4-Aminopyridine Decreases Progesterone Production by Porcine Granulosa Cells

Yan Li, Ganta Suhasini, Von, Mason, Mitchell, Freeman

Description

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 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 DiBAC 4 (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 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. Conclusion 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.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2003
• Nombre de lectures: 2
• Langue: English

Extrait

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Address: 1 Departments of Anatomy & Physiology Colleg e of Veterinary Medicine, Kansas State University, Manhattan, Kansas 66506-5802, USA and 2 Clinical Sciences, College of Veterinary Medicine , Kansas State University, Manhattan, Kansas 66506-5802, USA Email: Yan Li* - [e-mail]; Suhasini Ganta - sganta @vet.k-state.edu; Fred B von St ein - [e-mail]; Diane E Mason - [e-mail]; Bria nna M Mitchell - [e-mail]; Lisa C Freeman - [e-mail] * Corresponding author

Research 4-Aminopyridine Decreases Prog esterone Production by Porcine Granulosa Cells Yan Li , Suhasini Ganta 1 , Fred B von Stein 1 , Diane E Mason 2 , * 1 Brianna M Mitchell 1 and Lisa C Freeman 1

Bio Med  Central

Open Access

Abstract Background: Ion channels occur as large families of related genes with cell-specific expression patterns. Granulosa cells have been shown to e xpress voltage-gated potass ium 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 17 β , and DIDS. Complimentary experiments determined the effects of 4-AP on the spontaneously establis hed pig granulosa cell line PGC-2. Granulosa cell or PGC-2 function was assessed by radio-immunoassay of media progesterone accumulation. Cell viability was as sessed by trypan blue exclusio n. Drug-induced changes in cell membrane potential and intracellular pota ssium concentration were documented by spectrophotometric determination of DiBAC 4 (3) and PBFI fluorescence , respectively. Expression of proliferating cell nuclear antigen (PCNA) and steroidogenic acute regulatory protein (StAR) was assessed by immunoblotti ng. Flow cytometry was also used to examine granulosa cell viability and size. Results: 4-AP (2 mM) decreased progesterone accumu lation in the media of serum-supplemented and serum-free granulosa cultur es, but inhibited cell prolif eration only under serum-free conditions. 4-AP decreased the ex pression of StAR, the production of cAMP and the synthesis of estradiol by PGC-2. Addition of either 8-CPT-cAMP or estradiol 17 β  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 hy perpolarization 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. Conclusion: 4-AP inhibits basal and FSH-stimulated pr ogesterone production by pig granulosa cells via drug action at multiple interacting st eps in the steroidogenic pathway. These inhibitory effects of 4-AP on steroidogenesi s may reflect drug-induced change s in intracellular concentrations of K + and Cl - as well as granulosa cell resting membrane potential.

Published: 1 April 2003 Received: 5 February 2003 Reproductive Biology and Endocrinology 2003, 1 :31 Accepted: 1 April 2003 This article is available from: http://www.RBEj.com/content/1/1/31 © 2003 Li et al; licensee BioMed Central Ltd. This is an Open Access arti cle: verbatim copying and re distribution of this artic le are permitted in all media e for any purpose, provided this notice is pr eserved along with the articl 's original URL.