Mannosylation of glycoproteins and dolichol derivatives in fibroblasts from patients with cystic fibrosis

Increased incorporation of mannose into endogenous glycoprotein fractions has been found in whole cell lysates and crude membrane preparations of cultured skin fibroblasts from patients with cystic fibrosis (1.3–2.3-times normal) when GDP[¹⁴C]mannose served as the mannosyl donor. In contrast, the incorporation of mannose from GDPmannose into lipid fractions containing dolichol phosphate and dolichol pyrophosphate oligosaccharides as well as the incorporation of mannose from dolichol phospho[³H]mannose into both glycoproteins and dolichol derivatives were not significantly different among cell preparations from patients with cystic fibrosis and normal controls. Mannosyltransferase activity toward exogenous glycoproteins as well as the activities of soluble and membranous α-mannosidase and β-mannosidase appeared to be normal and could not account for the observed differences. The altered incorporation of mannose into endogenous glycoprotein may reflect changes in glycosylation processes other than mannosylation.     

The effect of phentolamine on synaptosomal phosphatidylinositol in experimental galactose toxicity

Abnormal myo-[2-³H]inositol incorporation into phosphatidylinositol has been found in phentolamine-treated synaptosomes that were isolated from the cerebral hemispheres of galactose toxic rats and incubated with [³³P]P_i and myo-[2-³H] inositol. In galactose toxic rats phentolamine-stimulated myo-[2-³H]inositol labeling of phosphatidylinositol was 70% greater than in normal animals. This enhanced labeling of synaptosomal phosphatidylinositol in galactose toxic rats during stimulation with phentolamine is in marked contrast to the depressed myo-inositol labeling of phosphatidylinositol reported with acetylcholine stimulation.     

The influence of ionic strength on potassium contractures and calcium movements in frog muscle

Frog toe muscles were bathed in isotonic, sodium-free Tris chloride, methanesulfonate, or sulfate solutions containing sucrose or mannitol and varying in ionic strength from 0.006 to 0.291. By decreasing the ionic strength the curve relating the peak tension of the K contractures to the log [K] was reversibly shifted to lower [K]. Increasing the [Ca] from 1 to 4 mM almost abolished this effect. The resting uptake of ⁴⁵Ca was increased more than two times by decreasing the ionic strength from 0.125 to 0.039. It was not increased significantly by raising [Ca] from 1 to 4 mM at low or normal ionic strength. The additional uptake of ⁴⁵Ca during contractures provoked by 120 mM K was not significantly different at the two levels of ionic strength. The rate of emergence of ⁴⁵Ca from muscles loaded with ⁴⁵Ca at reduced ionic strength, was decreased. The effects of low ionic strength are discussed in terms of changes in the potential difference across a membrane with fixed negative charges on the outer surface.     

The role of calmodulin in the regulation of (Mg + Ca)-ATPase activity in erythrocyte membranes of cystic fibrosis patients and controls

(Mg²⁺ + Ca²⁺)-ATPase activity has been found to be significantly reduced in EDTA-washed erythrocyte membrane preparations from cystic fibrosis patients compared to aged-matched controls. Calmodulin was found to be present in erythrocytes from cystic fibrosis patients and characterized similarly to calmodulin isolated from control preparations. Calmodulin from control erythrocyte preparations stimulated the (Mg²⁺ + Ca²⁺)-ATPase activity of EDTA-washed erythrocyte membranes derived from cystic fibrosis patients to the same extent as those membranes derived from controls. Similarly, calmodulin obtained from erythrocytes of cystic fibrosis patients stimulated the (Mg²⁺ + Ca²⁺)-ATPase activity of control and cystic fibrosis erythrocyte membrane preparations to a similar extent. These results indicate that this decrease in (Mg²⁺ + Ca²⁺)-ATPase activity in erythrocytes from cystic fibrosis patients is not due to an alteration in the regulatory function of calmodulin.     

