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. 1999 Mar 1;515(Pt 2):489–499. doi: 10.1111/j.1469-7793.1999.489ac.x

Calcium responses induced by acetylcholine in submucosal arterioles of the guinea-pig small intestine

Hiroyasu Fukuta 1, Hikaru Hashitani 1, Yoshimichi Yamamoto 1, Hikaru Suzuki 1
PMCID: PMC2269150  PMID: 10050015

Abstract

  1. Calcium responses induced by brief stimulation with acetylcholine (ACh) were assessed from the fluorescence changes in fura-2 loaded submucosal arterioles of the guinea-pig small intestine.

  2. Initially, 1–1.5 h after loading with fura-2 (fresh tissues), ACh increased [Ca2+]i in a concentration-dependent manner. This response diminished with time, and finally disappeared in 2–3 h (old tissues).

  3. Ba2+ elevated [Ca2+]i to a similar extent in both fresh and old tissues. ACh further increased the Ba2+-elevated [Ca2+]i in fresh tissues, but reduced it in old tissues. Responses were not affected by either indomethacin or nitroarginine.

  4. In fresh mesenteric arteries, mechanical removal of endothelial cells abolished the ACh-induced increase in [Ca2+]i, with no alteration of [Ca2+]i at rest and during elevation with Ba2+.

  5. In the presence of indomethacin and nitroarginine, high-K+ solution elevated [Ca2+]i in both fresh and old tissues. Subsequent addition of ACh further increased [Ca2+]i in fresh tissues without changing it in old tissues.

  6. Proadifen, an inhibitor of the enzyme cytochrome P450 mono-oxygenase, inhibited the ACh-induced changes in [Ca2+]i in both fresh and Ba2+-stimulated old tissues. It also inhibited the ACh-induced hyperpolarization.

  7. In fresh tissues, the ACh-induced Ca2+ response was not changed by apamin, charybdotoxin (CTX), 4-aminopyridine (4-AP) or glibenclamide. In old tissues in which [Ca2+]i had previously been elevated with Ba2+, the ACh-induced Ca2+ response was inhibited by CTX but not by apamin, 4-AP or glibenclamide.

  8. It is concluded that in submucosal arterioles, ACh elevates endothelial [Ca2+]i and reduces muscular [Ca2+]i, probably through the hyperpolarization of endothelial or smooth muscle membrane by activating CTX-sensitive K+ channels.

Many types of agonist produce vasodilatation, indirectly, through the release of endothelial products such as the endothelium-derived relaxing factor (EDRF), prostanoids and endothelium-derived hyperpolarizing factor (EDHF) (Furchgott, 1984; Vanhoutte et al. 1986; Moncada et al. 1991). EDRF has been identified as nitric oxide (NO) or related nitro-containing substances metabolized from L-arginine (Moncada et al. 1991), and this factor stimulates guanylate cyclase to increase cyclic GMP in smooth muscle cells. Intracellular cyclic GMP dilates blood vessels either by acceleration of the efflux of Ca2+ or the inhibition of Ca2+ release from intracellular stores, or by phosphorylation of contractile proteins (Ignarro & Kadowitz, 1985; Lincoln & Cornwell, 1993). The prostanoid released from vascular endothelial cells is mainly prostacyclin, which increases cyclic AMP in smooth muscle through the activation of adenylate cyclase (Gryglewski et al. 1991). Similar mechanisms to those of cyclic GMP may be involved in the vasodilatation by intracellular cyclic AMP (Gryglewski et al. 1991).

The endothelium-dependent hyperpolarization produced by acetylcholine (ACh) is insensitive to inhibitors of the actions of EDRF (Chen et al. 1988; Suzuki & Chen, 1990) or NO synthase inhibitors (Suzuki et al. 1992), and is suggested to be mediated by EDHF. EDHF is reportedly epoxyeicosatrienoic acids (EETs), which are metabolized from arachidonic acid with the activation of cytochrome P450 mono-oxygenase. This factor hyperpolarizes the membrane by activating Ca2+-sensitive K+ channels (Hecker et al. 1994; Campbell et al. 1996). Hyperpolarization reduces [Ca2+]i by either inhibiting the open probability of voltage-sensitive Ca2+ channels (Nelson et al. 1990) or inhibiting the production of second messenger inositol trisphosphate (InsP3) in the case of the agonist-induced contraction (Itoh et al. 1992).

The contribution of endothelial vasodilators EDRF and EDHF varies between vascular beds. EDRF is a predominant factor in large vessels; conversely, EDHF plays a major role in peripheral circulation (Garland et al. 1995; Shimokawa et al. 1996). The systemic blood pressure is mostly determined by peripheral vascular resistance. It is, therefore, important to investigate the mechanisms of vasodilatation in arterioles. However, the cellular mechanisms of vasodilatation in arterioles, in particular the role of endothelium, are not yet as well understood as those in large vessels. We aimed to investigate the calcium responses produced by ACh in submucosal arterioles of the guinea-pig to determine whether the ACh-induced vasodilatation in arterioles is generated by similar mechanisms to those seen in large arteries.

