A store-operated nonselective cation channel in lymphocytes is activated directly by Ca(2+) influx factor and diacylglycerol.

Agonist-receptor interactions at the plasma membrane often lead to activation of store-operated channels (SOCs) in the plasma membrane, allowing for sustained Ca(2+) influx. While Ca(2+) influx is important for many biological processes, little is known about the types of SOCs, the nature of the depletion signal, or how the SOCs are activated. We recently showed that in addition to the Ca(2+) release-activated Ca(2+) (CRAC) channel, both Jurkat T cells and human peripheral blood mononuclear cells express novel store-operated nonselective cation channels that we termed Ca(2+) release-activated nonselective cation (CRANC) channels. Here we demonstrate that activation of both CRAC and CRANC channels is accelerated by a soluble Ca(2+) influx factor (CIF). In addition, CRANC channels in inside-out plasma membrane patches are directly activated upon exposure of their cytoplasmic side to highly purified CIF preparations. Furthermore, CRANC channels are also directly activated by diacylglycerol. These results strongly suggest that the Ca(2+) store-depletion signal is a diffusible molecule and that at least some SOCs may have dual activation mechanisms.

Adult alveolar type II cells lack cAMP and Ca(2+)-activated Cl-channels.

We investigated the presence of Ca(2+)-activated Cl-channels in adult rat alveolar type II (ATII) using patch-clamp techniques. Only one active channel each, with a single channel conductance of 50 pS and an opening probability (Po) of 0.76 was found among 130 successful cell-attached and 5 inside-out patches. Addition of CPT-cAMP into the bath (500 microM) induced one active patch from 33 silent cell-attached patches. Incubation of 9 ATII cells, with ionomycin (1 microM), failed to elicit chloride single currents in 9 cell-attached patches. Cl- currents were also absent from 35 whole cell patches, even after the addition of 10 microM terbutaline in the bath or 1 mM ATP and 5 mM MgCl2 in the pipette. These results indicate that only a very small fraction of adult rat ATII cells express CFTR and suggest that Cl- ions are passively transported across the cell junctions.

Calcium-sensitive chloride channels in vascular smooth muscle cells.

Chloride (Cl-) channels were characterized in vascular smooth muscle cells (VSMC) using radioisotope flux and patch-clamp electrophysiological techniques. Transmembrane 125iodine (125I) efflux from subcultured (Passage 1-5) rat aortic VSMCs was used as an indicator of Cl- movements to study the relationship between intracellular calcium concentration ([Ca2+]i) and Cl- channel activity. Angiotensin II (Ang II) (10(-7) M) and adenosine 5'-triphosphate (ATP) (10(-4) M) induced rapid increases (9.7- and 14.9-fold, respectively) in 125I efflux rates. We found that both Ang II- and ATP-stimulated 125I efflux and [Ca2+]i increases were completely abolished after brief incubation (20 microM, 20 min) with the acetoxymethyl ester of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), a membrane-permeable Ca2+ chelator. However, when external EGTA was used to blunt agonist-stimulated Ca2+ influx, 125I efflux was still increased in response to Ang II and ATP. These data suggest that Ca2+ release from intracellular sites is sufficient to activate Cl- channels in response to Ang II and ATP. Using standard patch-clamp electrophysiological techniques, we found that Ang II, a Ca(2+)-mobilizing agonist, stimulated outward Cl- currents (gCl = 75 pS) in cell-attached (C/A) patches of primary and subcultured VSMCs. Collectively, these data suggest that Ang II and other vasoconstrictor agents stimulate Cl- channel activity via increases in [Ca2+]i. Cl- channel activation may help to depolarize the VSMC membrane leading to increased Ca2+ influx during agonist stimulation.

Differentiation of immortalized epithelial cells derived from cystic fibrosis airway submucosal glands.

