Volume 122, Issue 2, Pages 340-351 (February 2002) Opioid agonists inhibit excitatory neurotransmission in ganglia and at the neuromuscular junction in guinea pig gallbladder  Fay A. Guarraci, Maria J. Pozo, Sara M. Palomares, Tracy A. Firth, Gary M. Mawe  Gastroenterology  Volume 122, Issue 2, Pages 340-351 (February 2002) DOI: 10.1053/gast.2002.31037 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Lack of direct effects of opioid receptor agonists on gallbladder neurons. The delta, kappa, and mu opiate receptor agonists (DPDPE, U-50488H, and DAMGO) failed to alter the membrane potential or action potential generating properties of the gallbladder neurons. Furthermore, each neuron fired a single action potential at the onset of the depolarizing current pulse, and this pattern continued in the presence of each antagonist. Resting membrane potentials: A, −52 mV; B, −48 mV; C, −50 mV. Gastroenterology 2002 122, 340-351DOI: (10.1053/gast.2002.31037) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 The delta opioid receptor agonist, DPDPE, causes a naloxone-sensitive, concentration-dependent inhibition of excitatory neurotransmission in gallbladder ganglia. (A) Graph demonstrating the concentration-effect relationship for the DPDPE-induced inhibition of the fast EPSP, and a suppression of the agonist effect by 300 nmol/L naloxone. Each point represents a mean of 5–10 cells, and the error bars represent SEM. (B) Representative responses of fast synaptic potentials to DPDPE at progressively increasing concentrations. Each trace represents an average of 10 consecutive events. Resting membrane potentials: −50, −46, and −50 mV, respectively. (C) The depolarizing response to pressure microinjection of acetylcholine (arrow; 1 mmol/L, 100 msec, 10 PSI) was unaffected by application of DPDPE. Resting membrane potential = −49 mV. Gastroenterology 2002 122, 340-351DOI: (10.1053/gast.2002.31037) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 The kappa opioid receptor agonist, U-50488, causes a naloxone-sensitive, concentration-dependent inhibition of excitatory neurotransmission in gallbladder ganglia. (A) Graph demonstrating the concentration-effect relationship for the U-50488–induced inhibition of the fast EPSP, and a suppression of the agonist effect by 300 nmol/L naloxone. Each point represents a mean of 5–10 cells, and the error bars represent SEM. (B) Representative responses of fast synaptic potentials to U-50488 at progressively increasing concentrations. Each trace represents an average of 10 consecutive events. Resting membrane potentials: −51, −45, and −50 mV, respectively. (C) The depolarizing response to pressure microinjection of acetylcholine (arrow; 1 mmol/L, 100 msec, 10 PSI) was unaffected by application of U-50488. Resting membrane potential = −48 mV. Gastroenterology 2002 122, 340-351DOI: (10.1053/gast.2002.31037) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 The mu opioid receptor agonist, DAMGO, causes a naloxone-sensitive, concentration-dependent inhibition of excitatory neurotransmission in gallbladder ganglia. (A) Graph demonstrating the concentration-effect relationship for the DAMGO-induced inhibition of the fast EPSP, and a suppression of the agonist effect by 300 nmol/L naloxone. Each point represents a mean of 5–10 cells, and the error bars represent SEM. (B) Representative responses of fast synaptic potentials to DAMGO at progressively increasing concentrations. Each trace represents an average of 10 consecutive events. Resting membrane potentials: −47, −51, and −52 mV, respectively. (C) The depolarizing response to pressure microinjection of acetylcholine (arrow; 1 mmol/L, 100 msec, 10 PSI) was unaffected by application of DAMGO. Resting membrane potential = −52 mV. Gastroenterology 2002 122, 340-351DOI: (10.1053/gast.2002.31037) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 The MOR agonist, DAMGO, inhibits the excitatory effect of CCK on synaptic transmission in gallbladder ganglia. These are recordings from a gallbladder neuron that responded to interganglionic fiber tract stimulation with an EPSP leading to an action potential when the stimulus intensity was high enough to elicit a response. At lower intensities, no response was detected. (A) With the stimulus intensity set at a subthreshold level (1.5 V), brief pressure microejection of CCK (250 msec, 0.1 mmol/L) led to a transient activation of synaptic events. (B) With the stimulus