METHODS

Male albino guinea-pigs, weighing 200-250 g, were exsanguinated after CO2 anaesthesia. Preparations of the submucosal arterioles (outer diameter, 50-80 μm) were made by the methods reported by Hirst (1977). Briefly, a segment (2-3 cm long) of the ileum was dissected, slit opened along the mesenteric border, and pinned out in a dissecting chamber with the mucosal layer uppermost. The mucosal layer was removed and the sheet of submucosal connective tissue containing arterioles was separated from the underlying smooth muscle layer using fine forceps. In some experiments, segments (about 1 mm long) of small mesenteric arteries (diameter, 150-200 μm) were dissected, and vessels with and without endothelial cells were prepared by the methods reported previously (Yamamoto et al. 1998). Briefly, the segment of the artery was reverted inside out using a fine wire (diameter, 100 μm), and endothelial cells were mechanically removed by rubbing the surface with filter paper.

The arterioles were loaded with the fluorescent dye fura-2 by incubating with medium containing 5 × 10−6 M fura-2 AM (Dojindo, Kumamoto, Japan) and 0.02% pluronic F-127 (Funakoshi, Tokyo) for 1 h at room temperature (22°C). Preparations were then washed with dye-free medium for 30 min. The dye-loaded tissue was pinned out in a recording chamber which was made from a Lucite plate with a capacity of about 0.5 ml, and the bottom of which was made of transparent glass plate (0.1 mm thick) and Sylgard 184 (silicone elastomer, Dow Corning). The recording chamber was mounted on the stage of an inverted microscope (TMD-2S, Nikon Instec, Tokyo), and the tissue was superfused with warmed (35°C) Krebs solution at a constant flow (about 3 ml min−1) using a peristaltic pump (MP-A, Tokyo Rikakikai, Tokyo). A segment of the arterioles was visualized through a square window (about 50 μm length), and was stimulated alternately with two wavelengths of ultraviolet light (340 and 380 nm). The ratio of the emission fluorescence (F340/F380) of all the view field was measured through a barrier filter of 510 nm every 200 ms, using a micro-photoluminescence measurement system (EXS-1000, Nikon Instec, Tokyo). The concentration of Ca2+ was calibrated from the intensity of fluorescence according to the methods of Grynkiewicz et al. (1985) with the Kd value of the fura-2-Ca2+ complex of 225 nM (at 35°C). In fresh tissues, the [Ca2+]i determined separately was 68.1 ± 5.5 nM (n = 29) at rest and 128.3 ± 10.7 nM (n = 9) in the presence of 5 × 10−4 M Ba2+.

For recording membrane potentials of smooth muscle, a sheet of submucosal arterioles was pinned out at the bottom of the recording chamber (capacity, about 1 ml) which was made from Lucite plate with a transparent window at the centre, and mounted on the stage of the inverted microscope (TMD-300, Nikon Instec, Tokyo). The arteries were superfused with warmed (35°C) Krebs solution at a constant flow rate (3 ml min−1) using a peristaltic pump (Minipulse 2, Gilson Medical Inst. Co., France). Membrane potentials were measured from arteriolar smooth muscle cells using a conventional microelectrode technique. Microelectrodes were made from cored capillary with an outer diameter of 1.2 mm (Hilgenberg, Germany) and filled with 1 M KCl (tip resistance, 100-250 MΩ). Membrane potential changes were recorded using a high input impedance amplifier (Axoclamp-2B, Axon Instruments), and displayed on a cathode-ray oscilloscope (SS-9622, Iwatsu, Tokyo). After low-pass filtering (cut-off frequency, 40 Hz), the potential changes were digitized and stored on a personal computer for later analysis.

The ionic composition of the Krebs solution was as follows (mM): 137.4 Na+, 5.9 K+, 2.5 Ca2+, 1.2 Mg2+, 15.5 HCO3−, 1.2 H2PO4−, 134 Cl−, 11.5 glucose. High-K+ solution ([K+]o= 38.8 mM) was prepared by replacing a part of NaCl with equimolar KCl. The solution was aerated with 95% O2-5% CO2, and the pH of the solution was maintained at 7.2-7.3.

Drugs used were acetylcholine chloride (ACh), 4-aminopyridine (4-AP), apamin, clotrimazole, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), glibenclamide, indomethacin, pirenzepine dihydrochloride, proadifen hydrochloride (SKF 525a) (all from Sigma), and Nω-nitro-L-arginine (nitroarginine) and charybdotoxin (CTX, Peptide Institute, Osaka, Japan). These drugs were dissolved in distilled water (ACh, apamin, 4-AP, 4-DAMP, pirenzepine, proadifen, nitroarginine, CTX), dimethyl sulphoxide (clotrimazole, glibenclamide) or 5 × 10−3 M Na2CO3 solution (indomethacin), and added to the Krebs solution to obtain the desired concentration. The final concentration of these solvents in the Krebs solution did not exceed 0.001%, and no alteration of the pH of the solution was detected by these procedures.

Values are presented as means ± standard deviation (s.d.), and n indicates the number of tissues examined. Statistical analysis was performed by Student's t test (two-tailed) or multiple analysis of variance. Statistical significance was determined when the probabilities were less than 5% (P < 0.05).

