Human lymphocytes stimulate prostacyclin synthesis in human umbilical vein endothelial cells. Involvement of endothelial cPLA2.

Prostacyclin (PGI2) contributes to the maintenance of a nonadhesive luminal surface in blood vessels due to its anti-platelet and vasodilatory properties. Here, we sought to determine whether peripheral blood lymphocytes (PBL) may regulate the PGI2 production of human umbilical vein endothelial cells (HUVEC). Cell-cell contact between HUVEC and lymphocytes markedly enhanced PGI2 synthesis as a function of the number of lymphocytes added. This stimulated synthesis was totally suppressed when lymphocytes and HUVEC were separated by a microporous insert. It was not due to prostaglandin H synthase up-regulation. The pretreatment of lymphocytes with the PGI2 synthase inhibitor tranylcypromine partially inhibited PGI2 synthesis (47%), suggesting a transcellular metabolism of the endothelial prostaglandin endoperoxide PGH2 by the lymphocyte PGI2 synthase. Experiments using [14C]arachidonate-labeled lymphocytes coincubated with unlabeled HUVEC, and [14C]arachidonate-labeled HUVEC coincubated with unlabeled lymphocytes showed that the arachidonic acid used for PGI2 synthesis was totally of endothelial origin. Furthermore, the PGI2 synthesis was strongly inhibited by the cytosolic phospholipase A2 inhibitor, MAFP and totally suppressed by the combination of the calcium chelators, BAPTA and EGTA. Collectively, these results suggest that lymphocytes trigger an outside-in signaling in endothelial cells involving cPLA2 activation. Overall, the switch-on for PGI2 synthesis induced by lymphocytes might serve as a protection against atherothrombogenesis.

Activation of a cyclic nucleotide phosphodiesterase 4 (PDE4) from rat thymocytes by phosphatidic acid.

The cytosolic cyclic nucleotide phosphodiesterase (PDE) activity from rat thymocytes was resolved into five peaks by HPLC. Only two forms of the cAMP-specific PDE4 were found to be sensitive to physiologically relevant phosphatidic acid (PA) concentrations. PA activated the PDE4-peak 3 form, the fatty acid composition and unsaturation degree determining the efficiency of PA. The PDE4-peak 2 form was inhibited only by PA with saturated fatty acyl groups. PDE4 activation was specific of anionic phospholipids, a free phosphate group in the phospholipid molecule being required for maximum activation. These results suggest that PA may contribute to the lowering of cAMP level required in the early steps of a lympho-proliferative response, thus regulating immune functions through PDE4 activation.

Relationship between the multiple forms of rat gastric histidine decarboxylase: effects of conditions favouring phosphorylation and dephosphorylation.

Conditions favouring protein phosphorylation and dephosphorylation are examined for their effects on activity and charge heterogeneity of the rat gastric mucosal histidine decarboxylase. Incubation of gastric supernatant with various combinations of ATP, Mg2+, cyclic AMP and protein kinase under the blockade of endogenous phosphodiesterase and phosphatase fails to alter significantly enzyme activity as assayed with or without pyridoxal 5'-phosphate. Similar results are found with the purified enzyme. No change occurs in the distribution of activity between the charged forms. In contrast, treatment with alkaline phosphatase both inactivates the enzyme with preservation of heterogeneity, full reactivation being achieved by pyridoxal 5'-phosphate, and reduces the number of forms and converts forms II and III to form I with preservation of the catalytic potentialities. The data suggest that the enzyme heterogeneity may be related in part to the phosphorylation state; the possibility that the gastric enzyme is susceptible to several post-translational modifications is discussed.

Inactivation of rat gastric mucosal histidine decarboxylase by phosphatase.

Histidine decarboxylase of supernatants as well as of purified preparations from rat gastric mucosa is inactivated by a non-specific phosphatase in the absence of pyridoxal 5'-phosphate. The inactivation is a time and concentration-dependent process. Pyridoxal 5'-phosphate, but not histidine, protects the enzyme against phosphatase action. The inactivation is reversible, only pyridoxal 5'-phosphate reactivates the inactivated enzyme. Pyridoxamine 5'-phosphate is ineffective for histidine decarboxylase, but is converted into an active coenzyme only in gastric supernatant. Evidence for the occurrence of an active phosphatase in gastric tissue is also presented; its properties are those of an acid phosphatase and are similar to those of phosphatases hydrolyzing pyridoxal 5'-phosphate in other tissues. The data indicate that phosphatase promotes apoenzyme formation and may play a role in the regulation of histamine synthesis.

Polymorphism of rat gastric mucosal histidine decarboxylase: effects of the coenzyme and sulfhydryl groups.

