Materials and Methods

General.

The study design was approved by the Ethics Committee of the Göteborg University. Colon tissue was obtained from male Sprague-Dawley rats (B & K Universal AB, Sollentuna, Sweden; 210–400 g body weight). The animals were sacrificed by exsanguination during pentobarbital anesthesia (60 mg/kg i.p.). A segment of the distal colon (40-mm length; anal end situated ∼40 mm proximal to the anal orifice) was removed and immediately placed in chilled oxygenated (95% O₂/5% CO₂) Krebs’ solution containing 115.5 mM NaCl, 4.6 mM KCl, 1.2 mM KH₂PO₄, 21.9 mM NaHCO₃, 1.2 mM MgSO₄, 11.5 mM glucose, and 2.5 mM CaCl₂. To block noradrenergic neurotransmission, the Krebs’ solution also contained guanethidine (3.4 μM; Broadley, 1996). Each segment was gently flushed with the Krebs’ solution and then divided in two halves of 20-mm length. A silk thread was tied around either end of each preparation, thus occluding the lumen. (SeeBörjesson et al., 1997, for the rationale for the use of “closed” instead of the conventional, “open” colon segments.)

The segments (mounted so as to monitor contractile activity of the longitudinal muscle layer) were allowed to equilibrate under an initial load of 15 mN for 30 min with a washout every 15 min. Isometric contractions were recorded using a Grass FT 03 transducer on a Grass polygraph (Grass Instruments Co., Quincy, MA).

All drug concentrations reported refer to the final ones in the organ baths. The hypertonic K⁺-Krebs’ solutions (final K⁺ concentration, 10.8–50.8 mM) used for nerve activation were prepared by the addition of selected volumes (18.8–168.8 μl) of a stock solution containing 4 M KCl to the organ baths (cf. Ishii and Shimo, 1979). In experiments in which the effects of two concentrations of K⁺ (20.8 and 50.8 mM) were to be evaluated, K⁺ was administered cumulatively at 3-min intervals.

Experimental Protocol

Effect of K⁺ on Preparations at Basal Tone.

K⁺ was administered without prior precontraction of the tissues.

Analysis of K⁺-Induced Relaxation on Preparations Contracted with Carbachol.

K⁺ was added either noncumulatively or cumulatively to the preparations. The pharmacological analyses of the responses to cumulative administration of K⁺ was conducted according to the following. After obtaining a control response to carbachol and K⁺ followed by washouts, the following agents were incubated and, after equilibration, carbachol followed by K⁺ (as above) was again added: the nerve-blocking agent TTX (Broadley, 1996); the nicotinic ganglionic receptor blocker hexamethonium (Broadley, 1996); the inhibitor of Na⁺,K⁺-ATPase, ouabain (Thomas, 1972); first messenger inhibitors [ 1) apamin, a blocker of a class of low-conductance Ca²⁺-dependent K⁺channels in smooth muscle (Castle et al., 1989) that is assumed to function as a selective blocker of ATP-induced effects (Maas et al., 1980); 2) reactive blue 2, a putative P_2y purinoceptor antagonist (see Dalziel and Westfall, 1994, for references); 3)N^G-nitro-l-arginine (L-NNA), an inhibitor of NOS (Tøttrup et al., 1991) or its biologically inactived-enantiomerN^G-nitro-d-arginine (D-NNA;Tøttrup et al., 1991); 4) the combination of reactive blue 2 and L-NNA; and 5) the combination of reactive blue 2, L-NNA, and vasoactive intestinal peptide (VIP)_10–28, a VIP-receptor antagonist (Grider and Rivier, 1990)]; and second messenger inhibitors [ 1) methylene blue, a proposed inhibitor of soluble guanylyl cyclase (seeWard et al., 1992, for references); 2) 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a specific inhibitor of soluble guanylyl cyclase (Garthwaite et al., 1995); and 3)N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), an inhibitor of cAMP-dependent protein kinase (Chijiwa et al., 1990)].

Effect of VIP or Sodium Nitroprusside (SNP) and of Putative Inhibitors of These Compounds on Carbachol-Precontracted Preparations and Effect of K⁺ and Papaverine on Tone Induced by Lidocaine.

For details, see Results.

Evaluation of Results.

In preliminary experiments, it was found that there were neither qualitative nor quantitative differences between the two segments of the distal colon of each animal investigated with regard to contractile activity, being either spontaneous or resulting from the administration of drugs to the tissue. The segments of each rat were subjected to different treatments. The n value signifies the number of animals investigated. Data are presented as mean ± S.E. Relaxations are expressed as percent reduction from the tone prevailing immediately before each administration of K⁺, VIP, or SNP. Only the nadir of relaxations monitored immediately after the addition of any of these compounds was evaluated quantitatively, except for the series in which K⁺ was added noncumulatively, on carbachol-induced tone; in these experiments, the complete response to added K⁺ was evaluated.