RESULTS

Ca2+ responses of submucosal arterioles loaded with fura-2

In resting conditions, the submucosal arterioles exhibited a stable intensity of fluorescence with a ratio of 0.42 to 0.57 (mean value, 0.48 ± 0.04; n = 29). Stimulation of the arterioles with 10−6 M ACh for 2-3 min elevated the fluorescence ratio to a sustained level of 0.51-0.65 (mean value, 0.57 ± 0.04; n = 29), which reverted to the resting level immediately after withdrawal of ACh. Reproducible responses could be elicited 3-5 times by stimulation of arterioles with ACh at intervals of 5-7 min (Fig. 1A). The ACh-induced change in fluorescence was not altered by 10−7 M nifedipine (0.56 ± 0.01, n = 5, P > 0.1), suggesting that the response did not involve the activation of voltage-sensitive L-type Ca2+ channels. However, the amplitude of the ACh-induced response diminished gradually and finally disappeared 2-3 h after starting the experiments, with substantial elevation of the resting fluorescence ratio (0.57 ± 0.05, n = 29, P < 0.1; Fig. 1B). Experiments were therefore carried out on tissues in two different conditions: within 1.5 h (a fresh tissue) and 2-3 h (an old tissue) after starting experiments.

Figure 1. ACh-induced fluorescence responses recorded from fresh and old tissues of submucosal arteriole.

Figure 1

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Submucosal arterioles were stimulated with 10−6 M ACh both at 1 h (fresh tissue, A and C) and 3.5 h (old tissue, B and D) after loading with fura-2. ACh increased the ratio of fluorescence in fresh tissue (A) but not old tissue (B). Ba2+ (5 × 10−4 M) increased the ratio of fluorescence in both fresh tissue (C) and old tissue (D). In the presence of Ba2+, ACh further increased the ratio of fluorescence in fresh tissue (C), while reducing that in old tissue (D). All traces were recorded from the same tissue. Vertical axes represent the ratio of fluorescence stimulated by 340 and 380 nm UV light (F340/F380), measured through a filter of 510 nm.

Ba2+ (5 × 10−4 M) elevated the fluorescence ratio in either fresh (0.78 ± 0.03, n = 6) or old tissues (0.80 ± 0.06, n = 9), to a similar extent (P > 0.1). In fresh tissues, ACh (10−6 M) further increased the Ba2+-elevated fluorescence ratio to form a sustained phase (Fig. 1C) with a mean value of 0.85 ± 0.02 (n = 6). In old tissues, ACh produced a reduction of the Ba2+-elevated fluorescence, with a mean value of 0.68 ± 0.04 (n = 9); this reduction recovered immediately after withdrawal of ACh (Fig. 1D).

Figure 2 shows the relationship between fluorescence and concentrations of ACh (10−9-10−4 M) in both fresh and Ba2+-stimulated old tissues. In fresh tissues, ACh in concentrations over 10−8 M elevated the fluorescence in a concentration-dependent manner to reach a maximum value of about 0.55 at 10−6 M. In old tissues, the Ba2+-elevated fluorescence was reduced by ACh (10−6-10−4 M) again in a concentration-dependent manner, with a maximum reduction in the fluorescence ratio of about 0.1 at 10−6 M ACh.

Figure 2. The relationship between the ACh-induced changes in fluorescence and the concentration of ACh in submucosal arterioles.

Figure 2

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Submucosal arterioles were stimulated with increasing concentrations of ACh (10−9-10−4 M), and the fluorescence responses were recorded from fresh (○) and old (•) tissues. In fresh tissues, ACh increased the ratio in a concentration-dependent manner producing the maximum increase at 10−6 M. In old tissues which were stimulated by 5 × 10−4 M Ba2+, ACh reduced the elevated ratio in a concentration-dependent manner reaching the maximum inhibition at 10−6 M. Recordings were made at 1-1.5 h (fresh tissues) and 2-3.5 h (old tissues) after loading with fura-2. Vertical axis represents the ratio of fluorescence stimulated by 340 and 380 nm UV light (F340/F380), measured through a filter of 510 nm. Horizontal axis represents the concentration of ACh. Data are means ±s.d. (n = 3-6, from 12 tissues). Significant change (P < 0.1) was detected at > 10−8 M ACh in both tissues.

Experiments were carried out to test the effects of removal of endothelial cells on the ACh- and Ba2+-induced fura-2 fluorescence using the small mesenteric arteries. Although the fluorescence responses elicited by ultraviolet light with wavelengths of 340 and 380 nm varied between arteries, either in the intact state or after removal endothelial cells, the responses evoked by ACh appeared only in the intact artery whereas the responses evoked by Ba2+ appeared in both arteries (Fig. 3A and B). The ratio of fluorescence (F340/F380) indicated that responses to ACh appeared only in the intact artery while those to Ba2+ appeared in intact arteries and those with endothelial cells removed (Fig. 3C and D). The pooled data indicated that in fresh arteries with intact endothelial cells, ACh (10−6 M) and Ba2+ (5 × 10−4 M) elevated fluorescence ratio to about 0.6 and 0.8, respectively. In fresh arteries with endothelial cells removed, ACh failed but Ba2+ elevated the fluorescence ratio to a level similar to that seen in arteries with intact endothelial cells (Fig. 3E). Thus, we confirmed that the ACh-induced elevation of fluorescence ratio observed in fresh tissue was the response of endothelial cells. These results suggest that the ACh-induced inhibition of the Ba2+-elevated fluorescence found in old tissue was likely to have been the response of smooth muscle cells.

