Nona R. McSherry

Sodium Transport: Inhibitory Factor in Sweat of Patients with Cystic Fibrosis

A factor inhibitory to sodium transport exists in the sweat of patients with cystic fibrosis of the pancreas. When the duct system of the rat parotid was perfused with sweat from patients, marked inhibition of sodium reabsorption was observed. Perfusion with sweat from normal subjects caused no change in sodium reabsorption. The factor thus demonstrated may be responsible for the increased sodium concentrations in the sweat of patients with cystic fibrosis.

A Sodium Transport Inhibitory Factor in the Saliva of Patients with Cystic Fibrosis of the Pancreas

Extract: The saliva of patients with cystic fibrosis of the pancreas (CFP) contains higher concentrations of sodium than the saliva of normal subjects. The hypothesis that the salivary electrolyte abnormality in CFP may be due to a sodium transport inhibitory factor (or factors) present in the saliva of these patients was investigated. Such a factor would decrease sodium reabsorption in the salivary glands and cause increased sodium concentrations in the final saliva. Retrograde perfusion of the rat parotid gland was used to determine this factor. Retrograde perfusion with normal saline or saliva from normal children did not affect the rate of sodium reabsorption in the rat parotid gland. Retrograde perfusion with ouabain containing saline or saliva from patients with CFP resulted in a marked decrease of the rate of sodium reabsorption. It was concluded that saliva from patients with CFP contains a factor that causes inhibition of sodium transport in the rat parotid gland. The similarity of the effects of ouabain and saliva from patients with CFP led us to the investigation of the possibility of a common mode of action. It was demonstrated that the saliva of patients had no effect on the in vitro activity of membrane ATPase preparations obtained from beef parotid, human erythrocytes and rat kidneys. This indicates that the saliva of patients with CFP causes inhibition of sodium reabsorption in the rat parotid by an action other than inhibition of the activity of ATPase, an enzyme known to be involved in sodium transport across biological membranes.Speculation: A factor that inhibits sodium reabsorption in the rat parotid gland has been demonstrated in the saliva of patients with CFP. The same factor may be causing the salivary electrolyte abnormality of CFP. If this is true, this factor should be present in the sweat of patients with CFP since the elctrolyte abnormality is maximally expressed in the sweat glands. It is hoped that study of this factor may lead us closer to the nature of the molecular defect of CFP.

Response of sweat electrolyte concentrations to 9 alpha-fluorohydrocortisone in patients with cystic fibrosis and their families

Parents and siblings of patients with cystic fibrosis were found to have decreases in concentrations of sweat sodium and chloride proportionate to those of control adults and children after the administration of 3.0 mg. per square meter of 9-alpha-fluorohydrocortisone for 2 days. Thus the heterozygous state was not detected. Patients with cystic fibrosis, including 1 adult, showed no significant decrease in these sweat electrolytes with the same challenge. This lack of response to corticosteroids could be used to confirm the diagnosis of cystic fibrosis in adults since abnormally high sweat electrolyte concentrations are more difficult to detect in this age group.

Micropuncture study of sodium and potassium excretion in rat parotid saliva: role of aldosterone.

Recent studies (1, 2) of salivary gland function have demonstrated that the rat parotid gland, like the parotids of man and dog (3, 4), produces saliva hypotonic to plasma. Micropuncture and microanalytical techniques have been used to demonstrate that salivary hypotonicity is produced in the striated ducts of the gland by reabsorption of sodium in excess of water from a plasma-like primary secretion (2). The cellular structure of the striated ducts is similar to the structure of the distal tubules of the kidney (5). Since previous studies have indicated that the major action of aldosterone is on the distal tubules of the mammalian nephron (6), we undertook the present study of the role of aldosterone on the function of the rat parotid gland. The effects of this hormone on the handling of sodium and potassium by the parotid in general and the striated ducts in particular were investigated before and after adrenalectomy. The ability of exogenous aldosterone to reestablish normal handling of sodium and potassium by the rat parotid in the adrenalectomized state was investigated. Methods and Materials. Male, albino rats of the F.W. 49 and Sprague-Dawley strains, weighing 150–200 g, were used in these experiments. The animals were fasted for 24 hr prior to the experiments but were allowed free access to water. They were anesthetized with either Inactin (sodium 5-ethyl-5-methyl-propyl-2-thiobarbiturate) or sodium pentobarbital in doses of approximately 8.0 mg/100 g of body weight. The methods of surgical preparation of the animals, cannulation of the main ducts, and micropuncture of the intercalated and lobular ducts have been described in previous publications (1, 2).

