- Email: [email protected]
Intracellular mechanism responsible for VIP action on gastric relaxation: Interaction between cyclic AMP and prostaglandin pathways
April 1 9 9 5
• EFFECT OF OCTREOTIDE, GRANISETRON AND PHLOROGLUCINOL ON THE RECTO-COLONIC REFLEX INDUCED BY INTRARECTAL INJECTION OF GLYCEROL IN IBS PATIENTS. L.ouvel D, Delvaux M, Lagier E, Staumont G, Fioramonti J*, Budno L*, Frexinos J. Lab. Dig. Motility, CHU Rangueil, *Dep. Pharmacology INRA*, Toulouse, France. Rectal irritation by glycerol elicits a rectoeolonic reflex with a rapid increase in colonic motility and a long-lasting decrease in colonic tone. With these changes, occurs abdominal colic in most of the patients, the characteristics of which are closely similar to abdominal pain, they related to IBS. The aim of this study was to evaluate the effects of drugs acting on afferent ner)/e pathways (octreotide and granisetron) or as an antispasmodic on smooth muscle (phloroglucinol) on the rectocolonic reflex induced by glycerol in IBS patients. Patients and Method. In 20 IBS patients, (12 F, 8 M; mean age 38_+12 y.) a probe provided with a barostat bag (10 em length, vol. 700 rnl) was placed in the descending colon and its position controlled radiographyeally. On days 1 and 2 following eolonoseopy, after an overnight fast, colonic tone was recorded ever 8 h with a barostat (INRA, Toulouse). lntra-bag pressure was constantly maintained at 11-+0.8 mmRg. Volume variations of the bag reflected changes in tone and adjustements were made for pressure variations of 0.4 mm Hg. Baseline tone was recorded during 90 min. Then glycerol 15 ml was injected intr~trectally, Glycerol test was repeated trice in each patient during the recording time. 30 rain before each intrarectal injection of glycerol, either placebo (P1) (n=20), placebo (P2) (n=10), granisetron (G) 3 mg IV (n=10), octreotide (O) 1.25 ~tg/kg SC (n=10) or phloroglucinol (PH) 40 mg IV (n=10) was randomly administered, Results._ 11 AC BV (mll Vol. Var. CN CAmt)l P1 20 16 140_+20 +70% 10_+1 P2 10 7 135_+36 + 75 % 9+1 -8% O 10 7 128+26 + 64 % 11+_1 +6% G 10 8 138_+44 + 71% 10_+1 -5% PH 10 2* 138_+42 + 66 % 4_+1" -56%* N (number of patient), AC (patients with abdominal colics), BV (basal vol of the bag), Vol. Var. (Volume variations of the bag after glycerol-% of basal bag vol), CN (number of phasic contractions), CAmpl (amplitude of contractions-% from P1), *: p<0.05. C o n c l u e [ p n . The rectocolonic reflex induced by intrarectal injection of glycerol is reproducible. Phloroglucinol reduces pain; frequency and intensity of colonic contractions induced by glycerol, while octreotide and granisetron have no effect on it.
•
ROLE OF cGMP IN THE EFFECT OF NITRIC OXIDE ON CALCIUMACTIVATED K+ CHANNELS OF COLONIC SMOOTHMUSCLE. G. Lu, B. Mazet, M. G. Sarr and J. H. Szurszewski, Department of Physiology and Biophysics and Department of Surgery, Mayo Foundation, Rochester, MN 55905. We have previously shown in rabbit colonic myocytes that n i t r i c oxide (NO) increases an outward K current via calcium-activated K+ (Kca) channels. The purpose of this study was to determine whether this effect results from a direct action of NO on Kca channels or whether the effect is mediated through the cGMP. Isolated circular smooth muscle cells (CSM) were used either in the cell-attached patch or inside-out patch configuration. In the cell-attached patch configuration (command voltage -100 mV), I% NO increased the open probability (NPo) of KCa from 0.11 ± 0.04, (mean ± SD), to 0.71 ± 0.48 (n = 6, p < 0.05). In the inside-out patch configuration (command voltage -40 mV), NO did not affect NPo of Kca. In the cell-attached patch configuration (command voltage -100 mV). superfusion of methylene blue (i #aM) or cystamine (5 mM), inhibitors of guanylate cyclase a c t i v i t y , significantly reduced the effect of I% NO on NPo from 0.71 ± 0.48 to 0.20 ± 0.09, (n = 5, p < 0.05) and from 0.78 ± 0.30 to 0.28 ± 0.09, (n = 6, p < 0.01), respectively of Kca. In contrast, zaprinast (100 ~M), a selective cGMP-phosphodiesterase inhibitor, increased NPo KCa from 0.07 ± 0.04 to 0.13 ± 0.07 (n=4, p < 0.05). Zaprinast also significantly enhanced the effect of 0.5% NO on NPo of KCa. NPo was 0.20 ± 0.07 and 0.43 ± 0.07 (n = 4, p < 0.01) in the absence and presence of zaprinast, respectively. The addition of 8-Br cGMP (1 mM) increased NPo of KCa from 0.08 ± 0.04 to 0.41 ± 0.19 (n = 9, p < 0.01). These data suggest that the effect of NO in rabbit colonic myocytes was mediated, at least in part, through a cGMP pathway rather than by a direct effect on KCa channels. Supported by NIH Grants DK 17238 and DK 39337.
