of CF chromosomes (70%) have a single codon deletion that translates to a missing phenylalanine residue at position 508 (delta F508) of the protein. Unique for this family of proteins, the CFTR protein possesses an additional highly charged domain (the R domain) containing several consensus polypeptide sequences for kinase phosphorylation. Although CFTR bears structural resemblance to this family of ATP-dependent pumps, overexpression of the protein in a variety of different cell types is associated with the appearence of a cAMP-sensitive Cl- channel. We critically examine current information concerning the structure-function relationships of the CFTR protein obtained from both electrophysiological and biochemical approaches. We also summarize recent evidence suggesting that the CFTR protein may act as a pump and a channel, a hypothesis in keeping with the multifaceted nature of the disease.

Adenosine Triphosphate↗
PubMed1989

Secretagogue and second messenger-activated Cl- permeabilities in isolated pancreatic zymogen granules.

Previous studies of enzyme secretion from isolated pancreatic acinar cells and of isolated zymogen granules (ZG) have reported that both a Cl- and a K+ permeability are present on the ZG membrane. It has been suggested that ion influx via these permeability pathways, followed by water movement is required for granular swelling which appears to be intimately related to exocytosis. However, little is known about the regulation of these pathways by secretagogues. Evidence suggests that cAMP-protein kinase A and diacylglycerol-protein kinase C are second messengers in stimulation of exocytosis. In the present study we have examined ion permeability pathways in ZG isolated from control cells and from cells pretreated with the acetylcholine analog carbachol (Cch), with the peptide hormone cholecystokinin (CCK) and with second messengers of hormone action such as cAMP and the diacylglycerol analog 12-O-tetradecanoyl phorbol-13-acetate (TPA). Ion and water influx rates in ZG and consequent swelling and lysis of granules was monitored by measuring changes in optical densities of ZG suspensions at 540 nm following additions of the electrogenic or electroneutral ionophores valinomycin and nigericin, respectively. The data show that both a Cl- conductance and an anion exchange pathway are present in the granule membrane. Both pathways are activated by pretreatment of isolated cells with CCK or of isolated permeabilised cells with cAMP, whereas only the Cl- conductance is increased by pretreatment with Cch or with TPA.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

Ionic and osmotic dependence of secretion from permeabilised acini of the rat pancreas.

Many types of secretory granule have been observed to swell as a result of cell stimulation implying a degree of osmotic control, although the regulation of granule fusion with the apical plasma membrane is not clearly understood. In the present study we have investigated the ionic and osmotic dependency of basal and stimulated 3H-protein release from rat pancreatic acini, permeabilised by either digitonin or high voltage electric discharge. Acini were stimulated with either cholecystokinin-pancreozymin octapeptide (CCK-8), carbachol (CCh), or with phorbol ester (TPA) plus cAMP. Stimulated secretion was significantly reduced when 130 mmol/l Cl- in the buffer was replaced by I-, NO3-, SCN- or cyclamate-. Secretion in Cl- buffers was inhibited by the anion transport inhibitor 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), by 40% of the control response. Neither Na+ nor N-methyl-D-glucamine+ could replace K+ in the buffer. Ba2+ and quinine, which block K+ conductance pathways, inhibited stimulated secretion by 50%. Finally, stimulated secretion from leaky cells was nearly abolished by doubling buffer osmolarity. The data suggest that when the cell is stimulated, a Cl- and a K+ permeability appear in the zymogen granule membrane and the ions enter down their electrochemical gradients. The increased intragranular osmolarity results in granular swelling which is intimately associated with secretion.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1987

Cholinergic receptors regulate a voltage-insensitive but Na+-dependent calcium influx pathway in salivary acinar cells.

The calcium-sensitive fluorescent probe, quin 2, was employed to investigate the cholinergic receptor regulation of cytosolic free calcium concentration [( Ca2+]i) in isolated rat submandibular acinar cells. Cholinergic receptor stimulation results in a marked and sustained elevation [Ca2+]i. The major component of this response is shown to be due to activation of a calcium influx pathway. There is no evidence in K+-depolarized cells of any voltage-gated calcium influx. Acetylcholine failed to activate the calcium influx pathway in cells bathed in Na+-free solutions. The cholinergic receptors thus regulate a voltage-insensitive but Na+-dependent transport system for calcium entry in submandibular acinar cells.

Acetylcholine↗
PubMed1987

Cholinergic receptor-regulation of potassium channels and potassium transport in human submandibular acinar cells.

The cholinergic receptor-regulation of K+ transport was studied in human submandibular glands. Acetylcholine stimulation 10 mumol/L results in an increase in membrane permeability (86Rb+ efflux) for, and a net efflux of, K+ ions from the glandular tissue. In the post-stimulus period, there is a net re-uptake of K+ ions into the tissue. Patch-clamp electrophysiological techniques were employed to demonstrate the presence of a large conductance K+ selective ion channel in the basolateral membranes of isolated human submandibular acinar cells. The patch-clamp results indicate that this voltage- and calcium-activated K+ channel operates to regulate the K+ permeability in both the resting and acetylcholine-stimulated acinar cells. We discuss the role of the K+ channel, K+ efflux, and K+ re-uptake in relation to stimulus-secretion coupling.