Transductal fluxes of anions in the rat pancreas

Recent studies of the rat pancreas (1) have shown that during stimulation of the gland with exogenous secretin, the primary secretory fluid which was produced in the acini-intercalated ducts was isotonic to plasma. The concentrations of Na and K of this fluid were identical to those of plasma while the concentrations of Cl and HCO3 were higher. Simultaneous measurements of the concentrations of these ions in the primary secretory fluid and in the final pancreatic juice revealed that exchanges of Cl and HCO3 took place across the ducts of the gland while there were no net transductal fluxes of Na and K. The present experiments demonstrate that the transductal fluxes of anions in the rat pancreas during stimulation with exogenous secretin are regulated by this hormone. In the absence of exogenous secretin in the unstimulated gland or during stimulation of secretion with pilocarpine or pancreozymin (CCK-PZ) there are no transductal fluxes of anions both in the whole gland or in segments of the interlobular ducts perfused by the split-oil droplet method of stationary microperfusion. Under these conditions, the ionic composition of the final pancreatic juice is identical to that of the primary secretory fluid. During stimulation of secretion with porcine secretin, infused intravenously, activation of a transductal mechanism of exchange of anions against concentration gradients appears to regulate the final anionic composition of the rat pancreatic juice. Methods and Materials. Male, albino rats of the Sprague-Dawley strain, weighing approximately 200 g were used in these experiments. The methods of surgical preparation of the animals, stimulation of pancreatic secretion, collection of samples, and calculation of the flow rates have been described in previous publications (1, 2). The method of split-oil droplet stationary microperfusion developed by Shipp et al. (3) as modified by Gertz et al. (4) was used for the study of chloride fluxes in segments of the pancreatic ducts in situ. The ducts perfused were interlobular ducts of 60–90 μm external diameter.

Salivary Gland Enlargement and Functional Changes During Feeding of Pancreatin to Rats: (Possible Relation to Functional Changes in Cystic Fibrosis)

Extract: Enlargement and functionl alterations of salivary glands were sutdied in male rats of the Sprague-Dawley strain fed a diet contaning 4% pancreatin for 3 weeks. The weight of the parotid and submaxillary glands increased 1.7 and 2.7 times, respectively. When rats were fed 4% pancreation heated to 100% for 3 h no enlargement of the glands occurred; sililary, gastric intubation of the 4% pancreatin preparation had no effect on the size of the salivary glands. A marked increase in enlargement of the salivary gland occurred in rats fed 4% pancreatin and given subcutaneous injections of 5 mg theophylline twice daily. The functional alterations of the salivary glands included: a) parotid: salivary flow rates in response to the intravenous injection of a standard dose pilocarpine (0.1 mg/100 g body weight) were slightly lower in expermental rats than those observed in the parotid glands of control rats; sodium and calcium concentrations were higher in the saliva of expermenal rats than in the saliva of controls; potassium concentrations did not differ; rats fed heated pancreatin had no ministration of the standard dose of pilocarpine were significantly lower than those in the control rats; sodium, potassium and calcium concentraions were higher in the saliva of experimental rats than in the saliva of control rats at conmparable flow rates; the salivary protein concentrations were higher. In rats fed the 4% pancreatin preparation, the saliva of both glands exhibited marked changes in the electrophoretic patterns of excretion of basic secretory proteins.The similarities of these structural and functional changes observed in rats fed pancreation and those observed in patients with cystic fibrosis of the pancreas (CFP) suggest that a similar mechanism may be responsible in both cases. Study of this mechanism, particularly the biochemical events of intracelluar transmission of neurogenic stimuli and their chemical mediators such as cycle AMP, may help clarify the pathogenesis of CFP.Speculation: The sialadentrophic and functional effects observed in the salivary glands of rats when pancreatin was added to the feeds are similar to the change observed in the salivary glands of patients with cystic fibrosis of the pancreas (CFP). The action of these preparations depends on a neural reflex mechanism involving the autonomic nerve suplly to the glands. A similar alteration involving the transmission of stimuli from the autonomic nervous system to the secretory elements of exocrine glands may be pat of the mechanism of pathogenesis of CFP.

Secretory function of isolated parotid acinar cells

Functionally and anatomically intact acinar cells were obtained from the parotid gland of the rat by enzymatic dispersion. After exsanguination of the animal, the duct system of the gland was filled with a solution of hyaluronidase and collagenase (0.1%) in Ca±Mg free Hanks salt medium. The parotid was removed, minced and the tissue was incubated in the same enzyme solution for 60 min at 37°C. Then, the suspension was filtered through nylon mesh, the clumps of cells were separated by centrifugation, were suspended in a solution of trypsin (0.1%) and reincubated for 20 min. Finally, the cells were dispersed by pipetting the suspension 10 times, washed and then dispersed by pipetting the suspension 10 times, washed and then suspended in balanced Hanks solution containing bovine serum albumin (2 gm/100 ml). Yield of cells was ≃60% of the original gland tissue. The cells were anatomically intact and did not stain with 0.2% trypan blue for up to 8 hours. They showed normal respiration (O2 uptake: 21.4 ± 2.7 μl/hours·mg protein) and responded to stimulants of sectory activity (epinephrine, isoproterenol, dibutyryl cyclic. AMP, theophylline and NaF) by secreting amylase into the medim at rates approximately equal to those observed in the intact gland in situ.This method permits the investigation of aspects of the secretory processes of exocrine glands which cannot be studied in the intact glands of experimental animals in situ or in gland slices. Furthermore, as preliminary experimetns have shown, living acinar cells for similar secretory studies can be obtained from human salivary glands and pancreas immediately after death.