Excitatory regulation of angiotensin II on gastric motility and its mechanism in guinea pig.

In the present study, we investigated the effect of Ang II on gastric smooth muscle motility and its mechanism using intracellular recording and whole-cell patch clamp techniques. Ang II dose-dependently increased the tonic contraction and the frequency of spontaneous contraction in the gastric antral circular smooth muscles of guinea pig. ZD7155, an Ang II type 1 receptor (AT(1)R) blocker, completely blocked the effect of Ang II on the spontaneous contraction of gastric smooth muscle. In contrast, TTX, a sodium channel blocker, failed to block the effect. Furthermore, nicardipine, a voltage-gated Ca(2+)-channel antagonist, did not block the effect of Ang II on the tonic contraction of gastric smooth muscle, but external free-calcium almost completely blocked this effect. Both ryanodine, an inhibitor of calcium-induced Ca(2+) release (CICR) from ryanodine-sensitive calcium stores, and thapsigargin, which depletes calcium in calcium stores, almost completely blocked the effect of Ang II on tonic contraction. However, 2-APB, an inositol trisphosphate (IP(3)) receptor blocker, significantly, but not completely, blocked the Ang II effect on tonic contraction. We also determined that Ang II depolarized membrane potential and increased slow wave frequency in a dose-dependent manner. It also inhibited delayed rectifying potassium currents in a dose-dependent manner, but did not affect L-type calcium currents or calcium-activated potassium currents. These results suggest that Ang II plays an excitatory regulation in gastric motility via AT(1)R-IP(3) and the CICR signaling pathway. The Ang II-induced inhibition of delayed rectifying potassium currents that depolarize membrane potential is also involved in the potentiation of tonic contraction and the frequency of spontaneous contraction in the gastric smooth muscle of guinea pig.

The properties of spontaneous transient inward currents of interstitial cells in rabbit portal vein.

The present study was designed to investigate the properties of spontaneous transient inward currents generated by interstitial cells (ICs) in the rabbit portal vein. Single ICs were freshly isolated from smooth muscle of the rabbit portal vein enzymetically. Using whole-cell patch clamp techniques, the spontaneous transient inward currents (STICs) were recorded at -60 mV of holding potential in freshly dispersed ICs. Both gadolinium, a non-selective cation channel inhibitor, and niflumic acid, a calcium-activated chloride channel blocker, abolished the inward currents. Replacement of external Na(+) with N-methyl-d-glucamine (NMDG(+)) also blocked the inward currents. The inward currents were abolished by caffeine, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), thapsigargin and ryanodine, but were partly inhibited by 2-aminoethoxydiphenyl borate (2-APB). W-7, a calmodulin inhibitor, increased the amplitude of the inward currents. These results suggest that non-selective cation channels are involved in the generation of the spontaneous transient inward currents recorded from ICs. The currents are regulated by intracellular calcium and calmodulin. But in the present study, the involvement of the calcium-activated chloride channels in the generation of the currents cannot be excluded.

Spontaneous rhythmic inward currents recorded in interstitial cells of rabbit portal vein.

It is now well established that smooth muscle of the portal vein exhibits spontaneous rhythmic contraction in vitro. The present study was designed to investigate the pacemaking mechanism(s) underlying the spontaneous rhythmic contractions in the rabbit portal vein (RPV). Using whole-cell patch clamp techniques, spontaneous inward currents were recorded at -60 mV of holding potential in freshly dispersed c-Kit immunopositive interstitial cells (ICs) isolated from sections of RPV. The inward currents were abolished by caffeine, FCCP, thapsigargin, and ryanodine, but were partially inhibited by 2-APB. Both gadolinium, a non-selective cation channel inhibitor, and niflumic acid, a chloride channel blocker, inhibited the inward currents completely. Replacement of external Na(+) with NMDG(+) also blocked the inward currents. W-7, a calmodulin inhibitor, increased both the amplitude and frequency of the inward currents. Taken together, these results indicate that non-selective cationic channels are involved in the generation of spontaneous inward currents recorded from ICs. Intracellular calcium concentration and calmodulin regulate the spontaneous inward currents, which may account for spontaneous rhythmic contraction in the RPV, but a role of chloride channels may not be excluded in the present study.

Pacemaking activity is regulated by membrane stretch via the CICR pathway in cultured interstitial cells of Cajal from murine intestine.

