Adrenergic activation of electrogenic K+ secretion in guinea pig distal colonic epithelium: desensitization via the Y2-neuropeptide receptor

Abstract

Adrenergic activation of electrogenic K+ secretion in isolated mucosa from guinea pig distal colon was desensitized by peptide-YY (PYY). Addition of PYY or neuropeptide-Y (NPY) to the bathing solution of mucosae in Ussing chambers suppressed the short-circuit current (Isc) corresponding to electrogenic Cl- secretion, whether stimulated by epinephrine (epi), prostaglandin-E2 (PGE2), or carbachol (CCh). Neither peptide markedly inhibited the large transient component of synergistic secretion (PGE2 + CCh). Sustained Cl- secretory Isc was inhibited approximately 65% by PYY or NPY, with IC50s of 4.1 +/- 0.9 nM and 9.4 +/- 3.8 nM, respectively. This inhibition was eliminated by BIIE0246, an antagonist of the Y2-neuropeptide receptor (Y2-NpR), but not by Y1-NpR antagonist BVD10. Adrenergic sensitivity for activation of K+ secretion in the presence of Y2-NpR blockade by BIIE0246 was (EC50s) 2.9 +/- 1.2 nM for epi and 13.3 +/- 1.0 nM for norepinephrine, approximately fourfold greater than in the presence of PYY. Expression of mRNA for both Y1-NpR and Y2-NpR was indicated by RT-PCR of RNA from colonic mucosa, and protein expression was indicated by immunoblot. Immunoreactivity (ir) for Y1-NpR and Y2-NpR was distinct in basolateral membranes of columnar epithelial cells in the crypts of Lieberkühn as well as intercrypt surface epithelium. Adrenergic nerves in proximity with crypts were detected by ir for dopamine-beta-hydroxylase, and a portion of these nerves also contained NPY(ir). BIIE0246 addition increased secretagog-activated Isc, consistent with in vitro release of either PYY or NPY. Thus PYY and NPY were able to suppress Cl- secretory capacity and desensitize the adrenergic K+ secretory response, providing a direct inhibitory counterbalance against secretory activation.

Fig. 1.

Each mode of secretory activation was sensitive to peptide-YY (PYY). Isolated mucosae were stimulated sequentially by adding epinephrine (epi) (5 μM), prostaglandin-E₂ (PGE₂) (3 μM), and carbachol (CCh) (10 μM) to the serosal bath, from the standard basal condition (see materials and methods). Short-circuit current (I_sc) and transepithelial conductance (G_t) were measured in 4 adjacent mucosae (◊,▵, ▿, •) as PYY (1 μM) was added to the serosal bath during secretory activation (*). The time course was split into separate panels to aid viewing of each secretory mode, and the mucosae receiving PYY before secretagog stimulation (◊) was shown throughout. G_t was normalized by subtracting the basal value prior to stimulation (δG_t), except for the mucosae pretreated with PYY (◊) for which the normalization was made to the preaddition level. The basal G_t for the 4 mucosae was 9.47 ± 38 mS/cm². A and B: modulatory mode was activated by epi with one mucosa having PYY added before epi (◊) and the other after epi activation (▵). Each of the control mucosae (▵, ▿, •) had a transient positive I_sc at the onset of epi activation. The peak transient ΔI_sc and ΔG_t for epi addition alone were significantly larger than in the presence of PYY (P < 0.05, N = 10), respectively, +119 ± 13 μA/cm² and +20 ± 7 μA/cm² as well as +2.78 ± 28 mS/cm² and +0.93 ± 12 mS/cm². Before epi addition, the changes in I_sc and G_t attributable to PYY addition compared with control were significantly different from zero (P < 0.05, N = 10), respectively, +19 ± 3 μA/cm² and −1.12 ± 0.17 mS/cm². C and D: flushing mode activated by PGE₂ was shown in the presence (◊) and absence (•) of PYY, and a third mucosa was shown (▿) with PYY added ∼12 min after PGE₂ activation. E and F: synergistic mode was activated by adding CCh in the continued presence of PGE₂. The action of PYY was assessed further by comparing the response in the presence of PYY (◊) to the mucosa receiving PYY ∼26 min after CCh activation (•). G and H: PYY-sensitive components of I_sc (^PYYΔI_sc) and G_t (^PYYΔG_t) for each mode of secretory activation were calculated as the difference between mucosae with PYY absent and those with PYY present; these positive ΔI_sc and ΔG_t likely represented electrogenic Cl⁻ secretion. The responses were representative of 6 similar experiments.

