Volume 119, Issue 1, Pages 51-61 (July 2000) Gq-linked NK2 receptors mediate neurally induced contraction of human sigmoid circular smooth muscle  Weibiao Cao, Victor E. Pricolo, Liping Zhang, Jose Behar, Piero Biancani, Michael T. Kirber  Gastroenterology  Volume 119, Issue 1, Pages 51-61 (July 2000) DOI: 10.1053/gast.2000.8552 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Acetylcholine is not a major excitatory neurotransmitter in human sigmoid colon. (A) In the same strip of human sigmoid circular muscle, atropine (10−4 mol/L, 15-minute incubation) had no effect on the contraction in response to electrical stimulation (1 Hz, 0.5 milliseconds, 100 V) in the absence of L-NNA. (B) In the presence of 100 μmol/L L-NNA, 100 μmol/L atropine had no effect on EFS-induced contraction at 1-40 Hz in human sigmoid strips. Values are mean ± SE of 3 patients (7-10 strips). Gastroenterology 2000 119, 51-61DOI: (10.1053/gast.2000.8552) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 FK888 and NK-2ra are selective antagonists of NK1 and NK2 receptors, respectively, in human sigmoid colon. Intact muscle cells were isolated from human sigmoid circular smooth muscles by enzymatic digestion. The cells were contracted by exposure to tachykinin receptor agonists alone for 30 seconds (control) or after a 1-minute preincubation in a medium containing 1 μmol/L FK888 or 1 μmol/L NK-2ra. (A) GR73632 dose-dependently contracted the cells. GR73632-induced contraction was blocked by 1 μmol/L FK888, a potent and selective NK1 antagonist (P < 0.0001, ANOVA), but not by 1 μmol/L NK-2ra, a selective NK2 antagonist. (B) NK2 receptor agonist (β-Ala8)-NKA (4-10) dose-dependently contracted the cells and reached maximal contraction at 10−9 mol/L. (β-Ala8)-NKA (4-10)–induced contraction was blocked by 1 μmol/L NK-2ra (P < 0.0001, ANOVA), but not by 1 μmol/L FK888. Values are mean ± SE of 3 patients with 30 cells counted for each patient. Gastroenterology 2000 119, 51-61DOI: (10.1053/gast.2000.8552) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 NK2 receptors mediate neurally induced contraction in human sigmoid circular muscle strips. Sigmoid circular muscle strips were divided randomly into 3 groups: control, FK888, and NK-2ra. The contact time of antagonists was 15 minutes before EFS. In the presence of 100 μmol/L L-NNA, NK2-receptor antagonist NK-2ra (1 μmol/L) almost abolished the EFS-induced contraction (P < 0.0001, ANOVA), but NK1-receptor antagonist FK888 (1 μmol/L) had no effect. Values are mean ± SE of 6 patients (6 strips). Gastroenterology 2000 119, 51-61DOI: (10.1053/gast.2000.8552) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Contraction of human sigmoid circular muscle in response to NKA is mediated by NK2 receptors. (A) NKA dose-dependently contracted the muscle strips. NKA-induced contraction was blocked by 1 μmol/L NK-2ra, a potent and selective NK2 antagonist (P < 0.0001, ANOVA), but not by 1 μmol/L FK888, a selective NK1 antagonist. Values are mean ± SE of 7 patients (7 strips). (B) Intact muscle cells were isolated from human sigmoid circular smooth muscles by enzymatic digestion with collagenase. The cells were contracted by exposure to NKA alone for 30 seconds (control) or after a 1-minute preincubation in a medium containing 1 μmol/L FK888 or 1 μmol/L NK-2ra. NKA dose-dependently contracted the cells and reached maximal contraction at 10−9 mol/L. NKA-induced contraction was blocked by 1 μmol/L NK-2ra (P < 0.0001, ANOVA), but not by 1 μmol/L FK888. Values are mean ± SE of 3 patients with 30 cells counted for each patient. Gastroenterology 2000 119, 51-61DOI: (10.1053/gast.2000.8552) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 SP is a very weak agonist in human sigmoid circular muscle strips. (A) NKA and SP dose-dependently contracted the muscle strips. SP had a much weaker effect than NKA. Values are mean ± SE of 7 patients (7 strips) for NKA and of 2 patients (4 strips) for SP. (B) SP (10 μmol/L)-induced contraction was blocked by 1 μmol/L NK-2ra, a potent and selective NK2 antagonist (†P < 0.05, ANOVA), but not by 1 μmol/L FK888, a selective NK1 antagonist. Values are mean ± SE of 2 patients (4 strips). Gastroenterology 2000 119, 51-61DOI: (10.1053/gast.2000.8552) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 