Michael A. Bridges

The calcium hypothesis of cystic fibrosis.

Data have been presented which suggests that various CF cell types show evidence of alterations in calcium homeostasis. The significance of these observations and the exact nature of the putative calcium defect in CF remains to be elucidated. It must also be determined whether this possible defect is primary, or is secondary or tertiary to some more basic lesion. The data reviewed suggests that altered calcium homeostasis may play some focal role in the aetiology or the pathogenesis of CF.

Fluorometric determination of nanogram quantities of protein in small samples: application to calcium-transport adenosine triphosphatase

A fluorometric micro protein assay based on fluorescamine-labelling of homogeneous proteins in solution has been developed which is capable of accurately quantitating as little as 25 ng protein at a concentration of 1.25 micrograms/ml. This micro assay uses a flow-through HPLC fluorescence detector. Typical micro assays measuring bovine serum albumin standards (0-25 mg/l) yielded linear regression coefficients of r = 0.999. Assays of purified Ca2+-ATPase solutions determined by the micro fluorescamine procedure correlated well with measurements made using the deoxycholate-TCA-precipitation modification of the Lowry assay: 1.0 microgram ATPase by Lowry method = 1.1 microgram protein by fluorescamine microassay (when both procedures were standardized with bovine serum albumin) (r = 0.995). The assay proposed offers a 100-fold increase in sensitivity, compared to the Lowry procedure.

Relationship between Ca2+-transport and ATP hydrolytic activities in guinea-pig pancreatic acinar plasma membranes

Partially purified plasma membrane fractions were prepared from guinea-pig pancreatic acini. These membrane preparations were found to contain an ATP-dependent Ca2+-transporter as well as a heterogenous ATP-hydrolytic activity. The Ca2+-transporter showed high affinity for Ca2+ (KCa2+ = 0.04 ± 0.01 μM), an apparent requirement for Mg2+ and high substrate specificity. The major component of ATPase activity could be stimulated by either Ca2+ or Mg2+ but showed a low affinity for these cations. At low concentrations, Mg2+ appeared to inhibit the Ca2+-dependent ATPase activity expressed by these membranes. However, in the presence of high Mg2+ concentration (0.5–1 mM), a high affinity Ca2+-dependent ATPase activity was observed (KCa2+ = 0.08 ± 0.02 μM). The hydrolytic activity showed little specificity towards ATP. Neither the Ca2+-transport nor high affinity Ca2+-ATPase activity were stimulated by calmodulin. The results demonstrate, in addition to a low affinity Ca2+ (or Mg+)-ATPase activity, the presence of both a high affinity Ca2+-pump and high affinity Ca2+-dependent ATPase. However, the high affinity Ca2+-ATPase activity does not appear to be the biochemical expression of the Ca2+-pump.

Coeliac disease: An analysis of aetiological possibilities and re-evaluation of the enzymopathic hypothesis

Although it is well established that components of wheat gluten and structurally-related cereal proteins produce intestinal damage in Coeliac disease (CD), the primary defect which confers upon the host susceptibility to these dietary substances remains a mystery. To date, three main hypotheses have been framed to explain this susceptibility, but none has yet been proven. Diagrammatic representation of these aetiologic hypotheses facilitates their analysis and illustrates the potential importance of a relatively ignored possibility, i.e., that there may be a defect in the intraluminal phase of the in vivo processing of dietary gluten. We suggest that future work should be directed towards investigating the potential role of abnormalities of this phase in the aetiology of CD.

Regulation of calcium transport in pancreatic acinar plasma membranes from guinea pig

The regulation of the guinea-pig pancreatic acinar plasma membrane Ca2+ pump by protein kinase A, protein kinase C and calmodulin was investigated. The results were compared with the effects of these regulators on the high affinity Ca2+-ATPase found in this membrane preparation. The catalytic subunit of cyclic AMP-dependent protein kinase stimulated Ca2+ transport 2-fold, but had no effect on Ca2+-dependent ATPase activity. Purified protein kinase C, the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate and diacylglycerol derivative, 1-stearoyl-2-arachidonoyl-sn-glycerol, failed to stimulate the Ca2+-uptake but augmented the Ca2+-dependent ATPase activity. Exogenously added calmodulin failed to stimulate either activity. In addition, two antagonists of calmodulin activity, trifluoperazine and compound 48/80 produced a concentration-dependent inhibition of Ca2+-transport. These data suggest the presence of endogenous calmodulin within guinea-pig pancreatic acinar plasma membranes. Both calmodulin antagonists failed to influence the Ca2+-dependent ATPase activity. The ability of boiled extracts from guinea-pig pancreatic acinar plasma membranes to stimulate the Ca2+-ATPase activity in calmodulin-depleted erythrocyte plasma membranes confirmed the presence of endogenous calmodulin. Our results imply a role for calmodulin and cAMP-dependent protein kinase, but not protein kinase C, in the regulation of Ca2+ efflux from pancreatic acinar cells. These results also provide further evidence suggesting that the high affinity Ca2+-ATPase does not catalyze the plasma membrane Ca2+-transport activity observed in pancreatic acini.

