Korean J Urol.  2006 May;47(5):541-547. 10.4111/kju.2006.47.5.541.

Modulatory Role of Adenylyl Cyclase and Protein Kinase A (PKA) in 5-hydroxytriptamine3 Induced Intracellular Calcium Increase in Parasympathetic Neurons of Rat Major Pelvic Ganglia

• Kim DR
• Cha SK
• Park KS
• Song ES
• Park YH
• Song YS
• Kong ID

• KMID: 2294142
• DOI: http://doi.org/10.4111/kju.2006.47.5.541

Abstract

PURPOSE
Serotonin has effects on the bladder contraction or urethral sphincter tone. Different subtypes of 5-hydroxytriptamine (5-HT) receptors appear to mediate the effects of serotonin on voiding. 5-HT1 and 5-HT2, metamorphic receptors, are examined well. However 5-HT3, ionotrophic receptors, are not examined well. Pelvic ganglia provide the majority of the innervation of the lower urinary tract. Major pelvic ganglia (MPG) in rats are autonomic ganglia, containing both sympathetic and parasympathetic neurons related with voiding. We examined the modulatory role of adenylyl cyclase (AC) and protein kinase A (PKA) in 5-HT3 induced intra cellular calcium increase in rat MPG.
MATERIALS AND METHODS
The regulatory effects by AC and PKA were investigated in a single neuron of male rat major pelvic ganglia using patch clamp and fluorescence Ca2+ measurement techniques.
RESULTS
Inward currents were induced by 5-HT (10microM) at only parasympathetic neurons of MPG. MDL7222 (10(-6)M), selective 5-HT3 receptor antagonists, completely abolished the 5-HT induced inward currents. 5-HT (10microM) induced intracelluar increases of calcium. These increases were blocked by an AC inhibitor SQ22536 (2x10(-5M)) and myristoylated PKA inhibitor (10(-7)M). Furthermore, foskolin (10(-6)M), AC activator, augmented the 5-HT induced intracellular calcium increase.
CONCLUSIONS
The activation of AC/PKA-dependent pathway could enhance 5-HT3 induced intacellular calcium increase in parasympathetic neurons of rat MPG and these is helpful for the better understanding the mechanisms of the bladder contraction or urethral sphincter tone.

Keyword

Calcium; Parasympathetic ganglia

MeSH Terms

Adenylyl Cyclases*
Animals
Calcium*
Cyclic AMP-Dependent Protein Kinases*
Fluorescence
Ganglia*
Ganglia, Autonomic
Ganglia, Parasympathetic
Humans
Male
Neurons*
Protein Kinases*
Rats*
Receptors, Serotonin, 5-HT3
Serotonin
Urethra
Urinary Bladder
Urinary Tract
Calcium
Cyclic AMP-Dependent Protein Kinases
Protein Kinases
Receptors, Serotonin, 5-HT3
Serotonin

Figure

• 

  Fig. 1 Effects of 5-HT3 blocker on the inward currents induced by the treatment of 10 µM 5-HT in the parasympathetic MPG neurons held at -80 mV. (A, B) The inward currents are blocked by 1 uM MDL7222, 5-HT3 blocker. 5-HT: 5-hydroxytryptamine, MPG: major pelvic ganglia.

• 

  Fig. 2 Effects of 5-HT3 blocker on the intracellular increase of calcium induced by 10uM 5-HT. Intracelluar increases of calcium induced by the treatment of 10uM 5-HT are blocked by the treatment of 1uM MDL7222, 5-HT3 blocker, in parasympathetic MPG neurons. 5-HT: 5-hydroxytryptamine, MPG: major pelvic ganglia.

• 

  Fig. 3 Intracellular change of calcium ion concentration induced by 5-HT in current or voltage clamp mode in parasympathetic MPG neurons. (A) Changes of intracellular calcium ion concentration and membrane potential induced by the treatment of 10uM 5-HT in current clamp mode (I=0) are recorded simultaneously. (B) Changes of intracellular calcium ion concentration and membrane currents induced by the treatment of 10uM 5-HT in current clamp mode (holding potential=-80mV) are recorded simultaneously. 5-HT: 5-hydroxytryptamine, MPG: major pelvic ganglia.

• 

  Fig. 4 Effects of the adenylate cyclase activator on the increase of intracellular calcium induced by 5-HT in parasympathetic MPG neurons. (A, B) Intracellular calcium increase induced by 10uM 5-HT is augmented by the treatment of 1uM foskolin, adenylate cyclase activator. FSK: foskolin, Data are expressed mean±SE. *p<0.01. 5-HT: 5-hydroxytryptamine, MPG: major pelvic ganglia.