Nonparametric statistical analyses were made. Wilcoxon’s signed rank test was used for paired data, and the Kruskal-Wallis one-way ANOVA was used for group analyses; when relevant, Spearman’s rank correlation coefficient test was used (Siegel and Castellan, 1988). A value ofP < .05 was considered significant.

Drugs.

Apamin, L-NNA, carbamylcholine chloride (carbachol), guanethidine monosulfate, hexamethonium chloride, methylene blue, ouabain, SNP, TTX, VIP, and VIP_10–28 were obtained from Sigma Chemical Co. (St. Louis, MO). D-NNA was obtained from Serva Feinbiochemica GmbH (Heidelberg, Germany). Reactive blue 2 was obtained from Research Biochemicals Inc. (Natick, MA). Pentobarbital sodium (pentobarbitalnatrium) and papaverine sulfate were purchased from Apoteksbolaget (Umeå, Sweden). ODQ was purchased from Tocris Cookson Ltd. (Bristol, UK). H-89 was obtained from Calbiochem-Novabiochem Ltd. (Nottingham, UK). ODQ was dissolved in ethanol, and H-89 was dissolved in equal amounts of ethanol and distilled water. All other drugs were dissolved in distilled water. L-NNA and D-NNA required sonification to dissolve. The administration of a similar volume of pure solvent did not affect either the spontaneous activity of the muscle or the responses to drugs.

Results

Basal Contractile Activity.

The colonic segments investigated exhibited a low level of spontaneous contractile activity (Fig.1), as previously described (Börjesson et al., 1997).

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Figure 1

The spontaneous contractile activity of the longitudinal muscle layer of an intact segment from rat distal colon.

Effect of K⁺ on Preparations at Basal Tone.

K⁺ (20.8 and 50.8 mM) was administered to segments without previous precontraction (n = 3). The first concentration induced a persistent increase in tone of 7.0 ± 1.4 mN in all segments; a relaxation of ∼30-s duration preceded this response in two of three experiments. The second concentration induced a further increase in tone by an additional 24.0 ± 7.2 mN; also, immediately before this effect, there was a short-lasting (<30-s) relaxation observed in one of the three segments investigated. Thus, the results obtained could indicate that K⁺ may elicit inhibition of the tissue also at low tone. The magnitude of this effect, however, might be underestimated, partly due to a dominant excitatory response to K⁺ at low tone.

General Characteristics of Effect of K⁺ on Precontracted Preparations

Noncumulative Administration of K⁺.

The concentration-effect relationship for K⁺ (at 10.8, 15.8, 20.8, 25.8, or 45.8 mM, administered as a single injection to the bath) was investigated on segments precontracted with carbachol (1 μM;n = 6; see below for description of the effect of carbachol). Thus, the addition to the chamber of K⁺ was made 5 min after the administration of carbachol; K⁺ was then allowed to remain in the bath for 10 min before washouts (five complete changes of the Krebs’ solution at 5-min intervals) were performed. Thereafter, carbachol, followed by K⁺ (at the next higher concentration), was administered. The addition of K⁺ elicited an immediate relaxation. The latency of this response, defined as the duration from the addition of K⁺to half the nadir of this response, was estimated to be <1 s. The relaxation was followed by a contraction, reaching a peak that was not sustained but waned within 3 min before stabilizing at a steady-state level of tone. This “new” plateau was either the same or lower than that caused by carbachol (Fig. 2). In fact, at an approximate concentration of K⁺ of ≥25 mM, such a stable level of tone appeared as a second K⁺-induced relaxation. This was even more evident when K⁺ was added cumulatively (see below). Both the immediate response and the peak contraction were found to be maximal at 20.8 mM.

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Figure 2

Concentration-effect relationship for the response to K⁺ (mM), when added noncumulatively to precontracted segments from rat distal colon (n = 6). Data are expressed as percent of carbachol-induced tone. Inset, response to K⁺ (at 10.8, 25.8, or 45.8 mM; denoted by ∗) of the preparation under investigation has been included to define the different phases of the response. ▪, immediate relaxation (R). ●, peak contraction (P). ▴, stable level of tone as established after K⁺ administration (T). See also Fig. 3A.

Cumulative Administration of K⁺.

The effect of carbachol in the current preparation has been previously investigated (Börjesson et al., 1997). Thus, a concentration of 1 μM was shown to induce a response of ∼55 mN, being ∼70% of the maximal effect of this compound. The response consisted of a peak contraction that during a 5-min observation period leveled off to a fairly stable plateau. In the following, a concentration of carbachol of 1 μM is used throughout the study. The peak contraction to this compound was 47.4 ± 9.2 mN (first challenge) versus 57.5 ± 4.1 mN (second challenge; P < .05), which during the 5-min observation period (before the addition of K⁺, see below) leveled off to a plateau, being 37.4 ± 9.5 mN (79% of peak value; first challenge) versus 40.8 ± 9.3 mN (71% of peak value; second challenge; P < .05).

