Phorbol esters promote postsynaptic accumulation of Vesl-1S/Homer-1a protein.

We examined effects of phorbol esters on the amount and the subcellular distribution of the activity-regulated protein Vesl-1S/Homer-1a in cultured hippocampal neurons. Major Vesl-1S immunoreactivity (IR) was detected throughout neuronal somata under control conditions. Bath application of phorbol esters, PMA and PDBu resulted in the increase in the amount of Vesl-1S proteins and promoted punctate distribution of Vesl-1S IR at the cortical regions of the neuronal somata. Immunofluorescent observations using antisynaptophysin and anti-Vesl-1S antibodies, and electron microscopic observations, revealed that Vesl-1S accumulated at postsynaptic regions following PMA application. Membrane depolarization with high concentrations of external potassium also promoted the punctate distribution of Vesl-1S IR. These results demonstrate that phorbol-triggered reaction cascades result in the accumulation of Vesl-1S protein at postsynaptic regions, and suggest that these phorbol effects may mimic those caused by synaptic activities.

Regulation of the level of Vesl-1S/Homer-1a proteins by ubiquitin-proteasome proteolytic systems.

The vesl-1S/homer-1a gene is up-regulated during seizure and long term potentiation. Other members of the Vesl family, Vesl-1L, -2, and -3, are constitutively expressed in the brain. We examined the regulatory mechanisms governing the expression level of Vesl-1S protein, either an exogenously introduced one in COS7 or human embryonic kidney 293T cells or an endogenous one in rat brain neurons in cultures. In both cases, application of proteasome inhibitors increased the amount of Vesl-1S protein but not that of Vesl-1L, -2, or -3 protein. Deletion analyses revealed that the C-terminal 11-amino acid region was responsible for the proteolysis of Vesl-1S by proteasomes. Application of proteasome inhibitors promoted ubiquitination of Vesl-1S protein but not that of the Vesl-1S deletion mutant, which evaded proteasome-mediated degradation. These results indicate that ubiquitin-proteasome systems are involved in the regulation of the expression level of Vesl-1S protein.

Light and glutamate-induced degradation of the circadian oscillating protein BMAL1 during the mammalian clock resetting.

Recently discovered mammalian clock genes are believed to compose the core oscillator, which generates the circadian rhythm. BMAL1/CLOCK heterodimer is the essential positive element that drives clock-related transcription and self-sustaining oscillation by a negative feedback mechanism. We examined BMAL1 protein expression in the rat suprachiasmatic nuclei (SCN) by immunoblot analysis. Anti-BMAL1 antiserum raised against rBMAL1 recognized 70 kDa mBMAL1b and detected a similar immunoreactivity (IR) as a major band in rat brains. Robust circadian BMAL1-IR oscillations with nocturnal peaks were detected in the SCN during a light/dark cycle and under constant darkness. A short duration light exposure at night acutely reduced BMAL1-IR in the SCN during photoentrainment. This might be attributable to the degradation of BMAL1 protein. Application of glutamate and NMDA to the SCN slices at projected night, a procedure mimicking photic phase delay shift, also acutely reduced BMAL1-IR in a similar manner. A rapid decrease of BMAL1 protein suggests that BMAL1 protein might be implicated in the light-transducing pathway within the SCN.

Changes in circadian period and morphology of the hypothalamic suprachiasmatic nucleus in fyn kinase-deficient mice.

Protein tyrosine phosphorylation is involved in intracellular signal transduction and plays important roles in various physiological events. To understand the role of Fyn, a non-receptor type tyrosine kinase of Src family kinases, in the mechanism of circadian rhythms, we analyzed the circadian locomotor behavior and morphology of the hypothalamic suprachiasmatic nucleus (SCN), a master circadian oscillator in Fyn mutant mice, because Fyn-like immunoreactive substance was observed in the SCN. Under constant dark (DD) condition the Fyn (-/-) mutant mice showed a free-running circadian rhythm, and the period of the circadian rhythm of the locomotor activity was significantly longer than that of the control mice. Fyn (-/-) mutant mice had abnormal distribution of neurons containing vasoactive intestinal polypeptide (VIP)-like immunoreactive substance in the SCN. These findings suggest that Fyn is involved in the mechanism of circadian oscillation and morphological formation of the SCN. The mechanism of the implication of Fyn discussed with the Fyn's roles in neural network formation and cellular signal transduction pathway.

Little or no response to 24-hr water-deprivation of Fos-like immunoreactivity in vasopressinergic magnocellular neurons in the hypothalamus of hereditary microphthalmic rats.

In normal rats, 24-hr water-deprivation was found to cause a significant increase in the plasma concentration of arginine vasopressin (AVP), and marked induction of Fos in the AVP-positive magnocellular neurons in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the hypothalamus. On the other hand, 24-hr water-deprivation also caused a significant increase in the plasma AVP concentration in hereditary microphthalmic rats, but much less than in normal rats. Consistent with this, the extent of induction of Fos in the nuclei of the PVN and SON of these rats was lesser than in normal rats. These results suggest that hereditary microphthalmic rats have a defect or decrease in the response to water-deprivation of vasopressinergic magnocellular neurons in the PVN and SON.

Ultraweak biochemiluminescence detected from rat hippocampal slices.

From any kind of living organisms, very weak spontaneous light emission without excitation light (ultraweak biochemiluminescence) is observed. We succeeded to detect ultraweak biochemiluminescence from rat hippocampal slice preparation at the order of 10(-19) W/mm2 by a single-photon detector using a silicon avalanche photodiode. It was shown that depolarization induced by the high concentration of potassium caused an increase in the intensity of biochemiluminescence from the rat hippocampal slice, and that suppression of neural activity by tetrodotoxin elicited a decrease of its luminescence. These findings suggest that correlation between the intensity of ultraweak biochemiluminescence and neural metabolic activity.

Mazindol enhances glucose uptake by rat skeletal muscle.

The pharmacological action of mazindol (5-hydroxy-5-p-chlorophenyl-2,3-dihydro-5-imidazo[2,1-a]isoindo l) was examined by studying its effect on glucose uptake by rat tissues using radiolabelled 2-deoxy-D-glucose. The following results were obtained. (1) The rate constant (Ki) of net tissue 2-deoxy-D-glucose uptake increased in the cerebral cortex (4.7-fold, P < 0.05), the hypothalamus (4.6-fold, P < 0.05), heart (4.0 fold, P < 0.05) and skeletal muscles (gastrocnemius, 5.7-fold, P < 0.01; soleus, 4.7-fold, P < 0.05), but not in epididymal adipose tissue in vivo 90 min after intragastric administration of mazindol (20 mg/kg). (2) This increase in Ki values of net tissue 2-deoxy-D-glucose uptake was not observed after addition of mazindol to the diet (40 mg/100 g of diet) for 4 days. (3) Mazindol (16.7 ng/ml to 16.7 micrograms/ml) stimulated 2-deoxy-D-glucose transport into sarcolemmal vesicles of the gastrocnemius muscle in vitro (1.6-1.8-fold, P < 0.05) and this stimulation was blocked by cytochalasin B (10 microM). These findings suggest that mazindol stimulates glucose transport into skeletal muscles by acting on glucose transporters in the sarcolemmal membrane, and suggest that mazindol may stimulate glucose transport into the brain and heart by a similar mechanism.