Membrane stretch is an important stimulus in gastrointestinal (GI) motility regulation, but the relationship between membrane stretch and the pacemaking activity of GI smooth muscle is poorly understood. We examined the effect of intestinal distension on slow waves and the effect of membrane stretch on pacemaker currents in cultured intestinal interstitial cells of Cajal (ICCs) from murine small intestine. At organ level, intestinal distension significantly increased amplitude of slow and fast waves, and enhanced frequencies of fast but not slow waves. At the cellular level, membrane stretch-induced by hyposmotic cell swelling (MSHC) depolarized membrane potential and activated large inward holding current, but suppressed amplitude of pacemaker potential or pacemaking current. External Ca(2+)-free solution abolished pacemaker current and blocked MSHC-induced inward holding current. However, a sustained inward holding current was activated and the amplitude of pacemaker current was increased by high ethylene glycol tetraacetic acid (EGTA) in pipette. Then MSHC also potentiated the inward holding current. MSHC significantly increased amplitude of rhythmic Ca(2+) transients and basal intracellular Ca(2+) concentration ([Ca(2+)](i)). 2-APB blocked both pacemaker current and Ca(2+) transients but did not alter the effect of MSHC on pacemaker current and Ca(2+) transients. In contrast, ryanodine inhibited Ca(2+) transients but not pacemaker current, and completely blocked MSHC-induced inward holding current and MSHC-induced increase of basal [Ca(2+)](i). These results suggest that intestinal distension potentiates intestinal motility by increasing the amplitude of slow waves. Membrane stretch potentiates pacemaking activity via releasing Ca(2+) from calcium-induced calcium release (CICR) in cultured intestinal ICCs.

Actin microfilament involved in regulation of pacemaking activity in cultured interstitial cells of Cajal from murine intestine.

The present study investigated the effect of actin microfilament structure on pacemaker currents and calcium oscillation in cultured murine intestinal interstitial cells of Cajal (ICCs) by whole-cell patch-clamp technique and calcium imaging technique. Cytochalasin B, a disruptor of actin microfilaments, decreased the amplitude and frequency of pacemaker currents from 491.32 +/- 160.33 pA and 11.73 +/- 0.79 cycles/min to 233.12 +/- 92.00 pA and 10.29 +/- 0.76 cycles/min. Cytochalasin B also decreased the amplitude and frequency of calcium oscillation from 0.32 +/- 0.08 (DeltaF/F0) and 2.75 +/- 0.17 cycles/min to 0.02 +/- 0.01 (DeltaF/F0) and 1.20 +/- 0.08 cycles/min. Phalloidin, a stabilizer of actin microfilaments, increased the amplitude and frequency of pacemaker currents from 751.79 +/- 282.82 pA and 13.93 +/- 1.00 cycles/min to 1234.34 +/- 607.83 pA and 14.68 +/- 1.00 cycles/min. Phalloidin also increased the amplitude and frequency of calcium oscillation from 0.26 +/- 0.01 (DeltaF/F0) and 2.27 +/- 0.18 cycles/min to 0.43 +/- 0.03 (DeltaF/F0) and 2.87 +/- 0.07 cycles/min. 2-Aminoethoxydiphenyl borane (2-APB), an IP(3) receptor blocker, suppressed both pacemaker currents and calcium oscillations. 2-APB also blocked the phalloidin-induced increase in pacemaker currents and calcium oscillation. Ryanodine, an inhibitor of calcium-induced calcium release, did not affect pacemaker current but suppressed calcium oscillations. Ryanodine had no effect on altering phalloidin-induced increases in pacemaker current and calcium oscillation. These results suggest that actin microfilaments regulate pacemaker activity via the IP(3)-induced calcium release signaling pathway.

Atrial natriuretic peptide signal pathway upregulated in stomach of streptozotocin-induced diabetic mice.

AIM: To investigate atrial natriuretic peptide (ANP) secretion from gastric mucosa and the relationship between the ANP/natriuretic peptide receptor type A (NPR-A) pathway and diabetic gastroparesis. METHODS: Male imprinting control region (ICR) mice (4 wk old) were divided into two groups: control mice, and streptozotocin-induced diabetic mice. Eight weeks after injection, spontaneous gastric contraction was recorded by using physiography in control and streptozotocin-induced diabetic mice. The ANP-positive cells in gastric mucosa and among dispersed gastric epithelial cells were detected by using immunohistochemistry and flow cytometry, respectively. ANP and natriuretic peptide receptor type A (NPR-A) gene expression in gastric tissue was observed by using the reverse transcriptase polymerase chain reaction. RESULTS: The frequency of spontaneous gastric contraction was reduced from 12.9 +/- 0.8 cycles/min in the control group to 8.4 +/- 0.6 cycles/min in the diabetic mice (n = 8, P < 0.05). However, the amplitude of contraction was not significantly affected in the diabetic group. The depletion of interstitial cells of Cajal in the gastric muscle layer was observed in the diabetic mice. ANP-positive cells were distributed in the gastric mucosal layer and the density index of ANP-positive cells was increased from 20.9 +/- 2.2 cells/field in control mice to 51.8 +/- 2.9 cells/field in diabetic mice (n = 8, P < 0.05). The percentage of ANP-positive cells among the dispersed gastric epithelial cells was increased from 10.0% +/- 0.9% in the control mice to 41.2% +/- 1.0% in the diabetic mice (n = 3, P < 0.05). ANP and NPR-A genes were both expressed in mouse stomach, and the expression was significantly increased in the diabetic mice. CONCLUSION: These results suggest that the ANP/NPR-A signaling pathway is upregulated in streptozotocin-induced diabetic mice, and contributes to the development of diabetic gastroparesis.