Cystic fibrosis (CF) involves abnormalities in mucus production and secretion of the airway. Studies of the regulation of airway mucin production and secretion has been difficult due to the lack of in vitro models of the airway epithelial cells which express functional differentiation. Because the majority of the mucin in the airway is apparently produced by the submucosal glands, we have focused our attention on the development of cell culture models of human airway submucosal glands. This report describes the propagation of CF airway submucosal gland epithelial cells which continue to express mucin production. The CF bronchus was obtained from a 31-yr-old patient who received a double lung transplant. The glands were dissected out and primary cultures prepared by the explant/outgrowth procedure. The cells were immortalized by infection with Ad12-SV40 hybrid virus. The cultures are maintained in serum-free keratinocyte basal medium supplemented with insulin (5 micrograms/ml), hydrocortisone (0.5 microgram/ml), epidermal growth factor (10 ng/ml), bovine pituitary extract (25 micrograms/ml), and antibiotics. Cultures were passaged using 0.125% trypsin in Ca+2 and Mg(+2)-free Hanks', balanced salt solution. Polymerase chain reaction (PCR) analysis demonstrated that the cells were homozygous for the delta F508 mutation. Morphologic observations showed that the cells were epithelial and were interconnected by sparsely distributed desmosomes. Their cytoplasm contained secretory-type structures including abundant Golgi, rough endoplasmic reticulum, and secretory vesicles. Immunofluorescent studies determined that all cells were positive for cytokeratins, mucin glycoconjugates, and cystic fibrosis transmembrane conductance regulator. The cultures secreted substantial amounts of mucin glycoproteins and expressed the MUC-2 mucin gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of low-amiloride-affinity sodium channels in alveolar type II cells.

We determined the mechanisms by which beta-agonists increase sodium (Na+) currents across rat alveolar type II (ATII) cells grown in primary culture. When ATII cells were patched in the cell-attached mode using symmetrical Na+ solutions (150 mM Na(+)-glutamate), single-channel currents were observed for holding potentials between -80 and 30 mV (referenced to the pipette solution) with a single-channel conductance of 27 +/- 3 pS, a mean open time (tau 1) of 3.3 +/- 0.15 ms and an open probability (Po) of 0.36 +/- 0.06 (n = 7). Addition of 10 microM terbutaline into the bath increased tau 1 to 6.43 +/- 0.5 ms and Po to 0.62 +/- 0.06 (n = 7) without affecting channel conductance. Single-channel currents with a conductance of 25 +/- 2 pS were also recorded across ATII cells patched in the inside-out mode. Addition of 250 U/ml of protein kinase A (PKA), 1 mM ATP, and 5 mM MgCl2 in the bath solution (150 mM Na(+)-glutamate) increased the single channel tau 1 from 3.26 +/- 0.15 to 7.38 +/- 0.38 and Po from 0.41 +/- 0.06 to 0.72 +/- 0.07 (n = 6) without altering conductance. Addition of 1 microM amiloride or ethylisopropylamiloride (EIPA) in the pipette solution (150 mM Na(+)-glutamate) blocked single-channel activity almost completely. Ionic substitution experiments showed the relative permeability of Na+ to K+ and Na+ to Cl- to be 7:1 and 8:1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Differentiation of immortalized epithelial cells derived from cystic fibrosis airway submucosal glands.

Cystic fibrosis (CF) involves abnormalities in mucus production and secretion of the airway. Studies of the regulation of airway mucin production and secretion has been difficult due to the lack of in vitro models of the airway epithelial cells which express functional differentiation. Because the majority of the mucin in the airway is apparently produced by the submucosal glands, we have focused our attention on the development of cell culture models of human airway submucosal glands. This report describes the propagation of CF airway submucosal gland epithelial cells which continue to express mucin production. The CF bronchus was obtained from a 31-yr-old patient who received a double lung transplant. The glands were dissected out and primary cultures prepared by the explant/outgrowth procedure. The cells were immortalized by infection with Adl2-SV40 hybrid virus. The cultures are maintained in serum-free keratinocyte basal medium supplemented with insulin (5µg/ml), hydrocortisone (0.5µg/ml), epidermal growth factor (10 ng/ml), bovine pituitary extract (25µg/ml), and antibiotics. Cultures were passaged using 0.125% trypsin in Ca(+2) and Mg(+2)-free Hanks', balanced salt solution. Polymerase chain reaction (PCR) analysis demonstrated that the cells were homozygous for the ΔF508 mutation. Morphologic observations showed that the cells were epithelial and were interconnected by sparsely distributed desmosomes. Their cytoplasm contained secretory-type structures including abundant Golgi, rough endoplasmic reticulum, and secretory vesicles. Immunofluorescent studies determined that all cells were positive for cytokeratins, mucin glycoconjugates, and cystic fibrosis transmembrane conductance regulator. The cultures secreted substantial amounts of mucin glycoproteins and expressed the MUC-2 mucin gene. Patch clamp experiments revealed that the cells expressed defective Cl(-) channels which were not activated by Forskolin.

Culture-induced alterations in alveolar type II cell Na+ conductance.