Figure 3. The ACh-induced fluorescence response disappears after removal of endothelial cells.

Figure 3

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Small mesenteric arteries (outer diameter, 100-150 μm) intact (A) and with endothelial cells removed (B) were stimulated with 10−6 M ACh and 5 × 10−4 M Ba2+, and fluorescence responses evoked by ultraviolet light with wavelengths of 380 nm (upper trace) and 340 nm (lower trace) were measured through a filter of 510 nm. The ratio of fluorescence (F340/F380) with intact artery (C) and artery with endothelial cells removed (D) indicates that ACh elevates fluorescence in the intact artery but not in the artery with endothelium removed. E shows the summary of the fluorescence responses (peak values) measured at rest (□) and in the presence of 10−6 M ACh (Inline graphic) or 5 × 10−4 M Ba2+(▪) in intact arteries (EC(+)) and arteries with endothelial cells removed (EC(-)). Vertical axis indicates the ratio of fluorescence (F340/F380). Data are means ±s.d. (n = 4-6). * Significantly different from intact artery (P < 0.05).

Effects of muscarinic receptor antagonists on the ACh-induced fluorescence response

Experiments were carried out to test the effects of muscarinic antagonists on the ACh-induced responses, to determine the muscarinic receptor subtypes involved. The drugs tested were atropine, a non-selective antagonist, pirenzepine, a selective M1-receptor antagonist, and 4-DAMP, a selective M3-receptor antagonist (Eglen et al. 1996). The elevation of fluorescence ratio induced by ACh (10−6 M) observed in fresh tissues was abolished by 10−8 M 4-DAMP, but not by 10−8 M pirenzepine (Fig. 4A and B). In separate experiments, atropine (10−7 M) also abolished the ACh-induced fluorescence response (n = 3, data not shown). In old tissues, the ACh-induced inhibition of the Ba2+-elevated fluorescence was prevented by 4-DAMP but not by pirenzepine (Fig. 4C and D). These results suggest that in submucosal arterioles, the muscarinic receptors involved in the ACh-induced responses in endothelial cells are mainly of the M3-subtype.

Figure 4. The effects of muscarinic receptor antagonists on ACh-induced changes in fluorescence.

Figure 4

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Submucosal arterioles were stimulated with 10−6 M ACh before and during application of 10−8 M 4-DAMP (A and C) and 10−8 M pirenzepine (B and D). In fresh tissue, ACh-induced increases of the ratio were prevented by 4-DAMP (A) but not by pirenzepine (B). In old tissue which was stimulated by 5 × 10−4 M Ba2+, ACh-induced reductions of the elevated ratio were also prevented by 4-DAMP (C) but not by pirenzepine (D). Responses were recorded at 1 h (A), 1.2 h (B), 2.6 h (C) and 3 h (D) after loading the tissues with fura-2. A/C and B/D were recorded from different single tissues. Vertical axes represent the ratio of fluorescence stimulated by 340 and 380 nm UV light (F340/F380), measured through a filter of 510 nm.

Effects of inhibitors of biosynthesis of nitric oxide and arachidonic acid metabolites on the ACh-induced changes in fluorescence

Experiments were carried out to test the effects of (1) inhibition of nitric oxide (NO) synthase by 10−5 M nitroarginine, (2) inhibition of cyclo-oxygenase by 5 × 10−6 M indomethacin, and (3) inhibition of cytochrome P450 mono-oxygenase by proadifen or clotrimazole on the ACh-induced changes in fluorescence in submucosal arterioles. In fresh tissues, the resting fluorescence ratio (0.49 ± 0.05, n = 6) was not changed by the combined application of nitroarginine and indomethacin (0.50 ± 0.02, n = 6, P > 0.1). The ACh-induced change in fluorescence remained unaltered by nitroarginine and indomethacin (control, 0.58 ± 0.04, n = 6; in the presence of inhibitors, 0.56 ± 0.02, n = 6, P > 0.1), indicating that endogenous NO and prostanoids were not involved in these responses. In old tissues, stimulation of the arteriole with ACh for 5 min produced a reduction of fluorescence ratio with two phases, an initial sustained and following declining phase; the former lasted for about 2 min, while the latter decayed slowly and finally disappeared within 4-5 min (Fig. 5A). Nitroarginine did not change the ACh-induced response (Fig. 5B). Addition of indomethacin in the presence of nitroarginine abolished the second phase, with no significant alteration of the amplitude of the initial phase (Fig. 5C). In separate experiments, application of indomethacin in the absence of nitroarginine elicited alteration of the ACh-induced response similar to that seen in the presence of nitroarginine (n = 6, data not shown). Figure 5D summarizes the effects of indomethacin and nitroarginine on the fluorescence ratio in resting conditions, in the presence of Ba2+ alone and in the presence of both Ba2+ and ACh in submucosal arterioles. Both indomethacin and nitroarginine did not change the peak amplitude of these Ca2+ responses. These results suggest that in arteriolar smooth muscle, the slow declining phase of the ACh-induced Ca2+ response is mediated by prostanoids, whereas the initial sustained inhibition of [Ca2+]i is produced by neither NO nor prostanoids.

