Abstract-Recently, we found that vacuolar proton ATPase (VPATPase) operates in cardiomyocytes as a complementary proton-extruding mechanism. Its activity was increased by preconditioning with resultant attenuation of intracellular acidification during ischemia. In this study, we examined whether VPATPase-mediated proton efflux during metabolic inhibition/recovery may spare Na ϩ overload via Na ϩ -H ϩ exchange, attenuate Na ϩ -Ca 2ϩ exchange, and decrease apoptosis. Neonatal rat cardiomyocytes were subjected to 2-to 3-hour metabolic inhibition with cyanide and 2-deoxyglucose and 24-hour recovery. The effect of VPATPase inhibition by 50 nmol/L bafilomycin A 1 on apoptosis, pH i , and [Ca 2ϩ ] i was studied by flow cytometry with propidium iodide, seminaphthorhodafluor (SNARF)-1-AM, and indo-1-AM staining, respectively. VPATPase inhibition increased the amount of apoptosis measured after 24 hours of recovery and abrogated the protective effect of inhibition of Na

In the course of our studies, we have shown the presence of calcitonin gene related peptide (CGRP) by immunocytochemistry in cell bodies and nerve fibers of the murine thymus and in a sparse innervation of the spleen. Receptors for CGRP have been characterized within these glands, and their activation by physiological levels of CGRP was found to suppress Con A-stimulated proliferation of thymocytes and splenic T cells as well as antigen-specific T-cell proliferation. This suppression is blocked by the antagonist for CGRP (CGRP 8-37). Within the thymus cultures, the antagonist CGRP (8-37) alone enhanced proliferation of thymocytes during Con A stimulation, most likely by inhibiting the endogenous release of CGRP into the culture medium by resident thymocytes. Some of the CGRP-induced suppression of mitogenic stimulation of thymocytes, but not of splenocytes, was due to apoptosis. The antagonist, CGRP(8-37), did not block apoptosis caused by Con A or CGRP but rather enhanced it. Flow cytometric analysis of CGRP-treated cultures using antibodies to cluster determinates (CD) showed that the majority of thymocytes undergoing apoptosis induced by CGRP were of the CD4/CD8 double-positive type. These data indicate that apoptosis in the thymocytes is mediated by a CGRP receptor not sensitive to the antagonist CGRP(8-37). Because proliferation of thymocytes and splenocytes induced by Con A is blocked by this antagonist and splenocytes are refractory to CGRP induced apoptosis, CGRP appears to mediate at least two separate functions on subpopulations of thymocytes and T cells via two different CGRP receptors within the gland. These effects of a neuropeptide exemplify the phenomenon of differential regional regulation of immunity by the autonomic and neuroendocrine systems.

There is evidence suggesting that the diabetic state adversely affects replication of certain cell populations. We document that exposure to high ambient glucose (20 mM) induces delay in various phases of the cell cycle of human endothelial cells in primary culture. Cells in S phase were labeled with bromodeoxyuridine (an analogue of thymidine), and the cell-cycle position of the labeled cohort was analyzed by flow cytometry at successive time points. The movement of cells exposed to high glucose for 7-8 days was retarded both in S and G2 phases so that the increase in bromodeoxyuridine-positive cells over 24 h was 1.6-fold, versus 2.0-fold in control cultures. In experiments in which mitotic arrest with vinblastine was used to investigate the movement of cells out of G1 phase without interference from reentering cells, depletion of the G1 compartment was significantly inhibited in cultures grown in high glucose. The effects of chronic high glucose on cell cycle occurred while total protein synthesis was not diminished. Acute exposure to high glucose had no effect on cell-cycle traversal or cell generation time. Cell-cycle abnormalities observed in this study may relate to the DNA damage we have previously observed in endothelial cells exposed to high glucose and, if occurring in vivo, could be of pathogenetic importance for the vascular lesions and teratogenicity of diabetes.

The N-methyl-D-aspartate (NMDA) receptor is expressed on neural tissue where it gates calcium ion entry upon stimulation. Using immunohistochemistry, it has been demonstrated in this study that the NMDAR1 receptor is also expressed on keratinocytes (KCs) in normal human skin and inflamed psoriatic skin in vivo. Furthermore, the NMDA receptor was functional as demonstrated by the ability of this receptor to trigger Ca++ influx in KCs. Incubation of cultured, human KCs with MK-801 decreases the cell growth and induces an increase in apoptosis. These findings demonstrate that the KC expression of NMDA receptor is a mechanism through which the influx of Ca++ into the cell can be regulated and suggest that the expression of this receptor may play a role in the regulation of KC growth and differentiation.