store, but partially blocked by ryanodine or inhibition of lysosomerelated acidic organelles with bafilomycin A1. Simultaneous inhibition of both pathways was necessary to abolish the response. GRGDSP treatment increased cyclic ADP-ribose , the endogenous activator of ryanodine receptors, by 70%. GRGDSP also rapidly reduced Lysotracker Red accumulation, confirming direct modulation of acidic organelles. These data are the first demonstration of integrin-mediated Ca 2؉ regulation in PASMCs. The presence of an array of integrins, and activation of ryanodine-sensitive Ca 2؉ stores and lysosome-like organelles by GRGDSP suggest important roles for integrin-dependent Ca 2؉ signaling in regulating PASMC function. Extracellular matrix (ECM)2 protein receptors, or integrins, comprise a superfamily of structurally related heterodimeric transmembrane receptors that mediate cell-cell and cell-ECM interactions. Integrins physically bridge the ECM and cytoskeleton, and act as transducers of "outside-in" and "inside-out" signaling (1). Extracellular integrin ligation changes [Ca 2ϩ ] i in a variety of cell types, including platelets, neutrophils, monocytes, lymphocytes, fibroblasts, endothelial cells, osteoclasts, neurons, glomerulosa cells, epithelial and smooth muscle cells (2-5). Integrin ligation also activates other intracellular signaling molecules, including H ϩ and a plethora of protein kinases such as the focal adhesion kinase, Rac, and extracellular signal regulated kinases (1, 6). As a result, a myriad of cellular functions such as differentiation, proliferation, migration, apoptosis, and mechanosensing for shear and tension are affected (6, 7).Integrins participate in controlling systemic vascular tone by invoking changes in smooth muscle [Ca 2ϩ ] i , resulting in relaxation or contraction. Synthetic ligands carrying the prototypic integrin-binding tripeptide motif, arginine-glycine-aspartate (RGD), decrease [Ca 2ϩ ] i causing vasodilation in rat cremaster muscle arterioles (8, 9). On the other hand, RGD-containing peptides constrict rat renal afferent arterioles through increased [Ca 2ϩ ] i (10, 11). Other integrin binding sequences also exist, such as leucine-aspartate-valine (LDV), which elevates [Ca 2ϩ ] i and causes vasoconstriction of rat cremaster muscle arterioles (12). Integrins ␣ 5 ␤ 1 and ␣ v ␤ 3 are, furthermore, directly involved in the vascular myogenic behavior, where blockade of either integrin inhibits myogenic constriction of skeletal muscle arterioles (13).Apart from regulating tone, integrins play an important role in vascular pathogenesis. This is evidenced by the remodeling and deposition of ECM components seen in various vascular diseases such as atherosclerosis, hypertension, and restenosis (5). In the pulmonary vasculature, remodeling occurs in chronic obstructive pulmonary disease, asthma, scleroderma, and pulmonary hypertension (14). Interestingly, pulmonary vascular remodeling has been linked to integrin activation in rat models recapitulating pulmonary hypertension. Previo...

Background: Pulmonary hypertension is associated with vascular remodeling and increased extracellular matrix (ECM) deposition. While the contribution of ECM in vascular remodeling is well documented, the roles played by their receptors, integrins, in pulmonary hypertension have received little attention. Here we characterized the changes of integrin expression in endothelium-denuded pulmonary arteries (PAs) and aorta of chronic hypoxia as well as monocrotaline-treated rats. Methods and Results: Immunoblot showed increased α₁-, α₈- and α_v-integrins, and decreased α₅-integrin levels in PAs of both models. β₁- and β₃-integrins were reduced in PAs of chronic hypoxia and monocrotaline-treated rats, respectively. Integrin expression in aorta was minimally affected. Differential expression of α₁- and α₅-integrins induced by chronic hypoxia was further examined. Immunostaining showed that they were expressed on the surface of PA smooth muscle cells (PASMCs), and their distribution was unaltered by chronic hypoxia. Phosphorylation of focal adhesion kinase was augmented in PAs of chronic hypoxia rats, and in chronic hypoxia PASMCs cultured on the α₁-ligand collagen IV. Moreover, α₁-integrin binding hexapeptide GRGDTP elicited an enhanced Ca²⁺ response, whereas the response to α₅-integrin binding peptide GRGDNP was reduced in CH-PASMCs. Conclusion: Integrins in PASMCs are differentially regulated in pulmonary hypertension, and the dynamic integrin-ECM interactions may contribute to the vascular remodeling accompanying disease progression.

