Homeostasis of extracellular ATP in human erythrocytes.

We explored the intra- and extracellular processes governing the kinetics of extracellular ATP (ATPe) in human erythrocytes stimulated with agents that increase cAMP. Using the luciferin-luciferase reaction in off-line luminometry we found both direct adenylyl cyclase activation by forskolin and indirect activation through ß-adrenergic stimulation with isoproterenol-enhanced [ATP]e in a concentration-dependent manner. A mixture (3V) containing a combination of these agents and the phosphodiesterase inhibitor papaverine activated ATP release, leading to a 3-fold increase in [ATP]e, and caused increases in cAMP concentration (3-fold for forskolin + papaverine, and 10-fold for 3V). The pannexin 1 inhibitor carbenoxolone and a pannexin 1 blocking peptide ((10)Panx1) decreased [ATP]e by 75-84%. The residual efflux of ATP resulted from unavoidable mechanical perturbations stimulating a novel, carbenoxolone-insensitive pathway. In real-time luminometry experiments using soluble luciferase, addition of 3V led to an acute increase in [ATP]e to a constant value of ∼1 pmol × (10(6) cells)(-1). A similar treatment using a surface attached luciferase (proA-luc) triggered a rapid accumulation of surface ATP levels to a peak concentration of 2.4 pmol × (10(6) cells)(-1), followed by a slower exponential decay (t(½) = 3.7 min) to a constant value of 1.3 pmol × (10(6) cells)(-1). Both for soluble luciferase and proA-luc, ATP efflux was fully blocked by carbenoxolone, pointing to a 3V-induced mechanism of ATP release mediated by pannexin 1. Ecto-ATPase activity was extremely low (∼28 fmol × (10(6) cells min)(-1)), but nevertheless physiologically relevant considering the high density of erythrocytes in human blood.

Frequency spectrum of transepithelial potential difference reveals transport-related oscillations.

How epithelia transport fluid is a fundamental issue that is unresolved. Explanations offered include molecular engines, local transcellular osmosis, local paracellular osmosis, and paracellular fluid transport. On the basis of experimental and theoretical work done on corneal endothelium, a fluid transporting epithelium, we suggest electroosmotic coupling at the level of the intercellular junctions driven by the transendothelial electrical potential difference as an explanation of paracellular fluid transport. We collect frequency spectra of that potential difference in real-time. For what we believe is the first time for any epithelium, we report that, unexpectedly, the potential difference displays oscillations at many characteristic frequencies. We also show that on both stimulating cell activity and inhibiting ion transport mechanisms, there are corresponding changes in the oscillations amplitudes that mirror changes known previously in rates of fluid transport. We believe these findings provide a novel tool to study the kinetics of electrogenic elements such as channels and transporters, which from this evidence would give rise to current oscillations with characteristic periods going from 150 ms to 8 s.

Polycystin-2 cation channel function in the human syncytiotrophoblast is regulated by microtubular structures.

Polycystin-2 (PC2), encoded by PKD2, which is one of the genes whose mutations cause polycystic kidney disease, is abundantly produced in the apical domain of the syncytiotrophoblast (hST) of term human placenta. PC2, a TRP-type (TRPP2) non-selective cation channel, is present in primary cilia of renal epithelial cells, a microtubule-based ancillary structure with sensory function. The hST has abundant cytoskeletal structures, and actin filament dynamics regulate PC2 channel function in this epithelium. However, it is expected that the apical hST excludes microtubular structures. Here, we demonstrated by Western blot and immunocytochemical analyses that hST apical vesicles indeed contain microtubule structural components, including tubulin isoforms, acetylated alpha-tubulin, and the kinesin motor proteins KIF3A and KIF3B. PC2 and tubulin were substantially colocalized in hST vesicles. Treatment of hST vesicles with either the microtubular disrupter colchicine (15 microM) or the microtubular stabilizer paclitaxel (taxol, 15 microM) resulted in distinct patterns of microtubular re-organization and PC2 redistribution. We also observed that changes in microtubular dynamics regulate PC2 channel function. Addition of colchicine rapidly inhibited PC2 channel activity in lipid-bilayer reconstituted hST membranes. Addition of either tubulin and GTP, or taxol, however, stimulated PC2 channel activity in control hST membranes. Interestingly, we found that the kinesin motor protein KIF3A was capable of increasing PC2 channel activity in hST. We believe that the data are the first to provide a direct demonstration of a microtubular interaction with PC2 in the hST. This interaction thus plays an important regulatory role in the control of ion transport in the human placenta.

Effect of hydro-osmotic pressure on polycystin-2 channel function in the human syncytiotrophoblast.

Polycystin-2 (PC2), one of the gene products whose mutations cause autosomal dominant polycystic kidney disease is a transient receptor potential (TRP)-type (TRPP2) Ca(2+)-permeable, non-selective cation channel. PC2 is localized in the plasma membrane, the primary cilium, and other cellular organelles of renal epithelial and other cells. Recent studies indicate that PC2 is involved in signal transduction events associated with the transient increase in cytosolic Ca(2+). Proof of evidence now hinges on involvement of the PC2 channel in the transduction of environmental signals. PC2 is abundantly expressed in the apical membrane of human syncytiotrophoblast (hST), a highly intricate epithelial tissue, which is essential for the maternal-fetal transfer of solutes, including ions. Physical forces such as hydrostatic (H) and osmotic (Pi) pressure play important roles in placenta homeostasis. In this study, we provide new information on PC2 channel regulation in the hST by these environmental factors, and propose a model as to how they may trigger the activation of PC2. Using apical hST vesicles reconstituted in a lipid bilayer system, we found that a change in either H or Pi modified PC2 channel activity. This stimulatory effect was no longer observed in hST vesicles pre-treated with the actin cytoskeleton disrupter cytochalasin D. As shown by immunofluorescence analysis PC2 co-localized with actin filaments in the vicinity of the plasma membrane. This co-localization was disrupted by cytochalasin D. Taken together, our findings indicate that physical forces exerted on cells regulate PC2 channel activity by a sensory mechanism involving the actin cytoskeleton.

Cytoskeletal regulation of calcium-permeable cation channels in the human syncytiotrophoblast: role of gelsolin.

The human syncytiotrophoblast (hST) is the most apical epithelial barrier that covers the villous tree of the human placenta. An intricate and highly organized network of cytoskeletal structures supports the hST. Recently, polycystin-2 (PC2), a TRP-type nonselective cation channel, was functionally observed in hST, where it may be an important player to Ca2+ transport. Little is known, however, about channel regulation in hST. In this report, the regulatory role of actin dynamics on PC2 channels reconstituted from hST apical membranes was explored. Acute addition of cytochalasin D (CD, 5 microg ml-1) to reconstituted hST apical membranes transiently increased K+ -permeable channel activity. The actin-binding proteins alpha-actinin and gelsolin, as well as PC2, were observed by Western blot and immunofluorescence analyses in hST vesicles. CD treatment of hST vesicles resulted in a re-distribution of actin filaments, in agreement with the effect of CD on K+ channel activity. In contrast, addition of exogenous monomeric actin, but not prepolymerized actin, induced a rapid inhibition of channel function in hST. This inhibition was obliterated by the presence of CD in the medium. The acute (<15 min) CD stimulation of K+ channel activity was mimicked by addition of the actin-severing protein gelsolin in the presence, but not in the absence, of micromolar Ca2+. Ca2+ transport through PC2 triggers a regulatory feedback mechanism, which is based on the severing and re-formation of filamentous actin near the channels. Cytoskeletal structures may thus be relevant to ion transport regulation in the human placenta.