4-aminopyridine decreases progesterone production by porcine granulosa cells.
BACKGROUND
Ion 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.


METHODS
Granulosa cells were isolated from pig follicles and cultured with 4-AP, alone or in combination with FSH, 8-CPT-cAMP, estradiol 17beta, 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.


RESULTS
4-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 17beta 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.


CONCLUSIONS
4-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.
Departments of Anatomy & Physiology College of Veterinary Medicine, Kansas State University, Manhattan, Kansas 66506-5802, USA. yanli@vet.k-state.edu
Yan
Y
Li
Suhasini
S
Ganta
Fred B
FB
von Stein
Diane E
DE
Mason
Brianna M
BM
Mitchell
Lisa C
LC
Freeman
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D015761:4-Aminopyridine / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line / Q000187:drug effects / Q000378:metabolism; D048429:Cell Size / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002712:Chlorides / Q000378:metabolism; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D003864:Depression, Chemical; D004347:Drug Interactions; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D006107:Granulosa Cells / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D007424:Intracellular Fluid / Q000737:chemistry; D017136:Ion Transport / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects; D010750:Phosphoproteins / Q000096:biosynthesis; D011188:Potassium / Q000378:metabolism; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology; D011374:Progesterone / Q000096:biosynthesis*; D018809:Proliferating Cell Nuclear Antigen / Q000096:biosynthesis; D013552:Swine; D013873:Thionucleotides / Q000494:pharmacology; D000098682:Steroidogenic Acute Regulatory Protein
D002712:Chlorides; D016895:Culture Media, Serum-Free; D010750:Phosphoproteins; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D018809:Proliferating Cell Nuclear Antigen; D013873:Thionucleotides; D000098682:Steroidogenic Acute Regulatory Protein; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D011374:Progesterone; D004958:Estradiol; D005640:Follicle Stimulating Hormone; D015761:4-Aminopyridine; D000242:Cyclic AMP; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D011188:Potassium