Effect of parathyroid hormone on rat renal cAMP-dependent protein kinase and protein kinase C activity measured using synthetic peptide substrates.
The actions of parathyroid hormone (PTH) on the renal cortex are thought to be mediated primarily by cAMP-dependent protein kinase (PKA) with some suggestion of a role for protein kinase C (PKC). However, present methods for assaying PKA and PKC in subcellular fractions are insensitive and require large amounts of protein. Recently, a sensitive method for measuring the activity of protein kinases has been reported. This method uses synthetic peptides as substrates and a tandem chromatographic procedure for isolating the phosphorylated peptides. We have adapted this method to study the effect of PTH on PKA and PKC activity using thin slices of rat renal cortex. PTH (250 nM) stimulated cytosolic PKA activity four- to fivefold within 30 s, and PKA activity was sustained for at least 5 min. PTH also rapidly stimulated PKC activity in the membrane fraction and decreased PKC activity in the cytosol. These changes were maximal at 30 s, but unlike changes in PKA, they declined rapidly thereafter. PTH significantly activated PKC only at concentrations of 10 nM or greater. This study demonstrates that PTH does activate PKC in renal tissue, although the duration of activation is much less than for PKA. It also demonstrates that a combination of synthetic peptides with tandem chromatography can be used as a sensitive assay procedure for protein kinase activity in biological samples.
Nemani
R
R
Geriatric Research, Education, and Clinical Center, VA Medical Center, St. Louis, Missouri 63125.
Wongsurawat
N
N
Armbrecht
H J
HJ
D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D010281:Parathyroid Hormone / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D013379:Substrate Specificity
D010281:Parathyroid Hormone; D011494:Protein Kinases; D011493:Protein Kinase C