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First published online May 1, 2009
Journal of Experimental Biology 212, 1535-1543 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.030197
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Sympathetic outflow activates the venom gland of the snake Bothrops jararaca by regulating the activation of transcription factors and the synthesis of venom gland proteins

Milene S. A. Luna1, Thiago M. A. Hortencio1, Zulma S. Ferreira2 and Norma Yamanouye1,*

1 Laboratório de Farmacologia, Instituto Butantan, Av. Vital Brazil 1500, 05503-900, São Paulo, Brazil
2 Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, travessa 14, 05508-900, São Paulo, Brazil


Figure 1
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  		Fig. 1. Time course of NF{kappa}B activation in the venom gland of the snake Bothrops jararaca after venom removal. (A) Nuclear extracts from venom gland of male and female snakes in quiescent stage (0 min) and 30, 60 and 120 min after venom removal were assayed for the level of NF-{kappa}B binding to 32P end-labelled NF{kappa}B oligonucleotide probe by the electrophoretic mobility shift assay (EMSA). *Competition assays were performed using an excess of unlabelled specific NF{kappa}B oligonucleotide. (B) Densitometric analysis of NF{kappa}B activation above baseline level in EMSA using nuclear extracts from venom glands of male and female snakes 30, 60 and 120 min after venom removal. Note the difference in time of activation between males and females. Statistical analysis of differences between nuclear extracts was performed, and different lowercase letters indicate significant differences (ANOVA, Newman–Keuls, P<0.05).

 

Figure 2
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  		Fig. 2. Time course of AP-1 activation in the venom gland of the snake Bothrops jararaca after venom removal. (A) Nuclear extracts from venom gland of male and female snakes in quiescent stage (0 min) and 30, 60 and 120 min after venom removal were assayed for the level of AP-1 binding to 32P end-labelled AP-1 oligonucleotide probe by EMSA. *Competition assays were performed using an excess of unlabelled specific AP-1 oligonucleotide. (B) Densitometric analysis of AP-1 activation above baseline level in EMSA using nuclear extracts from venom glands of female and male snakes 30, 60 and 120 min after venom removal. Statistical analysis of differences between nuclear extracts was performed, and different lowercase letters indicate significant differences (ANOVA, Newman–Keuls, P<0.05).

 

Figure 3
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  		Fig. 3. Stimulation of {alpha}1- and β-adrenoceptors activates NF{kappa}B in secretory cells of the venom gland of female Bothrops jararaca. EMSA using nuclear extracts of quiescent secretory cells (labelled B) and cells incubated for 30 min with (A) phenylephrine (PHE, 0.3 mmol l–1), isoprenaline (ISO, 0.3 mmol l–1) or (B) noradrenaline (NA, 0.1 mmol l–1). *Competition assays were performed using an excess of unlabelled specific NF{kappa}B oligonucleotide over radiolabelled probe. (C) Densitometric analysis of NF{kappa}B activation in EMSA using nuclear extract of quiescent secretory cells incubated for 30 min with NA, PHE or ISO. Statistical analysis of differences between nuclear extracts was performed, and different lowercase letters indicate significant differences (ANOVA, Newman–Keuls, P<0.05).

 

Figure 4
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  		Fig. 4. Stimulation of {alpha}1- and β-adrenoceptors regulates AP-1 activation in secretory cells of the venom gland of female Bothrops jararaca. (A,C) Nuclear extracts from secretory cells of quiescent venom gland of female snakes (labelled B) or cells incubated with 0.1 mmol l–1 NA, 0.3 mmol l–1 phenylephrine or 0.3 mmol l–1 ISO for 30 or 60 min, respectively. *Competition assays were performed using an excess of unlabelled specific AP-1 oligonucleotide. (B,D) Densitometric analysis of AP-1 activation in EMSA using nuclear extract of quiescent secretory cells incubated for 30 or 60 min with NA, PHE or ISO. Statistical analysis of differences between nuclear extracts was performed, and different lowercase letters indicate significant differences (ANOVA, Newman–Keuls, P<0.05).

 

Figure 5
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  		Fig. 5. Effect of stimulation of {alpha}1- and β-adrenoceptors on venom composition in snakes treated with reserpine. The venom of snakes was manually removed twice, with a 15 day interval, and 10 µg of venom proteins were separated by SDS-PAGE (12%). Lanes 1, 3 and 5 show first samples, collected before treatment, and lanes 2, 4 and 6 show samples taken at 15 days from the same snakes: lane 2 untreated (no reserpine), lane 4 treated with reserpine plus isoprenaline, and lane 6 treated with reserpine and phenylephrine. Figure is representative of three independent experiments. M, marker lane (kDa).

 

Figure 6
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  		Fig. 6. Protein composition of venom gland tissue of Bothrops jararaca at different times after venom removal. (A) SDS-PAGE (12%) gel of female venom gland tissue (30 µg of protein) in the quiescent stage, and 4, 7 and 15 days after removal of venom (lanes 1, 2, 3 and 4, respectively). Four days after venom removal, bands of 81, 69, 47, 44 and 38 kDa were increased (arrows), and bands of 28 and 19 kDa were reduced 7 days after venom removal (arrowheads), when compared with quiescent cells. Figure is representative of five independent experiments. (B) SDS-PAGE (15%) gel of male venom gland tissue (30 µg of protein) in the quiescent stage, and 4, 7 and 15 days after removal of venom (lanes 1, 2, 3 and 4, respectively). Arrows indicate bands with increased (81, 69, 57, 54, 47 and 44 kDa) density, 7 days after venom removal and arrowheads indicate bands with reduced (38, 28 and 17 kDa) density, 4 days after venom removal, when compared with quiescent cells. Figure is representative of two independent experiments. M, marker lane (kDa).

 

Figure 7
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  		Fig. 7. Effect of reserpine and stimulation of {alpha}1- and β-adrenoceptors on the protein composition of venom gland tissue after venom removal. (A) SDS-PAGE (12%) gel shows female venom gland tissue (30 µg of protein) 4 days after removal of venom from a snake not treated with any drug (lane 1), and snakes treated with reserpine (lane 2), and reserpine plus isoprenaline plus phenylephrine (lane 3). Arrows indicate bands of 81, 69, 47, 44, 41 and 38 kDa that were reduced by reserpine. Figure is representative of two independent experiments. (B) SDS-PAGE (15%) gel shows male venom gland tissue (30 µg of protein) 7 days after removal of venom from a snake not treated with any drug (lane 1), and snakes treated with reserpine (lane 2), and reserpine plus isoprenaline plus phenylephrine (lane 3). Arrows indicate bands of 81, 69, 57, 54, 47 and 44 kDa that were reduced by reserpine. Figure is representative of two independent experiments. M, marker lane (kDa).

 

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© The Company of Biologists Ltd 2009