The effects of nimodipine and L-NAME on coronary flow and oxidative stress parameters in isolated rat heart.

The aim of this study was to assess the effects of Ca2+ channel antagonist nimodipine (in concentration which competitive inhibited phosphodiesterase 1--PDE1) on oxidative stress alone or under inhibition of nitric oxide synthase by L-NAME in isolated rat heart. The hearts from male Wistar albino rats (n=18, BM about 200 g, age 8 weeks) were retrograde perfused according to the Langendorff technique at gradually increased constant perfusion pressure conditions (CPP, 40-120 cm H2O). The experiments were performed under control conditions, in the presence of Nimodipine (2 microM) or Nimodipine (2 microM) plus L-NAME (30 microM). Coronary flow (CF) varied in the autoregulatory range from 3.7 +/- 0.4 ml/min/g wt at 50 cm H2O to 4.35 +/- 0.79 at 90 cm H2O. Basal nitrite outflow, index of lipid peroxidation (measured as TBARS release) and superoxide anion release (O2-) (at 60 cm H2O) were 0.64 +/- 0.18 nmol/min/g wt, 0.55 +/- 0.13 micromol/min/g wt and 19.72 +/- 3.70 nmol/min/g wt, respectively. Nimodipine induced significant vasodilation at all values of CPP (from 26% at 40 cm H2O to 36% at 120 cm H2O) accompanied with significant decrease of nitrite outflow (from 59% at 40 cm H2O to 40% at 120 cm H2O), significant increase of TBARS above autoregulatory range (about 40%) and significant increase of O2- release (from 186% at 40 cm H2O to 117% at 120 cm H2O). However, perfusion with L-NAME completely reversed the effects of Nimodipine. Nimodipine-induced flow changes were decreased under L-NAME (from 3% at 40 cm H2O to 11% at 120 cm H2O) without changes in the autoregulatory range, accompanied with significantly increased nitrite outflow (from 69% at 40 cm H2O to 36% at 120 cm H2O) and TBARS release (almost 50%), as well as significantly decreased O2- release (from 50% at 40 cm H2O to 43% at 120 cm H20). Our findings show that effect of nimodipine on coronary flow should be significantly influenced by NO, TBARS and O2- release in isolated rat heart.

The interconversion of protein phosphatase 2A between PP2A1 and PP2A0 during retinoic acid-induced granulocytic differentiation and a modification on the catalytic subunit in S phase of HL-60 cells.

Alterations in protein phosphatase 2A (PP2A) during retinoic acid-induced differentiation of HL-60 cells have been investigated. PP2A activity of HL-60 cells for phosphorylated myelin basic protein showed a sharp and transient increase after 18-h treatment with 1 microM retinoic acid, which corresponded to G1/S boundary of the cell cycle. This PP2A of the 18-h treated cells was eluted from a DEAE-Sepharose column with 0.13 M NaCl, while PP2A from control cells was eluted with 0.23 M NaCl. The phosphorylase phosphatase activity of PP2A in the 0.13 M eluate was greatly enhanced in the presence of protamine compared with that of the later eluting PP2A. Immunoblot analyses with antisera against B' and B alpha subunits showed that the PP2A in the 0.13 M NaCl eluate from 18-h retinoic acid-treated cells was PP2A0 (AC-B'), whereas the PP2A eluted with 0.23 M NaCl from 24-h retinoic acid-treated cells and 0-, 18-, and 24-h control cells was PP2A1 (AC-B alpha). These results strongly suggest that PP2A undergoes a transient and reversible interconversion of holoenzyme forms during the initial stage of retinoic acid-induced granulocytic differentiation. PP2A activity assayed after dissociation of the catalytic subunit, for phosphorylase as substrate, showed a sharp and transient decrease in S phase of HL-60 cells irrespective of the presence or absence of retinoic acid. Immunoblot analyses with antisera against C-terminus and N-terminus of the catalytic subunit of PP2A suggested that a modification at the C-terminus is responsible for the decrease in PP2A activity. Immunoreactivity to the C-terminal antibody was restored after treatments of the S-phase extract with alkali or ethanol, the conditions which remove the methyl group from the C-terminus. These results suggest that the C-terminus of PP2A catalytic subunit is transiently methylated in S phase of HL-60 cells.

The catalytic subunit of protein phosphatase 2A associates with the translation termination factor eRF1.

By a number of criteria, we have demonstrated that the translation termination factor eRF1 (eukaryotic release factor 1) associates with protein phosphatase 2A (PP2A). Trimeric PP2A1 was purified from rabbit skeletal muscle using an affinity purification step. In addition to the 36 kDa catalytic subunit (PP2Ac) and established regulatory subunits of 65 kDa (PR65) and 55 kDa (PR55), purified preparations contained two proteins with apparent Mrs of 54 and 55 kDa. Protein microsequencing revealed that the 55 kDa component is a novel protein, whereas the 54 kDa protein was identified as eRF1, a protein that functions in translational termination as a polypeptide chain release factor. Using the yeast two-hybrid system, human eRF1 was shown to interact specifically with PP2Ac, but not with the PR65 or PR55 subunits. By deletion analysis, the binding domains were found to be located within the 50 N-terminal amino acids of PP2Ac, and between amino acid residues 338 and 381 in the C-terminal part of human eRF1. This association also occurs in vivo, since PP2A can be co-immunoprecipitated with eRF1 from mammalian cells. We observed a significant increase in the amount of PP2A associated with the polysomes when eRF1 was transiently expressed in COS1 cells, and eRF1 immunoprecipitated from those fractions contained associated PP2A. Since we did not observe any dramatic effects of PP2A on the polypeptide chain release activity of eRF1 (or vice versa), we postulate that eRF1 also functions to recruit PP2A into polysomes, thus bringing the phosphatase into contact with putative targets among the components of the translational apparatus.

