Specific Protein Phosphorylation during Stimulation of Amylase Secretion by β‐Agonists or Dibutyryl Adenosine 3′,5′‐Monophosphate in the Rat Parotid Gland

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Abstract
The present study was undertaken in order to examine the possible involvement of protein phosphorylation during β-adrenergic stimulation in the rat parotid gland. Isolated parotid gland slices were stimulated by either isoproterenol or dibutyryl adenosine 3′,5′-monophosphate (Bt2cAMP) in the presence or absence of propranolol. Amylase output was measured as a parameter for the degree of stimulation of secretion. Stimulation of secretion by either isoproterenol or Bt2AMP was associated with phosphorylation of three protein bands as revealed by sodium dodecylsulfate/polyacryla:nide gel electrophoresis and autoradiography. The apparent molecular weights of the three proteins were 35100 (protein I), 25 700 (protein II) and 20400 (protein II). After cell fractionation by differential and gradient centrifugation, protein I was enriched in a light membrane fraction most likely corresponding to the plasma membrane as revealed by marker enzyme analysis. Proteins II and III were recovered in a denser fraction containing mainly mitochondria and rough micrcsomes. The effect of isoproterenol but not that of Bt2cAMP on phosphorylation of all three protein bands was completely abolished by propranolol. The different time course in the stimulation of amylase secretion by isoproterenol and Bt2cAMP respectively was reflected by corresponding differences in the time course of protein phosphorylation.