Changes in Na+ transport in rat alveolar type II (ATII) cells during culture were quantified and related to alterations in spatial distribution of proteins antigenically related to amiloride-sensitive Na+ channels. Adult rat ATII cells were cultured for periods ranging from 24 to 96 h. When patch clamped in the whole cell mode, both freshly isolated and cultured ATII cells exhibited outwardly rectified Na+ currents. At 0 and 24 h in culture, these currents were equally inhibited by amiloride, benzamil, and 5-(N-ethyl-N-isopropyl)-2',4'-amiloride (inhibitory constant approximately 1 microM). These conductive pathways were equally permeable to Na+ and K+. Immunocytochemical localization at 0 or 24 h in culture revealed the presence of plasma membrane antigenic sites; after 48 h, the appearance of intracellular antigenic sites increased significantly. A single band of molecular mass 135 kDa in membrane proteins of freshly isolated ATII cells was recognized in Western blots; at 48 h in culture, two lower bands with molecular masses of 75 and 65 kDa were detected in either membrane or cytoplasmic proteins. Photolabeling with 2'-methoxy-5'-nitrobenzamil showed that the 135-, 75-, and 65-kDa bands contained amiloride-binding sites. These results suggest the presence of low amiloride affinity conductive pathways in freshly isolated and cultured ATII cells. Culturing ATII cells resulted in internalization and possible breakdown of these pathways and decreased Na+ transport.

Parallel simulated annealing for emission tomography.

A method for implementing simulated annealing in parallel to speed up the execution of emission tomography (ET) image reconstruction is presented. A high degree of parallelism can be attained by using a parallel-acceptance partitioning strategy, in which perturbations to subsets of the estimate are evaluated in parallel. However because the point spread function in ET imaging systems is globally dependent, processors cannot update the current estimate independently. Consequently, processors must be synchronized each time a perturbation is accepted to avoid introducing error. This can produce excessive communications overhead, especially when the acceptance rate is high. In this paper an energy function is constructed to reduce the synchronization requirements by using a reformulation of the log-likelihood function from the expectation maximization (EM) algorithm. The approach is to change the global dependence in the energy function from the current estimate to the estimate generated during the last iteration. The synchronization requirements for guaranteed convergence are then significantly reduced from once per acceptance to once per iteration. This parallel implementation on 54 Inmos T800 transputers connected in a ring topology resulted in execution times that were almost 50 times faster than on a VAX 8600.

Histamine-induced chloride channels in apical membrane of isolated rabbit parietal cells.

The electrical properties of the apical membrane of isolated rabbit parietal cells were studied using the patch-clamp technique. The apical membrane of the parietal cells plated on Matrigel and maintained in culture conditions was identified by lectin-binding studies. Cell-attached and excised inside-out patches from 10(-4) M cimetidine-treated parietal cells infrequently contained Cl- channels (9% of the patches). A single class of voltage-dependent outwardly rectifying Cl- channels with 24 +/- 1-pS conductance was observed in 75% of the patches from cells stimulated (acid secreting) by 10(-4) M histamine. Other anions passed through these channels with a permeability sequence of I- (1.2) greater than Br- (1.1) greater than or equal to Cl- (1.0) greater than NO3- (0.7) greater than SO4(2-) (0.1), but there was a very low permeability for Na+ or K+ (PCl-/PNa+ or PCl-/PK+ greater than 5). In inside-out patch configurations the Cl- channel was insensitive to Ba2+ and stilbene derivatives but was inhibited by diphenylamine-2-carboxylic acid in a manner characteristic of a reversible open-channel blocker. It is concluded that H2-receptor agonist stimulation of acid secretion by rabbit parietal cells activates Cl- channels in the apical cell membrane.

Ca2(+)-sensitive K+ channel in aortic smooth muscle of rats.

We measured K+ channel activity in inside-out patches of cell membrane from aortic vascular smooth muscle cultured (Passages 1-3) from Wistar, Wistar-Kyoto, and spontaneously hypertensive rats (SHR). With [Ca2+]i between 25 and 100 nm and 150 mm K+ on both sides of the membrane, the conductance of this channel was 55 +/- 7 pS (slope of current-voltage curve through 0 mV) and the current was outwardly rectified. There was no difference in single-channel conductance among the three rat strains. Increasing negative holding voltages or increasing [Ca2+]i, increased the probability of this type channel being open (Npo; P less than 0.01); SHR had a larger NPo (P less than 0.01). Compared with cells from Wistar and Wistar-Kyoto, cells from SHR also had the longest mean open time. The increased NPo and mean open time we observed in this K+ channel of cells from SHR could contribute, at least in part, to the increased membrane K+ permeability, reported previously.

