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We investigated the substitution of 15(S)-hydroxyeicosatetraenoic acid (15(S)HETE) in phospholipid signaling pathways and its consequences on the growth of non-transformed (NT-) and spontaneously transformed (T-) rat liver epithelia cells (RLEC). 15(S)HETE was selectively incorporated into the sn-2 position of phosphatidylinositol (PI) and at a higher rate into T-RLEC. RLEC rapidly mobilized the resulting 15(S)HETE-containing PI (15(S)HETE-PI) and produced 1-acyl,2-[1(S)HETE]-glycerol. Although total diacylglycerol levels were similar in both cell types, the ratio 1-acyl,2-[15(S)HETE]-glycerol / 15(S)HETE-PI was higher in NT-RLEC, suggesting a lower mobilization of 15(S)HETE-PI in T-RLEC. Using rat brain protein kinase C, 1-stearoyl,2-[15(S)HETE]-glycerol was as potent an in vitro protein kinase C activator as 1-stearoyl,2-arachidonoyl-glycerol. Finally, selective substitution of 15(S)HETE in PI altered DNA synthesis in T-RLEC: whereas low concentrations of 15(S)HETE (1 nM and 10 nM) in these cells were mitogenic, higher concentrations resulted in a 30% inhibition of DNA synthesis.

The incorporation of 12-lipoxygenase metabolites into phospholipids (PLs) could modify second messengers such as diacylglycerols (DAG) and phosphatidic acids. Incubation of [14C]12(S)-HETE (1 μM) with bovine pulmonary artery endothelial cells (BPAEC), resulted in its incorporation in PLs with concentration-dependent kinetics. After a 4 h incubation, the proportion of radioactive phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) + phosphatidylinositol (PI) isolated by TLC, was 77.9%, 16.4% and 5.7%, respectively. In PC, [14C]12(S)-HETE was incorporated at the position 2 of the glycerol. Three major peaks of radioactive PC were isolated on RP-HPLC which were hydrolysed by phospholipase C (PLC). The resulting diacylglycerols were derivatized and identified by GC/MS as 1-oleyl-, 1-stearoyl- and 1-palmitoyl-2-[12-HETE] PC. BPAEC were incubated with [14C]12(S)-HETE (1 μM) before stimulation by bradykinin (1 μM). (A) 1-acyl-2-[12-HETE] diacylglycerols were isolated, derivatized and analysed by MS. We identified a major ion with m z = 926 that corresponds to the molecular ion of authentic 1-stearoyl-2-12(S)-HETE DAG, and 2 other ions with m z = 924 and 898 that correspond to the molecular ions of 1-oleyl- and 1-palmitoyl-2-12(S)-HETE DAG, respectively. (B) Radioactive PA was isolated and hydrolysed by alkaline phosphatase. The MS of resulting diacylglycerols identified 1-stearoyl-, 1-oleyl-, and 1-palmitoyl-2-12(S)-HETE phosphatidic acids. The quantities of 12-HETE PA and the 3 major 12-HETE diacylglycerols were shown to increase following bradykinin stimulation. Thus, the incorporation of 12(S)-HETE into PLs results in the production of altered phosphatidic acids and diacylglycerols. The time-course of increases in 1-acyl-2-(12-HETE) phosphatidic acids and 1-acyl-2-(12-HETE) diacylglycerols showed maximal concentrations 1 and 2 min after bradykinin stimulation, respectively, followed by the decrease of both compounds. Propranolol, an inhibitor of PA phosphohydrolase, totally abolished the bradykinin-induced increase in 12-HETE DAG while increasing the magnitude and duration of 12-HETE PA release. The inhibiting effect of propranolol on bradykinin-induced increase of 12-HETE DAG demonstrates that 12-HETE PA is the principal precursor for 12-HETE DAG. This affords a novel method for confirming the major role of phospholipase D in PC metabolic pathways triggered during cell signaling.