Figure 5. The effects of nitroarginine (L-NA) and indomethacin (Ind) on the ACh-induced changes in fluorescence recorded from old tissue.

Figure 5

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In old tissue (2.5-3 h after loading with fura-2) stimulated with 5 × 10−4 M Ba2+, ACh (10−6 M) reduced the elevated fluorescence ratio in control conditions (A) and in the presence of 10−5 M nitroarginine (B). In the presence of 10−5 M nitroarginine and 5 × 10−6 M indomethacin (C), ACh produced a transient reduction of fluorescence ratio. All traces were recorded from the same tissue. Vertical axes represent the ratio of fluorescence stimulated by 340 and 380 nm UV light (F340/F380), measured through a filter of 510 nm. D, summary of the effects of nitroarginine and indomethacin on fluorescence intensities observed in resting conditions (□), in the presence of Ba2+ (▪) and in the presence of ACh with Ba2+(Inline graphic). The ACh-induced responses were measured at the peak. Data are means ±s.d. (n = 5-12).

In smooth muscle of submucosal arterioles, ACh causes a hyperpolarization of the membrane which can be detected after inhibiting the inward rectifier K+ channel with Ba2+ (Hashitani & Suzuki, 1997). It has been reported that EDHF-induced hyperpolarization and Ca2+ responses were prevented by high-potassium solution (Fukuta et al. 1996, 1997). To investigate the contribution of the hyperpolarization to the ACh-induced changes in fluorescence in the submucosal arterioles, the effects of 38.8 mM [K+]o solution (high-K+ solution) on the ACh-induced Ca2+ responses were examined. The following experiments were carried out on the preparations where the production of NO and prostanoids had been blocked with nitroarginine and indomethacin, respectively. In fresh tissue (Fig. 6A), ACh produced a change in fluorescence either in the absence (0.085 ± 0.02, n = 6) or in the presence (0.089 ± 0.01, n = 6) of high-K+ solution, to a similar extent (P > 0.1). In old tissue (Fig. 6B), high-K+ solution elevated the fluorescence ratio to a sustained level (0.83 ± 0.03, n = 5), and subsequent application of ACh failed to produce any change of the ratio. These results indicate that in the absence of the production of NO and prostanoids, ACh-induced Ca2+ elevation in endothelial cells is independent of membrane potential while the ACh-induced inhibition of Ba2+-elevated fluorescence in old tissue is inhibited by shifting the equilibrium potential of potassium ions towards zero.

Figure 6. Changes in ACh-induced fluorescence responses in high-potassium containing solution.

Figure 6

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A, in fresh tissue (1.2 h after loading), 10−6 M ACh was applied in the absence and presence of high-potassium solution ([K+]o= 38.8 mM). B, in old tissue (3.0 h after loading), ACh was applied in the presence of high-potassium solution. Indomethacin (5 × 10−6 M) and nitroarginine (10−5 M) were present throughout. A and B were recorded from different tissues. Vertical axes represent the ratio of fluorescence stimulated by 340 and 380 nm UV light (F340/F380), measured through a filter of 510 nm.

EDHF is reportedly a metabolite of arachidonic acid through epoxygenase pathways which involve cytochrome P450 mono-oxygenase. This enzyme can be inhibited by proadifen or clotrimazole (Hecker et al. 1994; Campbell et al. 1996). In fresh tissue, proadifen (5 × 10−5 M) inhibited the ACh-induced increase in fluorescence ratio (Fig. 7A and D), with no significant alteration of the resting fluorescence ratio (Fig. 7D). However, clotrimazol had no effect on ACh-induced responses (Fig. 7D). In old tissue, proadifen reduced the Ba2+-elevated fluorescence ratio to about 60% of control and converted the sustained reduction of the fluorescence ratio by ACh to a transient form with reduced amplitude (Fig. 7B, C and E). Clotrimazole reduced the amplitude of Ba2+-induced elevation of fluorescence ratio and also reduced the amplitude of the ACh-induced reduction of fluorescence ratio (Fig. 7E). However, there was no significant alteration in the peak value of the ACh-induced reduction of the fluorescence ratio (P > 0.1).

Figure 7. Modulation by proadifen and clotrimazole of the ACh-induced fluorescence responses in submucosal arteriole.