intensity increased to a suprathreshold level (2 V), addition of DAMGO to the bathing solution inhibited synaptic transmission. (C) In the presence of DAMGO, with the stimulus intensity set at 2 V, CCK failed to facilitate synaptic activity. (D) After washout of DAMGO, with the stimulus intensity set at 1.5 V, the facilitory response to CCK recovered. Resting membrane potential, −51 mV. Gastroenterology 2002 122, 340-351DOI: (10.1053/gast.2002.31037) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Opioid receptor agonists do not alter the membrane potential or the spontaneous action potential of gallbladder smooth muscle cells. Spontaneous muscle potentials were unaffected by superfusion of the delta, kappa, and mu opiate receptor agonists, DPDPE, U-50488H, or DAMGO. Resting membrane potentials: A, −46 mV; B, −47 mV; C, −47 mV. Gastroenterology 2002 122, 340-351DOI: (10.1053/gast.2002.31037) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 The delta opioid receptor agonist, DPDPE, causes a concentration-dependent inhibition of neurogenic evoked contractions of gallbladder muscle strips. (A) Graph demonstrating the concentration-effect relationship for the DPDPE-induced inhibition of the contractile response, and a suppression of the agonist effect by 300 nmol/L naloxone. Each point represents a mean of 7 experiments, and the error bars represent SEM. (B) A representative trace demonstrating the reduction in amplitude of the muscle strip contraction in the presence of increasing concentrations of DPDPE. Gastroenterology 2002 122, 340-351DOI: (10.1053/gast.2002.31037) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 The kappa opioid receptor agonist, U-50488, causes a concentration-dependent inhibition of neurogenic-evoked contractions of gallbladder muscle strips. (A) Graph demonstrating the concentration-effect relationship for the U-50488–induced inhibition of the contractile response, and a suppression of the agonist effect by 300 nmol/L naloxone. Each point represents a mean of 7 experiments, and the error bars represent SEM. (B) A representative trace demonstrating the reduction in amplitude of the muscle strip contraction in the presence of increasing concentrations of U-50488. Gastroenterology 2002 122, 340-351DOI: (10.1053/gast.2002.31037) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 9 The mu opioid receptor agonist, DAMGO, causes a concentration-dependent inhibition of neurogenic-evoked contractions of gallbladder muscle strips. (A) Graph demonstrating the concentration-effect relationship for the DAMGO-induced inhibition of the contractile response, and a suppression of the agonist effect by 300 nmol/L naloxone. Each point represents a mean of 7 experiments, and the error bars represent SEM. (B) A representative trace demonstrating the reduction in amplitude of the muscle strip contraction in the presence of increasing concentrations of DAMGO. Gastroenterology 2002 122, 340-351DOI: (10.1053/gast.2002.31037) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 10 (A) Delta, (B) kappa, and (C) mu opioid receptor (DOR, KOR, and MOR) immunoreactivity in the ganglionated plexus of the guinea pig gallbladder. Immunoreactivity was particularly intense in gallbladder ganglia, but immunostaining could be detected with each of the antisera in interganglionic fiber bundles. (D) Leu-enkephalin (ENK) immunoreactivity was sparse in the ganglionated plexus of the gallbladder, but (E) was abundant in control preparations of the myenteric plexus. Gastroenterology 2002 122, 340-351DOI: (10.1053/gast.2002.31037) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 11 schematic diagram demonstrating the locations of opioid receptors in the neuromuscular axis of the guinea pig gallbladder. Opioid receptors are located on vagal nerve terminals in gallbladder ganglia and on the terminals of gallbladder postganglionic neurons. The vagal terminals are an important regulatory site in the gallbladder wall because opiates, cholecystokinin (CCK), and norepinephrine can act on presynaptic receptors to alter release of acetylcholine from vagal terminals onto gallbladder neurons. By acting on opioid receptors located on vagal terminals, opiate peptides could physiologically antagonize the excitatory effect of CCK on neuromuscular transmission. Gastroenterology 2002 122, 340-351DOI: (10.1053/gast.2002.31037) Copyright © 2002 American Gastroenterological Association Terms and Conditions