Acetylcholine↗
PubMed1986

Potassium uptake in the mouse submandibular gland is dependent on chloride and sodium and abolished by piretanide.

Nervous or hormonal stimulation of salivary secretion in vivo is associated with a pronounced efflux of K+ from the secretory, acinar cells into the blood. This K+ efflux is followed in the post-stimulus period by a reuptake of K+ into the glandular tissue. In the present study we monitor the changes in [K+] of physiological solutions perfusing a flow chamber containing isolated segments of mouse submandibular glands. Nervous stimulation or the application of exogenous acetylcholine (ACh, 10(-5) M) to the isolated glandular tissue results in characteristic changes in the [K+] of the superfusate, indicating net K+ release followed by K+ reuptake. The post-stimulus reuptake of K+ is shown to be susceptible to blockade by either ouabain (10(-3) M) or piretanide (10(-4) M). The reuptake was markedly attenuated if Cl- in the superfusate was replaced by either NO3- or SO4(2-). The K+ uptake was, however, unaffected when Br- replaced Cl- in the superfusate. Similar effects were observed in the unstimulated glandular tissues. The introduction of Cl-(-)free media containing either NO3- or SO4(2-) resulted in a loss of K+ from the tissue which was followed, upon reintroduction of Cl-, by a pronounced uptake of K+. When Br- was substituted for Cl- there was very little change in [K+] upon removal or reintroduction of Cl-. The uptake of K+ induced by reintroduction of Cl- after a period of NO3- or SO4(2-) superfusion was blocked by both ouabain and piretanide. This uptake of K+ was also dependent on the presence of extracellular Na+. Both Cl- and Na+ had to be present in the superfusing medium for K+ uptake to be fully manifest. These findings indicate that the K+ uptake observed in both the resting and stimulated submandibular gland cannot be explained as solely due to the activity of the Na+-K+-adenosine triphosphatase (Na+-K+-ATPase). The demonstrated anionic selectivity, dependence on extracellular Na+ and susceptibility to blockade by the diuretic piretanide would strongly suggest that a coupled Na+-K+-Cl- co-transport system operates in submandibular glands as it does in other transporting epithelia to achieve K+ uptake.

Acetylcholine↗
PubMed1985

Human pancreatic acinar cells: studies of stimulus-secretion coupling.

Elements of stimulus-secretion coupling were studied in human pancreatic acinar cells by using tissue samples obtained from cadaver organ donors. In pancreatic fragments, acetylcholine evoked amylase secretion as well as potassium release and increased the outflux of 45Ca and 86Rb from the prelabeled tissue. In patches of basolateral plasma membrane excised from acinar cell clusters, single-channel potassium currents were recorded. The inside of the plasma membrane faced the bath solution, allowing the effects of changes in the free ionized calcium concentration in contact with the membrane interior to be tested. Two types of calcium-activated potassium-selective channels were found with unit conductances of about 250 and 50 picosiemens (pS), respectively. In both cases channel opening was determined by the electrical potential difference across the plasma membrane and the free ionized calcium concentration in the bath solution. The probability of channel opening was markedly increased by elevation of the free ionized calcium concentration in contact with the membrane inside. The results suggest that the acetylcholine-evoked cellular potassium release occurs via selective membrane potassium channels opened by calcium released intracellularly after the action of the secretagogue.

Acetylcholine↗
PubMedDate not supplied

Bear meat trichinosis with a concomitant serologic response to Toxoplasma gondii.

A severe case of trichinosis occurred following the ingestion of raw meat from a common black bear, Ursus americanus, that was shot in Butte County, California. Examination of steaks cut from the bear revealed heavy infection with T. spiralis. A good clinical response was obtained following treatment with prednisone (1 mg/kg body weight per day) and thiabendazole (50 mg/kg body weight per day); no toxic effects were observed. In comparison with the pre-treatment biopsy, a more intense inflammatory response was seen following the administration of thiabendazole. Nevertheless, a muscle biopsy taken several months after discharge still showed well-encapsulated, morphologically intact larvae. In addition to the expected immunological responses to T spiralis, a marked rise in titer of antibodies to Toxoplasms gondii was observed by the fluorescent antibody and Sabin-Feldman dye test methods. Since toxoplasma infection of muscle is widespread in man, it is possible that an unrelated disease of muscle could result in stimulation of anti-toxoplasma antibodies similar to the findings in polymyositis and dermatomyositis. Alternatively, the possibility that a dual infection was acquired from the bear meat cannot be excluded.

Adult↗