Investigation of (Ca2++Mg2+)-ATPase phosphoprotein formation in erythrocyte membranes of patients with cystic fibrosis

The (Ca2+ + Mg2+)-ATPase present per mg of protein in erythrocyte membranes of controls and patients with cystic fibrosis (CF) was determined by estimation of the levels of its phosphoprotein. In the presence of 10 mM free Ca2+, which inhibits phosphoprotein decomposition, significantly less phosphoprotein intermediate, ECaP, was found in erythrocyte membranes from CF patients than in age- and sex-matched controls; this correlated with a significant decrease in (Ca2+ + Mg2+)-ATPase activity. These observations indicate a decrease in the number of functional (Ca2+ + Mg2+)-ATPase molecules in erythrocyte membranes from CF patients or an alteration in either the structure of the pump protein or the composition of its environment.

Isolation and characterization of erythrocyte membrane Ca2+-ATPase in cystic fibrosis

ABSTRACT. Ca2+-ATPase was purified from erythrocyte membranes prepared from cystic fibrosis (CF) blood samples (n=10) and from age/sex-matched control blood samples (n=10). The kinetics of calcium activation of the purified enzyme was investigated in the presence of asolectin phospholipids and found to be virtually identical for both CF and control preparations: VCa2+=3.01 ± 0.24 µmol ATP hydrolyzed/mg pure enzyme/min (mean ± SE) and 3.09 ± 0.20 for CF and control Ca2+-ATPase, respectively; KCa2+=0.328 ± 0.046 /µmolar free calcium and 0.333 ± 0.040 for CF and control enzyme, respectively. The preparative procedure used (one-step purification by calmodulin-affinity chromatography) allowed quantitative recovery of essentially 100% of the Ca2+-ATPase present in detergent-solubilized erythrocyte membranes, enabling expression of the yield of purified enzyme in terms of the quantity of starting membrane protein: 0.127% ± 0.006% (w/w) and 0.140% ± 0.007% for CF and control enzyme preparations, respectively. None of the parameters evaluated showed a statistically significant difference (p< 0.05) between the CF and control groups. Furthermore, when CF and control purified Ca2+-ATPase samples were analyzed by high-resolution gradient SDS-polyacrylamide gel electrophoresis, no differences in mobility were observed (mol wt=128 kdaltons). Thus, Ca2+-ATPase purified from CF erythrocyte membranes and assayed in the presence of asolectin appears to be quantitatively similar to control purified enzyme in amount, molecular weight, and kinetics of activation by calcium. These data suggest that Ca2+- ATPase may not be the defective gene product in CF. Previous reports of deficient Ca2+-ATPase hydrolytic and transport activities observed in situ within CF plasma membranes may reflect an abnormality of membrane components in close association with the Ca2+-ATPase (e.g. lipid or protein activators or inhibitors).

Is cystic fibrosis a disease confined to epithelial cells

We have reviewed evidence regarding the basic tissue involvement in cystic fibrosis (CF). CF is a disease almost exclusively confined to epithelial tissues, particularly those of endodermal origin. Exocrine glands containing such epithelium typically show both obstructive and electrolyte secretory defects. To date most basic CF research has centered on study of mesodermally-derived tissues in which little pathology is seen. The focus of CF research should be changed to concentrate on epithelial tissues.

Purification and analysis of erythrocyte membrane Ca2+-ATPase from small samples of patient blood: Application to cystic fibrosis

A method is presented for the micro-scale isolation and characterization of erythrocyte membrane Ca(2+)-ATPase from small samples (7 mL) of whole human blood. Ca(2+)-ATPase isolated by this technique was more than 92% pure and showed calcium-activation characteristics similar to enzyme purified by standard macroscale procedures--viz maximal velocity of activation (VCA2+) = 15.5 +/- 1.2 mumol ATP hydrolysed/mg/min, and reciprocal of apparent affinity (KCa2+) = 0.73 +/- 0.15 microM free calcium (mean +/- SEM; n = 9). Using the isolation procedure described, purified Ca(2+)-ATPase could be prepared and assayed in a single working day. When the calcium-activation kinetics of cystic fibrosis erythrocyte membrane Ca(2+)-ATPase were reassessed using enzyme purified by this technique, VCa2+ and KCa2+ were not significantly different from normal values.

Purification of Ca2+-ATPase from rat pancreatic acinar plasma membranes using calmodulin-affinity chromatography.

Although a great deal is known about the stimulatory role of calcium in stimulus-secretion coupling, little is known about the mechanism by which intracellular calcium is regulated. The free calcium concentration in the acinar cell cytoplasm is typically between 10−5–10−7 M, in contrast to the much higher levels of calcium (approximately 10−3 M) in the extracellular space (Peterson, 1982). Calcium therefore penetrates into cells continuously down its electrochemical gradient by passive diffusion. In order to maintain a homeostatic environment in the cell and assure proper cell function, free calcium must be continuously removed.