J. Biol. Chem., Vol. 265, Issue 2, 760-766, Jan, 1990
Histidine regulation of cyclic AMP metabolism in cultured renal epithelial LLC-PK1 cells
BS Dixon, R Breckon, MM Kaehny, MA Dillingham and RJ Anderson
Department of Medicine, Denver Veterans Affairs Medical Center, Colorado 80220.
L-Histidine and imidazole (the histidine side chain) significantly increase
cAMP accumulation in intact LLC-PK1 cells. This effect is completely
inhibited by isobutylmethylxanthine (IBMX). Histidine and imidazole
stimulate cAMP phosphodiesterase activity in soluble and membrane fractions
of LLC-PK1 cells suggesting that the IBMX-sensitive effect of these agents
to stimulate cAMP formation is not due to inhibition of cAMP
phosphodiesterase. Histidine and imidazole but not alanine (the histidine
core structure) increase basal, GTP-, forskolin- , and AVP-stimulated
adenylate cyclase activity in LLC-PK1 membranes. Two other amino acids with
charged side chains (aspartic and glutamic acids) increase AVP-stimulated
but neither basal- nor forskolin- stimulated adenylate cyclase activity.
This suggests that multiple amino acids with charged side chains can
regulate selected aspects of adenylate cyclase activity. To better define
the mechanism of histidine regulation of adenylate cyclase, membranes were
detergent-solubilized which prevents histidine and imidazole potentiation
of forskolin- stimulated adenylate cyclase activity and suggests that an
intact plasma membrane environment is required for potentiation. Neither
pertussis toxin nor indomethacin pretreatment alter imidazole potentiation
of adenylate cyclase. IBMX pretreatment of LLC-PK1 membranes also prevents
imidazole to potentiate adenylate cyclase activity. Since IBMX inhibits
adenylate cyclase coupled adenosine receptors, LLC-PK1 cells were incubated
in vitro with 5'-N- ethylcarboxyamideadenosine (NECA) which produced a
homologous pattern of desensitization of NECA to stimulate adenylate
cyclase activity. Despite homologous desensitization, histidine and
imidazole potentiation of adenylate cyclase was unaltered. These data
suggest that histidine, acting via an imidazole ring, potentiates adenylate
cyclase activity and thereby increases cAMP formation in cultured LLC- PK1
epithelial cells. This potentiation requires an intact plasma membrane
environment, occurs independent of a pertussis toxin-sensitive substrate
and of products of cyclooxygenase, and is inhibited by IBMX. This
IBMX-sensitive pathway does not involve either inhibition of cAMP
phosphodiesterase activity or a stimulatory adenosine receptor coupled to
adenylate cyclase.
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