Several factors are examined for their implication in the charge heterogeneity and form conversion of rat gastric mucosal histidine decarboxylase. The apoenzyme and the holoenzyme are undistinguishable with respect to their pI and to the distribution of enzyme activity in the three forms. The latter are not produced by differential coenzyme binding. Studies for glycoprotein characterization provide evidence that the heterogeneity does not arise from enzyme-bound carbohydrate. Oxidative or reductive environments change the distribution between forms without modifying the molecular weight. Conversion of form III to forms I and II can be effected by treatment with dithiothreitol. A similar loss of negatively charged form occurs upon ageing and is not prevented by an alkylating agent. All three forms show equal sensitivity to N-ethylmaleimide and dithiothreitol inhibitions. The oxidation-reduction state of exposed sulfhydryl groups may be responsible at least in part for the charge heterogeneity.

Histidine decarboxylase from rat gastric mucosa: heterogeneity and enzyme forms modification.

Rat gastric mucosal histidine decarboxylase is shown to exist in the crude extract as three active charged forms which are separable by isoelectric focusing. The distribution of enzyme activity in the three forms is independent of the homogenizing medium and of the isoelectric focusing procedure indicating that the heterogeneity does not arise during isolation. Multiple forms correspond to histidine decarboxylase and are related neither to 3,4-dihydroxyphenylalanine decarboxylase nor to the result of aggregation. This charge difference between the enzyme forms changes according to the time of storage and to the temperature, leading to the generation of less negatively charged species. The conversion cannot be attributed to proteolytic degradation nor to differences in stability between forms. The data indicate that these alternative charged states may really result from an in vivo post-translational modification of the enzyme.

Isolation and properties of multiple forms of histidine decarboxylase from rat gastric mucosa.

The heterogeneity of histidine decarboxylase from rat gastric mucosa was studied. The partially purified enzyme was fractionated by preparative isoelectric focusing on a flat-gel bed by using narrow pH-range carrier ampholytes and a short focusing time. The activity was resolved, with about 95% recovery, into three forms, designated I, II and III, with pI values of 5.90, 5.60 and 5.35 respectively. These three forms exhibited similar molecular weights, indicating that the forms were not the result of different degrees of polymerization. By preparative refocusing each form refocused as a single peak of enzyme activity with reproducible pI, but a high loss of activity occurred with repeated focusing. Forms I, II and III were purified by the combined use of preparative isoelectric focusing and gel chromatography and other fractionation methods. The active forms could be distinguished by electrophoresis and isoelectric focusing on polyacrylamide gels and displayed protein heterogeneity. These forms were found in the crude extract and in the partially purified preparations in the presence or absence of proteinase inhibitors. Form II had the highest specific activity, but all three forms had the same optimum pH and Km value for histidine.

Properties of histidine decarboxylase from rat gastric mucosa.

The properties of specific histidine decarboxylase from highly purified rat gastric mucosa preparations were studied. The kinetic parameters were pH dependent: the apparent Km value varied inversely with pH; the maximum reaction velocity was reached at pH 6.6; the optimum pH was related to substrate concentration. The enzyme was unstable below pH 5.5. The effect of temperature was investigated and the enzyme activity was optimum near 56 degrees C. The thermal inactivation of the enzyme showed the presence of several active forms displaying distinct thermostabilities. The effect of coenzyme and substrate on heat stability was established. A small amount of pyridoxal phosphate was required for maximum enzyme activity, and the Km was low. The cofactor appeared to be tightly bound to the apoenzyme; nevertheless there was a fraction more easily resolved by dialysis. With high pyridoxal phosphate concentrations non-competitive inhibition occurred. Histamine inhibited the enzyme at high concentrations, the inhibition being competitive with respect to the substrate. No metal ion was required for enzyme activity; the enzyme was inhibited by sulfhydryl reagents and heavy metal ions, and also by high concentrations of reducing agents. The tryptophan residue of the holoenzyme seemed to be essential for the catalytic process.

[Purification and detection of multiple forms of histidine decarboxylase in rat gastric mucosa (author's transl)]. / Purification et detection de plusieurs formes moleculaires de l'histidine decarboxylase de la muqueuse gastrique de rat.

A specific histidine decarboxylase from rat gastric mucosa has been obtained at high purity and good yield (purification about 600-fold). The purification procedure included double (NH4)2SO4 fractionation, ion-exchange chromatography, preparative isoelectric focusing in a granulated gel and gel filtration. Only the specific histidine enzyme was obtained by that procedure; DOPA decarboxylase, a non-specific enzyme, was absent in our final preparation. Each step of the purification was visualized by polyacrylamide gel electrophoresis and analytical isoelectric focusing. The purified enzyme was apparently homogenous by criteria of electrophoresis and gel filtration and has a molecular weight of 94 000. Several protein bands appeared after isoelectric focusing and the enzyme activity was localized in 3 distinct peaks. The gastric enzyme consists of 3 active forms which could be distinguished by their isoelectric points: 5.4, 5.75 and 6. Moleculare weights estimated by SDS polyacrylamide gel electrophoresis were 97 000, 93 000 and 90 000, and no subunits were observed. Pyridoxal phosphate was required as a coenzyme and resolution of the holoenzyme agreed with a portion of the coenzyme tightly bound to the apoenzyme. The purified enzyme was stable at low ionic strength, near neutral pH; concentrated reducing agents inhibit the enzyme.