Stimulation of mucosal secretion by lubiprostone (SPI-0211) in guinea pig small intestine and colon

Abstract

Actions of lubiprostone, a selective type-2 chloride channel activator, on mucosal secretion were investigated in guinea pig small intestine and colon. Flat-sheet preparations were mounted in Ussing flux chambers for recording short-circuit current (Isc) as a marker for electrogenic chloride secretion. Lubiprostone, applied to the small intestinal mucosa in eight concentrations ranging from 1-3000 nM, evoked increases in Isc in a concentration-dependent manner with an EC50 of 42.5 nM. Lubiprostone applied to the mucosa of the colon in eight concentrations ranging from 1-3000 nM evoked increases in Isc in a concentration-dependent manner with an EC50 of 31.7 nM. Blockade of enteric nerves by tetrodotoxin did not influence stimulation of Isc by lubiprostone. Antagonists acting at prostaglandin (PG)E2, EP1-3, or EP4 receptors did not suppress stimulation of Isc by lubiprostone but suppressed or abolished PGE2-evoked responses. Substitution of gluconate for chloride abolished all responses to lubiprostone. The selective CFTR channel blocker, CFTR(inh)-172, did not suppress lubiprostone-evoked Isc. The broadly acting blocker, glibenclamide, suppressed (P<0.001) lubiprostone-evoked Isc. Lubiprostone, in the presence of tetrodotoxin, enhanced carbachol-evoked Isc. The cholinergic component, but not the putative vasoactive intestinal peptide component, of neural responses to electrical field stimulation was enhanced by lubiprostone. Application of any of the prostaglandins, E2, F2, or I2, evoked depolarization of the resting membrane potential in enteric neurons. Unlike the prostaglandins, lubiprostone did not alter the electrical behavior of enteric neurons. Exposure to the histamine H2 receptor agonists increased basal Isc followed by persistent cyclical increases in Isc. Lubiprostone increased the peak amplitude of the dimaprit-evoked cycles.

Fig. 1.

Cumulative concentration-response curves for lubiprostone-evoked increases in short-circuit current (I_sc) in guinea pig ileum (A) and colon (B). Lubiprostone applied to the serosal side compartment of the Ussing chambers evoked increases in I_sc from 2.6 ± 0.9 to 50.26 ± 4.5 μA/cm² in a concentration-dependent manner, with an EC₅₀ of 227.2 nM, and an increase in I_sc from 0.78 ± 0.7 to 37.8 ± 7.9 μA/cm², with an EC₅₀ of 42.5 nM for mucosal side application. Lubiprostone applied to the serosal side of colonic preparations evoked increases in I_sc from 1.3 ± 0.7 to 26.8 ± 4.7 μA/cm² in a concentration-dependent manner, with an EC₅₀ of 31.7 nM for serosal side application, and an increase in I_sc from 3.4 ± 0.9 to 58.9 ± 12.3 μA/cm², with an EC₅₀ of 48.9 nM for mucosal side application. Values are expressed as means ± SE, n = 6 animals in each series.

Fig. 2.

Effects of tetrodotoxin (TTX), Cl-free media or bumetanide on lubiprostone-evoked I_sc responses in guinea pig ileum. Placement of 1 μM TTX in the serosal compartment evoked a small negative change in I_sc. Pretreatment with TTX (1 μM), applied to the serosal side, did not significantly alter the responses evoked by 300 nM lubiprostone (Lubi.) in the serosal compartment of the Ussing chamber (P = 0.30). Substitution of gluconate for Cl⁻ in the serosal or mucosal compartment evoked small increases in I_sc. Substitution of gluconate for Cl⁻ in both compartments abolished all responses to 300 nM lubiprostone when applied in the serosal or mucosal compartment. Bumetanide (100 μM) in the mucosal compartment, an inhibitor of the basolateral Na/K/2Cl cotransporter, suppressed increases in I_sc evoked by 300 nM lubiprostone when it was applied on either the mucosal (P < 0.01) or serosal side (P < 0.01). The responses to serosal and mucosal applications of lubiprostone were not different (P > 0.05) when bumetanide was present in the mucosal compartment. Bumetanide or TTX were applied 5 min before application of lubiprostone. Values are expressed as means ± SE, each mean is data for preparations from 6–12 animals. **P < 0.01; ***P < 0.001.

