CHO cells expressing the high affinity alpha(IIb)beta3 T562N integrin demonstrate enhanced adhesion under shear.

BACKGROUND: Alpha(IIb)beta3-mediated platelet adhesive interactions in the vasculature, which are dependent on the functional state of this receptor, may be sensitive to shear forces. OBJECTIVES: To evaluate the influence of the alpha(IIb)beta3 affinity state on cell attachment under flow, we compared Chinese hamster ovary cells expressing the low affinity alpha(IIb)beta3 wild-type (wt) receptor to those expressing the high affinity alpha(IIb)beta3 T562N receptor. MATERIALS AND METHODS: We designed a real-time videomicroscopy adhesion assay for von Willebrand factor (VWF) or fibrinogen under flow conditions. RESULTS: At 50 s(-1), alpha(IIb)beta3 T562N supported higher cell adhesion to fibrinogen (63.3 +/- 2.9 cells/field) than alpha(IIb)beta3 wt (38.7 +/- 2.4 cells/field, P < 0.0001). At 100 s(-1), alpha(IIb)beta3 T562N mediated cell adhesion (40.5 +/- 3.8 cells/field), while alpha(IIb)beta3 wt did not (5.3 +/- 1.4 cells/field, P < 0.001), allowing to discriminate the efficiency of each receptor. Similar findings were observed for adhesion to VWF. Complete inhibition of cell adhesion to fibrinogen was achieved with 800 microM fibrinogen gamma-chain dodecapeptide [HHLGGAKQAGDV (H12)], while Arg-Gly-Asp-Ser (RGDS) peptide (10-1000 microM) induced a dose-dependent cell detachment. These results suggest that the H12 motif allows initial attachment, in contrast to the RGDS site, which strengthens the stability of adhesion. Interestingly, compared with wt, a 10-fold lower concentration of RGDS was required to reach a similar reduction of cell adhesion mediated by alpha(IIb)beta3 T562N. CONCLUSIONS: Our data show that alpha(IIb)beta3 activation is associated with a stabilization of integrin binding to fibrinogen or VWF under shear.

Identification of lumbar spinal neurons controlling simultaneously the prostate and the bulbospongiosus muscles in the rat.

Lumbar spinothalamic neurons in the lamina X of the L3-L4 spinal cord segment have been proposed to constitute the spinal ejaculation generator in male rats. Lumbar spinothalamic cells are immunoreactive for galanin and neurokinin-1 receptors. We previously showed that after injection of pseudorabies virus either in the bulbospongiosus muscle or in the prostate, retrogradely labeled cells in the L3-L4 segment also displayed galanin or neurokinin-1 receptor immunoreactivities, demonstrating a direct link between lumbar spinothalamic cells and two anatomical structures involved in the two phases of ejaculation i.e. the emission and the expulsion phases. In order to provide with a more precise anatomical support for the role of lumbar spinothalamic cells in controlling ejaculation, we injected simultaneously in male adult rats two strains of recombinant pseudorabies virus, expressing either beta-galactosidase (PRV-BaBlu) or green fluorescent protein (PRV-152) in the prostate and in the bulbospongiosus muscle, respectively. After 5 days, we performed multiple immunofluorescence experiments to detect PRV-BaBlu, PRV-152 and galanin or neurokinin-1 receptors in transverse sections of the L1-S1 segment. Double- and triple-labeled cells were counted using confocal laser scanning microscope. Double-labeled neurons with the two strains of pseudorabies virus were mainly found at the L3-L4 segment lateral to the central canal in lamina X and represented about 60% of the total number of pseudorabies virus-labeled neurons. All the double pseudorabies virus-labeled neurons also expressed lumbar spinothalamic and most of them neurokinin-1 receptor, identifying them as lumbar spinothalamic neurons. The convergence of retrograde labeling from prostate and bulbospongiosus muscle on the same lumbar spinothalamic cells strongly reinforce their role in the spinal control and coordination of the emission and expulsion of sperm.