Fig. 2.

Neuroendocrine peptides inhibited secretory I_sc with high efficacy. Isolated mucosae were stimulated by sequential additions of epi (5 μM), PGE₂ (3 μM), and CCh (10 μM) as in Fig. 1. Neuroendocrine peptides were added at increasing concentrations to the serosal bath in adjacent mucosa either during PGE₂ (as in Fig. 1B) or CCh activation (as in Fig. 1C). Inhibitory responses were normalized to the I_sc before addition compared with the sustained I_sc during epi activation. Fractional inhibition was similar for both secretory modes so that the results were combined, neuropeptide-Y (NPY) (⧫, n = 3), PYY (•, n = 13), PYY(3-36) (○, n = 4). Antagonists to neuropeptide receptors [BVD10, Y1-neuropeptide receptor (Y1-NpR); □, n = 3] and (BIIE0246, Y2-NpR; ▿, n = 4) also were added (1 μM) for some mucosae before secretory activation and PYY addition. The fit of the data with a single binding site [NPY and PYY, solid line; PYY(3-36), dashed line; PYY + BVD10, gray dashed line] yielded IC₅₀s of 4.1 ± 0.9 nM (PYY), 6.2 ± 1.9 nM [PYY(3-36)], and 9.4 ± 3.8 nM (NPY); maximal fractional inhibitions were 0.67 ± 0.03, 0.70 ± 0.05, and 0.58 ± 0.10, respectively. Neither the IC₅₀s nor the maximal fractional inhibitions were significantly different among these peptides (P < 0.05). The Y1-NpR antagonist BIBP3226 (1 μM) also did not inhibit the action of PYY (data not shown).

Fig. 3.

NPY receptor mRNA detected by RT-PCR. RNA isolated from distal colonic mucosa was used to amplify Y1-NpR and Y2-NpR products by RT-PCR. Products were obtained at 546 base pairs (bp) for Y1-NpR and 442 base pairs for Y2-NpR as predicted from the position of the forward and reverse primers (indicated by asterisks), and amplification of GAPDH served as a positive control for RNA isolation. The negative control obtained by not including reverse transcriptase indicated the lack of contamination by genomic DNA. The faint band smaller than 100 bp likely was due to unused primers from the PCR.

Fig. 4.

NPY receptor proteins detected by immunoblot. Protein isolated from distal colonic epithelial cell membranes was immunoblotted with antibodies against the Y1-NpR and Y2-NpR proteins. Immunoreactive bands occurred at 43 kDa and 92 kDa for Y1-NpR and 55 kDa and 60 kDa for Y2-NpR (arrowheads), consistent with monomeric and possible oligomeric forms. Use of the secondary antibody alone eliminated all bands (data not shown), indicating that the primary antibodies were necessary for the observed results.

Fig. 5.

Y1-NpR proteins localized in the colonic epithelium. Y1-NpR was detected by immunofluorescence (anti-Y1-NpR) in distal colonic mucosa. A and B: surface epithelial cells had prominent immunoreactivity (Y1-NpR^ir) labeling of lateral membranes (arrowheads) without marked labeling of basal membranes (b). Labeling for apical membranes (a) was not apparent. Apically located goblet granule masses were apparent as dark voids (G). C and D: crypts showed distinct lateral membrane Y1-NpR^ir labeling (arrowheads), whereas the basal membrane lacked distinct labeling. Use of the secondary antibody alone eliminated all membrane labeling (data not shown), indicating that the primary antibodies were necessary for the observed results. Scale bars = 10 μm.

Fig. 6.

Y2-NpR proteins localized in the colonic epithelium. Y2-NpR was detected by immunofluorescence (anti-Y2-NpR) in distal colonic mucosa. A and B: surface epithelial cells had prominent Y2-NpR^ir labeling of lateral membranes (arrowheads) without marked labeling of basal membranes (b). Labeling for apical membranes (a) was not apparent. Apically located goblet granule masses were apparent as dark voids (G). C and D: crypts showed distinct lateral membrane labeling for Y2-NpR^ir (arrowheads), and luminal margins did not show labeling (lumen, L). Use of the secondary antibody alone eliminated all membrane labeling (data not shown), indicating that the primary antibodies were necessary for the observed results. Scale bars = 10 μm.

Fig. 7.

