Beat Hadorn

INTESTINAL ENTEROKINASE DEFICIENCY

Abstract An infant presenting with diarrhœa, failure to thrive, and hypoproteinaemic œdema was shown to have deficiency of intestinal enterokinase which resulted in failure to activate pancreatic proteolytic enzymes.

Partial purification and characterization of a chymotrypsin-like enzyme from human neutrophil leucocytes

Abstract A chymotrypsin-like enzyme with substrate specificity resembling pancreatic α-chymotrypsin was isolated in small quantities from human neutrophil leucocytes. The enzyme has activity against a variety of chymotrypsin substrates containing a tyrosine residue. It is inhibited by diisopropylphosphorofluoridate and by l -1-tosylamide-2-phenylethylchloromethylketone, suggesting the presence of serine and histidine in the active center. In addition the enzymatic activity is sensitive to the redox reagent 1,4-dithioerythritol. The behaviour of the enzyme in three different separation systems suggested that the molecular weight of the leucocyte enzyme and of its subunits obtained after treatment with 1,4-dithioerythritol is about twice as great as that of pancreatic α-chymotrypsin. The enzyme appears to be localised in similar granules to those containing peroxidase and arylsulfatase A.

Intestinal Enterokinase Deficiency: A Newly-recognized Disorder of Protein Digestion

Enterokinase is an intestinal enzyme necessary for the activation of pancreatic proteolytic zymogens. Two children with absence of enterokinase activity in the duodenal juice and intestinal mucosa are described. Both had diarrhoea from birth and failed to thrive; one developed hypoproteinaemic oedema. Oral administration of pancreatic extract resulted in prompt control of symptoms and acceleration of growth. A simple screening test for enterokinase deficiency is described.

PANCREOZYMIN ‐ SECRETIN TEST OF EXOCRINE PANCREATIC FUNCTION IN CYSTIC FIBROSIS AND THE SIGNIFICANCE OF THE RESULT FOR THE PATHOGENESIS OF THE DISEASE*

Pancreozymin‐secretin tests were performed on 10 children with cystic fibrosis of the pancreas, who did not show any clinical symptoms of exocrine pancreatic insufficiency. The results indicate that in this particular group of patients water and bicarbonate secretion from the pancreas is more severely affected by the disease than enzyme secretion. Thus these patients secrete a small amount of pancreatic juice with a very low bicarbonate content but an abnormally high concentration of enzymes.

CYSTIC FIBROSIS OF THE PANCREAS

Abstract From current knowledge of the normal secretory mechanisms in exocrine glands and their abnormal activity in patients with cystic fibrosis of the pancreas, a hypothesis has been developed which relates the disturbance in mucous and serous glands in the disease to a single biochemical abnormality—namely, inhibition of the movement of water and ions through the secretory cells in the glands affected. It is further suggested that this disturbance resides in the extracellular space, and is related to the structure or porosity of mucopolysaccharide-rich regions, either in the connective tissue proper or in a mucoid layer immediately adjacent to the cell plasma-membrane. This concept is illustrated in the accompanying figure. The primary genetic abnormality is still unidentified; however, the wide spectrum of changes observed in different patients may mean that the basic defect is a number of metabolic steps away from the final pathological manifestations of the disease. Mechanisms, possibly hormonal in nature, which control or can modify the movement of water and electrolytes in the extracellular regions of exocrine glands throughout the body should therefore be considered.

Enhanced adenosine 3':5' - monophosphate response to beta-adrenergic stimulation in cystic fibrosis fibroblasts after removal of conditioned medium.