Dual effect of exogenous hydrogen sulfide on the spontaneous contraction of gastric smooth muscle in guinea-pig.

Hydrogen sulfide (H(2)S) is produced endogenously in mammalian tissues and is important in both physiological and pathological processes. Despite its importance, little is known regarding the effect of H(2)S on gastrointestinal motility. We evaluated the effect of H(2)S on the spontaneous contraction of gastric antrum smooth muscle in the guinea pig (Cavia porcellus) using a physiograph. In addition, we investigated whether the effect of H(2)S was mediated by ionic channels by recording membrane currents in freshly dispersed gastric antrum myocytes using a whole-cell patch clamp. Sodium hydrogen sulfide (NaHS), an H(2)S donor, had a dual effect on the spontaneous contraction of gastric antrum muscle strips. At high concentrations (0.3-1.0 mM), NaHS suppressed the amplitude of spontaneous contraction. At low concentrations (0.1-0.3 mM), NaHS enhanced the resting tension of muscle strips while slightly reducing the contractile amplitude. The excitatory effect on spontaneous contraction, caused by low concentrations of NaHS, was abolished when the muscle strips were pretreated with 10 mM tetraethylammonium (TEA), a nonselective potassium channel blocker, or 0.5 mM 4-Aminopyridine (4-AP), a voltage-gated K(+) channel blocker. However, the excitatory effect of NaHS was not completely blocked by low concentrations of TEA (1 mM). Pretreatment with both TEA (1 mM) and 4-AP (0.5 mM) completely abolished the excitatory effect. The dose-response curve for the inhibitory effect of NaHS on the spontaneous contraction of gastric smooth muscle was shifted significantly to the left by TEA and 4-AP. Both Pinacidil, a K(ATP) channel opener, and NaHS significantly inhibited TEA-potentiated spontaneous contraction. Glibenclamide, a K(ATP) channel blocker, partially, but significantly, reversed the reduction in amplitude. NaHS enhanced the amplitude of the K(ATP) current, but inhibited the voltage-gated K(+) channel current (IK(V)) in a dose-dependent manner. NaHS had no effect on STOC at low concentrations (0.1-1.0 mM) but significantly inhibited STOC at high concentrations (4-10 mM). Our results suggest that H(2)S has multiple actions during the regulation of gastric motility in the guinea-pig. An excitatory effect is mediated via inhibition of the voltage-gated K(+) channel and an inhibitory effect is mediated via activation of the K(ATP) channel.

Role of calcium-activated potassium currents in CNP-induced relaxation of gastric antral circular smooth muscle in guinea pigs.

AIM: To investigate ion channel mechanism in CNP-induced relaxation of gastric circular smooth muscle in guinea pigs. METHODS: Spontaneous contraction of gastric smooth muscle was recorded by a four -channel physiograph. The whole cell patch-clamp technique was used to record calcium-activated potassium currents and membrane potential in the gastric myocytes isolated by collagenase. RESULTS: C-type natriuretic peptide (CNP) markedly inhibited the spontaneous contraction in a dose-dependent manner in gastric circular smooth muscle in guinea pigs. Ly83583, an inhibitor of guanylate cyclase, weakened CNP-induced inhibition on spontaneous contraction but Zaparinast, an inhibitor of cGMP sensitive phosphoesterase, potentiated CNP-induced inhibition in gastric circular smooth muscles. The inhibitory effects of CNP on spontaneous contraction were blocked by tetrathylammonium (TEA), a nonselective potassium channel blocker. CNP hyperpolarized membrane potential from -60.0 mV+/-2.0 mV to -68.3 mV+/-3.0 mV in a single gastric myocyte. CNP increased calcium-activated potassium currents (I(K(ca))) in a dose-dependent manner in gastric circular myocytes. CNP also increased the spontaneously transient outward currents (STOCs). Ly83583 partly blocked CNP-induced increase of calcium-activated potassium currents, but Zaparinast potented the effect. CONCLUSION: CNP inhibits spontaneous contraction, and potassium channel may be involved in the process in gastric circular smooth muscle of guinea pigs. CNP-induced increase of I(K(ca)) is mediated by a cGMP dependent pathway.

