M.D. Aragonés

Substance P stimulates translocation of protein kinase C in brain microvessels

The action of substance P on protein kinase C in cerebral microvessels, isolated from bovine, was investigated. We found that in untreated microvessels 85% of the total activity was localized to the cytosolic fraction. Substance P caused a shift of activity to the membrane fraction, accompanied by a loss of activity in the cytosolic fraction. This effect resulted in dose-dependence and it was evident after 5 min treatment. These data suggest that substance P may be involved in the regulation of processes underlying protein phosphorylation in the blood-brain barrier.

Insulin action on brain microvessels; Effect on alkaline phosphatase

The effects of insulin on brain alkaline phosphatase activity have been examined. Insulin inhibited the activity of alkaline phosphatase on brain microvessels in in vitro experiments. The inhibition observed was of the non-competitive type. These observations indicate that the hormone is able to induce neurochemical modifications revealed in this case as changes in the phosphate transfer enzymes in brain microvessels.

Effects of vanadate on the cyclic AMP-protein kinase system in rat liver

Abstract Vanadate produced a marked increase of cyclic AMP levels in rat liver slices; a parallel scheme of variation was found in the case of protein kinase activity. Similar trends of variations were observed in in vivo experiments. These results support the idea that vanadate can affect the cyclic AMP-protein kinase system and moreover, due to the function of liver (an organ considered to play a fundamental role in the general metabolism in mammals) the same results add more data in support for a general regulatory role for vanadate.

PAF-induced activation of polyphosphoinositide-hydrolyzing phospholipase C in cerebral cortex

The action of platelet-activating factor (PAF) on phosphoinositide hydrolysis was studied in rat brain slices. PAF produced a significant increase of 32P incorporation into phosphoinositides and phosphatidic acid (PA), in a dose- and time-dependent manner. Concomitantly, an increase of inositol phosphates and diacylglycerol (DAG) production was observed. Both inositol bisphosphate (IP2) and inositol trisphosphate (IP3) were detected as early as 5 s and they returned immediately to basal levels; concomitantly, formation of inositol monophosphate (IP) was detected. These findings demonstrated that PAF causes a rapid hydrolysis of polyphosphoinositides in cerebral cortex by a phospholipase C-dependent mechanism followed by subsequent resynthesis.

Identification of nitric oxide synthases in isolated bovine brain vessels

We have studied the presence of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) in parenchymal and pial bovine cerebral vessels by using western blot analysis. The different vessel structures were analysed by microscopic observation and their biochemical features determined by using gamma-glutamiltranspeptidase (gamma-GTP) as an endothelial marker and alpha-smooth muscle actin (alpha-SMA) as a smooth muscle cell marker. nNOS could not be found in parenchymal vessels, being present only in pial vessels; eNOS was present in all vessel-fractions studied, albeit a different distribution was found. Thus, the eNOS is more abundant in the parenchymal vessels which are associated with smooth muscle cells whereas both pial vessels and brain microvessels, almost devoid of actin, show extremely low levels. Two main conclusions can be obtained: first, nNOS is exclusively restricted to pial vessels and, second, eNOS is present in endothelial cells which are in association with smooth muscle cells.

Evidence for a role of somatostatin in lipid metabolism of liver and adipose tissue

We have studied the effects of somatostatin on lipid metabolism in liver and adipose tissue of fasted mice. The animals were injected subcutaneously with 8 micrograms somatostatin and killed 5 min after injection. In vivo incorporation of [14C]acetate into triglycerides in both tissues and into hepatic cholesterol was significantly enhanced by somatostatin. Concomitantly, a decrease of triglyceride lipase activity was observed, which corresponds well with the variation undergone by cyclic AMP-protein kinase system. In addition, a marked increase of serum cholesterol levels was observed. Additionally, in vitro experiments were also performed by employing 2.4 X 10(-6) M somatostatin. The results showed that the direct effect of somatostatin on liver seems to be a decrease in acetate uptake. The results obtained with the adipose tissue were similar to those obtained in in vivo conditions. On the other hand, when somatostatin was administered in vivo, the ability to incorporate ortho[32P]phosphate into phospholipids was enhanced in both tissues. Likewise in the in vitro experiments with [14C]acetate, the somatostatin seems to act by decreasing the ortho[32 P]phosphate uptake in liver. While in adipose tissue the somatostatin only caused a strong increase in the specific activity of phosphatidylcholine. These data demonstrate in fasted mice that somatostatin is able to counteract the lipolytic manifestations of the fasted state.

Involvement of sphingolipids in the endothelin-1 signal transduction mechanism in rat brain

In cerebral cortex, endothelin-1 (ET-1) evoked a decrease of 40% in sphingomyelin (SM) levels together with an increase in both ceramide and glycosphingolipid (GSL) levels (100 and 56% respectively). These facts indicate that ET-1 increases sphingomyelinase activity and, possibly, activates the synthesis of GSL. By contrast, in cerebellum ET-1 seems to activate the hydrolysis of both SM and GSL, since the peptide evoked a decrease (near 30%) of their levels concomitantly with an increased production of ceramides (200%). These ceramides are clearly different from those produced in cerebral cortex which come from the SM hydrolysis only. It is suggested that ETB receptor subtype is involved in these responses.

Effect of ecdysterone on the cyclic AMP-protein kinase system in mouse liver.

Abstract In vivo administration of ecdysterone produced a decrease in cyclic AMP levels and cyclic AMP-binding protein activity in mouse liver 40 min after injection. These changes were accompanied by a concomitant decrease in cyclic AMP-dependent protein kinase. The effect on phosphoprotein phosphatase was the opposite pattern of that on protein kinase. These results support the idea that the cyclic AMP-protein kinase system may be involved in the heterophylic action of ecdysterone.

SELECTIVE TIME-DEPENDENT EFFECTS OF INSULIN ON BRAIN PHOSPHOINOSITIDE METABOLISM

The effect of insulin on phosphoinositide metabolism in the cerebral cortex was examined using 32P as precursor. A maximal increase was detected as early as 15 s; phospholipid labeling declined after this initial peak but then increased to another maximum at 30 min. The levels of these phospholipids were unchanged at the earliest time examined, but at 30 min insulin caused an increase in the content of all phospholipids tested. In pulse-chase experiments, insulin stimulated depletion of 32P-labeled phosphoinositides only at 15 s. On the other hand, insulin treatment caused a biphasic diacyglycerol (DAG) production. We conclude that in cerebral cortex, insulin has a dual mechanism of action on phosphoinositide metabolism. First, insulin causes a rapid but transient hydrolysis of phosphoinositides by a phospholipase C-dependent mechanism, followed by subsequent resynthesis; thereafter, insulin increases de novo phospholipid synthesis.

Effect of insulin on acetylcholinesterase activity

Abstract The effects of insulin on acetylcholinesterase activity have been examined. Insulin increased the activity in rat brain and electric eel acetylcholinesterases in in vitro experiments. These observations indicate that the hormone induces neurochemical alterations revealed as changes in the acetylcholine hydrolyzing enzyme.