Metabotropic Signal Transduction for Bradykinin in Submucosal Neurons of Guinea Pig Small Intestine

Abstract

Intracellular recording methods with "sharp" microelectrodes were used to study signal transduction mechanisms underlying the excitatory action of bradykinin (BK) in morphologically identified neurons in the small intestinal submucosal plexus. Exposure to BK evoked slowly activating membrane depolarization and enhanced excitability associated with increased input resistance in AH-type and decreased input resistance in S-type neurons. Preincubation with pertussis toxin did not affect the BK-evoked responses. Pretreatment with the cyclooxygenase inhibitors indomethacin or piroxicam suppressed or abolished the BK-evoked responses. Application of prostaglandin (PG) E 2 or PG analogs evoked BK-like depolarizing responses in the submucosal plexus with a potency order of PGE 2 Ͼ PGE 1 Ͼ 17-phenyl trinor-PGE 2 Ͼ PGI 2 Ͼ sulprostone Ͼ PGF 2␣ . Depolarizing responses to bradykinin or PGE 2 in S-type neurons were suppressed in the presence of the phospholipase C inhibitor U73122 [(1-6-The inositol-1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenylborane and the calmodulin inhibitor W-7, but not ryanodine, suppressed both bradykinin-and PGE 2 -evoked responses. KN-62, an inhibitor of calmodulin kinases, or GF109203X, a specific protein kinase C inhibitor, suppressed both BK-and PGE 2 -evoked depolarizing responses. Selective protein kinase A inhibitors did not alter BKor PGE 2 -evoked depolarizing responses in S neurons. The results suggest that BK stimulates synthesis and release of PGE 2 , which acts at EP 1 receptors to evoke depolarizing responses in submucosal neurons. The postreceptor transduction cascade includes activation of phospholipase C, inositol-1,4,5-trisphosphate production, intraneuronal Ca 2ϩ mobilization, activation of protein kinase C and/or calmodulin kinases, and phosphorylation of cationic channels.Exposing neurons in the guinea pig small intestinal myenteric or submucosal plexus to bradykinin (BK) in vitro evokes slowly activating depolarization of the membrane potential and enhanced excitability characterized by increased firing frequency during intraneuronal injection of depolarizing current pulses in both AH-and S-type neurons and the appearance of anodal break excitation at the offset of hyperpolarizing current pulses in AH neurons (Hu et al., 2003(Hu et al., , 2004. The depolarizing responses are associated with increased input resistance (i.e., decreased conductance) in AH neurons and with decreased input resistance (i.e., increased conductance) in S neurons. Selective BK B 2 receptor antagonists, but not BK B 1 antagonists, suppress the actions of BK in both myenteric and submucosal plexuses (Hu et al., 2003(Hu et al., , 2004. Binding studies with a fluorescently labeled, selective BK B 2 receptor antagonist reveal expression of BK B 2 receptors on a majority of the ganglion cells in the myenteric and submucosal plexuses. RT-PCR and Western blot analysis confirms the expression of BK B 2 receptor mRNA and protein in both plexuses (Hu et al., 2003(Hu et al., , 2004.Inhibition...

Neurophysiological mechanisms of bradykinin-evoked mucosal chloride secretion in guinea pig small intestine

“…Our previous studies found the expression of B2R mRNA and protein in submucosal neurons. BK increased excitatory in both AH-and S-type neurons in the submucosal plexus [9,10] . The S-type neurons are primarily secretomotor neurons that receive excitatory synaptic input from their AHtype neighbors [24] .…”

“…BK receptors belong to the family of G-protein-coupled receptors with seven transmembrane helices. BK and kallidin are ligands for the constitutively expressed B2R, whereas des-Arg 9 -BK (in rodents) and des-Arg 10 -kallidin (in human) are ligands for the inducible B1R [6,7] . Previously, we demonstrated that B2R is expressed on a majority of the ganglion cells in the myenteric and submucosal plexuses in the small intestine of guinea pigs [8][9][10] .…”

“…BK and kallidin are ligands for the constitutively expressed B2R, whereas des-Arg 9 -BK (in rodents) and des-Arg 10 -kallidin (in human) are ligands for the inducible B1R [6,7] . Previously, we demonstrated that B2R is expressed on a majority of the ganglion cells in the myenteric and submucosal plexuses in the small intestine of guinea pigs [8][9][10] . Exposing neurons in the guinea pig small intestinal myenteric or submucosal plexus to BK in vitro evokes slow activation of depolarization of the membrane potential and enhanced excitability characterized by increased firing frequency during intraneuronal injection of depolarizing current pulses in both AH-and S-type neurons and the appearance of anodal break excitation at the offset of hyperpolarizing current pulses in AH neurons [8,9] .…”

Stimulation of adenosine A1and A2Areceptors by AMP in the submucosal plexus of guinea pig small intestine

“…The preparations were microdissected as described in MATERIALS AND METHODS, washed three times with sterile PBS, and incubated in culture media (DMEMϩ10% fetal bovine serumϩ1% penicillin-streptomycin) at 37°C in a CO 2 incubator. Viability of the tissue after incubation was verified by recording typical excitatory responses to application of bradykinin as reported by Hu et al (33). Incubation in PTX did not alter the responses to AMP in seven neurons (data not shown).…”

“…Because it was known that increased conductance in nonselective cation channels accounts for the membrane depolarization during metabotropic slow EPSPs and slow EPSP-like responses to excitatory agonists in S-type submucosal neurons, we examined the effects of Na ϩ depletion on the depolarizing responses to AMP (33,49). Depletion of Na ϩ in the bathing medium suppressed the depolarizing responses to AMP (Figs.…”