Human NPPB peptide

Regulatory volume decrease (RVD) occurs after hypotonicity-caused cell swelling. RVD is caused by activation of ion channels and transporters, which cause effluxes of K+, Cl-, and H2O, leading to cell shrinkage. Recently, we showed that hypotonicity stimulated transepithelial Na+ reabsorption via elevation of epithelial Na+ channel (alpha-ENaC) expression in renal epithelia A6 cells in an RVD-dependent manner and that reduction of intracellular Cl- concentration ([Cl-](i)) stimulated the Na+ reabsorption. These suggest that RVD would reveal its stimulatory action on the Na+ reabsorption by reducing [Cl-](i). However, the reduction of [Cl-](i) during RVD has not been definitely analyzed due to technical difficulties involved in halide-sensitive fluorescent dyes. In the present study, we developed a new method for the measurement of [Cl+](i) change during RVD by using a high-resolution flow cytometer with a halide-specific fluorescent dye, N-(6-methoxyquinolyl) acetoethyl ester. The [Cl+] i in A6 cells in an isotonic medium was 43.6 +/- 3.1 mM. After hypotonic shock (268 to 134 mosmol/ kgH(2)O), a rapid increase of cell volume followed by RVD occurred. The RVD caused drastic diminution of [Cl-] i from 43.6 to 10.8 mM. Under an RVD- blocked condition with NPPB (Cl- channel blocker) or quinine (K+ channel blocker), we did not detect the reduction of [Cl-] i. Based on these observations, we conclude that one of the physiological significances of RVD is the reduction of [Cl-] i and that RVD shows its action via reduction of [Cl-] i acting as an intracellular signal regulating cellular physiological functions.

Volume-regulated anion channels (VRAC) are widely expressed chloride channels that are critical for the cell volume regulation. In the mammalian central nervous system, the physiological expression of neuronal VRAC and its role in cerebral ischemia are issues largely unknown. We show that hypoosmotic medium induce an outwardly rectifying chloride conductance in CA1 pyramidal neurons in rat hippocampal slices. The induced chloride conductance was sensitive to some of the VRAC inhibitors, namely, IAA-94 (300 mu M) and NPPB (100 mu M), but not to tamoxifen (10 mu M). Using oxygen-andglucose deprivation (OGD) to simulate ischemic conditions in slices, VRAC activation appeared after OGD induced anoxic depolarization (AD) that showed a progressive increase in current amplitude over the period of post-OGD reperfusion. The OGD induced VRAC currents were significantly inhibited by inhibitors for glutamate AMPA (30 mu M NBQX) and NMDA (40 mu M AP-5) receptors in the OGD solution, supporting the view that induction of AD requires an excessive Na+-loading via these receptors that in turn to activate neuronal VRAC. In the presence of NPPB and DCPIB in the post-OGD reperfusion solution, the OGD induced CA1 pyramidal neuron death, as measured by TO-PRO-3-I staining, was significantly reduced, although DCPIB did not appear to be an effective neuronal VRAC blocker. Altogether, we show that rat hippocampal pyramidal neurons express functional VRAC, and ischemic conditions can initial neuronal VRAC activation that may contribute to ischemic neuronal damage.