Motility and Nerve-Gut Interactions
A641
• CANINE JEJUNOILEAL TRANSPLANTATION: EFFEC'qS ON ELECTRICAL AND MECHANICAL PROPERTIES OF CIRCULAR SMOOTH MUSCLE. G. Lu, M.G. Sarr, J.H. Szurszewski. Depts. of Surgery and Physiology,Mayo Clinic, Rochester, MN Little is know about the acute and chronic effects of intestinal transplantation on smooth muscle contractile physiology. AIM: To determine the effects of jejunoileal autotransplantation (complete extrinsic denervation) on membrane potential and contractile activity. METHODS: Six dogs underwent a model of jejunoileal autotransplantation that specifically avoids isehemia/reperfusioninjury (by maintainingblood flow to the gut during the "transplantation" procedure). Strips of jejunal circular muscle were prepared sequentially at 0 (before), and 2 and 8 wk after autotransplantation. Mechanical and intracellular electrical activities were recorded simultaneouslyin a special superfused bath under controlled conditions. RESULTS: The amplitude of spontaneous contractions (~±SD) was increased (p<0.05) at 2 wk but markedly decreased at 8 wk (Table). Contraction frequency, resting membrane potential, and amplitude of stow waves were unchanged across these time points. Bethanechol (10"7-10-4 M) and substance P (10"8-10"6M) dose-dependently increased contractile activity at all time points, but the absolute change in amplitude was decreased at 8 wk. The amplitude of inhibitory junction potentials (IJPs) and duration of relaxation in presence of cholinergic/adrenergic inhibitors (NANC conditions) increased at 2 and 8 wk; off-contraction amplitude was decreased at 8 wk (p <0.05). CONCLUSIONS:Amplitude of jejunal circular muscle contractions are increased initially after extrinsic denervation. Later adaptation (8 wk) to denervation leads to a decrease in contractile activity. These effects may occur via changes in neurotransmitter release, change in regulation of membrane receptors, or alteration in characteristics of membrane threshold potential. (Support: NIH DK 39337) Contractions Amp slow Time A m p a Freqb Mem Potc wavec IJPd Dur relaxe 0 wk 77±32 12±1 -61±2 16±5 14.9±2.1 75±25 2 wk 126±19" 1 2 ± 1 -62±3 12±4 20.0±2.8* 380±228* 8 wk 7~2"* 11±1 58±5 11±5 20.1±5.1" 540+454* %/g (force/mass), n=4; bcont/min, n=6; emV, n=6; dmV at 15 Hz, n-6; emsec, NANC conditions at 15 Hz for 1 rain, n=4; *p<0.05 and **p<0.01 compared to 0 wk.