Fig. 3.

Effects of 5-nitro-2-2(-henylpropylamino) benzoic acid (NPPB) and DIDS on lubiprostone-evoked I_sc in guinea pig ileum. NPPB (200 μM), a nonspecific chloride channel blocker, or DIDS (250 μM), a specific inhibitor of cellular anion permeability, was applied into the serosal or mucosal compartment of the Ussing chamber 5 min before adding 300 nM lubiprostone. NPPB abolished lubiprostone-evoked ΔI_sc. DIDS did not alter lubiprostone-evoked increases in I_sc. DIDS or NPPB was applied 5 min before application of lubiprostone. Values are expressed as means ± SE, n = 6 animals. ***P < 0.001.

Fig. 4.

Effects of CTFR(inh)-172 and glibenclamide on lubiprostone-evoked I_sc in guinea pig ileum. CTFR(inh)-172 (10 μM), is an inhibitor of CFTR with a K_i = 300 nM, or glibenclamide (100 μM), a nonspecific blocker of CFTR and ClC-2 channels, was applied into the serosal or mucosal compartment of the Ussing chamber 5 min before adding 300 nM lubiprostone. CTFR(inh)-172 did not significantly reduce the amplitude of lubiprostone-evoked I_sc. Glibenclamide suppressed lubiprostone-evoked I_sc by 59% and 47% when applied to the serosal or mucosal side, respectively. Values are expressed as means ± SE, n = number of animals. *P < 0.001.

Fig. 5.

Effects of lubiprostone on carbachol or vasoactive intestinal peptide (VIP) stimulation of I_sc in guinea pig ileum. In the presence of TTX, application of the muscarinic receptor agonist carbachol or VIP evoked increases in I_sc, which reflected the direct actions at receptors for acetylcholine or VIP on the intestinal epithelium. Lubiprostone (300 nM) or vehicle was applied in the serosal compartment of the Ussing chamber 5 min after 1 μM TTX. Carbachol (1 μM) or VIP (10 nM) was applied in the serosal side of the chamber 6 min after application of lubiprostone. A: sample traces illustrate I_sc responses to carbachol (A₃, A₄) or VIP (A₁, A₂) in the presence of TTX with or without lubiprostone pretreatment. B: lubiprostone augmented the stimulatory action of carbachol. Lubiprostone increased baseline I_sc but suppressed the stimulatory action of VIP at its receptors on the epithelium. Values are expressed as means ± SE, n = 6 animals. **P < 0.01; ***P < 0.001.

Fig. 6.

Action of lubiprostone on I_sc evoked by transmural electrical field stimulation (EFS) in guinea pig ileum. Transmural EFS in evoked biphasic increases in I_sc. The first phase 1 and the second phase 2 represent cholinergically evoked and putative peptidergic/cholinergic-evoked chloride secretion, respectively. Lubiprostone (300 nM) was applied into either the serosal or mucosal compartment of the Ussing chambers 20 min after EFS. When baseline I_sc returned to a stable level, EFS was repeated. A: sample traces illustrating the I_sc response to EFS before and after application of lubiprostone. B: lubiprostone significantly increased the magnitude of phase 1 of the response to EFS but had no significant effect on phase 2. Values are expressed as means ± SE, n = 6 animals. **P < 0.01; ***P < 0.001 (compared with response to EFS before lubiprostone).

Fig. 7.

