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4-aminopyridine decreases progesterone production by porcine granulosa cells.

Li Y, Ganta S, von Stein FB, Mason DE, Mitchell BM, Freeman LC - Reprod. Biol. Endocrinol. (2003)

Bottom Line: Complimentary experiments determined the effects of 4-AP on the spontaneously established pig granulosa cell line 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Anatomy & Physiology College of Veterinary Medicine, Kansas State University, Manhattan, Kansas 66506-5802, USA. yanli@vet.k-state.edu

ABSTRACT

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.

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.

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4-AP hyperpolarizes granulosa cell resting potential and increases intracellular potassium. A) Granulosa cells (GC) were cultured in the basic media, alone (Con), or with 4AP (2 mM), FSH (200 ng/mL) or FSH+4AP. After 24 hours, resting membrane potentials were compared using DiBAC4(3) fluorescence. Decreased DiBAC4(3) fluorescence indicates hyperpolarization of resting membrane potential. Data were obtained from 16 (Con, 4-AP) or 17 (FSH, FSH+4AP) culture wells from 4 GC isolations. B) GC were cultured in the basic media, alone, or with 4AP, FSH or FSH+4AP. After 24 hours, intracellular K+ concentrations were compared by PBFI fluorescence. Increased PBFI fluorescence indicates increased intracellular potassium concentration. Data were obtained from 5 (FSH, FSH+4-AP) or 7 (Con, 4-AP) culture wells from 3 GC isolations. In both panels, asterisk indicates P < 0.05 compared with GC cultured under similar conditions in the absence of 4-AP.
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Figure 7: 4-AP hyperpolarizes granulosa cell resting potential and increases intracellular potassium. A) Granulosa cells (GC) were cultured in the basic media, alone (Con), or with 4AP (2 mM), FSH (200 ng/mL) or FSH+4AP. After 24 hours, resting membrane potentials were compared using DiBAC4(3) fluorescence. Decreased DiBAC4(3) fluorescence indicates hyperpolarization of resting membrane potential. Data were obtained from 16 (Con, 4-AP) or 17 (FSH, FSH+4AP) culture wells from 4 GC isolations. B) GC were cultured in the basic media, alone, or with 4AP, FSH or FSH+4AP. After 24 hours, intracellular K+ concentrations were compared by PBFI fluorescence. Increased PBFI fluorescence indicates increased intracellular potassium concentration. Data were obtained from 5 (FSH, FSH+4-AP) or 7 (Con, 4-AP) culture wells from 3 GC isolations. In both panels, asterisk indicates P < 0.05 compared with GC cultured under similar conditions in the absence of 4-AP.

Mentions: Samples (10,000 cells) of GC from 24 hour cultures associated with 4 GC isolations were analyzed by flow cytometry to determine if 4-AP treatment influenced GC size. Light scattering properties did not differ significantly with GC isolate. However, the average forward scatter (median, coefficient of variation) of GC cultured for 24 hours in the presence of 4-AP (768, 15%) was significantly greater than that of GC maintained similarly in the absence of drug (694, 15%). Representative dot plots are shown in Figure 6. Resting membrane potential (Figure 7A) and [K+]in (Figure 7B) were also found to be increased in GC exposed to 4-AP, based on DiBAC4(3) and PBFI fluorescence, respectively.


4-aminopyridine decreases progesterone production by porcine granulosa cells.

Li Y, Ganta S, von Stein FB, Mason DE, Mitchell BM, Freeman LC - Reprod. Biol. Endocrinol. (2003)

4-AP hyperpolarizes granulosa cell resting potential and increases intracellular potassium. A) Granulosa cells (GC) were cultured in the basic media, alone (Con), or with 4AP (2 mM), FSH (200 ng/mL) or FSH+4AP. After 24 hours, resting membrane potentials were compared using DiBAC4(3) fluorescence. Decreased DiBAC4(3) fluorescence indicates hyperpolarization of resting membrane potential. Data were obtained from 16 (Con, 4-AP) or 17 (FSH, FSH+4AP) culture wells from 4 GC isolations. B) GC were cultured in the basic media, alone, or with 4AP, FSH or FSH+4AP. After 24 hours, intracellular K+ concentrations were compared by PBFI fluorescence. Increased PBFI fluorescence indicates increased intracellular potassium concentration. Data were obtained from 5 (FSH, FSH+4-AP) or 7 (Con, 4-AP) culture wells from 3 GC isolations. In both panels, asterisk indicates P < 0.05 compared with GC cultured under similar conditions in the absence of 4-AP.
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Figure 7: 4-AP hyperpolarizes granulosa cell resting potential and increases intracellular potassium. A) Granulosa cells (GC) were cultured in the basic media, alone (Con), or with 4AP (2 mM), FSH (200 ng/mL) or FSH+4AP. After 24 hours, resting membrane potentials were compared using DiBAC4(3) fluorescence. Decreased DiBAC4(3) fluorescence indicates hyperpolarization of resting membrane potential. Data were obtained from 16 (Con, 4-AP) or 17 (FSH, FSH+4AP) culture wells from 4 GC isolations. B) GC were cultured in the basic media, alone, or with 4AP, FSH or FSH+4AP. After 24 hours, intracellular K+ concentrations were compared by PBFI fluorescence. Increased PBFI fluorescence indicates increased intracellular potassium concentration. Data were obtained from 5 (FSH, FSH+4-AP) or 7 (Con, 4-AP) culture wells from 3 GC isolations. In both panels, asterisk indicates P < 0.05 compared with GC cultured under similar conditions in the absence of 4-AP.
Mentions: Samples (10,000 cells) of GC from 24 hour cultures associated with 4 GC isolations were analyzed by flow cytometry to determine if 4-AP treatment influenced GC size. Light scattering properties did not differ significantly with GC isolate. However, the average forward scatter (median, coefficient of variation) of GC cultured for 24 hours in the presence of 4-AP (768, 15%) was significantly greater than that of GC maintained similarly in the absence of drug (694, 15%). Representative dot plots are shown in Figure 6. Resting membrane potential (Figure 7A) and [K+]in (Figure 7B) were also found to be increased in GC exposed to 4-AP, based on DiBAC4(3) and PBFI fluorescence, respectively.

Bottom Line: Complimentary experiments determined the effects of 4-AP on the spontaneously established pig granulosa cell line 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Anatomy & Physiology College of Veterinary Medicine, Kansas State University, Manhattan, Kansas 66506-5802, USA. yanli@vet.k-state.edu

ABSTRACT

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.

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.

Show MeSH
Related in: MedlinePlus