Oxytocin microinjected into dorsal motor nucleus of the vagus excites gallbladder motility via NMDA receptor-NO-cGMP pathway.

Our recent study indicated that, in the dorsal motor nucleus of the vagus (DMV), the N-methyl-D-aspartic acid (NMDA) receptor-nitric oxide (NO)-cGMP pathway participated in the regulation of gallbladder motility in rabbits. Oxytocin (OT) is involved as a neurotransmitter in autonomic regulation. The aim of the present experiments is to investigate the effect of OT microinjected into DMV on the gallbladder motility and the involvement of NMDA receptor-NO-cGMP pathway. A frog bladder connected with transducer was inserted into the gallbladder to record the gallbladder pressure. Microinjection of OT (10-50 nmol/L, 100 nl) dose dependently increased the strength of gallbladder phasic contraction. The excitatory effect of OT (10 nmol/L, 100 nl) was completely abolished by atosiban (10 mmol/L, 100 nl), the specific OT receptor antagonist, but was not influenced by [deamino-Pen(1), O-Me-Tyr(2),Arg(8)]-vasopressin (10 mmol/L, 100 nl), the V(1) receptor antagonist. Pretreatment of ketamine (10 mmol/L, 100 nl), the NMDA receptor antagonist, suppressed the gallbladder motor response to OT; but pretreatment of 6-Cyaon-7-Nitroquinoxaline-2,3-(1H,4H)-Dione (CNQX; 10 mmol/L, 100 nl), the non-NMDA receptor antagonist, did not affect it. Pretreatment of L-NAME (10 mmol/L, 100 nl), the nitric oxide synthase (NOS) inhibitor, or methyl blue (10 mmol/L, 100 nl), the guanylyl cyclase inhibitor, inhibited the excitatory effect of OT on gallbladder motility. Hence, we deduced that the microinjection of OT into the DMV enhanced the gallbladder motility through binding specific OT receptors and activating the NMDA receptor-NO-cGMP pathway.

Participation of caudal ventrolateral medulla in the regulation of gallbladder motility in rabbits.

To investigate whether the caudal ventrolateral medulla (CVLM) participates in the regulation of gallbladder motility, we studied the effects of microinjection of L-glutamate and other agents into the CVLM on gallbladder pressure (GP) in anesthetized rabbits. A frog bladder connected with a force transducer was inserted into the gallbladder to record the change of GP. Microinjection of L-glutamate into the CVLM decreased GP, While micnoinjection of gamma-amino-butyric acid (GABA) increased GP. Microinjection of ketamine, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, into CVLM increased GP, while microinjection of 6-cyano-7-nitroquinoxaline-2,3-(1H,4H)-dione (CNQX), a competitive (+/-)-a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor antagonist, had no significant effect on GP. The effects of L-glutamate was abolished by ketamine, but not by CNQX. Intravenous injection of phentolamine or transection of the spinal cord eliminated the effects of L-glutamate on GP. These results indicate that [1] CVLM participated in the regulation of gallbladder motility; [2] endogenous L-glutamate in CVLM is involved in the regulation mediated by NMDA receptors, the output of which is sent through sympathetic nerve and alpha-adrenergic receptors.

Effect of oxytocin on contraction of rabbit proximal colon in vitro.

AIM: To investigate the effects of oxytocin (OT) on isolated rabbit proximal colon and its mechanism. METHODS: Both longitudinal muscle (LM) and circular muscle (CM) were suspended in a tissue chamber containing 5 mL Krebs solution (37 degrees ), bubbled continuously with 950 mL x L(-1) O(2) and 50 mL x L(-1) CO(2). Isometric spontaneous contractile responses to oxytocin or other drugs were recorded in circular and longitudinal muscle strips. RESULTS: OT (0.1 U x L(-1)) failed to elicit significant effects on the contractile activity of proximal colonic smooth muscle strips (P>0.05). OT (1 to 10 U x L(-1)) decreased the mean contractile amplitude and the contractile frequency of CM and LM. Hexamethonium (10 micromol x L(-1)) partly blocked the inhibition of oxytocin (1 U x L(-1)) on the contractile frenquency of CM. N(omega))-nitro-L-arginine-methylester (L-NAME, 1 micromol x L (-1)), progesterone (32 micromol x L(-1)) and estrogen (2.6 micromol x L(-1)) had no effects on OT-induced responses. CONCLUSION: OT inhibits the motility of proximal colon in rabbits. The action is partly relevant with N receptor, but irrelevant with that of NO, progesterone or estrogen.

Anatomical and functional study of localization of originating neurons of the parasympathetic nerve to gallbladder in rabbit brain stem.