The role of calmodulin in the regulation of (Mg2+ + Ca2+)-ATPase activity in erythrocyte membranes of cystic fibrosis patients and controls

(Mg²⁺ + Ca²⁺)-ATPase activity has been found to be significantly reduced in EDTA-washed erythrocyte membrane preparations from cystic fibrosis patients compared to aged-matched controls. Calmodulin was found to be present in erythrocytes from cystic fibrosis patients and characterized similarly to calmodulin isolated from control preparations. Calmodulin from control erythrocyte preparations stimulated the (Mg²⁺ + Ca²⁺)-ATPase activity of EDTA-washed erythrocyte membranes derived from cystic fibrosis patients to the same extent as those membranes derived from controls. Similarly, calmodulin obtained from erythrocytes of cystic fibrosis patients stimulated the (Mg²⁺ + Ca²⁺)-ATPase activity of control and cystic fibrosis erythrocyte membrane preparations to a similar extent. These results indicate that this decrease in (Mg²⁺ + Ca²⁺)-ATPase activity in erythrocytes from cystic fibrosis patients is not due to an alteration in the regulatory function of calmodulin.     

Secretory activity of hamster tracheal explants and isolated tracheal epithelial cells and the effects of cystic fibrosis serum

Serum from cystic fibrosis patients has been shown by scanning electron microscopy to cause release of large quantities of mucus from the cultured tracheal rings of 3–4-month-old male Golden Syrian hamsters. In order to study this phenomenon on single cells, an epithelial (HTE) cell culture has been established from the hamster tracheal rings using the cell rescue method of Goldman and Baseman (1980a, In Vitro, 16:313). The cells were demonstrated to be epithelial by histochemical staining and immunofluorescent detection of laminin. Proteins secreted by HTE cells were partially characterized and shown to consist, at least in part, of acidic glycoproteins. The proteins were precipitated by addition of buffered alcian blue (AB) to the cell-free medium under conditions in which all of a polyanionic protein [³H]-labeled mucin, was precipitated without carrier. [¹⁴C] galactosamine-labeled AB precipitate was β-eliminated and, after neutralization and centrifugation, the material in the supernatant was sized by chromatography on a calibrated Bio-Gel P₂ column. The label eluted with a molecular weight close to a disaccharide. HTE cells pulse--labeled for 1.0 hr with [³H] leucine or [¹⁴C] galactosamine secreted increasing amounts of labeled glycoprotein during the chase. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography of labeled AB precipitates revealed three major bands, two with molecular weights greater than 100 kd. Secretion was stimulated by retinoate (50% increase), but not by retinol. Exposure of HTE cells to whole sera from cystic fibrosis patients resulted in heightened secretion rates as compared to results obtained with normal sera. Heterozygote sera produced secretion rates intermediate between the two extremes.     

Is cytoplasmic Ca2+ in lymphocytes elevated in cystic fibrosis

An increased cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) has been implicated in the pathogenesis of cystic fibrosis. We compared the [Ca²⁺]_i levels of normal and cystic fibrosis peripheral blood lymphocytes and Epstein-Barr virus-transformed lymphoblasts using quin 2, an internally trapped indicator. The [Ca²⁺]_i levels of normal and cystic fibrosis cells were not significantly different. The ionophore-releasable intracellular Ca²⁺ stores were also comparable in both types of individual.     