Figure 7

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In fresh tissue (1.2 h after loading), ACh (10−6 M)-induced increases of the ratio were inhibited by proadifen (5 × 10−5 M, A). In old tissue (2.5 h after loading), 5 × 10−4 M Ba2+ increased the ratio to a sustained level and subsequent ACh reduced the elevated ratio (B). In old tissue (3 h after loading) treated with 5 × 10−5 M proadifen, Ba2+ still increased the ratio, but the ACh (10−6 M)-induced reduction of the elevated ratio was inhibited (C). A-C were recorded from the same tissue. Vertical axes represent the ratio of fluorescence stimulated by 340 and 380 nm UV light (F340/F380), measured through a filter of 510 nm. D summarizes the fluorescence response produced by 10−6 M ACh (Inline graphic) in fresh tissue (1-1.5 h after loading), in the absence and presence of 5 × 10−5 M proadifen or 10−5 M clotrimazole; □, the resting fluorescence intensity. Data are means ±s.d. (n = 5-15). E summarizes the fluorescence response produced by 10−6 M ACh (Inline graphic) in old tissue (2.5-3.5 h after loading), in the absence (Cont.) and presence of 5 × 10−5 M proadifen or 10−5 M clotrimazole; ▪, fluorescence intensity produced by 5 × 10−4 M Ba2+. Data are means ±s.d. (n = 3-10). *Significantly different from control (P < 0.1).

The effects of proadifen and clotrimazole on hyperpolarization produced by ACh were also observed in smooth muscle of the submucosal arterioles. Experiments were carried out in the presence of 5 × 10−4 M Ba2+ to block the inward rectifier K+ channels. Ba2+ depolarized the membrane from -70.5 ± 1.2 mV (n = 10) to -42.5 ± 2.5 mV (n = 10), and subsequent application of ACh (10−6 M) hyperpolarized the membrane by about 23 mV (peak potential, -65.5 ± 3.5 mV; n = 10; Fig. 8A). The ACh-induced hyperpolarization was changed to a transient form with reduced amplitude in the presence of 10−4 M proadifen (peak potential, -50.6 ± 0.5 mV; n = 3, P < 0.05; Fig. 8B), with no significant alteration of the membrane potential (-40.1 ± 1.2 mV; n = 3, P > 0.1). Clotrimazole (3 × 10−5 M) slightly inhibited the sustained phase of the ACh-induced hyperpolarization, with no significant inhibition of the peak amplitude (-65.8 ± 1.8 mV; n = 3, P > 0.1; Fig. 8C) or the membrane potential (-40.7 ± 1.4 mV; n = 3, P > 0.1).

Figure 8. The inhibition of acetylcholine (ACh)-induced hyperpolarization by proadifen and clotrimazole in submucosal arteriole.

Figure 8

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In the presence of 5 × 10−4 M Ba2+, hyperpolarizations produced by 10−6 M ACh were recorded from submucosal arteriole in the absence (A, Control) and presence of 5 × 10−5 M proadifen (B) or 10−5 M clotrimazole (C). All responses were recorded from the same preparation. The scale on the right represents the membrane potential.

Effects of K+ channel inhibitors on the ACh-induced fluorescence response

The effects of apamin, an inhibitor of the small conductance Ca2+-sensitive K+ channels, charybdotoxin (CTX), an inhibitor of the large conductance Ca2+-sensitive K+ channels, glibenclamide, an inhibitor of the ATP-sensitive K+ channels, and 4-aminopyridine (4-AP), an inhibitor of the voltage-dependent K+ channels (Nelson & Quayle, 1995), on ACh-induced changes in fluorescence were examined in both fresh and Ba2+-stimulated old tissues. In fresh tissues, the fluorescence ratio in the resting condition was elevated by 10−3 M 4-AP but not by 10−7 M apamin, 5 × 10−8 M CTX, or 10−5 M glibenclamide. ACh elevated the fluorescence ratio to similar levels in the presence of any of these K+ channel inhibitors. The summarized results obtained from different tissues (Fig. 9A) showed that the ACh-induced Ca2+ responses in the fresh tissues were not changed by any of the K+ channel inhibitors used in the present experiments. In old tissue, the elevation of fluorescence ratio by Ba2+ was reduced by glibenclamide without being affected by 4-AP, apamin or CTX (Fig. 9B). The ACh-induced reduction of the Ba2+-elevated fluorescence ratio was greatly inhibited by CTX but not by apamin or 4-AP. When the ratio of fluorescence was measured at the peak and 1 min after application of ACh, CTX was found to abolish the latter component of the ACh-induced response (Fig. 9B). Although glibenclamide reduced the ACh-induced response, this was associated with a reduction of the Ba2+-elevated fluorescence ratio (Fig. 9B), and as a consequence the relative value of the ACh-induced inhibition remained unaltered by glibenclamide (control, 49.5 ± 6.8%, n = 29; in glibenclamide, 43.3 ± 4.7%, n = 5, P > 0.1).

Figure 9. Summary of the effects of K+ channel inhibitors on the ACh-induced changes in fluorescence in submucosal arteriole.