Characterization of epithelial cell cultures derived from human tracheal glands.

Cultures of normal human tracheal gland epithelial cells that exhibit functional differentiation have been propagated in serum-free medium supplemented with insulin (5 micrograms/ml), epidermal growth factor (10 ng/ml), hydrocortisone (0.5 micrograms/ml), and bovine pituitary extract (25 micrograms/ml). The cells retain many characteristics of epithelial cells including microvilli on cell surfaces, desmosomes between cells, and tonofilaments in the cytoplasm. In addition, they exhibit keratin-positive titers and react positively with Peanut agglutinin, which is specific for the disaccharide beta-D-galactose-(1----3)N-acetyl D-galactosamine, a major component of mucin glycoprotein. The cells also exhibit normal Cl- channel activity which was enhanced by the cAMP agonist Forskolin. The major component of the cellular secretion was hyaluronic acid; approximately 10% of the void volume material was resistant to hyaluronidase and may contain material similar to mucin glycoprotein. Some of the cell cultures have been maintained in serum-free conditions for 6 to 7 passages. This model will be important to study regulation of ion-channel activities and mucous glycoprotein secretion and to compare such regulations with the tracheal mucosal epithelial cells already established.

Peroxisomal fatty acyl-CoA oxidase is not regulated by triiodothyronine.

In studies using primary cultures of adult rat hepatocytes in serum-free medium, peroxisomal fatty acyl-CoA oxidase activity was not altered by the presence of 3,5,3'-triiodothyronine, whereas time- and dose-dependent increases in the thyroid hormone-responsive enzyme mitochondrial glycero-3-phosphate dehydrogenase were seen. Activity of peroxisomal oxidase was stimulated with clofibric acid in the absence of 3,5,3'-triiodothyronine. The results demonstrate that hepatic peroxisomal fatty acyl-CoA oxidase activity is not directly regulated by 3,5,3'-triiodothyronine and that stimulation of peroxisomal fatty acyl-CoA oxidase activity by clofibric acid does not require thyroid hormone.

Image understanding system for histopathology.

An image understanding machine vision system for histological diagnoses is based on three interacting expert systems: a diagnostic expert system utilizing terms familiar to pathologists, an interpretive expert system relating human diagnostic concepts to computable histometric features and a scene segmentation expert system which extracts the diagnostic information from the imagery. The control software for the image understanding system resides on a multiprocessor computer. This article details measures to maintain system efficiency and to accommodate the requirements of interprocess communication and processing task scheduling.

Phosphorylation fails to activate chloride channels from cystic fibrosis airway cells.

Chloride impermeability of epithelial cells can account for many of the experimental and clinical manifestations of cystic fibrosis (CF). Activation of apical-membrane Cl- channels by cyclic AMP-mediated stimuli is defective in CF airway epithelial cells, despite normal agonist-induced increases in cellular cAMP levels. This defect in Cl- channel regulation has been localized to the apical membrane by exposing the cytoplasmic surface of excised membrane patches to the catalytic subunit (C subunit) of cAMP-dependent protein kinase and ATP. In membranes from normal cells, C-subunit activated Cl- channels with properties identical to those stimulated by cAMP-dependent agonists during cell-attached recording. Activation by the C subunit was not observed in CF membranes, but the presence of Cl- channels was verified by voltage-induced activation. The failure of the C subunit to activate the Cl- channels of CF membranes indicates that the block in their cAMP-mediated activation lies distal to induction of cAMP-dependent protein kinase activity and focuses our attention on the Cl- channel and its membrane-associated regulatory proteins as the probable site of the CF defect.

Design for a fast fluorescence laser scanning microscope.

The design of a fast fluorescence laser scanning microscope is described and illustrated, with discussion of the design consideration of the principal components, including the optical elements. The system, now under construction at the Optical Sciences Center of the University of Arizona, is expected to provide very-high-speed scanning, at a high spatial sampling density, of large object areas while retaining a flexibility of applications. The projected scanning rate approximates the rate achieved by flow cytometry; the projected rates of information generation should be orders of magnitude higher.

Chloride channel regulation in secretory epithelia.

