Changes in tubular reabsorption of
amino acids and other solutes are characteristic of the immature renal tubule and of various hereditary nephropathies. The cellular mechanisms governing these aberrations in renal
amino acid transport have not been established.
Calcium (Ca2+)-dependent
protein kinases are known to phosphorylate membrane-bound
carrier proteins, thereby modulating transport of various solutes by the proximal tubule. The role of these
enzymes in regulating renal tubular
amino acid transport, particularly during kidney development, is unknown. We investigated: (1) the effect of Ca(2+)- and
phospholipid-dependent
protein kinase [
protein kinase C (PKC)] and Ca2+/
calmodulin-dependent protein kinase II (
CaMKII) on
sodium chloride (NaCl)-linked
proline transport by renal brush border membrane vesicles (BBMV) from adult rats using the "hypoosmotic
shock" technique (lysis of vesicles); (2) the activity, expression and subcellular distribution (cytosol, particulate, BBM) of Ca(2+)-dependent
protein kinases in kidneys from 7-day-old and adult rats using MBP 4-14 and
autocamtide II phosphorylation assays for PKC and
CaMKII, respectively, endogenous
protein phosphorylation (using gel electrophoresis and autoradiography) and Western immunoblot analysis to detect PKC and
CaMKII. The studies showed: (1) endogenous (membrane-bound)
CaMKII and PKC as well as exogenous, highly purified PKC inhibit
proline uptake by phosphorylated, lyzed/resealed BBMV when compared with control vesicles; the voltage-clamped, nonelectrogenic component of
proline transport was inhibited by PKC- but not
CaMKII-mediated phosphorylation; (2) a Ca(2+)-dependent activity of both
kinases was evident in all subcellular fractions tested in immature and adult kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)