Role of actin microfilament in hyposmotic membrane stretch-induced increase in muscarinic current of guinea-pig gastric myocytes.

To investigate the relationship between cytoskeleton and hyposmotic membrane stretch-induced increase in muscarinic current, the role of actin microfilament in hyposmotic membrane stretch-induced increase in muscarinic current was studied with the whole-cell patch clamp technique in guinea-pig gastric myocytes. In this study, the muscarinic current was induced by carbachol (50 micromol/L) or GTPgammaS (0.5 mmol/L). The results showed that hyposmotic superfusate (202 mOsmol/L) increased carbachol-induced current (I(CCh)) by 145+/-27% and increased GTPgammaS-induced current by 183+/-30%; but in the presence of cytochalasin-B (Cyt-B, 20 micromol/L), an actin cytoskeleton disruptor, hyposmotic membrane stretch increased I(CCh) by 70+/-6%. However, hyposmotic membrane stretch induced increase in I(CCh) was potentiated to 545+/-81% by phalloidin (20 micromol/L), an actin microfilament stabilizer. The results demonstrated that hyposmotic membrane stretch increased the muscarinic currents induced by carbachol or GTPgammaS and that the actin microfilament is involved in the process in guinea-pig gastric myocytes.

Comparative study in the effect of C-type natriuretic peptide on gastric motility in various animals.

AIM: To investigate the effect of natriuretic peptides on gastric motility in various animals, and the effect of C-type natriuretic peptide (CNP) on spontaneous contraction of gastric smooth muscle in rat, guinea-pig and human in vitro was compared. METHODS: Spontaneous contraction of gastric smooth muscle was recorded by four channel physiograph. RESULTS: In the guinea-pig and rat gastric antral circular smooth muscle, CNP markedly decreased the amplitude of spontaneous contraction but it didn't affect the frequency, however, the contractile activity was completely inhibited by CNP in gastric antral longitudinal smooth muscle. In the human gastric antral circular and longitudinal smooth musle, CNP completely inhibited spontaneous contraction. In the circular smooth muscle of guinea-pig and rat gastric fundus, CNP obviously decreased the amplitude of spontaneous contraction but it didn't affect the frequency, however, the contractile activity was completely inhibited by CNP in smooth muscle of fundus longitudinal. In the circular and longitudinal smooth muscle of guinea-pig gastric body, CNP at first induced a relaxation and then an increase in amplitude of spontaneous contraction (rebound contraction), but the frequency was not changed. After the circular smooth muscle of gastric body was pretreated with atropine, an M receptor blocker, the rebound contraction was abolished; In circular and longitudinal smooth muscle of rat gastric body, CNP induced a transient and slight relaxation and successively followed by the recovery in amplitude of spontaneous contraction but it also didn't affect the frequency. After the smooth muscle was pretreated with atropine, the transient and slight relaxation was replaced by long term and complete inhibition; The percentage of CNP-induced inhibition was 76.77+/-6.21 % (fundus), 67.21+/-5.32 % (body) and 58.23+/- 6.21 % (antral) in the gastric circular muscle, however, the inhibitory percentage was 100+/-0.00 % (fundus), 68.66+/- 3.55 % (body) and 100+/-0.00 % (antrum) in the gastric longitudinal smooth muscle of guinea-pigs; In the rat, the percentage of CNP-induced inhibition was 95.87+/-4.12 % (fundus), 94.91+/-5.08 % (body) and 66.32+/-7.32 % (antrum)in the gastric circular smooth muscle, but in the longitudinal smooth muscle, CNP completely inhibited the spontaneous contraction. Using LY83583, a guanylate cyclase inhibitor, and zaparinast as a phosphoesterase inhibitor to inhibit the generation of cGMP, the effect of CNP on the spontaneous contraction was markedly weakened by LY83583, however, the inhibitory effect was enhanced by zaparinast. CONCLUSION: (1) CNP can obviously inhibit the spontaneous contraction of gastric antral circular and longitudinal smooth muscle in the rat, guinea-pig and human. The order of inhibitory potency is human >rat> guinea-pig. (2) In the same animals, the inhibitory effect of CNP on spontaneous contraction is the most powerful in fundus and the weakest in antrum, in the same position, the inhibitory effect on the circular smooth muscle is more powerful than that on longitudinal smooth muscle. (3) The inhibitory effect of CNP on spontaneous contraction in the gastric smooth muscle is mediated by a cGMP dependent pathway.