ATP and adenosine activated the modulatory mode of secretion. Isolated mucosae were stimulated by secretory agonists, from the standard basal condition as in Fig 1. A: I_sc was measured in 3 adjacent mucosae with ATP (100 μM) added to the serosal (▾) or mucosal (▵) bath, or epi (shaded circles, 1 μM) added to the serosal bath. The responses were representative of 3 similar experiments (^agonistΔI_sc: transient ATP_mucosal, −22 ± 2 μA/cm²; sustained ATP_mucosal, −13 ± 4 μA/cm²; sustained ATP_serosal, −31 ± 2 μA/cm²; sustained epi, −72 ± 9 μA/cm²; each response was significantly different from zero, ATP responses were significantly smaller than epi, and sustained ATP_serosal response was significantly more negative than sustained ATP_mucosal response, P < 0.05). B: I_sc was measured in 2 adjacent mucosae as ATP (100 μM) and epi (1 μM) were added sequentially to the serosal bath with pretreatment either by PYY (○, 0.3 μM) or BIIE0246 (BIIE) (•, 1 μM). Difference in I_sc between mucosae (▵) revealed the PYY-sensitive components. The responses were representative of 3 similar experiments (sustained ^ATPΔI_sc for serosal addition was not significantly different with BIIE0246 compared with PYY, P < 0.05). C: I_sc was measured in 3 adjacent mucosae as ATP (100 μM) was added either to the serosal (▾) or mucosal (▵) bath with pretreatment by the adenosine receptor antagonist CGS15943 (5 μM) (shaded circles, no addition control). The small positive change in I_sc during the initial addition of CGS15943 may have resulted from blunting of a response to adenosine present in the bath (^CGSΔI_sc: +6 ± 1 μA/cm², significantly different from zero, P < 0.05). The responses were representative of 3 similar experiments (^agonistΔI_sc: sustained ATP_mucosal, −1 ± 1 μA/cm²; sustained ATP_serosal, −4 ± 1 μA/cm²; ATP_mucosal response was not significantly different from zero, ATP_serosal response was significantly different from zero, but also significantly smaller than without CGS15943, P < 0.05). D: I_sc was measured in 3 adjacent mucosae with adenosine (100 μM) added to the serosal (▾) or mucosal (▵) bath, or epi (shaded circles, 1 μM) added to the serosal bath. The responses were representative of 4 similar experiments (^agonistΔI_sc: mucosal adenosine, −12 ± 2 μA/cm²; serosal adenosine, −25 ± 8 μA/cm²; epi, −79 ± 13 μA/cm²; each response was significantly different from zero, and adenosine responses were significantly smaller than epi, P < 0.05).

Fig. 8.

Adrenergic and NPY-containing nerve types localized in the mucosa. Adrenergic and peptidergic nerves were detected in distal colon by ir for dopamine-β-hydroxylase (DβH) and NPY (anti-DβH, anti-NPY). A, B, and C: prominent immunoreactive labeling for DβH (green) and NPY (red) was apparent in the mucosa and muscularis mucosa with numerous points of colocalization (C, yellow). An apparent nerve bundle (arrowhead) crossed the muscularis mucosa into the mucosa. Nonspecific labeling of immune cells apparent in the interstitium were marked with asterisks. Scale bar = 40 μm. D: submucosa situated between the muscularis mucosa (mm) and the circular muscle had prominent points of DβH^ir and NPY^ir labeling with colocalization distinct around blood vessels (bv). Other nerve bundles did not show colocalization (arrowhead). Scale bar = 40 μm. E: myenteric plexus had prominent DβH^ir and NPY^ir labeling without colocalization. Scale bar = 40 μm. F: apparent nerve bundle lay between the muscularis mucosa and the base of crypts and extended between crypts (arrowhead) with points of colocalized DβH^ir and NPY^ir labeling seen throughout the nerve bundle course. Another nerve bundle was apparent crossing the muscularis mucosa (asterisk). Scale bar = 40 μm. G: crypt from C is shown enlarged with apparent nerve fibers parallel to the crypt axis. Scale bar = 20 μm. H–K: nerve fibers were apparent often parallel to the crypt axis with either colocalization of DβH^ir and NPY^ir labeling or only labeling for one of these markers (arrowheads). Muscularis mucosa is at the bottom of each panel with the crypt axis vertical; scale bar = 20 μm. L: region near the yellow puncta indicated in K (arrowhead) was enlarged. The z-axis profiles through the puncta confirmed colocalization. A line scan of fluorescence intensity across this puncta (inset) indicated overlap of the peaks for both wavelengths, as well as a neighboring red puncta with only NPY labeling that lacked overlap. Use of each primary antibody alone, together with both secondary antibodies led to loss of labeling from the unmatched secondary antibody (data not shown), indicating that the primary antibodies were necessary for the observed labeling and that overlap did not occur between the fluorescence channels. Labeling of apparent immune cells (asterisks, C) was nonspecific because it occurred in the absence of primary antibodies.

