Extracellular nucleotides regulate NaCl transport in some epithelia. However, the effects of nucleotide agonists on NaCl transport in the renal inner medullary collecting duct (IMCD) are not known. The objective of this study was to determine whether ATP and related nucleotides regulate NaCl transport across mouse IMCD cell line (mIMCD-K2) epithelial monolayers and, if so, via what purinergic receptor subtypes. ATP and UTP inhibited Na(+) absorption [measured via Na(+) short-circuit current (I(Na)(sc))] and stimulated Cl(-) secretion [measured via Cl(-) short-circuit current (I(Cl)(sc))]. Using selective P2 agonists, we report that P2X and P2Y purinoceptors regulate I(Na)(sc) and I(Cl)(sc). By RT-PCR, two P2X receptor channels (P2X(3), P2X(4)) and two P2Y G protein-coupled receptors (P2Y(1), P2Y(2)) were identified. Functional localization of P2 purinoceptors suggest that I(Cl)(sc) is stimulated by apical membrane-resident P2Y purinoceptors and P2X receptor channels, whereas I(Na)(sc) is inhibited by apical membrane-resident P2Y purinoceptors and P2X receptor channels. Together, we conclude that nucleotide agonists inhibit I(Na)(sc) across mIMCD-K2 monolayers through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane, whereas extracellular nucleotides stimulate I(Cl)(sc) through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane.

We have studied human melanoma cell (C8161) adhesion and migration in response to stimulation by soluble collagen IV (CIV) using a modified Boyden chamber. In this modified chamber, shear flow can be introduced over the cell-substrate interface, affecting tumor cell chemotactic migration through a microporous filter. A relatively high level of intercellular adhesion molecule-1 (ICAM-1) was found on C8161 cells. In contrast, levels of ␤ 2 -integrins (e.g., LFA-1 and Mac-1), the molecules that would be necessary for C8161 stable adhesion to the endothelium substrate, were found to be very low on these melanoma cells. As a result, C8161 transendothelial migration under a flow condition of 4 dyn/cm 2 decreased by 70% as compared to static migration. When human neutrophils (PMNs) were present in the tumor cell suspension, C8161 migration recovered by 85% over C8161 cells alone under the 4 dyn/cm 2 flow condition. Blocking ICAM-1 on C8161 cells or Mac-1 on PMNs significantly inhibited C8161-PMN adhesion and subsequent C8161 migration through the endothelium under flow conditions. In addition, increased interleukin-8 production and Mac-1 expression by PMNs were detected when they were co-cultured with C8161 melanoma cells. These results suggest that transmigration of C8161 cells under flow conditions can be influenced by PMNs, mediated by Mac-1/ICAM-1 adhesive interactions and enhanced by altered cytokine production.

Previously, we found polymorphonuclear neutrophils (PMNs) increased melanoma cell extravasation under flow conditions (Intl J Cancer 106: 713-722, 2003). In this study, we characterized the effect of hydrodynamic shear on PMN-facilitated melanoma extravasation using a novel flow-migration assay. The effect of shear stress and shear rate on PMN-facilitated melanoma extravasation was studied by increasing the medium viscosity with dextran to increase shear stress independently of shear rate. Under fixed shear rate conditions, melanoma cell extravasation did not change significantly. In contrast, the extravasation level increased at a fixed shear stress but with a decreasing shear rate. PMN-melanoma aggregation and adhesion to the endothelium via beta(2)-integrin/intracellular adhesion molecule-1 (ICAM-1) interactions were also studied. Lymphocyte function-associated molecule-1 (LFA-1; CD11a/CD18) influenced the capture phase of PMN binding to both melanoma cells and the endothelium, whereas Mac-1 (CD11b/CD18) affected prolonged PMN-melanoma aggregation. Blockage of E-selectin or ICAM-1 on the endothelium or ICAM-1 on the melanoma surface reduced PMN-facilitated melanoma extravasation. We have found PMN-melanoma adhesion is correlated with the inverse of shear rate, whereas the PMN-endothelial adhesion correlated with shear stress. Interleukin-8 (IL-8) also influenced PMN-melanoma cell adhesion. Functional blocking of the PMN IL-8 receptors, CXCR1 and CXCR2, decreased the level of Mac-1 upregulation on PMNs while in contact with melanoma cells and reduced melanoma extravasation. We have found PMN-facilitated melanoma adhesion to be a complex multistep process that is regulated by both microfluid mechanics and biology.

Previous studies have shown that neutrophils (PMNs) facilitate melanoma cell extravasation [M.J. Slattery, C. Dong, Neutrophils influence melanoma adhesion and migration under flow conditions, Intl. J. Cancer 106 (2003) 713-722] Little is known, however, about the specific interactions between PMNs, melanoma and the endothelium (EC) or the molecular mechanism involved under flow conditions. The aim of this study is to investigate a "two-step adhesion" hypothesis that involves initial PMN tethering on the EC and subsequent melanoma cells being captured by tethered PMNs. Different effects of hydrodynamic shear stress and shear rate were analyzed using a parallel-plate flow chamber. Results indicate a novel finding that PMN-facilitated melanoma cell arrest on the EC is modulated by shear rate, which is inversely-proportional to cell-cell contact time, rather than by the shear stress, which is proportional to the force exerted on formed bonds. Beta2 integrins/ICAM-1 adhesion mechanisms were examined and the results indicate LFA-1 and Mac-1 cooperate to mediate the PMN-EC-melanoma interactions under shear conditions. In addition, endogenously produced IL-8 contributes to PMN-facilitated melanoma arrest on the EC through the CXC chemokine receptors 1 and 2 (CXCR1 and CXCR2) on PMN. These results provide new evidence for the complex role of hemodynamic forces, secreted chemokines and PMN-melanoma adhesion in the recruitment of metastatic cancer cells to the EC.

Adhesion to and subsequent extravasation through the endothelial lining of blood vessels is critical for tumor cells to establish metastases. Recent studies have indicated that polymorphonuclear neutrophils (PMNs) may enhance melanoma adhesion to the endothelium (EC) and subsequent extravasation under dynamic flow conditions. However, little is known about hydrodynamics involved in the tumor microenvironment within the microcirculation. In this study, effects of hydrodynamic flow on regulating melanoma cell adhesion to the EC have been investigated. Results indicate that under flow conditions, interactions between melanoma cells and the EC are distinctly different from PMN-EC interactions. Without expressions of surface integrins or sialylated molecules, most melanoma cells that express a high-level of intercellular adhesion molecule (ICAM-1) are not able to effectively adhere to the inflamed EC by themselves. Binding of melanoma cells and PMNs through ICAM-1 on melanoma cells and β 2 integrins on PMNs has been shown to enhance melanoma cell arrest on the EC. Although PMN tethering on the EC is regulated by both the shear rate and shear stress, melanoma cell adhesion to the EC and subsequent extravasation via tethering PMN on the EC is predominantly regulated by shear rate, which partly is due to the shearrate-dependent PMN-melanoma aggregation in shear flow. These findings provide a rationale and mechanistic basis for understanding of leukocyte-tumor cell interactions under flow conditions during tumor cell extravasation and metastasis.