Control of neurosecretion of neurotransmitters appears to be regulated through second messengers that change the phosphorylation state of critical enzymes and proteins. The effect of protein phosphorylation promoting agents on secretion, desensitization and Na$\sp+$/Ca$\sp{2+}$ exchange activity were investigated and protein phosphatases were identified in bovine chromaffin cells. Cells exposed to 8-Br-cAMP, PDBu or okadaic acid alone show slightly decreased rates of desensitization. Okadaic acid plus 8-Br-cAMP potentiated secretion with repeated stimulations. The protein kinase inhibitor H7 increased the desensitization rate. These phenomena are observed during secretion evoked with elevated K$\sp+$ as well as by a nicotinic agonist. Thus, the effect of phosphorylation is at a post-receptor site. Cells treated with dbcAMP, PDBu, okadaic acid or calyculin A show lowered Na$\sp+$/Ca$\sp{2+}$ exchange activity and prolong cytosolic Ca$\sp{2+}$ transients caused by depolarization. Conversely, H7 enhances Na$\sp+$/Ca$\sp{2+}$ exchange activity. Na$\sp+$/Ca$\sp{2+}$ exchange activity in isolated membrane vesicles is inhibited by PKA and PKC. The results indicate that reversible protein phosphorylation modulates Na$\sp+$/Ca$\sp{2+}$ exchange activity and suggest that modulation of the exchanger may play a role in the regulation of secretion. Four distinct peaks of phosphatase activity were observed in homogenized bovine adrenal medulla cells when fractionated using an HPLC ion exchange DEAE column. Of these phosphatases, peaks II, III and IV show preferential dephosphorylation of the $\alpha$ subunit of phosphorylase kinase relative to the $\beta$ subunit and therefore are classified as protein phosphatases type 2. These phosphatases have broad substrate specificity and distinctly different relative specific activities toward the different substrates. Phosphatase 2A is likely to be the major enzyme in bovine adrenal medulla cells. ATP induced a greater secretory response than did acetylcholine without causing preferential secretion of norepinephrine or epinephrine. These data show that the response to ATP found in cultured cells is not an artifact of cell culture, and that ATP corelease with catecholamines from the storage vesicles has a significant physiological role. Freshly isolated cells were separated on a density gradient; the lower density cells develop a much stronger response to ATP than do the higher density cells.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-8976 |
| Date | 01 January 1994 |
| Creators | Lin, Lih Fang |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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