The present study was to investigate the localization of preganglionic parasympathetic neurons of gallbladder in brain stem by anatomical and functional approaches. Male or female rabbits (n = 11) were anesthetized with sodium pentobarbital (30 mg/kg, i.v.). Cholera toxin B conjugated to horseradish peroxidase (CB-HRP) was injected into the gallbladder wall. Four days later, animals were re-anesthetized and perfused transcardially with paraformaldehyde solution in a 0.1 M phosphate buffer. The rabbit brain was then frozenly sectioned. The sections were processed for HRP label and stained with neutral red. Another group of rabbits (n = 54) were anesthetized by urethane (1 g/kg) after fasting for 18-24 hours, Gallbladder pressure (GP) was measured by inserting a frog bladder filled with normal saline into the gallbladder. Myoelectrical activity of the sphincter of Oddi (SO) was induced by a pair of copper electrodes. A glass tube (30 microm tip diameter) connected with a microsyringe was directed to the dorsal vagal complex (DVC) for microinjection. Majority of retrogradely labeled cells was found bilaterally in dorsal motor nucleus of the vagus nerve (DMV) throughout the length, except the rostral and caudal part. These cells were distributed in subnuclei parvicellularis or mediocellularis of DMV. Some labeled perikarya located in the medial subnucleus of the solitary tract (mNTS). Thyrotropin-releasing hormone (TRH, 1.3 mmol/L, 0.2 microl) microinjected into the rostral portion of the DVC (including DMV and NTS) enhanced the motility of gallbladder and SO. Microinjection of TRH at the middle part of DVC seldom induces excitatory effects on the gallbladder or SO. TRH microinjected into the caudal portion of the DVC elicited weaker response of gallbladder and SO than rostral portion. Our results indicated that DMV is one of the most important original nuclei of gallbladder's vagus nerves and mNTS may be also involved in the control of gallbladder's parasympathetic activity. Neurons that innervate the gallbladder distribute at most part of DVC, and are relatively dense at rostral and caudal position of DMV.

Nucleus raphe obscurus participates in regulation of gallbladder motility through vagus and sympathetic nerves in rabbits.

The aim of the present study is to investigate if the nucleus raphe obscurus (NRO) participate in regulating the gallbladder motility in rabbits. Rabbits were fasted for about 20-24 hours. After anesthetization with urethane, an incision was made at the middle of the abdomen and the gallbladder was exposed. A frog bladder connected with force transducer was inserted into the gallbladder through a small incision at the funds to record gallbladder motility (tonic contraction and phasic contraction). Glutamate and other chemicals were microinjected into NRO through a vitreous tube attached to a microsyringe. We found both the tonic contraction and phasic contraction of the gallbladder were enhanced after the glutamate was injected into NRO. GABA inhibited gallbladder motility if administrated in the same way. Microinjection of ketamine, NMDA (N-methyl-D-aspartate) receptor antagonist, into NRO inhibited the phasic contraction of gallbladder. Administration of CNQX (6-cyano-7-nitroquinoxaline-2, 3-dione), a non-NMDA receptor antagonist, enhanced the gallbladder tonic contraction. Pretreatment of ketamine into NRO attenuated the effect of glutamate, while pretreatment of CNQX had no effect on it. Intravenous injection of atropine or vagotomy completely abolished the effect of glutamate on gallbladder phasic contraction, while intravenous injection of phentolamine or transecting the spinal cord at T3-4 inhibited that on tonic contraction. Intravenous injection of propranolol did not influence the glutamate effect. These results suggested that glutamate in NRO participates in regulating the motility of the gallbladder through NMDA receptor. When excited, the NMDA receptors in NRO enhance the phasic contraction of the gallbladder through vagus nerve and peripheral M-receptors, and enhance the tonic contraction of the gallbladder through sympathetic nerve and peripheral alpha-receptors. GABA in NRO is also involved in the regulation of gallbladder motility.

TRH microinjection into DVC enhances motility of rabbits gallbladder via vagus nerve.

AIM:To investigate the effects of TRH in DVC on motility of the gallbladder in rabbits.METHODS:fter fasted for 15h-18h, rabbits were anesthetized with urethane (1.0g/kg).Gallbladder pressure (GP) was measured by a frog bladder perfused with normal saline.RESULTS:After microinjection of TRH (8.8nmol,1&mgr;l) into DVC,GP was raised and the frequency of phasic contraction of gallbladder (FPCGB) increased. All the doses of TRH (0.13, 0.25, 0.50, 0.80, 1.30nmol, 1&mgr;l) injected into DVC could excite the motility of gallblader. As the dose of TRH was enlarged, the amplitude and duration of the reaction increased. Effects of TRH in DVC on motility of the gallbladder could be completely abolished by atropine (0.2mg/g, i.v.) or vagotomy, but could not be inhibited by phentolamine iv (1.5mg/g) or propranolol iv (1.5mg/g)or by transecting the spinal cord.CONCLUSION: Thyrotropin-releasing hormone in DVC can excite motility of gallbladder. This effect was mediated by vagus nerves and peripheral M receptor. Its physiological significance may be related to maintaining the phasic contraction of gallbladder in interdigestive period.