Selective modulation of PKC isozymes by inflammation in canine colonic circular muscle cells  Irshad Ali, Sushil K. Sarna  Gastroenterology  Volume 122, Issue 2, Pages 483-494 (February 2002) DOI: 10.1053/gast.2002.31215 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Stimulation of PKC activity by ACh and TPA in (A) normal and (B) inflamed colon cells. (A) PKC was significantly activated by 10−6 mol/L ACh and TPA in normal colon cells (P < 0.05, n = 5). (B) It was not activated by these agonists in inflamed colon cells (n = 4–7). Gastroenterology 2002 122, 483-494DOI: (10.1053/gast.2002.31215) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Western immunoblots of classic PKC isozymes (A) α, (B) β, and (C) γ in whole cell lysates of circular smooth muscle cells of 2 normal (lanes 1 and 2) and 2 inflamed (lanes 3 and 4) canine colons. The expression of PKC α and β was significantly attenuated in the inflamed colon cells (P < 0.03, n = 4–6), but that of PKC γ was not affected (n = 5). □, Normal colon cells; ■, inflamed colon cells. Gastroenterology 2002 122, 483-494DOI: (10.1053/gast.2002.31215) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Western immunoblots of novel PKC isozymes (A) δ and (B) ϵ in whole cell lysates of circular smooth muscle cells of 2 normal (lanes 1 and 2) and 2 inflamed (lanes 3 and 4) canine colons. The expression of PKC ϵ was significantly attenuated in the inflamed colon cells (P < 0.025, n = 3), but that of PKC δ was not affected (n = 7). Gastroenterology 2002 122, 483-494DOI: (10.1053/gast.2002.31215) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Western immunoblots of atypical PKC isozymes (A) ι, (B) λ, and (C) ζ in whole cell lysates of circular smooth muscle cells of 2 normal (lanes 1 and 2) and 2 inflamed (lanes 3 and 4) canine colons. The expression of PKC ι and λ was significantly up-regulated in the inflamed colon cells (P < 0.05, n = 3), but that of PKC ζ was not affected (n = 3–4). Gastroenterology 2002 122, 483-494DOI: (10.1053/gast.2002.31215) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 The activation and translocation of classic PKC isozymes (A) α, (B) β, and (C) γ by ACh in circular smooth muscle cells of normal (lanes 1–4) and inflamed (lanes 5–8) colon. Lanes 1 and 2 represent cytosolic and membrane factions, respectively, of normal untreated cells. Lanes 3 and 4 represent cytosolic and membrane fractions of ACh-treated normal cells, respectively. Lanes 5 and 6 represent inflamed untreated cytosolic and membrane fractions, respectively, whereas lanes 7 and 8 represent cytosolic and membrane fractions, respectively, from ACh-treated inflamed colon cells. PKC α and β, but not PKC γ, translocated to the membrane in normal cells in response to ACh as indicated by increase in the intensity of the representative bands (n = 3–4). None of these isozymes was affected in the inflamed colon cells. Gastroenterology 2002 122, 483-494DOI: (10.1053/gast.2002.31215) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 The activation and translocation of novel PKC isozymes (A) δ and (B) ϵ by ACh in circular smooth muscle cells of normal (lanes 1–4) and inflamed (lanes 5–8) colon. Lanes 1 and 2 represent cytosolic and membrane fractions, respectively, of normal untreated cells. Lanes 3 and 4 represent cytosolic and membrane fractions, respectively, of ACh-treated normal cells. Lanes 5 and 6 represent inflamed untreated cytosolic and membrane fractions, respectively, whereas lanes 7 and 8 represent cytosolic and membrane fractions, respectively, from ACh-treated inflamed cells. PKC ϵ but not PKC δ translocated to the membrane in normal cells in response to ACh as indicated by increase in the intensity of the representative bands (n = 3–4). These isozymes were not affected by ACh in the inflamed cells. Gastroenterology 2002 122, 483-494DOI: (10.1053/gast.2002.31215) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 The activation and translocation of atypical PKC isozymes (A) ι, (B) λ, and (C) ζ by ACh in circular smooth muscle cells of normal (lanes 1–4) and inflamed (lanes 5–8) colons. Lanes 1 and 2 represent cytosolic and membrane fractions, respectively, of normal untreated cells. Lanes 3 and 4 represent cytosolic and membrane fractions, respectively, of ACh-treated normal cells. Lanes 5 and 6 represent inflamed untreated cytosolic and membrane fractions, respectively, whereas lanes 7 and 8 represent cytosolic and membrane fractions, respectively, from ACh-treated inflamed cells. PKC ι, λ, and ζ did not translocate to the membrane in either the normal or the inflamed cells in response to ACh (n = 2–3). Gastroenterology 2002 122, 483-494DOI: (10.1053/gast.2002.31215) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 Immunofluorescence imaging of PKC α and λ in normal and inflamed colon cells before and after their treatment with ACh. (A) PKC α was diffused throughout cytosol in untreated normal cells, (C) but it was associated with membranes in untreated inflamed colon cells. ACh induced the translocation of PKC α in (B) normal and not in the (D) inflamed colon cells. (E) PKC λ was found primarily in the perinuclear area in untreated normal cells, but (G) it was found both at the perinuclear area and the nucleus in the untreated inflamed colon cells. PKC λ was not affected by ACh treatment in both (F) normal and (H) inflamed colon cells. Gastroenterology 2002 122, 483-494DOI: (10.1053/gast.2002.31215) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 9 The effect of PKC inhibitors, (A) chelerythrine, (B) myristoylated PKC α and β peptide inhibitor, and (C) myristoylated PKC ϵ peptide inhibitor on ACh-mediated contraction of normal and inflamed colon cells. All inhibitors concentration-dependently reduced the ACh-stimulated contraction of normal colon cells. The inhibitors were ineffective in the inflamed colon cells. Gastroenterology 2002 122, 483-494DOI: (10.1053/gast.2002.31215) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 10 TPA concentration-dependently reduced the length of normal and inflamed colon cells. The response was significantly attenuated in the inflamed cells. Gastroenterology 2002 122, 483-494DOI: (10.1053/gast.2002.31215) Copyright © 2002 American Gastroenterological Association Terms and Conditions