Korean J Physiol Pharmacol.  2014 Feb;18(1):47-53. 10.4196/kjpp.2014.18.1.47.

Characteristics of Diprophylline-Induced Bidirectional Modulation on Rat Jejunal Contractility

• Liu FF
• Chen DP
• Xiong YJ
• Lv BC
• Lin Y

• KMID: 2285490
• DOI: http://doi.org/10.4196/kjpp.2014.18.1.47

Abstract

In this study, we propose that diprophylline exerts bidirectional modulation (BM) on the isolated rat jejunal segment depending on its contractile state. The results supported the hypothesis. Diprophylline (20 microM) exerted stimulatory effects on the contractility of jejunal segment in six low contractile states while inhibitory effects in six high contractile states, showing the characteristics of BM. Diprophylline-induced stimulatory effect was significantly blocked by atropine, indicating the correlation with cholinergic activation. Diprophylline-induced inhibitory effect was partially blocked by phentolamine, propranolol, and L-N-Nitro-Arginine respectively, indicating their correlation with sympathetic activation and nitric oxide-mediated relaxing mechanisms. Diprophylline-induced BM was abolished by tetrodotoxin or in a Ca2+ free condition or pretreated with tyrosine kinase inhibitor imatinib, suggesting that diprophylline-induced BM is Ca2+ dependent, and that it requires the presence of enteric nervous system as well as pacemaker activity of interstitial cells of Cajal. Diprophylline significantly increased the reduced MLCK expression and myosin extent in constipation-prominent rats and significantly decreased the increased MLCK expression and myosin extent in diarrhea-prominent rats, suggesting that the change of MLCK expression may also be involved in diprophylline-induced BM on rat jejunal contractility. In summary, diprophylline-exerted BM depends on the contractile states of the jejunal segments, requires the presence of Ca2+, enteric nervous system, pacemaker activity of interstitial cells of Cajal, and MLCK-correlated myosin phosphorylation. The results suggest the potential implication of diprophylline in relieving alternative hypo/hyper intestinal motility.

Keyword

Bidirectional modulation; Contractile state; Diprophylline; Jejunal segment; Phosphorylation

MeSH Terms

Animals
Atropine
Dyphylline
Enteric Nervous System
Gastrointestinal Motility
Interstitial Cells of Cajal
Myosins
Phentolamine
Phosphorylation
Propranolol
Protein-Tyrosine Kinases
Rats*
Tetrodotoxin
Imatinib Mesylate
Atropine
Dyphylline
Myosins
Phentolamine
Propranolol
Protein-Tyrosine Kinases
Tetrodotoxin

Figure

• 

  Fig. 1 Diprophylline-induced bidirectional modulation (BM) on jejunal contractility. (A1) Representative traces and (A2) statistical analysis of concentration-response relationship for diprophylline-indued effects on jejunal contractility. (B1) Representative traces and (B2) statistical analysis of diprophylline-exerted stimulatory effects on the contractility of jejunal segments in 6 low contractile states (LCS). (C1) Representative traces and (C2) statistical analysis of diprophylline-exerted inhibitory effects on the contractility of jejunal segments in 6 high contractile states (HCS). Contractile amplitude of jejunal segment in normal contractile state is set to 100% (NC). Data are expressed as the mean±SEM (% NC, n=6). ##p<0.01 compared with NC. **p<0.01 compared with the contractile amplitudes in LCS and the contractile amplitudes in HCS before diprophylline treatment respectively. CP, constipation-prominent; DP, diarrhea-prominent; SNP, sodium nitropr usside; ACh, acetylcholine.

• 

  Fig. 2 Effects of diprophylline on jejunal contractility pretreated with tetrodotoxin (TTX) and imatinib respectively. (A1) Representative traces and (A2) statistical analysis of diprophylline (20 µM)-induced effects on jejunal segment pretreated with tetrodotoxin (0.1µM). (B1) Representative traces and (B2) statistical analysis of diprophylline on the contractility of jejunal segment pretreated with c-Kit tyrosine kinase blocker imatinib. Contractile amplitude of jejunal segment in normal contractile state is set to100% (normal control, NC). Data are expressed as the mean±SEM (% NC, n=6). **p<0.01 compared with contractile amplitude of jejunal segment after treatment with TTX or imatinib. RLCS, representative low contractile state; RHCS, representative high contractile state.

• 

  Fig. 3 Links between Ca2+ and diprophylline-induced effects. (A) Representative traces of diprophylline (5~80 µM)-induced effects on the contractility of jejunal segment pre-treated with verapamil (0.1 µM) in normal contractile state (NC), representative low contractile state (RLCS) and representative high contractile state (RHCS). (B) Representative traces of the effects of diprophylline (5~80 µM) on the contractility of jejunal segment assayed in Ca2+-free Krebs buffer.

• 

  Fig. 4 Receptors and enzymes potentially involved in diprophylline-induced bidirectional modulation. Effects of diprophylline on the jejunal contractility pretreated with (A) 10 µM atropine and (B) 10 µM diphenhydramine respectively in normal contractile state (NC) and in representative low contractile state (RLCS). Effects of diprophylline on jejunal contractility, pretreated with (C) 10 µM phentolamine, (D) 5 µM propranolol, and (E) 10 µM L-NG-nitroargenine (L-NNA) respectively in NC and representative high contractile state (RHCS).

• 

  Fig. 5 Effects of diprophylline on myosin phosphorylation. (A1) Representative traces and (A2) statistical analysis of the phosphorylation of 20-kDa regulatory light chain of myosin (p-MLC20). (B1) Representative traces and (B2) statistical analysis of the protein content of myosin light chain kinase (MLCK). (C) Statistical analysis of the expression of MLCK mRNA. Data obtained from normal control (NC) is set to 100%. Data are expressed as the mean±SEM (% NC, n=6). **p<0.01 compared with the NC. ##p<0.01 compared with the jejunal contractility before diprophylline treatment. CP, Constipation-prominent; DP, diarrhea-prominent; DIP, diprophylline.

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