The variable subunit associated with protein phosphatase 2A0 defines a novel multimember family of regulatory subunits.

Two protein phosphatase 2A (PP2A) holoenzymes were isolated from rabbit skeletal muscle containing, in addition to the catalytic and PR65 regulatory subunits, proteins of apparent molecular masses of 61 and 56 kDa respectively. Both holoenzymes displayed low basal phosphorylase phosphatase activity, which could be stimulated by protamine to an extent similar to that of previously characterized PP2A holoenzymes. Protein micro-sequencing of tryptic peptides derived from the 61 kDa protein, termed PR61, yielded 117 residues of amino acid sequence. Molecular cloning by enrichment of specific mRNAs, followed by reverse transcription-PCR and cDNA library screening, revealed that this protein exists in multiple isoforms encoded by at least three genes, one of which gives rise to several splicing variants. Comparisons of these sequences with the available databases identified one more human gene and predicted another based on a rabbit cDNA-derived sequence, thus bringing the number of genes encoding PR61 family members to five. Peptide sequences derived from PR61 corresponded to the deduced amino acid sequences of either alpha or beta isoforms, indicating that the purified PP2A preparation was a mixture of at least two trimers. In contrast, the 56 kDa subunit (termed PR56) seems to correspond to the epsilon isoform of PR61. Several regulatory subunits of PP2A belonging to the PR61 family contain consensus sequences for nuclear localization and might therefore target PP2A to nuclear substrates.

The phospho-opsin phosphatase from bovine rod outer segments. An insight into the mechanism of stimulation of type-2A protein phosphatase activity by protamine.

The vertebrate visual transduction system involves a cycle of phosphorylation and dephosphorylation of a transmembranous photoreceptor (rhodopsin). Upon illumination, the activated photoreceptor (metarhodopsin-II) is phosphorylated by a specific kinase on up to seven serine and threonine residues. A dephosphorylation process must then be undertaken to return the photoreceptor to its ground state. Initial work, along with studies using the rabbit skeletal muscle catalytic subunit of protein phosphatase 2A, indicated that the phosphatase responsible was a member of the type-2 family. The work has been further extended and using 1000 bovine retinae, the catalytic subunit and a holoenzyme form of phospho-opsin phosphatase were purified 2100-fold and 550-fold respectively. The stimulation of the activities of both these fractions with protamine sulphate and the inhibition by okadaic acid are consistent with the fact that these phosphatases belong to the type-2A family. Western blotting using a variety of specific antibodies established that the catalytic subunit (36 kDa, C subunit) was indeed of type 2A, while the holoenzyme was a heterotrimer comprising the preceding catalytic subunit complexed to two other polypeptides of 55 kDa (B subunit) and 65 kDa (A subunit), both of which were of alpha subtype; phospho-opsin phosphatase may thus be described as a trimeric enzyme containing the ABC subunits of type-2A protein phosphatase, i.e. PP2A1. The dephosphorylation of phospho-opsin by both fractions was found to be stimulated (4-8-fold) by the presence of protamine sulphate (250 micrograms/ml; 50 microM). However, when phospho-peptides corresponding to the C-terminal region of opsin were used, these were maximally dephosphorylated without requiring the presence of protamine; at equivalent concentrations of substrates the phospho-peptides were dephosphorylated (in the absence of protamine) at rates which were approximately equal to those obtained with phospho-opsin (in the presence of protamine). It was shown that type-1 phosphatases had little activity against these phospho-peptides. Furthermore, if phospho-opsin was treated with protamine, the activity of the phosphatase assumed an elevated level and was not significantly stimulated by the addition of exogenous protamine. This effect could be reversed by washing the protamine-treated substrate with 1 M NaCl, whence the protamine-dependent stimulation returned to normal levels. To this end, studies revealed that protamine was binding to the particulate substrate in a ratio of protamine/opsin of 0.7:1. The cumulative finding may be rationalised by suggesting that the effect of protamine is a substrate-directed phenomenon and a hypothetical mechanism for this effect is considered.

Drosophila mutants in the 55 kDa regulatory subunit of protein phosphatase 2A show strongly reduced ability to dephosphorylate substrates of p34cdc2.

The 55 kDa regulatory subunit of Drosophila protein phosphatase 2A is located in the cytoplasm at all cell cycle stages, by the criterion of immunofluorescence. We are unable to detect significant change in protein phosphatase activity during the nuclear division cycle of syncytial embryos. However, cell cycle function of the enzyme is suggested by the mitotic defects exhibited by two Drosophila mutants, aar1 and twinsP, defective in the gene encoding the 55 kDa subunit. The reduced levels of the 55 kDa subunit correlate with the loss of protein phosphatase 2A-like, okadaic acid-sensitive phosphatase activity of brain extracts against caldesmon and histone H1 phosphorylated by p34cdc2/cyclin B kinase, but not against phosphorylase a. Thus the mitotic defects of aar1 and twinsP are likely to result from the lack of dephosphorylation of specific substrates by protein phosphatase 2A.