INTRACELLULAR MECHANISM RESPONSIBLE FOR VIP ACTION ON GASTRIC RELAXATION: INTERACTION BETWEEN CYCLIC AMP AND PROSTAGLANDIN PATHWAYS. Y Lu. Y Tsunoda, C Owyang. Dept of Int Med, University of Michigan, Ann Arbor, MI. We have recently shown that secretin inhibits gastric motility by stimulation of presynaptic cholinergic neurons in a vagally mediated pathway (GE 106:A553, 1994). Through nicotinic synapses, secretin stimulates the release of VIP from gastric postganglionic neurons which induces gastric relaxation via an indomethacin-sensitive pathway. To explain the in vivo observation that VIP-induced gastric relaxation is blunted by indomethacin, we hypothesized that VIP receptors on gastric smooth muscle are coupled to two intracellular pathways mediated by adenylate cyclase and phospholipase A2 (PLA2). PLA2 activation triggers the formation of prostaglandins (PG) which potentiate cyclic AMP accumulation elicited by VIP. To test this hypothesis, we obtainerJ isolitted gastric smooth muscle from the rat and showed that VIP (10-5-10-aM) dose-dependently stimulated cyclic AMP formation which was abolished by the adenylate cyclase inhibitor, dideoxyadenosine (10-4M). At 10"6M, VIP increased cAMP from 7.5+1 to 18.2+_2 pmol/mg cell extract protein. Addition of indomethacin (10-5M) reduced cAMP formation to 13.1±1.5 pmol/mg cell extract protein (P<0.05). VIP (10-8-10-3M) also stimulated the formation of prostaglandin E2 (PGE2) with an ED50 of 10-°M. Addition of the PLA2 inhibitor ONO-RS-082 (10-aM) but not the phospholipase C injaibitor, U-73122 (10-aM) abolished the increase in PGE2 by VIP (10-OM). This indicates that, in addition to the adenylate cyclase system, VIP receptors are coupled to the PLA2 pathway which is responsible for the formation of PG via the arachidonic acid cascade. To examine the interaction I~etwegnPG and VIP-evoked cAMP formation, we showed that PGE2 (10"6-10"°M) dose-dependently shifted the VIP dose response curve for cAMP formation to the right indicating synergism between the two signal transduction systems. To investigate the site of interaction, we showed that forskolin (10-6M), an activator of adenylate cyclase, and PGE2 produced 400% and 150% increases in cAMP over basal, respectively. The combination of forskolin (10-OM) and PGE2 (10DM) evoked a 730% increase in cAMP over basal. This suggests that PGE2 acts at a postreceptor level to potentiate the action of adenylate cyclase in the formation of cAMP. In conclusion, we have discovered a novel pathway where VIP receptors are coupled to both the adenylate cyclase and PLA2 signal transduction systems. The formation of PG via the PLA2/arachidonic acid cascade potentiates the increase in cAMP elicited by VIP. This helps to explain the in rive observation that VIP evokes gastric relaxation by a indomethacin-sensitive pathway.
• EFFECT OF OCTREOTIDE, GRANISETRON AND PHLOROGLUCINOL ON THE RECTO-COLONIC REFLEX INDUCED BY INTRARECTAL INJECTION OF GLYCEROL IN IBS PATIENTS. L.ouvel D, Delvaux M, Lagier E, Staumont G, Fioramonti J*, Budno L*, Frexinos J. Lab. Dig. Motility, CHU Rangueil, *Dep. Pharmacology INRA*, Toulouse, France. Rectal irritation by glycerol elicits a rectoeolonic reflex with a rapid increase in colonic motility and a long-lasting decrease in colonic tone. With these changes, occurs abdominal colic in most of the patients, the characteristics of which are closely similar to abdominal pain, they related to IBS. The aim of this study was to evaluate the effects of drugs acting on afferent ner)/e pathways (octreotide and granisetron) or as an antispasmodic on smooth muscle (phloroglucinol) on the rectocolonic reflex induced by glycerol in IBS patients. Patients and Method. In 20 IBS patients, (12 F, 8 M; mean age 38_+12 y.) a probe provided with a barostat bag (10 em length, vol. 700 rnl) was placed in the descending colon and its position controlled radiographyeally. On days 1 and 2 following eolonoseopy, after an overnight fast, colonic tone was recorded ever 8 h with a barostat (INRA, Toulouse). lntra-bag pressure was constantly maintained at 11-+0.8 mmRg. Volume variations of the bag reflected changes in tone and adjustements were made for pressure variations of 0.4 mm Hg. Baseline tone was recorded during 90 min. Then glycerol 15 ml was injected intr~trectally, Glycerol test was repeated trice in each patient during the recording time. 30 rain before each intrarectal injection of glycerol, either placebo (P1) (n=20), placebo (P2) (n=10), granisetron (G) 3 mg IV (n=10), octreotide (O) 1.25 ~tg/kg SC (n=10) or phloroglucinol (PH) 40 mg IV (n=10) was randomly administered, Results._ 11 AC BV (mll Vol. Var. CN CAmt)l P1 20 16 140_+20 +70% 10_+1 P2 10 7 135_+36 + 75 % 9+1 -8% O 10 7 128+26 + 64 % 11+_1 +6% G 10 8 138_+44 + 71% 10_+1 -5% PH 10 2* 138_+42 + 66 % 4_+1" -56%* N (number of patient), AC (patients with abdominal colics), BV (basal vol of the bag), Vol. Var. (Volume variations of the bag after glycerol-% of basal bag vol), CN (number of phasic contractions), CAmpl (amplitude of contractions-% from P1), *: p<0.05. C o n c l u e [ p n . The rectocolonic reflex induced by intrarectal injection of glycerol is reproducible. Phloroglucinol reduces pain; frequency and intensity of colonic contractions induced by glycerol, while octreotide and granisetron have no effect on it.
