Effect of hypoxia on expression of selected proteins involved in regulation of apoptotic activity in striatum of newborn piglets.

The levels of selected neuroregulatory proteins that inhibit or promote apoptotic cell death were measured in the striatum of piglets subjected to precisely controlled 1 h hypoxic insult followed by 0, 2 and 4 h recovery and compared to sham operated animals. The anti-apoptotic proteins: there were increases in Survivin at 0 (157%, P = 0.031) and 4 h (171%, P = 0.033), in Bcl-XL at 0 (138%, P = 0.028) and 4 h (143%, P = 0.007), in VEGF at 4 h (185%, P = 0.019) and Hsp27 at 2 h (144%, P = 0.05) and 4 h (143%, P = 0.05). The pro-apoptotic proteins: caspases-1 and 7 increased at 4 h (135%, P = 0.05) and (129%, P = 0.038), respectively. Bim increased after 4 h (115%, P = 0.028), Apoptosis Inducing Factor after 2 h (127%, P = 0.048) and Calpain after 4 h (143% of control, P = 0.04). Hypoxia causes increase in levels of both anti- and pro-apoptotic proteins. Their relative activity determines the outcome in terms of cell damage and neuronal deficit.

Activation of tyrosine hydroxylase in striatum of newborn piglets in response to hypocapnic ischemia and recovery.

The present study describes the effect of hypocapnic ischemia caused by hyperventilation on striatal levels of dopamine, DOPAC, HVA and activity of tyrosine hydroxylase in striatal synaptosomes isolated from the brain of newborn piglets. Hyperventilation did not result in statistically significant changes in the striatal level of dopamine and its major metabolites; however, it was observed that after 20 min of recovery the levels of striatal tissue dopamine, DOPAC and HVA increase by 195%, 110% and 205%, respectively. The level of DOPA (3,4-dihydroxyphenylalanine), which was used as an index of tyrosine hydroxylase activity, also increased after recovery. The rate of dopamine synthesis was 32 pmoles/mg protein/10 min in control piglets and after recovery this increased to 132 pmoles/mg protein/10 min. Measurement of the tyrosine hydroxylase activity in Triton X-100 treated synaptosomes showed that, after 20 min of recovery, there was an increase in Vmax with no change in K(m) for pteridine cofactor, compared to control. This is consistent with the enzyme having been covalently modified (activated) during tissue ischemia caused by hyperventilation and remaining activated well into the recovery period. We postulate that ischemia can induce long lasting alterations in dopamine synthesis, which may play some role in mediation of hypoxic cell injury in immature brain.

Activation of striatal tyrosine hydroxylase by neurocatin, a neuroregulator from mammalian brain.

Neurocatin, a neuroregulatory factor isolated from mammalian brain, is a powerful affector of dopamine synthesis in striatal rat synaptosomes. Incubation of intact synaptosomes with neurocatin caused an increase in the rate of dopamine synthesis measured by accumulation of DOPA. The increase is rapid (within two minutes) and dependent on the concentration of added neurocatin. The stimulatory effect of neurocatin on dopamine synthesis occurred only in intact synaptosomes and was almost completely abolished by lysis of the synaptosomes with Triton X-100 or sonification prior to neurocatin addition. The kinetic parameters of tyrosine hydroxylase were measured in lysates prepared from synaptosomes preincubated with neurocatin. These showed that with increasing neurocatin concentration there was an increase in Vmax with no significant change in KM for the pteridine cofactor, compared to control. Activation of tyrosine hydroxylase by neurocatin is at least partially caused by a receptor mediated increase in phosphorylation of the enzyme. Protein kinase C and protein kinase II may be involved in this process.

Purification of neurocatin, a neuroregulatory factor from brain.

A neuroregulatory factor (neurocatin) has been isolated from bovine brain. Neurocatin is a powerful affector of catecholamine metabolism in synaptosomes isolated from rat brain, causing increased formation of norepinephrine (NE) and decreased formation of 3,4-dihydroxyphenylacetic acid (DOPAC). The ratio NE/DOPAC in synaptosomes has been used to measure the neurocatin content of samples during isolation. Neurocatin has now been purified to a single chromatographic peak by high-resolution HPLC and a preliminary amino acid content measured. It appears to be a small peptide with a molecular weight in the range of 2,000-2,500 Da.