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Protein phosphorylation at a postreceptor site can block desensitization and induce potentiation of secretion in
L F Lin1, K T Kim, E W Westhead
1Program in Molecular and Cellular Biology, University of Massachusetts, Amherst 01003.
Abstract:
Desensitization or habituation to repeated or prolonged stimulation is a common property of secretory cells. Phosphorylation of receptors mediates some desensitization processes, but the relationship of phosphorylation to desensitization at postreceptor sites is not well understood. We have tested the effect of protein phosphorylation on desensitization in bovine chromaffin cells. To increase protein phosphorylation, we have used the protein phosphatase inhibitor okadaic acid at 12.5 nM, 100 microM 8-bromo-cyclic AMP to activate protein kinase A, and 10 nM phorbol 12,13-dibutyrate to activate protein kinase C. During repeated 6-s stimulation at 5-min intervals, catecholamine secretion from control cells decreases. Cells exposed to 8-bromo-cyclic AMP or okadaic acid alone show slightly decreased rates of desensitization. In cells pretreated with phorbol 12,13-dibutyrate, desensitization is blocked. Okadaic acid-treated cells stimulated in the presence of 8-bromo-cyclic AMP show potentiation of secretion with repeated stimulation. The protein kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) increases the desensitization rate. Because these phenomena are observed during secretion evoked with elevated K+ as well as by a nicotinic agonist, the effect of phosphorylation is at a postreceptor site. In contrast to desensitization to the repeated stimulations, desensitization to prolonged stimulation with high K+ is not altered by the above protocols in chromaffin cells.
Insights
Protein phosphorylation influences secretory cell desensitization by affecting postreceptor sites. Modulating protein kinases and phosphatases can block or alter desensitization rates during repeated stimulation.
Area of Science:
- Cellular biology
- Neuroendocrinology
Background:
- Secretory cells commonly exhibit desensitization to repeated stimulation.
- Receptor phosphorylation is known to mediate some desensitization, but postreceptor mechanisms remain unclear.
Purpose of the Study:
- To investigate the role of protein phosphorylation in desensitization at postreceptor sites in bovine chromaffin cells.
Main Methods:
- Used okadaic acid (protein phosphatase inhibitor), 8-bromo-cyclic AMP (protein kinase A activator), and phorbol 12,13-dibutyrate (protein kinase C activator) to modulate protein phosphorylation.
- Stimulated cells repeatedly and measured catecholamine secretion.
- Utilized protein kinase inhibitor H7.
Main Results:
- Repeated stimulation led to decreased catecholamine secretion in control cells.
- 8-bromo-cyclic AMP or okadaic acid alone showed slightly reduced desensitization.
- Phorbol 12,13-dibutyrate blocked desensitization, while combined okadaic acid and 8-bromo-cyclic AMP potentiated secretion.
- Protein kinase inhibitor H7 increased desensitization rates.
- Effects were observed for both high K+ and nicotinic agonist stimulation, indicating postreceptor involvement.
Conclusions:
- Protein phosphorylation plays a significant role in regulating desensitization at postreceptor sites in secretory cells.
- Specific modulation of protein kinases and phosphatases can control the rate and extent of desensitization to repeated secretory stimuli.