•
ROLE OF cGMP IN THE EFFECT OF NITRIC OXIDE ON CALCIUMACTIVATED K+ CHANNELS OF COLONIC SMOOTHMUSCLE. G. Lu, B. Mazet, M. G. Sarr and J. H. Szurszewski, Department of Physiology and Biophysics and Department of Surgery, Mayo Foundation, Rochester, MN 55905. We have previously shown in rabbit colonic myocytes that n i t r i c oxide (NO) increases an outward K current via calcium-activated K+ (Kca) channels. The purpose of this study was to determine whether this effect results from a direct action of NO on Kca channels or whether the effect is mediated through the cGMP. Isolated circular smooth muscle cells (CSM) were used either in the cell-attached patch or inside-out patch configuration. In the cell-attached patch configuration (command voltage -100 mV), I% NO increased the open probability (NPo) of KCa from 0.11 ± 0.04, (mean ± SD), to 0.71 ± 0.48 (n = 6, p < 0.05). In the inside-out patch configuration (command voltage -40 mV), NO did not affect NPo of Kca. In the cell-attached patch configuration (command voltage -100 mV). superfusion of methylene blue (i #aM) or cystamine (5 mM), inhibitors of guanylate cyclase a c t i v i t y , significantly reduced the effect of I% NO on NPo from 0.71 ± 0.48 to 0.20 ± 0.09, (n = 5, p < 0.05) and from 0.78 ± 0.30 to 0.28 ± 0.09, (n = 6, p < 0.01), respectively of Kca. In contrast, zaprinast (100 ~M), a selective cGMP-phosphodiesterase inhibitor, increased NPo KCa from 0.07 ± 0.04 to 0.13 ± 0.07 (n=4, p < 0.05). Zaprinast also significantly enhanced the effect of 0.5% NO on NPo of KCa. NPo was 0.20 ± 0.07 and 0.43 ± 0.07 (n = 4, p < 0.01) in the absence and presence of zaprinast, respectively. The addition of 8-Br cGMP (1 mM) increased NPo of KCa from 0.08 ± 0.04 to 0.41 ± 0.19 (n = 9, p < 0.01). These data suggest that the effect of NO in rabbit colonic myocytes was mediated, at least in part, through a cGMP pathway rather than by a direct effect on KCa channels. Supported by NIH Grants DK 17238 and DK 39337.