An iodoazido[125I]prazosin analogue was employed to photoaffinity label alpha 1-adrenergic receptors in rat liver plasma membranes. Labeled proteins were separated by gradient polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and (-)-epinephrine displacement of [3H]prazosin binding was concurrently measured in the presence or absence of guanosine 5'-O-(gamma-thiotriphosphate) (GTP[gamma S]). Inclusion of EGTA and/or proteinase inhibitors during membrane preparation and incubation increased the effect of GTP[gamma S] on alpha 1-adrenergic agonist binding and this could be correlated with increased concentrations of a 78 kDa photoaffinity labeled protein. In contrast, omission of EGTA or addition of exogenous Ca2+ diminished or abolished the effect of GTP[gamma S] on binding and caused loss of the 78 kDa form and the appearance of lower molecular weight labeled proteins. Age-dependent differences in GTP[gamma S] effects on alpha 1-adrenergic agonist binding were abolished when membranes were prepared and incubated in the presence of EGTA and proteinase inhibitors. However, the 78 kDa photoaffinity labeled protein observed in adult rats (over 225 g body weight) was not apparent in membranes from younger rats (50-75 g), even when the membranes were prepared and incubated in the presence of EGTA and proteinase inhibitors. Instead, a 68 kDa species was the major labeled protein. These data suggest that GTP effects on alpha 1-adrenergic agonist binding in rat liver membranes require the presence of either a 68 or 78 kDa alpha 1-adrenergic binding protein. Failure to inhibit proteolysis in the membranes leads to the generation of lower-molecular-weight binding proteins and the loss of GTP effects on alpha 1-adrenergic agonist binding, although [3H]prazosin binding characteristics are not changed. It is suggested that either the proteolyzed forms of the alpha 1-adrenergic receptor are unable to couple to a putative guanine nucleotide-binding regulatory protein, or that such a protein is concurrently proteolyzed and is thus unable to couple to the receptor.

Summary: We investigated with an in vivo single pass perfusion technique net absorption and the lumen to mucosa flux of zinc in the proximal and distal small intestine and in the colon segments of suckling (2-wk-old), weanling (3-wk-old) and adolescent (6-wk-old) rats. The concentration of zinc in the isotonic sodium chloride solution perfused through the intestinal segments was either below (10 μM) or above (25, 50, and 100 μM) serum zinc level. In suckling rats, the relationship between luminal zinc concentration and net absorption and lumen to mucosa flux was curvilinear in the proximal segment only; however, the relationship was linear in the distal small intestine and colon. In weanling rats, the relationship between luminal zinc concentration and net absorption and lumen to mucosa flux was curvilinear in the proximal and distal small intestinal segments and linear in the colon segments. In the adolescent rats, the relationship between luminal zinc concentration, net absorption and lumen to mucosa flux was curvilinear in all intestinal segments studied. The curvilinear relationships noted fits a Michaelis-Menten kinetics. Analysis of the kinetic data indicate a progressive rise in mean Km values with age in the proximal segments of all age groups. Similarly, there was a rise in mean Km values in the distal segments of the weanling and adolescent rats. These findings suggest that intestinal zinc transport is characterized by a maturational pattern which evolves with age.

Incubation of rat liver phosphorylase kinase in the presence of MgATP results in a time-dependent increase in activity, i.e., activation. Determination of the magnitude of activation depends, in large part, on the relative concentrations of Mg2+ and ATP used in the phosphorylase kinase activity assay, such that as the Mg2+ to ATP ratio increases less activation is detectable. Prior to activation, maximal activity of nonactivated phosphorylase kinase requires a 2–3 fold molar excess of Mg2+ (i.e., free Mg2+) over ATP. MgATP-dependent activation of the enzyme results in an alteration in the free Mg2+ requirement such that the activity of the activated enzyme is sharply inhibited by the free cation. Inhibition by free Mg2+ of the activated enzyme is rapidly reversed by removal of free Mg2+ but is not affected by addition of Ca2+. Both nonactivated and activated forms of enzyme appear to be inhibited by free ATP4–. The results show that the use of high concentrations of free Mg2+ in the phosphorylase kinase activity assay can blunt or completely obscure changes in enzyme activity following activation of the enzyme.