Summary: Skin fibroblasts derived from 6 patients with cystic fibrosis (CF), 1–6 months old, and from 6 age matched donors were investigated for their ability to accumulate cyclic adenosine 3‘:5’-monophosphate (c-AMP) in response to isoproterenol and prostaglandin E1 (PGE1) using strictly defined culture conditions. In order to obtain, as far as possible,constant protein content and cell number, cultures were synchronized in the early G. phase of the cell cycle by growing them in serum free medium before adding stimulating drugs. There were no statistically significant differences both in basal c-AMP or after incubation with theophylli-ne alone. When cultures were thoroughly washed prior to stimulation, c-AMP accumulation in response to isoproterenol was consistently higher (p <0.001) in CF than in normal fibroblasts, whereas response to PGE1 did not differ significantly. This difference in response cannot be attributed to differences in dose-or time-response curves, or to differential escape of cAMP into the culture medium. Returning the conditioned media (CM) to the cultures after the washing procedure, or omitting the washing procedure altogether, normalized the cAMP response of CF cells. These data indicate that CF fibroblasts “delete” or “add to” the conditioned medium a substance when washed out of the cultures leave the cells hypersensitized to β-adrenergic stimulation.Speculation: The enhanced cAMP response to isoproterenol stimulation of CF-cells is most likely related to the washing off of a soluble substance which is secreted or deleted into the culture medium in excess of normals. This “factor” could exert its effect in the manner of an antagonist regulating catecholamine responsiveness as a part of a negative feed back loop.Water, electrolyte and macromolecular secretions of exocrine cells are controlled by the action of neurotransmitters of the autonomic nervous system (ANS) (11). Since in CF the disturbance of the exocrine secretions is one of the main symptoms, ANS regulated functions have repeatedly been investigated. Clinical observations in CF patients have demonstrated abnormal pupillary reactions (28) and a clearing of the abnormal turbidity of the saliva after guanethidine treatment, which is an antiadrenergic agent (9). Furthermore a paradoxical discordance between the pulmonary response of the CF-patients to isoproterenol and the response to oral theophylline has been shown (30). In rats, chronically treated with isoproterenol or reserpine, morphological and secretory changes of the salivary glands were reported that strongly resembled the ones observed in CF (31, 21, 22, 23). All these observations lend support to the concept that the primary defect in CF could be linked to a derangement of the ANS in the patients. Adrenergic neurotransmitters stimulate their target cells through specific binding to α and β-receptor sites on the surface of the cells. As a consequence of the binding to β-receptors, cAMP is formed from ATP by the activation of the enzyme adenylate cyclase present in the plasma membranes of the cells. The synthesis of cAMP secondarily induces α chain of biochemical events of the cell to the external stimulus (e.g.) the secretion of amylase in the salivary glands (7) or the sodium and the bicarbonate transport in the exocrine pancreas (8). The presence of β-receptors and adenylate cyclase has been demonstrated in many eucariotic cells, including cultured human diploid fibroblasts. Therefore it seems likely that a genetic disturbance of this mechanism would also be expressed in cultured fibroblasts. On the cellular level in vitro the measurement of cAMP formation after β-adrenergic stimuli might be as relevant as the measurement of the final cellular response induced by the cAMP in vivo.Hitherto, studies on cAMP responses to isoproterenol stimulation with cultured fibroblasts from CF patients have yielded contradictory results (5, 6, 10). It is difficult, however, to compare the results of the different studies since many factors can modify the cyclic nucleotide synthesis in cultured cells. Culture conditions such as passage number (14), cell density (16), time after subculture (20), serum concentration (19) and stage of the cell cycle (25) have been shown to influence significantly the response of the cAMP system. We therefore reinvestigated cAMP response to β-adrenergic stimuli in cultured fibroblasts from CF-patients and from normal controls using strictly defined culture conditions and examined the contribution of the conditioned culture medium to this system.

Effect of secretin on human rectal mucosa in vivo.

IT is now more than 60 years since secretin was discovered and since its significance with respect to pancreatic and biliary secretion was recognized1. Later investigations have led to the conclusion that pancreatic juice2 and bile3 can both be considered to be a mixture of two fluid components which are probably secreted by different cells and under independent physiological control; that is, they are made up of a fluid which is rich in electrolytes and a fraction containing a high concentration of the macromolecular secretory products of the exocrine cells. The principal effect of secretin seems to be to stimulate formation of the “electrolyte fraction”, but its precise mode of action is still unknown.

ISOLATION OF APICAL MEMBRANES FROM HORMONE-SENSITIVE RAT COLONIC CRYPT CELLS

In order to study hormone-dependent transport properties of VIP-sensitive colonic crypt cells were prepared using chelators and mechanical forces. Thymidine kinase activities in crypt cells and absorptive cells were 77±8 and 9±2 nmol/min/mg protein respectively. Hormone sensitivity was shown by a dose-dependent increase of cAMP levels up to 18-fold over basal with 10−6M VIP. From this preparation apical membranes were isolated by standard procedures. Membrane purity was assessed by electron microscopy and marker enzyme assays: In the final membrane preparation increase of relative specific activity (nmol/min/mg protein) - as compared to the intact cells – was 10-fold for alkaline phosphatase, 2-fold for Na–K–ATPase. Succinate dehydrogenase was decreased by 50%.Conclusions: 1.Thymidine kinase concentrat ion in isolated colonic crypt cells is 8-fold higher than in absorptive cells. 2.This preparation is VIP-sensitive. 3. Apical crypt cell membranes were partially purified (ALP: 10-fold) with only minor contamination with basolateral membranes and mitochondria.

“Big chymotrypsin” from human leucocytes

A chymotrypsin-like enzyme with substrate specificity resembling pancreatic α-chymotrypsin was isolated in small quantities from human polymorphonuclear leucocytes and compared with pancreatic chymotrypsin. The enzyme had activity against a variety of chymotrypsin substrates containing tyrosin residues. It was inhibited by DFP and by TPCK suggesting the presence of serine and histidine residues in the active center. However, the inhibition by TPCK was slower for the leucocyte enzyme than for pancreatic chymotrypsin. The behaviour of the enzyme in three different separation systems suggested that the molecular weight of the leucocyte enzyme and of its subunits obtained after treatment with DTT was about twice as great as for pancreatic chymotrypsin. The enzyme was localized in the cytosol and no association with the leucocyte granules could be demonstrated. The physiological role of “big leucocyte chymotrypsin” is at the present time unknown. It may be involved in phagocytosis or in the immunological response of the cell.DFP = DiisopropylphosphofluorideTPCK=L-1-tosylamide-2-phenylethychloromethylketoneDTT=Dithioerythrite