Increased pulmonary artery endothelial cell (PAEC) endotheliumdependent nitric oxide synthase (eNOS) activity mediates perinatal pulmonary vasodilation. Compromised eNOS activity is central to the pathogenesis of persistent pulmonary hypertension of the newborn (PPHN). Voltage-derived anion channel (VDAC)-1 was recently demonstrated to bind eNOS in the systemic circulation. We hypothesized that VDAC isoforms modulate eNOS activity in the pulmonary circulation, and that decreased VDAC expression contributes to PPHN. In PAECs derived from an ovine model of PPHN: (1) there is eNOS activity, but not expression; and (2) VDAC1 and -2 proteins are decreased. Immunocytochemistry, coimmunoprecipitation, and in situ proximity ligation assays in human PAECs (hPAECs) demonstrate binding between eNOS and both VDAC1 and -2, which increased upon stimulation with NO agonists. The ability of agonists to increase the eNOS/VDAC interaction was significantly blunted in hypertensive, compared with normotensive, ovine PAECs. Depletion of VDAC2, but not VDAC1, blocked the agonist-induced increase in eNOS activity in hPAECs. Overexpression of VDAC2 in hypertensive PAECs increased eNOS activity. Binding of VDAC2 enhances eNOS activity in the pulmonary circulation, and diminished VDAC2 constrains eNOS in PAECs derived from fetal lambs with chronic intrauterine pulmonary hypertension. We speculate that decreases in VDAC2 may contribute to the limited eNOS activity that characterizes pulmonary hypertension.Keywords: pulmonary hypertension; vasodilation; fetal; protein-protein interactions At birth, the pulmonary vasculature dilates in response to ventilation, oxygenation, and an increase in shear stress (1-4). Each of these stimuli acts, to a significant degree, through an increase in endothelium-dependent nitric oxide synthase (eNOS) activity and elaboration of nitric oxide (NO) (5). NO, in turn, causes activation of pulmonary artery smooth muscle cell soluble guanylate cyclase (6) and subsequent vasodilation through activation of a calcium-sensitive K 1 channel (7). NO production from pulmonary artery endothelial cells (PAECs) is essential for the successful transition from fetal to air-breathing life (5, 8).In the absence of sufficient NO production, pulmonary vascular resistance remains elevated and blood is shunted away from the lungs, resulting in severe central hypoxemia and a syndrome called persistent pulmonary hypertension of the newborn (PPHN), a significant cause of neonatal morbidity and mortality (9).Clinical and experimental evidence demonstrates that impaired eNOS activity contributes to the pathogenesis of PPHN. However, the molecular mechanisms leading to insufficient NO production are incompletely understood. In PPHN, constrained NO production may result not only from diminished eNOS expression (10, 11), but also from modulation of eNOS activity by posttranslational modifications, protein-protein interactions, and sequestration into subcellular compartments. For example, myristoylation and palmitoylation enhance eNOS act...

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in insect central and peripheral nervous systems. Although much work has focused on the downstream targets of GABA, signal termination at insect GABAergic synapses has received very little attention. One of the major mechanisms of terminating synaptic transmission involves transport of the neurotransmitter molecules into presynaptic neurons or surrounding glia. Here we report the immunolocalization of a GABA transporter in the tobacco hornworm, Manduca sexta (MasGAT), using an affinity-purified antibody developed to the C-terminus. This is the first demonstration of an insect neurotransmitter transporter immunolocalization study. Results showed strong staining in the neuropil regions of embryonic, larval, and pharate adult central nervous system. Expression pattern in the pharate adult brain mostly mimicked that observed for GABA, with staining in parts of the optic and antennal lobes, mushroom body, lateral protocerebrum, and central complex. Certain longitudinal and lateral connectives of ganglia were observed to have immunostained fibers representing axons. These data support the view that GABA is involved in visual and olfactory processing in the insect brain.