Impairment of pancreatic acinar function by reserpine in vivo and in vitro

Summary Chronic reserpine treatment of animals, an experimental model for cystic fibrosis (CF), results in generalized exocrinopathy, impaired pancreatic secretion, and decreased pancreatic content of amylase. The mechanisms of altered acinar function and decreased amylase content in both CF and the reserpine-treated rat are unknown. To examine this alteration, the rate of [³H]phenylalanine (phe) incorporation into cellular protein was determined in pancreatic acinar cells after reserpine treatment of rats in vivo (7 d) and of cells in vitro (1 to 24 h). Acinar cells isolated from control, chronic reserpine-treated, and pair-fed rats were incubated in vitro with 0, 30, 50, or 100 μM reserpine. Reserpine treatment in vitro for 24 h of acinar cells from control rats significantly decreased amylase activity (20 to 70%), an effect similar to that of reserpine treatment in vivo. In vivo, reserpine treatment decreased [³H]phe incorporation (disintegrations per minute per milligram protein) 56% in freshly isolated cells, but did not alter intracellular specific activity (disintegrations per minute per nanomole phe, SA) of [³H]phe. Reserpine treatment (30 and 50 μM) in vitro for 1 h also decreased [³H]phe incorporation by freshly isolated cells from control (53 to 85%) and pair-fed (40 to 68%) rats. Reserpine treatment for 24 h in vitro significantly decreased [³H]phe incorporation by cells from control (82 and 98%), pair-fed (80 and 95%), and chronic reserpine-treated (90 and 97%) rats as compared with cells from respective in vivo treatments cultured with no reserpine. In vitro reserpine treatment also decreased the intracellular SA of [³H]phe in freshly isolated cells from control (14 and 36%) and pair-fed (35 and 39%) rats and in cultured cells from control (11 and 86%), pair-fed (60 and 88%), and chronic reserpine-treated (49 and 76%) rats. However, these alterations of SA by reserpine did not account for the decreased incorporation of [³H]phe into acinar protein, which remained significantly lower (70 to 88%) when expressed as total phe incorporation. These results suggest (a) that reserpine acts directly on acinar cells to alter function and (b) that the ability of the pancreas to synthesize digestive enzymes may be impaired in this model of cystic fibrosis. This study was supported in part by the Cystic Fibrosis Foundation, Bethesda, MD.     

Glycosylation of VSV glycoprotein is similar in cystic fibrosis,heterozygous carrier,and normal human fibroblasts

The single envelope glycoprotein of vesicular stomatitis virus was used as a specific probe of glycosyltransferase activities in fibroblasts from two cystic fibrosis patients, an obligate heterozygous carrier and a normal individual. Gel filtration of pronasedigested glycopeptides from both purified virions and infected cell-associated VSV glycoprotein which had been labeled with [³H] glucosamine did not reveal any significant differences in the glycosylation patterns between the different cell cultures. All 4 cell lines were apparently able to synthesize the mannose- and glucosamine-containing core structure and branch chains terminating in sialic acid which are characteristic of asparagine-linked carbohydrate side chains in cellular glycoproteins. Analysis of tryptic glycopeptides by anion-exchange chromotography indicated that the same 2 major sites on the virus polypeptide were recognized and glycosylated in all 4 VSV-infected cell cultures. These studies suggest that the basic biochemical defect(s) in cystic fibrosis is not an absence or deficiency in enzymes responsible for the biosynthesis of complex carbohydrate side chains.     

Synthesis of sulfated oligosaccharides by cystic fibrosis trachea epithelial cells

The mucin glycoproteins in tracheal mucus of patients with cystic fibrosis is more highly sulfated than the corresponding secretions from healthy individuals [16]. In order to further characterize these differences in sulfation and possibly also glycosylation patterns, we compared the structures of sulfated mucin oligosaccharides synthesized by continuously cultured human tracheal cells transformed by siman virus 40. The synthesis of highly sulfated oligosaccharide chains in mucins secreted by normal human epithelial and submucosal cell lines were compared with mucins formed by cystic fibrosis tracheal epithelial and submucosal cell lines.The epithelial cell lines from cystic fibrosis trachea showed a higher rate of sulfate uptake and a significantly higher rate of synthesis and sulfation of high molecular weight chains. Mucins synthesized by each cell line in the presence of ³⁵SO₄ were isolated and oligosaccharide chains were released by beta-elimination and separated by ion exchange chromatography and gel filtration. The sulfated high molecular weight chains synthesized by the cystic fibrosis cell lines were characterized by methylation analysis and sequential glycosidase digestion before and after desulfation. Carbohydrate analysis yielded Fuc, Gal and GlcNAc in a ratio of 1:2:2.2 and only one galactosaminitol residue for about every 150-200 sugar residues present. The average molecular size of oligosaccharide chains in these fractions was between 30,000-40,000 daltons.These studies show that increased sulfation of oligosaccharides in mucins synthesized by cells from cystic fibrosis trachea is accompanied by a significant increase in the extension of a basic branched structure present in many of the lower molecular weight oligosaccharides.     