• 

  Fig. 5 Effects of AC or PKA on the increase of intracellular calcium induced by 5-HT in parasympathetic MPG neurons. (A, B) Intracellular calcium increase induced by 10uM 5-HT is inhibited by the treatment of 200uM SQ22536, AC inhibitor and recovered without treatment. (C, D) Intracellular calcium increase induced by 10uM 5-HT is inhibited by the treatment of 100nM myristolylated PKA inhibitor and recovered without treatment. Data are expressed mean±SE. AC: adenylate cyclase, PKA: protein kinase A, WO: without treatment of SQ22536. *p<0.01, †p<0.05. 5-HT: 5-hydroxytryptamine, MPG: major pelvic ganglia.

Reference

1. de Groat WC. Influence of central serotonergic mechanisms on lower urinary tract function. Urology. 2002. 59:5 Suppl 1. 30–36.

2. Keast JR. Unusual autonomic ganglia: connections, chemistry, and plasticity of pelvic ganglia. Int Rev Cytol. 1999. 193:1–69.

3. de Groat WC, Booth AM, Yoshimura N. Maggi CA, editor. Neurophysiology of micturation and its modification in animal models of human disease. The autonomic nervous system. 1993. 1st ed. London, UK: Harwood Academic Publisher;227–290.

4. Barnes NM, Sharp T. A review of central 5-HT receptors and their function. Neuropharmacology. 1999. 38:1083–1152.

5. Raymond JR, Mukhin YV, Gelasco A, Turner J, Colinsworth G, Gettys TW, et al. Multiplicity of mechanisms of serotonin receptor signal transduction. Pharmacol Ther. 2001. 92:179–212.

6. van Hooft JA, Vijverberg HP. 5-HT₃ receptors and neurotransmitter release in the CNS: a nerve ending story? Trends Neurosci. 2000. 23:605–610.

7. Hapfelmeier G, Tredt C, Haseneder R, Zieglgansberger W, Eisensamer B, Rupprecht R, et al. Co-expression of the 5-HT_3B serotonin receptor subunit alters the biophysics of the 5-HT₃ receptor. Biophys J. 2003. 84:1720–1733.

8. Hill B. Hill B, editor. Calcium dynamics, epithelial trasport, and intercellular coupling. Ion channels of excitable membranes. 2001. 3rd ed. Saunderland: Sinauer Associates, Inc.;269–306.

9. Morales M, Wang SD. Differential composition of 5-hydroxytryptamine3 receptors synthesized in the rat CNS and peripheral nervous system. J Neurosci. 2002. 22:6732–6741.

10. Glaum SR, Proudfit HK, Anderson EG. 5-HT₃ receptors modulate spinal nociceptive reflexes. Brain Res. 1990. 510:12–16.

11. Morales M, McCollum N, Kirkness EF. 5-HT₃ receptor subunits A and B are co-expressed in neurons of the dorsal root ganglion. J Comp Neurol. 2001. 438:163–172.

12. Dail WG. Maggi CA, editor. Autonomic innervation of male reproductive genitalia. The autonomic nervous system. Nervous control of the urogenital system. 1992. London: Harwood Academic Publishers;69–101.

13. Keast JR. Unsusal autonomic ganglia: connections, chemistry, and plasticity of pelvic ganglia. Int Rev Cytol. 1999. 193:1–69.

14. Zhu Y, Zboran EL, Ikeda SR. Phenotype-specific expression of T-type calcium channels in neurons of the major pelvic ganglion of the adult male rat. J Physiol. 1995. 489:363–375.

15. Akasu T, Hasuo H, Tokimasa T. Activation of 5-HT₃ receptor subtypes causes rapid excitation of rabbit parasympathetic neurones. Br J Pharmacol. 1987. 91:453–455.

16. Sun H, Hu XQ, Moradel EM, Weight FF, Zhang L. Modulation of 5-HT₃ receptor-mediated response and trafficking by activation of protein kinase C. J Biol Chem. 2003. 278:34150–34157.

17. Hubbard PC, Thompson AJ, Lummis SC. Functional differences between splice variants of the murine 5-HT_3A receptor: possible role for phosphorylation. Brain Res Mol Brain Res. 2000. 81:101–108.

18. Knight AR, Bowery NG. The pharmacology of adenylyl cyclase modulation by GABA_B receptors in rat brain slices. Neuropharmacology. 1996. 35:703–712.

19. Kubota H, Katsurabayashi S, Moorhouse AJ, Murakami N, Koga H, Akaike N. GABA_B receptor transduction mechanisms, and cross-talk betwen protein kinase A and C, in Gabaergic terminals synapsing onto neurons of the rat nucleus basalis of Meynert. J Physiol. 2003. 551:263–276.

20. Taniyama K, Niwa M, Kataoka Y, Yamashita K. Activation of protein kinase C suppresses the gamma-aminobutyric acid B receptor-mediated inhibition of the vesicular release of noradrenaline and acetylcholine. J Neurochem. 1992. 58:1239–1245.