gms | German Medical Science

The reduced efficiency to control the alternative pathway of the complement system by complement proteins with polymorphisms associated with AMD risk led to the hypothesis that the constant attack of the RPE by the terminal complement complex (TCC) is a major disease mechanism. To test this hypothesis we exposed ARPE-19 cells to high amount of complement protein (25% standardized human serum; NHS) and investigated changes in the membrane conductance and membrane permeability by means of patch-clamp techniques and measurements of intracellular free Ca2+ by means of fluorescence imaging with Ca2+-sensitive fluorescence dyes. Exposure of ARPE-19 cells with NHS led to a transient increases in the membrane conductance which lasted for 3 min and returned back to the basic values. Using either heat-inactivated NHS, Ca2+-free solutions or solutions without extracellular K+ abolished this reaction. In recordings of the membrane potential NHS led to transient hyperpolarizations for 1 min which also returned back to the basal level of -40 mV. These changes in membrane conductance and voltage were accompanied by a biphasic increase in intracellular free Ca2+ consisting of a peak followed by a sustained elevation. The Ca2+ rises were inhibited by blockers against L-type Ca2+ channels, TRPV2 Ca2+ channels, maxiK K+ channels, ryanodine receptors and sarcolemmal Ca2+ ATPase (SERCA). Using complement depleted NHS we found that all three active complement components, C3a, C5a and C5b-9, specifically contributed to different parts of the Ca2+ signal:

C3a contributed more to the initial peak, the following sustained phase was driven by C5a and C5b-9. In conclusion we found by using the highest sensitive methods to detect unspecific pores in the cell membrane that the complement proteins rather activate an orchestrated Ca2+ signal as second messenger by activation of endogenously expressed ion channels than leading to RPE cell damage.