Ove Ceder

The Reserpinized Rat in the Study of Cystic Fibrosis: X-ray Microanalysis of Submandibular Gland and Pancreas

The chronically reserpinized rat has been suggested as an animal model for cystic fibrosis. X-ray microanalysis of thick and thin cryosections was carried out to assess elemental redistribution in the submandibular glands and the pancreas of reserpinized rats at the cellular and subcellular level. In the submandibular gland of reserpinized rats, calcium and magnesium concentrations were significantly elevated. Mucus globules, secretory granules, and endoplasmic reticulum were the primary sites of the localization of excess calcium and magnesium. A significant potassium loss from the gland had occurred, particularly from the serous cells. Electron microscopy of conventionally prepared tissue showed marked swelling of the endoplasmic reticulum, especially in mucous cells. The elemental changes in the pancreatic acinar cells of reserpinized rats were reminiscent of elemental redistribution connected with cell death: increased levels of sodium, chlorine, and calcium and decreased levels of magnesium and potassium. Ultrastructural changes included swelling of the endoplasmic reticulum and obstruction of the acinar lumen. It is concluded tha elemental redistribution in chronically reserpinized rats presents interesting parallels with cystic fibrosis.

Electrolyte redistribution in cystic fibrosis fibroblasts studied by electron probe X-ray microanalysis.

The elemental composition of fibroblasts from cystic fibrosis (CF) patients and from healthy controls was compared by means of electron probe X-ray micro-analysis. Significantly lower sodium levels (p < .01) and higher calcium levels (p < .02) were found in CF fibroblasts, indicating a disturbance of ion regulation in these cells. Treatment with Staphylococcus aureus protein A and gammaglobulin (proteins that may influence the ciliotoxic CF factor) did not change the elemental composition of the fibroblasts.

Acid hydrolases in sera and plasma from patients with cystic fibrosis.

The enzyme activities of alpha-fucosidase (pH 4.0 and pH 5.5), alpha-galactosidase, beta-galactosidase, alpha-glucosidase (pH 4.5 and pH 6.0), beta-glucosidase, beta-glucuronidase, beta-hexosaminidase, and alpha-mannosidase (pH 4.5 and pH 5.5) were investigated in sera from cystic fibrosis (CF) patients. Several of these activities were significantly increased in sera from patients compared to age-matched control children. CF-patients in a more advanced stage of the disease had a tendency to higher values of some of these hydrolases than those in better condition. No new isoenzymes of these hydrolases were found. Only minor differences could be detected in the pH-profiles of alpha-mannosidase and acid phosphatase from age-matched normal controls, heterozygotes and homozygotes for CF. With our technique, alpha-mannosidase and acid phosphatase showed the same thermostability in CF-patients. CF-heterozygotes and age-matched controls, except at 56 degrees C, when the activity of acid-phosphatase in the plasma from adult CF-heterozygotes decreased more than that from adult controls

Ribonuclease in Different Types of Saliva from Cystic Fibrosis Patients

ABSTRACT. The flow rate, the activity of ribonuclease (RNAase) and the concentration of protein were determined in whole saliva and in parotid and submandibular saliva from patients with cystic fibrosis (CF) and from healthy controls, both before and after stimulation of the salivary secretion. Lower flow rates were found in all types of saliva from CF‐patients than in control saliva. Increased activity of RNAase was found in CF saliva, both before and after stimulation of the secretion. The concentration of protein was also increased, but to a lesser degree. No correlation was found between either RNAase activity or protein concentration and severity of the disease or age of the children. It is therefore unlikely that these disturbances are secondary to progression of the disease. Considerable variations in RNAase activity and protein concentration were observed, especially within the CF group, and therefore despite the significant increase in these variables in CF their estimation is of limited diagnostic value.

Salivary Ribonuclease in Cystic Fibrosis and Control Subjects

ABSTRACT. Alkaline acid‐ and thermostable ribonuclease (RNAase) was assayed in whole mixed saliva from controls and from cystic fibrosis (CF) heterozygotes and homozygotes. There was a significant difference in salivary RNAase activity between control adults (n= 99) and CF heterozygotes (n= 77) (12.6 ± 0.60 and 36.6 ± 2.2 U/l, mean ± SE respectively; p < 0.001) and between control children (n= 1834) and CF patients (n= 60) (7.9 ± 0.15 and 47.0 ± 5.4 U/l respectively; p < 0.001). A statistically significant difference in salivary RNAase activity was also found between control children and control adults (p < 0.001) and between CF homozygotes and CF heterozygotes (p < 0.001). The protein concentration was significantly increased by about 50–60 % in saliva both from CF heterozygotes and from CF homozygotes (p < 0.001 for both groups). It is concluded that in view of the great overlapping in values between the groups, these tests can only be of limited use for diagnostic purposes.

Effects of Cystic Fibrosis Serum and Fibroblast Culture Medium on Ion Distribution in Rat Submandibular Gland

The effects of cystic fibrosis (CF) serum and culture medium from CF fibroblasts on ion distribution in rat submandibular gland cells were investigated by X-ray microanalysis. These effects were compared to the effects of normal serum and culture medium from normal fibroblasts, of cholinergic and adrenergic agonists, and of the uncoupler 2,4-dinitrophenol. Incubation of gland tissue with CF serum or normal serum caused a significant decrease in potassium and calcium concentrations and an increase in sodium in mucous acinar and serous granular duct cells. CF serum gave a significantly larger decrease of the potassium level than normal serum. Culture medium from CF fibroblasts altered the cellular ion content in a way similar to CF serum. Exposure to medium from cultured normal fibroblasts did not affect the elemental composition of the gland cells significantly, compared to incubation with fresh medium or buffer. Hence, fibroblast culture medium is more suitable than serum to test specific effects of CF-associated factors. The changes in elemental composition of gland cells caused by CF serum or CF fibroblast culture medium mimic some of the effects of the agonist carbachol. They could, however, also in part result from nonspecific changes in membrane permeability.

