Membrane expression of platelet calpain

Platelet calpain has many platelet substrates, including external membrane proteins. We thus investigated whether platelet calpain II was associated with platelet membranes in unstimulated and thrombin- activated platelets. A monospecific, goat polyclonal antibody was reared to purified platelet calpain II. Sixteen whole platelet lysates were found to contain 4.5 +/- 0.7 micrograms calpain antigen II per 10(8) platelets (mean +/- SEM) as determined by a competitive enzyme- linked immunosorbent assay. Using the dipeptide fluorogenic substrate, Suc-Leu-Tyr-MCA, 17 human platelet lysates contained 3.6 +/- 0.4 micrograms calpain activity per 10(8) platelets. Platelet calpain II was associated with the Triton X-100 insoluble platelet cytoskeletons from both unstimulated and thrombin-activated platelets. When compared with the total cell content of platelet calpain II, calpain antigen (10% to 13%) and calpain activity (24% to 28%) was associated with platelet cytoskeletons in unstimulated and thrombin-activated platelets, respectively. On immunoblot, the heavy chain (80 Kd) of calpain II was detected in platelet cytoskeletons. Subcellular fractionation studies on both unstimulated and thrombin-activated platelets, revealed that half of the total platelet calpain II antigen was associated with cytosol, and the other half was associated with the membrane fraction. Platelet calpain II was not seen on the surface of unstimulated, paraformaldehyde fixed platelets by immunofluorescence. However, on thrombin-activated platelets, rim immunofluorescence was seen, indicating that activated platelets externalize their calpain. This observation was confirmed by the finding that about 2,000 molecules per platelet of an 125I-anti-calpain II Fab' specifically bound to thrombin-activated but not unstimulated platelets. Both dibucaine (1 mmol/L) and platelet activating factor (1.86 mumol/L) in the absence of external Ca++, but not collagen (5 micrograms/mL) or ionophore A23187 (2.5 mumol/L) in the absence of external Ca++, were also able to externalize platelet calpain II antigen, as indicated by a similar level of specific 125I-anti-calpain II Fab'-platelet binding. These combined studies indicate that platelet calpain II is a major protein, comprising 2% of total platelet protein, a substantial portion of which is membrane-associated. When platelets are activated by thrombin and platelet activating factor, calpain II antigen also becomes present on the external platelet surface.     

Involvement of the parietal cortex in perceptual learning (Eureka effect): An interference approach using rTMS

The neural mechanisms underlying perceptual learning are still under investigation. Eureka effect is a form of rapid, long-lasting perceptual learning by which a degraded image, which appears meaningless when first seen, becomes recognizable after a single exposure to its undegraded version. We used online interference by focal 10-Hz repetitive transcranial magnetic stimulation (rTMS) to evaluate whether the parietal cortex (PC) is involved in Eureka effect, as suggested by neuroimaging data. RTMS of the PC did not affect recognition of degraded pictures when displayed 2 s after the presentation of their undegraded version (learning phase). However, rTMS delivered over either right or left intraparietal sulcus simultaneously to the undegraded image presentation, disrupted identification of the degraded version of the same pictures when displayed 30 min after the learning phase. In contrast, recognition of degraded images was unaffected by rTMS over the vertex or by sham rTMS, or when rTMS of either PC was delivered 2 s after the presentation of the undegraded image. Findings strongly support the hypothesis that both PC at the level of the intraparietal sulcus play a pivotal role in the Eureka effect particularly in consolidation processes, and contribute to elucidate the neural network underlying rapid perceptual learning.     

ANGIOTENSIN II RECEPTORS AND RENIN IN THE PORCINE UTERUS: MYOMETRIAL AT2 AND ENDOMETRIAL AT1 RECEPTORS ARE DOWN-REGULATED DURING GESTATION

1. The aim of the present study was to characterize the angiotensin II (AngII) receptor subtypes in the porcine uterus and the variation of receptor densities and renin concentrations during gestation. 2. In myometrium from non-pregnant sows, the AngII receptors were almost exclusively AT₂ receptors. During gestation, the AngII receptor density was decreased and the AT₁ receptor became predominant in the last part of gestation as a result of a down-regulation of the AT₂ receptor. 3. In the endometrium, the AT₁ receptor was predominant both in non-pregnant sows and throughout gestation. The AngII receptor density was decreased during gestation as a consequence of down-regulation of the AT₁ receptor. 4. The renin concentrations in the myometrium and endometrium of pregnant sows did not differ from those in non-pregnant animals. 5. The finding of enzymatically active renin and high densities of AngII receptors in the porcine uterus is in accordance with a functional renin-angiotensin system (RAS), which may be important for an increased vascular permeability and stimulated angiogenesis in early pregnancy and for contraction of the myo-metrial smooth muscle cells during parturition. The predominance of ATi receptors in the endometrium of non-pregnant sows differs from an earlier finding in non-pregnant women, where AT2 receptors were predominant in the endometrium. This is in accordance with earlier studies, indicating species differences in the expression and possibly also the physiological roles of the RAS in reproductive tissues.     

Bidirectional transport of NMDA receptor and ionophore in the vagus nerve

Accumulations of binding sites for [3H]3-[+-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic ([3H]CPP) and [3H]-N-(1-[2-thienyl]cyclohexyl)-3,4-piperidine ([3H]TCP) were demonstrated both proximal and distal to 24-h ligatures of the central and peripheral vagus nerve in the rat using radioligand binding and autoradiography. Peaks of label occurred at the proximal and distal extremities of double ligations but not between the ligatures, or in sham-operated or colchicine-treated nerves. Together these results suggest that both the primary acceptor site and the ionophore of the N-methyl-D-aspartate receptor undergo bidirectional axonal transport in central and peripheral branches of the vagus nerve.