Amiloride is a cholinergic antagonist in the rabbit pancreas

The effect of amiloride on fluid and protein secretion in the isolated rabbit pancreas and on amylase secretion in rabbit pancreatic acini has been studied. Amiloride (1 mM) has no effect on the pancreatic fluid secretion either in a normal incubation medium (143 mM Na⁺), or in a medium containing only 25 mM Na⁺. The carbachol-induced enzyme secretion is inhibited by amiloride in both systems, whereas the enzyme secretion induced by the C-terminal octapeptide of cholecystokinin (PzO) is not affected. Amiloride also inhibits the carbachol-induced ⁴⁵Ca efflux from rabbit pancreatic acini, but again not that induced by PzO. The amiloride concentrations for half-maximal inhibition of carbachol-induced amylase secretion and ⁴⁵Ca efflux are 40 and 80 μM, respectively. Amiloride also competitively inhibits the specific binding of [³H]quinuclidinyl benzylate ([³H]QNB) to rabbit pancreatic acini, suggesting that the amiloride effect is due to competition on the level of the muscarinic acetylcholine receptor.     

Effects of amino acids on calcium uptake by glial and neuroblastoma cells

The uptake of [⁴⁵Ca] has been studied in clonal glial and neuronal cells. It was somewhat more efficient in the neuroblastoma clone M₁ compared to glial clones. In all cases [⁴⁵Ca] uptake was shown to depend on the phosphate concentration in the incubation medium. It was decreased by the ionophore A 23187 at 200 μM concentration in both neuronal and glial clones. The influence of amino acids some of which are putative neurotransmitters was investigated; the interactions between [⁴⁵Ca] uptake and these amino acids were related to their concentration and the type of cells used (neuronal or glial). L-aspartate and taurine for example had two opposite effects on [⁴⁵Ca] uptake by the glial clone NN at two different concentrations; they could therefore play a role in the control of calcium level in the synaptic cleft.     

Effect of elevated potassium on phospholipid and inositol metabolism of isolated nerve endings

Synaptosomes were isolated from rat cerebra, and incubated in the presence of labelled phosphate and inositol. When the potassium concentration of the medium was increased by replacing NaCl with KCl, there was a marked increase in phosphate labeling of phosphatidic acid (PA) and phosphatidylinositol (PI). This was evident with [K⁺] above 12 mM and peaked at about 40 mM KCl. In normal calcium buffers, phosphate labeling of PI but not PA declined sharply with [KCl] above 40 mM. In low calcium buffers, the phosphate labeling response was greatly attenuated for both lipids, but PI labeling did not decline at higher [K⁺].The phosphate labeling response was confined to PA and PI, and was specific for the increase in [K⁺]₀. The same response was seen in constant (105 mM) sodium buffers, and atropine had no effect. The specific radioactivity of ATP was increased by elevated potassium, but not enough to account for the increased labeling of PA. Further, this appeared to be a result of the loss of stored ATP rather than an increase in turnover.Increasing [K⁺]₀ produced a decline in [³H]inositol incorporation into PI in parallel with the increase in its labeling by ³³PO₄. This was the same in constant sodium and in low calcium buffers. It could be attributed to an inhibition of synaptosomal uptake of labelled inositol from the medium. Synaptosomal inositol content was unaffected.Elevated potassium had a greater effect on PA labeling than on PI, and it was more effective in increasing phosphate labeling of PA than was acetylcholine (ACh). When ACh and elevated potassium were combined at their maximally effective concentration, they acted synergistically to stimulate phosphate incorporation into PA but elevated potassium blocked the increase in [³H]inositol incorporation into PI normally produced by ACh. These results indicate that elevated potassium and ACh act upon the same population of synaptosomes, but affect different biochemical steps. Elevated potassium probably effects phospholipid labeling by a calcium dependent increase in diglyceride production from lipids other than PA or PI.     

