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The role of intracellular free calcium as a second messenger in human airway epithelia Harris, Robert Arthur


The role of [Ca²⁺]i as a second messenger controlling electrolyte transport in human airway epithelia was investigated using cultured human nasal epithelial cells as a model. Human nasal epithelial tissue was obtained as a by-product of surgery and put into monolayer cell culture. After 2-5 days in culture, cells were loaded with fura-2 and intracellular free Ca²⁺ measured using wavelength emission ratio cytofluorometry. Initially, bradykinin increased Ca²⁺]; in all cells tested, whereas isoproterenol increased Ca²⁺]i; in only half the cells tested. Epinephrine, norepinephrine, and prostaglandin E2 had no stimulatory effect on Ca²⁺]i. However, nucleotide receptor agonists, such as adenosine 5'-triphosphate, uridine 5'-triphosphate, and adenosine 5'-diphosphate, stimulated increases in Ca²⁺]i in a manner similar to bradykinin. Other related compounds such as guanidine 5'-triphosphate and cyclic 3'-5'-adenosine monophosphate had no detectable stimulatory effect on [Ca²⁺]i. Verapamil, a voltage-sensitive Ca²⁺ channel blocker, had no effect on bradykinin-induced increases in Ca²⁺]i. Removal of extracellular Ca²⁺ for periods as long as 30 minutes only slightly attenuated the increase in Ca²⁺]i which resulted from both bradykinin stimulation and stimulation with a variety of exogenous nucleotides. Histamine also increased Ca²⁺]i in a dose dependent manner; however, the kinetics of theCa²⁺]i response to histamine differed markedly from stimulation with either bradykinin or exogenous nucleotides. In order to elicit a change in Ca²⁺]i, cells required at least a 30 second exposure to histamine. Bathing cells in Ca²⁺-free saline for 5 minutes had no effect on the histamineinduced change in Ca²⁺]i. However, bathing the cells in Ca²⁺-free saline for 30 minutes completely abolished the histamine-induced change in Ca²⁺]i, yet subsequent stimulation with bradykinin or exogenous nucleotides resulted in a dramatic increase in Ca²⁺]i. Over the course of this study a change in the responsiveness of the cultured cells to bradykinin-mediated increases in Ca²⁺]i was observed. All aspects of the cell culture procedure were investigated and virtually ruled out as the agent which caused the observed change in physiological response to bradykinin. During the critical time frame (February-March 1990), new topical steroid treatments consisting of budesonide became popular with the local ENT surgeons from whom I obtained tissue samples. These steroid treatments may be good candidates for the agent which changed the cellular physiology. Subsequent experiments demonstrated that, after introduction of this steroid treatment, human nasal epithelial cells retained a Ca²⁺]i response to bradykinin and exogenous nucleotides for the first 2 days in cell culture only. After 2 days in cell culture, the number of responsive cells decreased rapidly such that after 6 days in culture only about 15% of the cells responded to bradykinin. A model for the second messenger pathways mediating CI" secretion in human airway epithelial cells is proposed. Briefly, 8-adrenergic agonists activate the cAMP second messenger pathway independent of Ca²⁺]i. Adenosine 5'-triphosphate and bradykinin stimulate the production of D-myo-inositol 1,4,5-trisphosphate, which in turn releases sequestered calcium from intracellular stores. Histamine also releases sequestered calcium; however, the calcium released by histamine comes from a pool which is separate from that released by D-myo-inositol 1,4,5- trisphosphate. In addition, histamine may act directly as the second messenger which releases calcium from this hitherto undescribed intracellular store.

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