Figure 9

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Fluorescence intensities which were determined as the ratio of the intensity excited by 340 and 380 nm UV light were recorded from fresh (A) and old tissues (B). In fresh tissues, intensities of fluorescence both in resting conditions (□) and those evoked by 10−6 M ACh (Inline graphic) were not inhibited by 5 × 10−8 M charybdotoxin (CTX), 10−7 M apamin, 10−3 M 4-aminopyridine (4-AP), or 10−5 M glibenclamide (Glb). Data are means ±s.d. (n = 5-12). In old tissues, intensities of fluorescence produced by both Ba2+ (▪) and Ba2+ plus subsequent ACh (Inline graphic) were not inhibited by either apamin or 4-AP. Glibenclamide (Glb) inhibited the Ba2+-induced response. CTX did not inhibit the peak amplitude of the ACh-induced response but abolished the ACh-induced responses at 1 min (CTX, 1 min). Data are means ±s.d. (n = 5-15). * Significantly different from control (P < 0.1).

DISCUSSION

In most larger arteries, the hyperpolarization produced by ACh is an endothelium-dependent phenomenon, which possibly involves the humoral factor EDHF (Furchgott & Vanhoutte, 1989; Suzuki & Chen, 1990; Garland et al. 1995). Although this is assumed to also be the case in arterioles, it has not been experimentally confirmed, since the removal of endothelial cells causes damage to the arteriolar smooth muscle (Neild et al. 1990; Kotecha & Neild, 1995; Hashitani & Suzuki, 1997). The present experiments used fura-2 fluorescence to examine changes in [Ca2+]i induced by ACh in submucosal arterioles. Responses to ACh changed with time; ACh elevated fluorescence ratio in fresh tissues and reduced the Ba2+-elevated fluorescence ratio in old tissues. This difference may reflect fluorescence changes being recorded from two different cell types, since Kuroiwa et al. (1995) showed that in porcine coronary artery the fluorescence dye fura-2 leaks out from endothelial cells rapidly while the dye remains much longer in smooth muscle cells. This possibility was tested using arteries which were larger in diameter but still had properties similar to those of submucosal arterioles (Yamamoto et al. 1998). The results indicated that the ACh-induced increase in fluorescence is the response of endothelial cells, while the ACh-induced inhibition of the Ba2+-elevated fluorescence response found in old tissue may be the response of smooth muscle cells produced indirectly via the ACh-induced endothelial products. Thus, the present experiments might show Ca2+ responses elicited by ACh separately in arteriolar endothelial and smooth muscle cells.

Endothelial cells lack voltage-sensitive Ca2+ channels and Ca2+ influx may occur through non-selective cation channels (Takeda et al. 1990; Yamamoto et al. 1992; Adams, 1994); thus depolarization of the membrane may reduce the influx of Ca2+ due to reduction in driving force for the influx of Ca2+ (Laskey et al. 1989; Schlling, 1989). The absence of voltage-activated L-type Ca2+ channels in the endothelial membrane was confirmed in the present experiments in which the ACh-induced elevation of fluorescence level was not changed by nifedipine. This may be extrapolated to indicate that in the submucosal arterioles, the elevation of fluorescence level by high-K+ solution reflects the Ca2+ response of smooth muscle cells. Stimulation of endothelial cells by ACh enhances the influx of Ca2+ through non-selective cation channels and also increases production of second messenger inositol trisphophate (InsP3) to enhance the release of Ca2+ from the endoplasmic reticulum (ER) (Karaki et al. 1997). The ACh-induced fluorescence change in fresh arteriole was not changed by high-K+ solution. This suggests that the increase in endothelial [Ca2+]i by ACh is largely a voltage-independent phenomenon, and the results are in good agreement with those reported in endothelial cells of the porcine pulmonary valve (Kuroiwa et al. 1995). In vascular smooth muscles, on the other hand, membrane potential modulates the production of second messenger, e.g. hyperpolarization reduces the production of InsP3 (Itoh et al. 1992). Thus, the regulation system of Ca2+ release from intracellular store sites may differ between endothelial and smooth muscle cells.

In submucosal arterioles, ACh increased the fluorescence ratio only in fresh tissues, and in old tissues the fluorescence ratio was elevated by Ba2+ but not by ACh. This means that ACh may not directly modulate [Ca2+]i in smooth muscle. The ACh-induced hyperpolarization of smooth muscle in submucosal arterioles may be produced indirectly by the release of endothelial products (Hashitani & Suzuki, 1997). The estimation of endothelial factors involved in the ACh-induced Ca2+ response using nitroarginine, an inhibitor of NO (EDRF) production, indomethacin, an inhibitor of prostanoid production, and high-K+ solution, which prevents EDHF-induced hyperpolarization of the membrane, revealed that EDHF may be the major factor to reduce [Ca2+]i in arterioles. Endothelial prostanoids were found to contribute only to the sustained phase of the response. NO is a major endothelial factor in the ACh-induced vasodilatation of large arteries (Furchgott, 1984; Vanhoutte et al. 1986; Garland et al. 1995), but this factor is found to have no significant effect on the reduction of Ba2+-elevated [Ca2+]i in submucosal arterioles. These results are consistent with the observations made in muscular arteries in which the major endothelial factors to induce vasodilatation are EDRF in proximal arteries and EDHF in peripheral arteries (Garland et al. 1995; Shimokawa et al. 1996).