Patch-clamp techniques were applied for single-channel recording to cultured cells from Cl secretory epithelia: human airway cells and the T84 cell line. Epinephrine or cyclic AMP (cAMP) stimulated single-channel activity in human airway cells during cell-attached recording. Similarly, prostaglandin E2 and cAMP stimulated single-channel activity in T84 cells. Ion substitution experiments with patches in the inside-out configuration indicated greater than 10:1 selectivity for Cl over Na in channels from both cell types, which confirms the identity of these events as Cl channel openings. The Ca ionophore A23187 stimulated these Cl channels to open in both cell types. Human airway cells from patients with cystic fibrosis (CF) did not respond to epinephrine or cAMP, but A23187 treatment elicited Cl channel activity. Changes in bath Ca activity in the inside-out configuration demonstrated that increased Ca could activate cAMP-insensitive Cl channels in CF cells. This indicates that the primary defect in CF is in the regulation of Cl channel opening rather than in conduction of Cl through the channel.

Altered regulation of airway epithelial cell chloride channels in cystic fibrosis.

In many epithelial cells the chloride conductance of the apical membrane increases during the stimulation of electrolyte secretion. Single-channel recordings from human airway epithelial cells showed that beta-adrenergic stimulation evoked apical membrane chloride channel activity, but this response was absent in cells from patients with cystic fibrosis (CF). However, when membrane patches were excised from CF cells into media containing sufficient free calcium (approximately 180 nanomolar), chloride channels were activated. The chloride channels of CF cells were similar to those of normal cells as judged by their current-voltage relations, ion selectivity, and kinetic behavior. These findings demonstrate the presence of chloride channels in the apical membranes of CF airway cells. Their regulation by calcium appears to be intact, but cyclic adenosine monophosphate (cAMP)-dependent control of their activity is defective.

Single chloride channel currents from canine tracheal epithelial cells.

Patch-clamp techniques were used to characterize the properties of anion-selective channels in canine tracheal epithelial cells that had been maintained in primary culture. Gigaohm seals (10-30 G omega) were obtained in single isolated cells or cells at the edge of a confluent sheet, and channels were studied in the cell attached or the inside-out, excised patch configuration. Pretreatment with isotonic KCl caused the cells to round-up and allowed us to have better success in obtaining good seals. Based on conductance, anion-cation selectivity and voltage-dependent kinetic properties, four anion channel types could be detected in symmetrical solutions of 0.15 M NaCl: (i) a 30-50 pS Cl- channel of high selectivity, active at negative potentials and inactivated by large positive potentials; (ii) an approx. 20 pS Cl- channel of high selectivity, active at positive potentials and inactivated at negative potentials; (iii) an approx. 250 pS channel of moderate selectivity (PCl/PNa = 4) that was not voltage-dependent, and (iv) an approx. 10 pS Cl- channel with characteristics similar to (iii) above, but remaining somewhat active at large negative voltages. All excised patches were exposed to relatively high calcium concentrations on the intracellular side. Channel activity was increased in tracheal cells treated with 1 mM cAMP, suggesting that at least one of these channels plays a role in the increase of the apical membrane Cl- conductance that is mediated by cAMP and elicited by agonists of active Cl- secretion.

Vascular smooth muscle membrane potential in rats with early and chronic one-kidney, one-clip hypertension.

It has been suggested that the cell membrane of vascular smooth muscle in one-kidney, one-clip hypertension, and other forms of volume-dependent, low-renin hypertension, is partially depolarized due to the effects of a circulating ouabain-like factor, and that this depolarization is an important mechanism of the hypertension. Levels of circulating ouabain-like factors in early stages of volume-dependent hypertension are reported equal to, or greater than, those in chronic hypertension. Therefore, we measured intracellular membrane potential (Em) in vitro (37 degrees C, physiological salt solution) in vascular smooth muscle of the caudal artery from normotensive control rats (1K) and rats in the early and chronic stages of one-kidney, one-clip hypertension (1K1C). In 20 chronic 1K1C (4-6 weeks of systolic pressure greater than 140 mm Hg) the resting Em's (M +/- SEM) were -46.7 +/- 0.7 mV, compared to -50.9 +/- 0.6 for 20 1K (P less than 0.01). The delta Em due to 1 mM ouabain was attenuated in 10 1K1C compared to 11 1K (+5.4 +/- 0.9 and +10.0 +/- 0.7 mV, respectively; P less than 0.01). The Em's of the two groups after ouabain were the same. In contrast, in 16 early 1K1C rats (less than 7 days hypertension, average 3 days) compared to 15 appropriate 1K, there were no significant alterations in resting Em (-50.1 +/- 0.4 mV, compared to -50.5 +/- 0.5, respectively) and there were no differences in ouabain response. These results suggest a temporal dissociation between levels of humoral inhibitors and depolarization, and between depolarization and hypertension, and thus fail to support the hypotheses that there are casual relationships between these variables in volume-dependent, low-renin hypertension.