Abstract
BackgroundIon channels occur as large families of related genes with cell-specific expression patterns. Granulosa cells have been shown to express voltage-gated potassium channels from more than one family. The purpose of this study was to determine the effects of 4-aminopyridine (4-AP), an antagonist of KCNA but not KCNQ channels.MethodsGranulosa cells were isolated from pig follicles and cultured with 4-AP, alone or in combination with FSH, 8-CPT-cAMP, estradiol 17β, and DIDS. Complimentary experiments determined the effects of 4-AP on the spontaneously established pig granulosa cell line PGC-2. Granulosa cell or PGC-2 function was assessed by radio-immunoassay of media progesterone accumulation. Cell viability was assessed by trypan blue exclusion. Drug-induced changes in cell membrane potential and intracellular potassium concentration were documented by spectrophotometric determination of DiBAC4(3) and PBFI fluorescence, respectively. Expression of proliferating cell nuclear antigen (PCNA) and steroidogenic acute regulatory protein (StAR) was assessed by immunoblotting. Flow cytometry was also used to examine granulosa cell viability and size.Results4-AP (2 mM) decreased progesterone accumulation in the media of serum-supplemented and serum-free granulosa cultures, but inhibited cell proliferation only under serum-free conditions. 4-AP decreased the expression of StAR, the production of cAMP and the synthesis of estradiol by PGC-2. Addition of either 8-CPT-cAMP or estradiol 17β to serum-supplemented primary cultures reduced the inhibitory effects of 4-AP. 4-AP treatment was also associated with increased cell size, increased intracellular potassium concentration, and hyperpolarization of resting membrane potential. The drug-induced hyperpolarization of resting membrane potential was prevented either by decreasing extracellular chloride or by adding DIDS to the media. DIDS also prevented 4-AP inhibition of progesterone production.Conclusion4-AP inhibits basal and FSH-stimulated progesterone production by pig granulosa cells via drug action at multiple interacting steps in the steroidogenic pathway. These inhibitory effects of 4-AP on steroidogenesis may reflect drug-induced changes in intracellular concentrations of K+and Cl- as well as granulosa cell resting membrane potential.
Highlights
Ion channels occur as large families of related genes with cell-specific expression patterns
These data provide additional evidence that 4-AP exerts neither an anti-proliferative nor a pro-apoptotic influence on serum-supplemented granulosa cells (GC) cultures, because proliferating cell nuclear antigen (PCNA) expression is a sensitive marker of GC proliferation and apoptosis [18,26,27]
The lack of 4-AP effect on GC viability is evident from the flow cytometric analysis; the percentages of cells staining positive for propidium iodide were 8 ± 2% and 7 ± 2% in the presence and absence of drug (n = 4 samples of 10,000 cells/each from 4 GC isolations)
Summary
Ion channels occur as large families of related genes with cell-specific expression patterns. Voltage-gated K+ channels in nonnerve, non-muscle cells play crucial roles in cell development, proliferation, migration, volume regulation, as well as maintenance of membrane potential and cell viability. This is in part because these channels regulate the cytoplasmic concentrations of K+, Ca2+, and Na+ ions [1,2,3,4,5,6,7,8,9,10]. Selective antagonism of GC K+ channels with distinct molecular correlates, electrophysiological properties and expression patterns can influence differentially GC proliferation, steroidogenic capability, and apoptosis [17,18]. We have shown that selective block of IKs enhances basal progesterone synthesis, while complete block of both IKs and IKur accelerates apoptosis [18]
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