Effect of cibacron blue and suramin on non-adrenergic non-cholinergic motor responses in rat anorectum

Galya Stavreva1, Radomir Radomirov1,2, Nikolai Lazarov3. 1Medical University-Pleven, Department of Experimental and Clinical Pharmacology, 5800, Bulgaria, 2Bulgarian Academy of Sciences, Institute of Neurobiology, 1113, Bulgaria, 3Medical University – Sofia, Department of Anatomy and Histology, Bulgaria

Objective: The involvement of P2-purinergic receptors in electrically-induced nonadrenergic noncholinergic (NANC) motor activity of rectal longitudinal and circular muscle and NANC activity of internal anal sphincter and anal canal were studied in a rat anorectum.

Methods: The study was conducted on isolated anorectal preparations from 16 male Wistar rats (weight 250-300 g, age 4 months). Mechanographic computerized on-line technique, partitioned organ bath, and electrical stimulation (EFS, 0.8 ms; 40 V; 5 Hz) were used to evaluate the effect of P2-receptor antagonists cibacron blue and suramin on nonadrenergic noncholinergic (NANC) local and ascending motor responses of rectal longitudinal and circular muscle and NANC local and descending motor activity of internal anal sphincter and anal canal. Immunohistochemical technique was used to study the presence and distribution of ATP-synthase in neuronal structures of the myenteric plexus of rat anorectum. Results are presented as mean ± S.E.M. The data were processed by one-way ANOVA, followed by LSD multiple comparison post-hoc test.

Results: Electrical field stimulation applied to proximal or distal part of segments elicited both tetrodotoxin (0.1 microM)-sensitive local motor responses of the stimulated part and ascending or descending motor responses of the contralateral, nonstimulated part of the preparations. Сibacron blue (0.01 mM) and suramin (1.0 mM) altered the pattern of NANC EFS-induced ascending motor responses of longitudinal and circular muscles of the rectum: contractions but no relaxations were recorded synchronously in both muscles. Cibacron blue significantly increased NANC responses. Recorded responses were 1.85±0.28 mN; n=8 in rectal longitudinal muscle and 2.08±0.12 mN; n=8 in circular muscle. The amplitudes of contractions of longitudinal (1.04±0.16 mN; n=8) and circular (1.12±0.21 mN; n=8) muscles in the presence of suramin were significantly lower than those in presence of sibacron blue. Descending NANC relaxation of internal sphincter in the presence of cibacron blue was significantly suppressed to -0.47±0.11 mN; n=8 at 5 Hz EFS (p <0.05). Suramin inhibited NANC relaxation (-0.60 ± 0.17 mN; n=8). The structure of recorded descending NANC motor responses of the anal canal was maintained against the background of purinergic antagonists. Cibacron blue increased contractile response (1.57±0.24 mN/10 Hz; n=8) and inhibited relaxation (-0.30±0.06 mN; n=8) without a statistically significant difference, as compared to NANC responses. Suramin inhibited both contraction (0.90±0.17 mN; n=8), and relaxation (-0.42±0.12 mN; n=8). ATP-synthase-positive nerve fibers were found in myenteric ganglia of anorectum.

Conclusion: The effects of selective Р2Y antagonist сibacron blue and nonselective P2-antagonist suramin confirm the involvement of ATP or ATP-related purine in NANC neurotransmission in rat anorectum, determined by P2 receptors.