Motility and Nerve-Gut Interactions
A641
• CANINE JEJUNOILEAL TRANSPLANTATION: EFFEC'qS ON ELECTRICAL AND MECHANICAL PROPERTIES OF CIRCULAR SMOOTH MUSCLE. G. Lu, M.G. Sarr, J.H. Szurszewski. Depts. of Surgery and Physiology,Mayo Clinic, Rochester, MN Little is know about the acute and chronic effects of intestinal transplantation on smooth muscle contractile physiology. AIM: To determine the effects of jejunoileal autotransplantation (complete extrinsic denervation) on membrane potential and contractile activity. METHODS: Six dogs underwent a model of jejunoileal autotransplantation that specifically avoids isehemia/reperfusioninjury (by maintainingblood flow to the gut during the "transplantation" procedure). Strips of jejunal circular muscle were prepared sequentially at 0 (before), and 2 and 8 wk after autotransplantation. Mechanical and intracellular electrical activities were recorded simultaneouslyin a special superfused bath under controlled conditions. RESULTS: The amplitude of spontaneous contractions (~±SD) was increased (p<0.05) at 2 wk but markedly decreased at 8 wk (Table). Contraction frequency, resting membrane potential, and amplitude of stow waves were unchanged across these time points. Bethanechol (10"7-10-4 M) and substance P (10"8-10"6M) dose-dependently increased contractile activity at all time points, but the absolute change in amplitude was decreased at 8 wk. The amplitude of inhibitory junction potentials (IJPs) and duration of relaxation in presence of cholinergic/adrenergic inhibitors (NANC conditions) increased at 2 and 8 wk; off-contraction amplitude was decreased at 8 wk (p <0.05). CONCLUSIONS:Amplitude of jejunal circular muscle contractions are increased initially after extrinsic denervation. Later adaptation (8 wk) to denervation leads to a decrease in contractile activity. These effects may occur via changes in neurotransmitter release, change in regulation of membrane receptors, or alteration in characteristics of membrane threshold potential. (Support: NIH DK 39337) Contractions Amp slow Time A m p a Freqb Mem Potc wavec IJPd Dur relaxe 0 wk 77±32 12±1 -61±2 16±5 14.9±2.1 75±25 2 wk 126±19" 1 2 ± 1 -62±3 12±4 20.0±2.8* 380±228* 8 wk 7~2"* 11±1 58±5 11±5 20.1±5.1" 540+454* %/g (force/mass), n=4; bcont/min, n=6; emV, n=6; dmV at 15 Hz, n-6; emsec, NANC conditions at 15 Hz for 1 rain, n=4; *p<0.05 and **p<0.01 compared to 0 wk.
INTRACELLULAR MECHANISM RESPONSIBLE FOR VIP ACTION ON GASTRIC RELAXATION: INTERACTION BETWEEN CYCLIC AMP AND PROSTAGLANDIN PATHWAYS. Y Lu. Y Tsunoda, C Owyang. Dept of Int Med, University of Michigan, Ann Arbor, MI. We have recently shown that secretin inhibits gastric motility by stimulation of presynaptic cholinergic neurons in a vagally mediated pathway (GE 106:A553, 1994). Through nicotinic synapses, secretin stimulates the release of VIP from gastric postganglionic neurons which induces gastric relaxation via an indomethacin-sensitive pathway. To explain the in vivo observation that VIP-induced gastric relaxation is blunted by indomethacin, we hypothesized that VIP receptors on gastric smooth muscle are coupled to two intracellular pathways mediated by adenylate cyclase and phospholipase A2 (PLA2). PLA2 activation triggers the formation of prostaglandins (PG) which potentiate cyclic AMP accumulation elicited by VIP. To test this hypothesis, we obtainerJ isolitted gastric smooth muscle from the rat and showed that VIP (10-5-10-aM) dose-dependently stimulated cyclic AMP formation which was abolished by the adenylate cyclase inhibitor, dideoxyadenosine (10-4M). At 10"6M, VIP increased cAMP from 7.5+1 to 18.2+_2 pmol/mg cell extract protein. Addition of indomethacin (10-5M) reduced cAMP formation to 13.1±1.5 pmol/mg cell extract protein (P<0.05). VIP (10-8-10-3M) also stimulated the formation of prostaglandin E2 (PGE2) with an ED50 of 10-°M. Addition of the PLA2 inhibitor ONO-RS-082 (10-aM) but not the phospholipase C injaibitor, U-73122 (10-aM) abolished the increase in PGE2 by VIP (10-OM). This indicates that, in addition to the adenylate cyclase system, VIP receptors are coupled to the PLA2 pathway which is responsible for the formation of PG via the arachidonic acid cascade. To examine the interaction I~etwegnPG and VIP-evoked cAMP formation, we showed that PGE2 (10"6-10"°M) dose-dependently shifted the VIP dose response curve for cAMP formation to the right indicating synergism between the two signal transduction systems. To investigate the site of interaction, we showed that forskolin (10-6M), an activator of adenylate cyclase, and PGE2 produced 400% and 150% increases in cAMP over basal, respectively. The combination of forskolin (10-OM) and PGE2 (10DM) evoked a 730% increase in cAMP over basal. This suggests that PGE2 acts at a postreceptor level to potentiate the action of adenylate cyclase in the formation of cAMP. In conclusion, we have discovered a novel pathway where VIP receptors are coupled to both the adenylate cyclase and PLA2 signal transduction systems. The formation of PG via the PLA2/arachidonic acid cascade potentiates the increase in cAMP elicited by VIP. This helps to explain the in rive observation that VIP evokes gastric relaxation by a indomethacin-sensitive pathway.