Fig. 9.

Secretory I_sc suppressed by endogenous sources. Isolated mucosae were stimulated by secretory agonists, from the standard basal condition as in Fig 1. A: I_sc was measured in 2 adjacent mucosae after secretory activation by CCh (10 μM) followed by PGE₂ (3 μM) with either no pretreatment (○) or BIIE0246 (1 μM) preaddition to the serosal bath (shaded diamond); time was from the addition of CCh. BIIE0246 was added at asterisk. Differences of I_sc between these mucosae (▵) revealed the Y2-NpR-dependent component. The ΔI_sc and ΔG_t responses to BIIE0246 during synergistic secretion were +80 ± 28 μA/cm² and +1.63 ± 0.36 mS/cm² (significantly different from zero; P < 0.5, N = 6, n = 13). B: I_sc was measured in 3 adjacent mucosae (shaded circle, ◊) after secretory activation by epi (5 μM) followed by PGE₂ (3 μM). One mucosa (◊) was pretreated with veratridine (10 μM), and two (shaded circle, ◊) had tetrodotoxin (TTx) added at 1 μM (*); time was from the addition of veratridine. PYY (1 μM) was added subsequently to all mucosae. Difference of I_sc between mucosae (▵) revealed the neural-dependent component. The veratridine-inhibited ΔI_sc and ΔG_t in the presence of TTx were +45 ± 2 μA/cm² and +2.36 ± 0.69 mS/cm² (significantly different from zero; P < 0.5, N = 4). C: I_sc was measured in 2 adjacent mucosae (shaded circle, ◊) after secretory activation by epinephrine (5 μM) followed by PGE₂ (3 μM). One mucosa was pretreated with veratridine (10 μM) (◊); time was from the addition of veratridine. After TTx (1 μM) addition (shaded circle, ◊), BIIE0246 (1 μM) was added (*) to the veratridine-treated mucosa (◊). Difference of I_sc between mucosae (▵) revealed a Y2-NpR-dependent component. The responses were representative of 3 similar experiments (response to BIIE0246 was significantly different from zero, P < 0.05).

Fig. 10.

PYY altered the secretory sensitivity to epi and norepi. Isolated mucosae were stimulated by increasing concentrations of either epi or norepi, from the standard basal condition as in Fig 1. A: 4 mucosae from each animal (N = 5) were paired for pretreatment with either BIIE0246 (◊, ○; 1 μM) or PYY (⧫, •; 0.3 μM), and then I_sc was recorded during addition of either norepi (◊, ⧫) or epi (○, •). The fit of the data with a single binding site (epi, black lines; norepi, gray lines; PYY, solid lines; BIIE0246, dashed lines) yielded EC₅₀s of 2.9 ± 1.2 nM (BIIE/epi), 13.3 ± 1.0 nM (BIIE/norepi), 12.8 ± 1.3 nM (PYY/epi), and 39.8 ± 8.4 nM (PYY/norepi). Maximal secretory I_sc values were −119 ± 12 μA/cm² (BIIE/epi), −123 ± 13 μA/cm² (BIIE/norepi), −133 ± 9 μA/cm² (PYY/epi), and −144 ± 10 μA/cm² (PYY/norepi). The EC₅₀s for epi and norepi in the presence of PYY were significantly larger than those with BIIE0246 (P < 0.05), respectively, +9.9 ± 2.9 nM and +26.5 ± 8.7 nM. The EC₅₀s for epi compared with norepi were significantly larger with either BIIE0246 or PYY (P < 0.05), respectively, +10.4 ± 1.6 nM and +27.0 ± 9.5 nM. Maximal I_scs for epi and norepi in the presence of PYY were significantly more negative than those with BIIE0246 (P < 0.05), respectively, −14 ± 3 μA/cm² and −21 ± 6 μA/cm². Maximal I_scs for epi compared with norepi were not significantly different with either BIIE0246 or PYY (P < 0.05). B: EC₅₀s of the I_sc responses to norepi and epi in the presence of BIIE0246 and PYY were compared with those from untreated guinea pig distal colonic mucosa (shaded circle; 52), rabbit distal colonic mucosa (shaded square; 42), and the three β-adrenergic receptors (▵,▿, ◊; 41).

