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.

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.

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.

Diffusion resistance of endothelium and stroma of bullfrog cornea determined by potential response to K+.

Corneas of bullfrog (Rana catesbeiana) were mounted between lucite chambers. A four-electrode system was used and the potential difference (PD) and the electrical resistance were measured. In intact corneas, the PD averaged 25 mV (acqueous side positive) and the electrical resistance 1.5 kQ - cm2. perfusion of the aqueous side with high K+ solutions resulted in a marked decrease in PD and a drop in the electrical resistance. Scraping the epithelium (leaving the stroma plus endothelium) resulted in a drop of the PD to about zero and a decrease in electrical resistance to about 0.1 kQ - CM2 and a very small PD response to a marked elevation of the K+ concentration on the aqueous side. On the basis of the above, it is obvious that the large delta PD in intact corneas, due to elevation of the K+ concentration, must be due to K+ diffusing from the aqueous side across the endothelium and stroma and reaching the epithelium. The duration of the PD response is therefore a measure of the resistance to diffusion of the stroma plus endothelium. A quantitiative analysis shows that under in vitro conditions the resistance of the endothelium plus stroma to the diffusion of ions is very low.

Barium inhibition of sodium ion transport in toad bladder.

The effect of Ba2+ on Na+ transport and electrical characteristics of toad bladder was determined from change produced in short circuit current (Isc), epithelial, apical and basal-lateral potentials (psit, psia, psib), epithelial and membrane resistances (Rt, Ra, Rb) and shunt resistance (Rs). Mucosal Ba2+ had no effect. Serosal Ba2+ reduced Isc, psit, psia, and psib, but had no effect on Rt, Ra, Rb and Rs. Minimal effective Ba2+ concentration was 5-10(-5) M. The phenomenon was reversed by Ba2+ removal, but not by 86 mM serosal K+. Ba2+ inhibition of Isc did not impair the response to vasopressin which was quantitatively the same as controls. Psia with Ba2+ equalled psib. After Ba2+ inhibition, ouabain produced no further decrease in psit and Isc. Ba2+ exposure after ouabain did not decrease psit and Isc. The results suggest that Ba2+ inhibits the basal-lateral electrogenic Na+ pump.

Quantitation of conductance pathways in antral gastric mucosa.

The magnitude of cellular and shunt conductance of Necturus gastric antral mucosa was studied by (a) comparing the cellular PD response to transepithelial PD response during changes of ionic activity in the serosal bathing solution and (b) by measurement of current spread within the epithelial sheet. Using constant product KCl changes cellular resistance was 6,788 omegacm2 and shunt resistance was 1,803 omegacm2. Deletion of HCO3- from the serosal solution produced similar but quantitatively smaller changes in PD. Using HCO3- deletion cellular resistance was 7,338 omegacm2 and shunt resistance was 1,973 omegacm2. Measurement of current spead within the mucosa avoids changing ionic gradients yet gave very similar results; cellular resistance was 8,967 omegacm2 and shunt resistance was 2,947 omegacm2. The shunt contribution to transepithelial conductance ranged from 75.2 to 79.0%. Shunt selectivity was assessed using KCl dilution potentials, where mucosal dilution gave a small change in tissue PD compatible with an anion/cation selectivity ratio of 1.16 across the shunt, whereas serosal dilution effect was dominated by a PD change across the serosal membrane of the cell.

Cell-to-cell communication of osteoblasts.

Osteoblasts were investigated by two methods, electrical conductance and dye injection. Current injection into one cell caused a change in the recorded transmembrane potential of a second cell, indicating high conductance pathways between the two cells. Dyes injected into a single osteoblast were transmitted to numerous surrounding cells.

Transmembrane potentials of osteoblasts.

Transmembrane potentials were recorded in situ from osteoblasts on the parietal bones of albino rat pups, 3 to 24 days of age. Osteoblasts exhibit a uniquely low polarization of their cell membranes (3.93 mV, inside negative). Osteoblasts respond rapidly, but transiently, to parathyroid hormone by depolarization, and to thyrocalcitonin by hyperpolarization.

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.

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.

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)

Simple laser pulse energy monitor.

A simple and inexpensive relative energy monitor for short laser pulses is described. The three basic units of this system are an integrating detector circuit, an amplifier, and a peak detector and hold circuit. With this device one can measure optical pulse energies (at 1.06 microm) as small as 1.0 nJ with an electronic accuracy of approximately 1%.

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.

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.

Inhibition of H+ secretion in frog gastric mucosa by somatostatin.

Somatostatin added to the serosal bathing solution of the isolated gastric mucosa of Rana pipiens significantly inhibited pentagastrin- and histamine-stimulated H+ secretion. The decrease in H+ secretion rate was accompanied by an increase in the transmucosal potential difference and resistance. Somatostatin (10(-5) M) had no effect on the N6,O2-dibutyryl adenosine 3'-5'-cyclic monophosphate (DBcAMP)-stimulated H+ secretion rate. The mucosa exposed to somatostatin secreted H+ on stimulation by DBcAMP or histamine, but did not respond to 2.8 X 10(-7) M pentagastrin. However, pentagastrin added to the serosal solution stimulated H+ secretion after the somatostatin was washed away. Calcium inophore (3 X 10(-5) M) alone or 10(-2) M Ca2+ plus calcium ionophore temporarily increased the H+ secretion rate inhibited by somatostatin. The data suggest that somatostatin has a direct effect on the oxyntic cells in the gastric mucosa.