ENHANCED ADENOSINE 3': 5' - MONOPHOSPHATE RESPONSE TO BETAADRENERGIC STIMULATION IN CYSTIC FIBROSIS FIBROBLASTS AFTER REMOVAL OF CONDITIONED MEDIUM

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Abstract
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 1 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.