Cytoplasmic free Ca in isolated snail neurons as revealed by fluorescent probe fura-2: Mechanisms of Ca recovery after Ca load and Ca release from intracellular stores

Summary Using the fluorescent probe fura-2, we measured the cytoplasmic concentration of free Ca²⁺ ([Ca] _i ) and its changes in isolated, nonidentified neurons of the snailHelix pomatia. [Ca] _i increased during membrane depolarization due to opening of Ca channels in the surface membrane. When the membrane potential returned to the resting level, [Ca] _i recovered monoexponentially, with the time constant ranging from 10 to 30 sec. The rate of recovery remained unchanged after treatments that interferred with the normal functioning of both Ca/Na exchange and Ca-ATPase in the surface membrane or mitochondria. [Ca] _i recovery slowed down upon cooling according to Q₁₀=2.3 and after intracellular injection of vanadate. The data obtained suggest that the rate of [Ca] _i recovery after membrane depolarization is mainly determined by Ca pump of intracellular stores (presumably by the endoplasmic reticulum). Ca release from these stores could be induced in the presence of millimolar caffeine or theophylline in the external medium when [Ca] _i increased up to a certain threshold level. This depolarization-induced Ca load triggered further transient increase in [Ca] _i , which was accompanied by membrane hyperpolarization due to the development of Ca-activated potassium conductance. 1mm procaine or tetracaine, but not lidocaine, inhibited this Ca-induced Ca release. In some cases stable oscillations of [Ca] _i were observed. They could be induced by producing a steady Ca influx by membrane depolarization.     

Cytosolic free calcium concentration and intracellular calcium distribution in lymphocytes from cystic fibrosis patients

Lymphocytes prepared from normal individuals and patients with cystic fibrosis (CF) were compared with regard to intercellular Ca²⁺ concentration, distribution, and handling. No difference between control and CF was found in the concentration of cytosolic free Ca²⁺ ($\text{98 ± 5}\text{vs}\text{102 ± 7}\text{nM}$), and no difference was observed in the kinetics with which control and CF cells restored cytoplasmic Ca²⁺ toward normal following a perturbation induced by cold-exposure. However, total intracellular Ca²⁺ is about 25% higher in CF lymphocytes than in control. Of this excess Ca²⁺, about 50% appears to be sequested in mitochondria. This suggests that some difference in Ca²⁺ handling does exist, but the significance of this cystic fibrosis remains to be determined.     

Stimulation of the incorporation of 32Pi and myo-(2-3H)inositol into the phosphoinositides in polymorphonuclear leukocytes during phagocytosis

The effect of phagocytosis on the incorporation of ³²P_i and myo-[2-³H]inositol into the phosphoinositides (phosphatidylinositol, diphosphoinositide, and triphosphoinositide) by polymorphonuclear leukocytes from guinea pig peritoneal exudates has been studied. The results show that phagocytosis enhanced the incorporation of ³²P_i and myo-[2-³H]inositol into all three inositides in polymorphonuclear leukocytes. Pulse-chase experiments revealed that phagocytosis did not stimulate the loss of the label from the inositides. The findings indicate that the increased radioactivity of the phosphoinositides in polymorphonuclear leukocytes during phagocytosis is due to a greater rate of synthesis of these phospholipids at the time of labeling, rather than due to an increase in the rate of their turnover.     

Lack of Differences in Serum Binding of Calcium in Cystic Fibrosis,Carriers and Controls

Attempts have been made to detect carriers for cystic fibrosis (CF) by some means other than genetic inference from affected children to their otherwise normal parents¹. The ability to detect CF carriers would result in substantial advances in genetic counselling. Moreover, an assay by which heterozygotes are identifiable must involve a parameter close to the elusive primary gene abnormality. Accordingly the paper by Fitz-patrick et al.² in which serum binding of tracer amounts of calcium (determined by equilibrium dialysis) of CF > heterozygotes > controls was a potentially significant report. We were, however, sceptical about data in which healthy carrier individuals demonstrated so great a difference from normal (an increase in serum binding of ⁴⁵Ca in heterozygotes over controls of 65 ± 11%) for a clearly nonspecific characteristic. As we have substantial serum and salivary electrophoretic data³ in which no consistent protein differences were apparent in carriers and because we wondered about the biological explanation of the findings of Fitzpatrick et al.², we decided to repeat their serum binding of calcium experiments. We repeated the reported procedures as closely as possible. We also assayed calcium binding with the additional buffer and salt concentration conditions described below. Sera from different individuals [N (controls = 30; N (heterozygotes) = 31; N (CF) = 33] were used in 4 experiments.     