NKA-induced contraction is mediated by Gq G proteins in human sigmoid circular cells. Muscle cells were permeabilized by brief exposure to saponin to allow diffusion of antibodies into the cytosolic side of the cell membrane. Cells were contracted with NKA (10−9 mol/L) after 60 minutes of preincubation in cytosolic medium containing G-protein antibodies (1/200 dilution). NKA-induced contraction of human sigmoid cells was significantly inhibited by Gq (††P < 0.0001, ANOVA), but not by Go, or Gi1–Gi2, or Gi3 antibodies. Values are the mean ± SEM of 3 patients with 30 cells counted for each patient. Gastroenterology 2000 119, 51-61DOI: (10.1053/gast.2000.8552) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 [35S]GTPγS binding to NKA-activated membranes of human sigmoid colon. Purified membranes were exposed to NKA (10−6 mol/L) in the presence of [35S]GTPγS for 5 minutes. NKA-induced activation of specific G proteins was reflected by the amount of [35S]GTPγS bound to wells that were precoated with Gi3, Gq, or Gi1–Gi2 antibodies. G-protein activation was measured as percent increase in [35S]GTPγS binding in membranes exposed to NKA compared with unstimulated membranes. Exposure to NKA (10−6 mol/L) caused significant activation of Gq (††P = 0.0001, ANOVA) in sigmoid muscle membranes. Values are the mean ± SEM of 4 patients with each sample performed in triplicate. Gastroenterology 2000 119, 51-61DOI: (10.1053/gast.2000.8552) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 NKA-induced contraction of sigmoid muscle requires release of intracellular Ca2+. Intact muscle cells were isolated from human sigmoid circular smooth muscles by enzymatic digestion with collagenase and incubated in HEPES-buffered solution (control) or in solution containing 3 μmol/L thapsigargin for 30 minutes. The cells were contracted by exposure to NKA for 30 seconds. Contraction of sigmoid cells in response to NKA was abolished by thapsigargin (P < 0.0001, ANOVA). Values are the mean ± SEM of 3 patients with 30 cells counted for each patient. Gastroenterology 2000 119, 51-61DOI: (10.1053/gast.2000.8552) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 9 NKA application causes transient increase in cytosolic Ca2+ concentration and subsequent contraction of freshly isolated colonic myocytes. Pseudocolor ratiometric images of Fura 2 fluorescence (see Materials and Methods section) indicate the change in cytosolic Ca2+ and track the shape of the cell as it contracts. The numbers below the images indicate the point in time when the images were acquired. Images were acquired at a rate of 30 Hz, and a ratiometric image was computed at 15 Hz (every 67 milliseconds). The images of intracellular Ca2+ were filtered with a 9-point digital median filter. The entire time course of the Ca2+ transient is shown in the trace below the images; the bar indicates the time when 10 μmol/L NKA was applied via a pressure ejection micropipette. This cell shortened 33% of its resting length. The bathing solution consisted of 117 mmol/L NaCl, 3 mmol/L KCl, 1 mmol/L MgCl2, 2 mmol/L CaCl2, and 10 mmol/L HEPES (sodium salt); the pH level was set at 7.2. The pipette solution was identical to the bathing solution with 10 μmol/L NKA added. Gastroenterology 2000 119, 51-61DOI: (10.1053/gast.2000.8552) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 10 Increase in intracellular calcium in the normal calcium medium and calcium-free medium induced by 10 μmol/L NKA. (A) In both normal calcium and calcium-free medium, NKA caused a significant increase in cytosolic Ca2+ concentration. The increase of intracellular calcium induced by NKA was abolished by preincubation in 1 μmol/L thapsigargin (TG) and 1 μmol/L ryanodine (Ryan). (B) The cell shortening was significantly reduced by preincubation in 1 μmol/L thapsigargin and 1 μmol/L ryanodine. Values are the mean ± SEM of 5-13 experiments. *P < 0.01, paired Student t test, compared with resting levels; **P < 0.001, Student t test, compared with normal calcium group. There was no significant difference in cell shortening between the normal calcium and the calcium-free group. Gastroenterology 2000 119, 51-61DOI: (10.1053/gast.2000.8552) Copyright © 2000 American Gastroenterological Association Terms and Conditions