Lubiprostone action on cyclical changes in I_sc evoked by the histamine H₂ receptor agonist, dimaprit, in full-thickness preparations of guinea pig colon. A: exposure to dimaprit, in the serosal compartment of the Ussing chamber, evoked an initial increase in baseline I_sc followed by cyclically occurring elevations of I_sc superimposed on the elevated baseline. Blockade of intramural neuronal excitability by TTX in the presence of dimaprit reduced I_sc to a subbaseline level and eliminated the cyclic changes in I_sc. Lubiprostone (Lubi.) applied in the presence of dimaprit and TTX reversed the effects of neural blockade and elevated I_sc beyond its previous baseline value. Carbachol, which was applied as a control for viability of the preparation, evoked maximal elevation of I_sc when applied in the presence of dimaprit, TTX, and lubiprostone. B: quantitative data for the action of dimaprit alone, TTX action on the dimaprit response, action of lubiprostone in the presence of dimaprit and TTX, and action of carbachol. Values are expressed as means ± SE, n = 7 animals. ***P < 0.001 (compared with response to dimaprit alone).

Fig. 8.

Action of lubiprostone to enhance I_sc responses evoked by the histamine H₂ receptor agonist, dimaprit, in whole-thickness preparations from guinea pig colon. A: exposure to dimaprit, in the mucosal-side compartment of the Ussing chamber, stimulated basal I_sc. Application of lubiprostone significantly enhanced the dimaprit-evoked stimulation of I_sc (*P < 0.05; data for preparations from 7 animals). Carbachol, which was applied as a control for viability of the preparation, evoked maximal elevation of I_sc when applied in the presence of dimaprit and lubiprostone. B: quantitative data for the amplitude of 7 successive “I_sc spikes” recorded after application of dimaprit in the mucosal-side compartment for preparations from 7 animals. Application of lubiprostone, in the presence of dimaprit, resulted in significant enhancement of the cyclically occurring “I_sc spikes”.

Fig. 9.

Neither resting membrane potential nor input resistance of submucosal secretomotor neurons was altered by lubiprostone as determined by microelectrode recording. A: control. B: absence of any action of lubiprostone on resting membrane potential or neuronal input resistance. C: application of prostaglandin E₂ resulted in depolarization of the membrane potential and increased input resistance in an AH-type neuron. Downward deflections are electrotonic potentials evoked by repetitive injection of hyperpolarizing current pulses. An increase in the amplitude of the electrotonic potentials reflects increased input resistance, which is a marker for closing of membrane channels and increased ionic conductance. Lack of effect of lubiprostone on the amplitude of the electrotonic potentials indicates that it does not alter the neuronal input resistance. Prostaglandin E₂ decreased membrane conductance in the neuron.

Fig. 10.

Concentration-response relationship for membrane depolarization evoked by prostaglandin E₂ and its analogs in submucous plexus neurons. A and B: concentration-response relations for PGE₂, PGF₂, and PGI₂. The curves were plotted with the least-squares fitting routine: V = V_max/[1 + (EC₅₀/C)^nH], where V is the observed membrane depolarization, EC₅₀ is the concentration which induces the half-maximal response, and n_H is the apparent Hill coefficient. Each point represents 5–12 neurons.

Fig. 11.

Actions of mucosal application of prostaglandin receptor antagonists on stimulation of I_sc by lubiprostone and prostaglandin E2 in full-thickness preparations of distal colon. A: neither the PGE₂ receptor antagonist SC-19220, the EP_1–3 receptor antagonist AH6809 nor the EP₄ receptor antagonist GW627368X suppressed stimulation of I_sc by lubiprostone when applied to the mucosa in preparations from 7 animals (P < 0.05). B: PGE₂ receptor antagonist SC-19220, the EP_1–3 receptor antagonist AH6890 and the EP₄ receptor antagonist GW627368X each significantly suppressed (P > 0.05) or abolished stimulation of I_sc by prostaglandin PGE₂ in preparations from 3 animals.