Specific uncoupling of excitation and contraction in mammalian cardiac tissue by lanthanum

Arterially cannulated rabbit interventricular septal tissue was exposed to 5–40 µM La in 2.5 mM Ca perfusate. Immediately following perfusion with La two concurrent events were consistently observed: (a) a rapid decline of active tension to a lesser steady-state value, and (b) an abrupt, release of short duration of tissue-bound Ca. The magnitude of both events was directly related to the [La]_o. If the duration of exposure to La was brief, contractility returned toward normal upon return to the La-free perfusate. Elevation of [Ca]_o during exposure to La counteracted its effect and induced a concurrent displacement of tissue-bound La. Cellular action potentials recorded during brief perfusion with La demonstrated that essentially normal regenerative depolarization was maintained. Analysis of the quantities of ⁴⁵Ca released following exposure to 10 µM La indicated that this La-susceptible Ca was being displaced from a homogeneous pool—the one previously shown by Langer to represent contractile dependent Ca. These data led to the following conclusions: During perfusion with 2.5 mM Ca contractile dependent Ca was derived primarily from "superficially" located sites. La effected the release of contractile dependent Ca by modifying the normal permselectivity of this "superficial" membrane for activator Ca. These and other data infer that contractile dependent Ca is derived primarily from superficially located sites.     

Incorporation of [3H]thymidine into DNA and of [35S]sulfate into sulfatides of oligodendroglial cells during development: Effect of malnutrition

Incorporation of [³H]thymidine into DNA and of [³⁵S]sulfate into sulfatides of oligodendroglial cells isolated from brain slices incubated with the radioactive precursor was studied in normal and malnourished rats at different ages. The pattern and the values of incorporation of [³H]thymidine into DNA were similar in both groups of animals. The maximum value of incorporation was observed at 7 days of age decreasing rapidly thereafter and leveling off between 18–21 days. In both groups of animals labeling of sulfatides attained a maximum at 18 days of age, showing similar values of incorporation up to that age. However, at 21 days of age; the values corresponding to malnourished rats were found to be 40% lower in comparison to controls. The results suggest that (a) proliferation of oligodendroglial cells stops at similar ages in normal and malnourished rats, (b) expression of sulfatide synthesis by oligodendroglial cells is similar in both groups of animals up to 18 days, and (c) the starved rats seem to be unable to maintain normal synthesis of these galactolipids throughout the entire period of active myelinogenesis.     

Catabolism of Tritiated Thymidine by Aquatic Microbial Communities and Incorporation of Tritium into RNA and Protein

The incorporation of tritiated thymidine by five microbial ecosystems and the distribution of tritium into DNA, RNA, and protein were determined. All microbial assemblages tested exhibited significant labeling of RNA and protein (i.e., nonspecific labeling), as determined by differential acid-base hydrolysis. Nonspecific labeling was greatest in sediment samples, for which ≥95% of the tritium was recovered with the RNA and protein fractions. The percentage of tritium recovered in the DNA fraction ranged from 15 to 38% of the total labeled macromolecules recovered. Nonspecific labeling was independent of both incubation time and thymidine concentration over very wide ranges. Four different RNA hydrolysis reagents (KOH, NaOH, piperidine, and enzymes) solubilized tritium from cold trichloroacetic acid precipitates. High-pressure liquid chromatography separation of piperidine hydrolysates followed by measurement of isolated monophosphates confirmed the labeling of RNA and indicated that tritium was recovered primarily in CMP and AMP residues. We also evaluated the specificity of [2-³H]adenine incorporation into adenylate residues in both RNA and DNA in parallel with the [³H]thymidine experiments and compared the degree of nonspecific labeling by [³H]adenine with that derived from [³H]thymidine. Rapid catabolism of tritiated thymidine was evaluated by determining the disappearance of tritiated thymidine from the incubation medium and the appearance of degradation products by high-pressure liquid chromatography separation of the cell-free medium. Degradation product formation, including that of both volatile and nonvolatile compounds, was much greater than the rate of incorporation of tritium into stable macromolecules. The standard degradation pathway for thymidine coupled with utilization of Krebs cycle intermediates for the biosynthesis of amino acids, purines, and pyrimidines readily accounts for the observed nonspecific labeling in environmental samples.