Development and characterization of stable cell lines constitutively expressing the porcine reproductive and respiratory syndrome virus nucleocapsid protein

Despite global efforts to control porcine reproductive and respiratory syndrome virus (PRRSV) infection, the virus continues to cause economic problems in the swine industry worldwide. In this study, we attempted to generate and characterize a panel of stable BHK cell lines that constitutively express the nucleocapsid (N) protein of type 1 or type 2 PRRSV. The established BHK cell lines were found to react well with N-specific antibodies as well as the hyperimmune serum of pigs raised against each genotype of PRRSV. Taken together, the data implicate a potential usefulness for the newly generated stable cell lines as a diagnostic reagent for PRRSV serology.

PKC-Independent Stimulation of Cardiac Na+/Ca2+ Exchanger by Staurosporine

[Ca2+]i transients by reverse mode of cardiac Na+/Ca2+ exchanger (NCX1) were recorded in fura-2 loaded BHK cells with stable expression of NCX1. Repeated stimulation of reverse NCX1 produced a long-lasting decrease of Ca2+ transients ('rundown'). Rundown of NCX1 was independent of membrane PIP2 depletion. Although the activation of protein kinase C (PKC) was observed during the Ca2+ transients, neither a selective PKC inhibitor (calphostin C) nor a PKC activator (PMA) changed the degrees of rundown. By comparison, a non-specific PKC inhibitor, staurosporine (STS), reversed rundown in a dose-dependent and reversible manner. The action of STS was unaffected by pretreatment of the cells with calphostin C, PMA, or forskolin. Taken together, the results suggest that the stimulation of reverse NCX1 by STS is independent of PKC and/or PKA inhibition.

Ca2+-dependent Long-term Inactivation of Cardiac Na+/Ca2+ Exchanger

Using BHK cells with stable expression of cardiac Na+/Ca2+ exchanger (BHK-NCX1), reverse mode (i.e. Ca2+ influx mode) of NCX1 current was recorded by whole-cell patch clamp. Repeated stimulation of reverse NCX1 produced a cytosolic Ca2+-dependent long-term inactivation of the exchanger activity. The degrees of inactivation correlated with NCX1 densities of the cells and were attenuated by reduced Ca2+ influx via the reverse exchanger. The inactivation of NCX1 was attenuated by (i) inhibition of Ca2+ influx with reduced extracellular Ca2+, (ii) treatment with NCX1 blocker (Ni2+), and (iii) increase of cytoplasmic Ca2+ buffer (EGTA). In BHK-NCX1 cells transiently expressing TRPV1 channels, Ca2+ influx elicited by capsaicin produced a marked inactivation of NCX1. We suggest that cytoplasmic Ca2+ has a dual effect on NCX1 activities, and that allosteric Ca2+ activation of NCX1 can be opposed by the Ca2+-dependent long-term inactivation in intact cells.