Proadifen (SKF 525a) reduces the endothelium-dependent relaxation or hyperpolarization by inhibiting the biosynthesis of EDHF in porcine coronary artery (Hecker et al. 1994), bovine coronary artery (Campbell et al. 1996) and rat mesenteric artery (Chen & Cheung, 1996). The present experiments revealed that in submucosal arterioles, proadifen inhibits Ca2+ responses produced by ACh in both endothelial and smooth muscle cells. The production of EDHF requires an increase in endothelial [Ca2+]i (Chen & Suzuki, 1990; Fukao et al. 1997b), and therefore it was likely that the inhibition by proadifen of the ACh-induced response in smooth muscle was a result of the reduction of endothelial [Ca2+]i elevation. Clotrimazole is also an inhibitor of the production of EDHF through inhibiting the enzyme cytochrome P450 (Hecker et al. 1994), and in fact this chemical is a potent inhibitor of the ACh-induced hyperpolarization in the rat mesenteric artery (Chen & Cheung, 1996). However, the selectivity of clotrimazole on the endothelium-dependent hyperpolarization is questionable due to differences in the inhibitory action of clotrimazole on the hyperpolarizations produced by ACh and those produced by epoxyeicosatrienoic acids (EETs), the final products of arachidonic acid metabolism (Fukao et al. 1997a; Vanheel & Van de Voorde, 1997). Furthermore, inhibition by clotrimazole of potassium channels has been reported in smooth muscle of the rat portal vein (Edwards et al. 1996). The present experiments showed that in submucosal arterioles, clotrimazole was not a potent inhibitor of the ACh-induced hyperpolarization, nor of the ACh-induced fluorescence response. Thus, it seems likely that clotrimazole has some regional differences in the inhibitory action on the endothelium-dependent hyperpolarization. As clotrimazole reduces the Ba2+-induced elevation of fluorescence ratio, this chemical may have actions to reduce [Ca2+]i in smooth muscle of the submucosal arterioles.

Endothelial membranes possess potassium channels similar to those in smooth muscle (Adams, 1994), and ACh hyperpolarizes the endothelial membrane through activation of the Ca2+-sensitive K+ channels (Busse et al. 1988; Sakai, 1990; Marchenko & Sage, 1996). In the present experiments, many types of K+ channel inhibitor did not modulate the ACh-induced elevation of endothelial [Ca2+]i in submucosal arterioles, indicating that the elevation of [Ca2+]i by ACh is not linked to the hyperpolarization in endothelial cells. Here again, we confirmed that the elevation of endothelial [Ca2+]i by ACh is independent of membrane potential. CTX inhibits the Ca2+-sensitive K+ channel (Nelson & Quayle, 1995), and the ACh-induced hyperpolarization is inhibited by CTX in submucosal arterioles (Hashitani & Suzuki, 1997). The present experiments found that the ACh-induced inhibition of the Ba2+-elevated fluorescence ratio is sensitive to CTX. Thus, the ACh-induced Ca2+ response of smooth muscle may be causally related to the hyperpolarization of the membrane due to activation of Ca2+-sensitive K+ channels. The CTX-sensitive K+ channel is found in many tissues including endothelial and vascular smooth muscle cells (Adams, 1994; Nelson & Quayle, 1995). In submucosal arterioles, electrical signals may be conducted between these different cell types through gap junctions (Yamamoto et al. 1998). These results suggest that the ACh-induced hyperpolarization is produced by activation of Ca2+-sensitive K+ channels either in smooth muscle or in endothelial cells. [Ca2+]i is elevated by ACh in endothelial cells and is reduced in smooth muscles, suggesting that the K+ channels activated by Ca2+ locate mainly in endothelial cells. All these data could be reasonably explained if the ACh-induced hyperpolarization in the submucosal arteriole is produced by electrotonic spread of endothelial hyperpolarization, rather than the humoral factor EDHF as in other arteries (Chen et al. 1988, 1991). Alternatively, EDHF may activate the Ca2+-sensitive K+ channels irrespective of a reduction of [Ca2+]i in smooth muscle cells.

It is concluded that in submucosal arterioles, Ca2+ responses elicited by ACh in endothelial and smooth muscle cells can be assessed using fura-2 fluorescence. Through the activation of muscarinic M3-subtype receptors, ACh elevates [Ca2+]i in fresh tissues which may represent endothelial cells, and reduces Ba2+-elevated [Ca2+]i in old tissue which may represent smooth muscle cells. The ACh-induced reduction of [Ca2+]i in old tissue may be produced mainly by hyperpolarization of the membrane through the activation of CTX-sensitive K+ channels; the hyperpolarization is produced either by EDHF or by electrotonic spread of endothelial hyperpolarization to smooth muscles. Inhibition of the ACh-induced response by proadifen appears to be non-specific through a reduction of [Ca2+]i in endothelial cells, while clotrimazole has little inhibitory action on the ACh-induced hyperpolarization and Ca2+ response.

Acknowledgments

This work was supported by a Grant-in-Aid from the Ministry of Culture and Education, Japan (no. 06670122) to H.S. The authors greatly appreciated Professor H. Kanaide, Kyushu University, for useful comments and suggestions on Ca2+ responses and also Dr N. J. Bramich, University of Melbourne, for her helpful comments and correction of the text.

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