Fig. 11.

β1 and β2 subtype selective antagonists inhibited I_sc synergistically. Isolated mucosae were stimulated by increasing concentrations of epi as in Fig 9. A: 4 mucosae from each animal were pretreated with PYY (0.3 μM) and a β-adrenergic antagonist (1 μM) either CGP20712A (▿, N = 10), ICI-118551 (◊, N = 10), SR59320A (▵, N = 4), or no antagonist control (•, N = 10). The ^epiEC₅₀s were 13.7 ± 2.5 nM (control), 18.6 ± 5.4 nM (CGP20712A), 104 ± 12 nM (ICI-118551), and 17.7 ± 5.2 nM (SR59320A). The ^epiEC₅₀ with ICI-118551 was significantly larger than control (P < 0.05). Inhibition constants (K_d) were calculated from ^epiEC₅₀s obtained with antagonists, assuming competitive antagonism (40); K_ds were 2800 ± 690 nM (CGP20712A), 152 ± 31 nM (ICI-118551), and 439 ± 89 nM (SR59320A). B: 4 mucosae from each animal were pretreated with BIIE0246 (1 μM) and a β-adrenergic antagonist (1 μM) as in A. The ^epiEC₅₀s were 4.4 ± 0.9 nM (control, N = 10), 3.6 ± 0.9 nM (CGP20712A, N = 10), 44.7 ± 3.8 nM (ICI-118551, N = 10), and 6.9 ± 2.4 nM (SR59320A, N = 4). The ^epiEC₅₀ with ICI-118551 was significantly larger than control (P < 0.05). The corresponding K_ds were 109 ± 9 nM (ICI-118551), 1,230 ± 420 nM (SR59320A), and for CGP20712A was indeterminate but likely >3 μM. C: 4 mucosae from each animal (N = 6) were pretreated with PYY (0.3 μM) and β-adrenergic antagonists (1 μM) either CGP20712A (▿), ICI-118551 (◊), CGP20712A + ICI-118551 (○), or no addition control (•). The ^epiEC₅₀s were 18.0 ± 5.5 nM (control), 22.0 ± 7.4 nM (CGP20712A), 115 ± 15 nM (ICI-118551), and 535 ± 65 nM (CGP + ICI). The predicted ^epiEC₅₀ (black dotted line) for a single binding site including competitive antagonism from CGP20712A and ICI-118551 (40) using the K_ds determined from A was 121 ± 13 nM. The measured ^epiEC₅₀ for combined addition of CGP20712A and ICI-118551 was significantly higher (4.41 ± 0.99 fold, P < 0.05) than this predicted value for these 2 competitive antagonists acting at a single site. D: 4 mucosae from each animal (N = 6) were pretreated with BIIE0246 (1 μM) and antagonists as in C. The ^epiEC₅₀s were 3.9 ± 1.1 nM (control), 2.8 ± 0.9 nM (CGP20712A), 46.0 ± 5.6 nM (ICI-118551), and 205 ± 66 nM (CGP + ICI). This measured ^epiEC₅₀ for combined addition of CGP20712A and ICI-118551 was significantly higher (4.33 ± 1.24 fold, P < 0.05) than the predicted value for these 2 competitive antagonists acting at a single site, 47.4 ± 9.9 nM.

Fig. 12.

Neural activation of modulatory mode electrogenic ion secretion. The colonic epithelium is shown schematically with an L cell (PYY releasing) of the enteroendocrine cell population between two columnar cells. The ion transport proteins necessary for electrogenic K⁺ and Cl⁻ secretion are indicated together with the receptor types examined in this study (β1-AdrR, β2-AdrR, Y2-NpR, A2B-AdoR, P2R). The upper columnar cell is secreting K⁺ and Cl⁻, as illustrated by shading of the channels. Receptors that can activate this modulatory K⁺ and Cl⁻ secretion are shaded. The lower columnar cell is secreting only K⁺ because of the activation of Y2-NpR that suppresses Cl⁻ secretion. Apical membrane A2B or P2 receptors also activate K⁺ secretion without accompanying Cl⁻ secretion. Nerve processes with neurotransmitter-containing varicosities near the epithelial cells are shown from three populations, sympathetic inhibitory neurons (norepi, NPY, ATP), sympathetic secreto-inhibitory neurons (norepi), and cholinergic secretomotor neurons (ACh, NPY). Other ion transport proteins and receptors are present but were omitted for clarity.