Fig 1.
Telokin phosphorylation is increased by incubation of non-contracted gastric fundus smooth muscles with compounds that elevate cGMP.
A. Representative recordings of the relaxation responses evoked by 10μM SNP in the absence or presence of 1μM ODQ (n = 6, each), or 50μM 8Br-cGMP (n = 6). B. Representative western blots of telokin S13 phosphorylation in untreated control muscles and muscles incubated with 50μM 8Br-cGMP for 5 min (n = 6) or 10μM SNP (n = 6) for 1 min. C. Average ratios ± SD of pS13:telokin in the absence or presence of 50μM 8Br-cGMP (n = 6) or 10μM SNP. *P<0.01 compared to untreated controls.
Fig 2.
Telokin phosphorylation in gastric fundus smooth muscles pre-contracted with 30mM KCl is increased by compounds that elevate cGMP.
A. Representative recordings of the contractile response to 30mM KCl, and the relaxation responses evoked by 50μM 8Br-cGMP (n = 6) or 10μM SNP (n = 6) in the presence of 30mM KCl. Average ratios ± SD of pS13:telokin, pT38:CPI-17, pT696:MYPT1, pT853:MYPT1, and pS19:MLC and representative western blots of the phosphorylation of (B) telokin S13, (C) CPI-17 T38, (D) MYPT1 T696 and T853, and (E) MLC S19 evoked by 50μM 8Br-cGMP (n = 6) or 10μM SNP (n = 6) in the presence of 30mM KCl. *P<0.01, #P<0.05, compared to untreated controls. ‡P< 0.05 compared to 30mM KCl treated muscles.
Fig 3.
Telokin phosphorylation in gastric fundus smooth muscles pre-contracted with 1μM CCh is increased by compounds that elevate cGMP.
A. Representative recordings of the contractile response to 1μM CCh, and the relaxation responses evoked by 50μM 8Br-cGMP (n = 6) or 10μM SNP (n = 6) in the presence of 1μM CCh. Average ratios ± SD of pS13:telokin, pT38:CPI-17, pT696: MYPT1, pT853:MYPT1, and pS19:MLC and representative western blots of the phosphorylation of (B) telokin S13, (C) CPI-17 T38, (D) MYPT1 T696 and T853, and (E) MLC S19 evoked by 50μM 8Br-cGMP (n = 6) or 10μM SNP (n = 6) in the presence of 30mM KCl. *P<0.01, #P<0.05, compared to untreated controls. ‡P<0.05 compared to 1μM CCh treated muscles.
Fig 4.
The contractile responses of telokin-/- gastric fundus smooth muscles are increased compared to wild-type controls.
A. Representative recordings of the contractile responses of wild-type and telokin-/- gastric fundus smooth muscles to 1μM CCh (n = 4). B. Representative recordings of the contractile responses of wild-type and telokin-/- gastric fundus smooth muscles to 15mM, 30mM, and 60mM KCl. C. Average integrals and peak amplitudes of contraction ± SD of wild-type and telokin-/- gastric fundus smooth muscles to 15mM, 30mM, and 60mM KCl (#P<0.05 compared to wild-type, n = 8). D. Representative recordings of the contractile responses of wild-type and telokin-/- gastric fundus smooth muscles incubated with 100μM LNNA and 1μM MRS2500 to 30 sec of 1Hz, 5Hz, 10Hz, or 20Hz EFS. E. Average integrals and peak amplitudes of contraction ± SD of wild-type and telokin-/- gastric fundus smooth muscles incubated with 100μM LNNA and 1μM MRS2500 to 30 sec of 1Hz, 5Hz, 10Hz, or 20Hz EFS KCl (#P<0.05 compared to wild-type, n = 4). F. Representative western blots of phosphorylated and non-phosphorylated MLC in wild-type and telokin-/- gastric fundus smooth muscles.
Fig 5.
The relaxation responses of pre-contracted telokin-/- gastric fundus smooth muscles are reduced compared to wild-type controls.
Representative recordings of the relaxation responses of wild-type and telokin-/- gastric fundus smooth muscles to (A) 10μM SNP, (B) 50μM 8Br-cGMP, and (C) 30 sec of 5Hz or 10Hz EFS. Representative recordings of the relaxation responses of wild-type and telokin-/- gastric fundus smooth muscles evoked by 10μM SNP in the presence of (D) 1μM CCh or (E) 30mM KCl. F. Representative recording of the relaxation responses evoked by 30 sec of 5Hz or 10Hz EFS in wild-type and telokin-/- gastric fundus smooth muscles incubated with 30mM KCl and 1μM atropine, 1μM phentolamine, and 1μM propranolol.
Fig 6.
Telokin phosphorylation in non-contracted gastric fundus smooth muscles is not increased by nitrergic neurotransmission.
A. Representative recording of the relaxation responses evoked by 30 sec of 5Hz or 10Hz EFS of muscles incubated in 1μM atropine, 1μM phentolamine, and 1μM propranolol. Representative western blots of the phosphorylation of telokin S13 (B), CPI-17 T38 (C), MYPT1 T853 and T696 (D), and MLC S19 (E) evoked by 30 sec of 5Hz or 10Hz EFS of muscles incubated in 1μM atropine, 1μM phentolamine, and 1μM propranolol. F. Average ratios ± SD of pS13:telokin, pT38:CPI-17, pT696:MYPT1, pT853:MYPT1, and pS19:MLC evoked by 30 sec of 5Hz or 10Hz EFS of muscles incubated in 1μM atropine, 1μM phentolamine, and 1μM propranolol (P>0.05, n = 6).
Fig 7.
SNP dose-dependently increases telokin S13 phosphorylation in non-pre-contracted gastric fundus smooth muscles.
A. Representative recording of the relaxation responses and representative western blot of phosphorylated and non-phosphorylated MLC evoked by 10Hz EFS, 0.1μM, 1μM, or 10μM SNP (n = 4). B. Average ratios ± SD of peak relaxation amplitudes evoked by 10Hz EFS, 0.1μM, 1μM, or 10μM SNP (*P<0.01, #P<0.05, n = 4). C. Average ratios ± SD and representative western blots of pS13:telokin evoked by 0.1μM, 1μM, or 10μM SNP (#P<0.05 compared to 0.1μM SNP or 1μM SNP, n = 4).
Fig 8.
Telokin S13 phosphorylation is not increased by nitrergic neurotransmission in W/WV gastric fundus smooth muscles.
Representative recordings of the relaxation responses evoked by 30 sec of 5Hz EFS and 10Hz EFS (A), or 10μM SNP (B) from wild-type and W/WV fundus muscles. B. Representative western blot of phosphorylated and non-phosphorylated MLC from wild-type and W/WV fundus muscles treated with 10μM SNP. C. Average ratios ± SD of pS13:telokin and representative western blots of phosphorylated telokin S13 and telokin from wild-type and W/WV muscles stimulated by 10Hz/30 sec EFS, or incubated with 10μM SNP. *P<0.01 compared to untreated wild-type or W/WV fundus muscle controls, n = 4.