Effects of Culture Medium on Cystic Fibrosis and Normal Fibroblasts Studied by X-Ray Microanalysis

The effect of culture medium from fibroblast cultures of cystic fibrosis (CF) patients and healthy controls on the elemental composition of fibroblasts was investigated by X-ray microanalysis. Exposure of normal fibroblasts to culture medium from CF fibroblasts caused an increase in calcium level. Exposure of CF fibroblasts to culture medium from normal cells caused an increase of the sodium content of CF cells to approximately normal levels; the calcium level of the CF fibroblasts, however, remained abnormally high. The results may indicate that CF fibroblasts lack a factor needed for the regulation of sodium transport. CF fibroblast medium apparently contains a factor that interferes with the regulation of calcium transport.

Effect of chronic treatment with cystic fibrosis fibroblast medium on rat submandibular gland acinar cells.

The effect of chronic treatment with cystic fibrosis (CF) fibroblast medium on rat submandibular gland and pancreas was investigated. Rats were injected for 8 days with conditioned medium from normal or CF fibroblasts. The elemental content of the acinar cells was measured by X-ray microanalysis of cryosections. A significant increase in cellular calcium, and a decrease in cellular sodium concentrations were found after treatment with CF medium. The ultrastructure of the submandibular acinar cells was not affected by the conditioned CF fibroblast culture medium. No effect of treatment with CF medium on ultrastructure and elemental content of pancreatic acinar cells could be demonstrated. The response to alpha-adrenergic, beta-adrenergic, cholinergic, and peptidergic stimulation in submandibular gland acinar cells of rats injected with normal or CF medium was investigated in vitro. With regard to changes in elemental composition after stimulation, no significant differences in response between the two groups could be found. Apparently, a factor in conditioned medium from cultured CF fibroblasts induces a net increase in calcium content of rat submandibular gland acinar cells. Possibly, this factor acts in a similar way in CF patients and may cause elevated calcium levels in CF cells.

Diagnosis of cystic fibrosis homozygotes and heterozygotes from plasma and fibroblast cultures. A three-generation family study

The diagnosis of cystic fibrosis (CF) homozygotes and heterozygotes and of individuals without the CF gene, based on differences in the thermal stability of acid phosphatase and a‐mannosidase, is reported. The residual activities at 36.5°C and 41.3°C were below 10% of the activity in unheated samples for homozygotes, 4&50% for heterozygotes and above 90% for normals. The intracellular alkaline phosphatase and extracellular β‐hexosaminidase activities after treatment with heparin and gammaglobulin were 500% and 200%, respectively, of the activities without this treatment in CF homozygotes, whereas for heterozygotes and normals the values were the same after treatment as before. Pedigrees of four CF families, covering 2–3 generations, are presented. The possible use of these tests as diagnostic tools is further discussed.

Microprobe Analysis in Studies and Diagnosis of Cystic Fibrosisa

Cystic fibrosis (CF) is a generalized exocrinopathy with diverse clinical symptoms, of which chronic obstructive lung disease is the most serious. With an incidence of 1 :2,000 to 1 :4,000 newborns, C F is the most common lethal, congenital, genetic disease among Caucasians. Despite considerable progress in the treatment of the symptoms, still about twenty percent of the patients do not survive beyond the age of twenty.’-3 Several exocrine secretions of C F patients have an abnormal ionic composition: elevated Na, CI, and K in sweat,24 elevated Ca, Na, and C1 in submandibular ~ a l i v a , ~ . ~ and elevated Ca in parotid ~ a l i v a . ~ There is also excessive salivary protein ~ e c r e t i o n . ~ . ~ The secretion of a viscous fluid of abnormal ionic composition by exocrine glands could well be the underlying factor in many of the clinical symptoms of CF, such as blocking of the airways by viscous mucus associated with recurrent and persistent infections, exocrine pancreatic insufficiency, meconium ileus, and infertility in males. The abnormal composition of sweat is routinely used in diagnosis of CF.4 The molecular basis of the disease is not known. Considerable attention has, of course, been given to possible abnormalities in the regulation of ion transport in CF. Decreased ductal reabsorption of Na+,8 possibly caused by increased primary secretion of Ca2+, and decreased ductal reabsorption of chloride ions,’ are among the suggested mechanisms. Also of interest is the activity of a “humoral C F factor” (or, possibly, several “CF factors”), present in serum, saliva, and urine from C F patients. Originally of interest because of its ciliostatic properties,” the C F factor may also be responsible for increased secretion of proteins’’ and Kf12 and decreased ductal reabsorption of N a + ” in salivary glands in experimental animal systems. The nature of this hypothetical C F factor, or its relationship to the molecular basis or the clinical symptoms of the disease, is, however, not known at present. The relevance of abnormal ion transport for a t least some of the clinical symptoms of CF, combined with the possibility of studying abnormalities in ion distribution at the