The effect of cumulative administration of K⁺(20.8 and 50.8 mM) was investigated on carbachol-precontracted preparations (n = 6), with the first concentration administered 3 min on carbachol-induced tone and the second concentration administered when the response to K⁺ had stabilized. After the lower concentration, tone remained at a more or less unchanged level of tone (Fig.3A). The higher concentration of K⁺ again caused an immediate relaxation, amounting to 27.8 ± 5% (being of about the same magnitude as the response to 20.8 mM), followed by a peak contraction of 98.2 ± 3% of the tone recorded before this second administration. This peak was followed by a slow relaxation, which leveled off to a plateau of 40.2 ± 5% of the prevailing tone immediately before the second administration of K⁺ (Fig. 3A). This complex effect to K⁺, when administered either noncumulatively or cumulatively, was qualitatively identical with that reported by Gabella (1978) on carbachol-precontracted guinea pigTenia coli and with that noted by Tøttrup et al. (1993) on feline lower esophageal sphincter. At 10 min after the addition of the second concentration of K⁺, washouts were performed. Thereafter, carbachol and K⁺ were again administered to the bath, identical to that described above (second challenge).

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Figure 3

A, effect of cumulative administration of K⁺ (∗, mM, representing final concentration in the organ bath chamber) on a colonic segment precontracted with carbachol (1 μM). B, K⁺ administration repeated after 20 min incubation with TTX (1 μM).

The response to K⁺ was 88.5 ± 16.7 (20.8 mM) and 85.5 ± 11.5% (50.8 mM), respectively, of that obtained by the first challenge with K⁺ (P> .05; n = 6). Therefore, despite the small but significant increase in tone induced by carbachol when administered the second time, the relaxation to K⁺ was considered to be reproducible. In the further analyses of the effects to K⁺, this was invariably administered cumulatively at the two concentrations: 20.8 and 50.8 mM.

Effect of K⁺ on Precontracted Preparations in Presence of TTX.

TTX (1 μM; n = 13) induced an increase in the phasic activity of the colonic muscle, suggesting a prevailing, neurogenic inhibition (cf. Wood, 1994; Börjesson et al., 1997). After an equilibration period of 20 min, basal tone immediately before the second addition of carbachol was 115.5 ± 12.8% of the corresponding value before the first administration of carbachol (P > .05). Carbachol-induced tone after TTX was 129.6 ± 7.2% of the effect obtained in the absence of this compound (P < .05). The immediate relaxation in response to K⁺ was almost abolished by TTX; in fact, in the presence of TTX, the immediate response seen after 20.8 mM K⁺ was replaced by a small contraction (∼3 mN; same latency as the immediate relaxation, see above). Conversely, the slow “secondary” relaxation in response to K⁺ as noted in the absence of TTX was seemingly unchanged by this compound. Thus, after the contractile effect, there was a slow relaxation (latency, ∼1 min), with a nadir of 61.3 ± 5.2% of the tone recorded immediately before K⁺ administration (Figs. 3B and 4, A and B).

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Figure 4

The following agents, known to interfere with neurotransmission mechanisms, were studied with respect to their blocking effect on immediate relaxations induced by K⁺ (A, 20.8 mM; B, 50.8 mM), expressed as percent of control: TTX (1 μM,n = 13); hexamethonium (Hexam.; 1 mM,n = 6); VIP_10–28 (3 μM,n = 6); L-NNA (100 μM, n = 8); D-NNA (100 μM, n = 7); apamin (0.5 μM,n = 6); and reactive blue 2 (RB 2; 50 μM,n = 7). The combination of RB 2 (50 μM) and L-NNA (100 μM, n = 7). ∗, statistically significant versus control (P < .05). §Statistically significant versus reactive blue 2 (P < .01). †Statistically significant versus L-NNA (P < .05).

Therefore, it may be concluded that only the immediate relaxation in response to K⁺ could be considered neurogenic in origin. The further analysis of the K⁺-induced relaxation (in the following, denoted only as “relaxation”) of the distal colon will be restricted to this immediate (i.e., TTX-sensitive) component.

Effect of K⁺ on Precontracted Preparations in Presence of Hexamethonium.

Hexamethonium (1 mM; n = 6) induced a marked increase in the phasic activity of the colonic segments, as also reported previously (Börjesson et al., 1997). This compound did not significantly affect either basal tone, tone induced by carbachol, or relaxations induced by K⁺ (Fig.4, A and B).

Effect of Ouabain on Relaxations to K⁺ on Precontracted Preparations.

After obtaining a control response to carbachol and K⁺ followed by washouts, the segments were thereafter incubated with ouabain (0.01, 0.1, 1, 10, or 100 μM) for 20 min, followed by another challenge with carbachol and K⁺(n = 6). After washouts, this experimental cycle was then repeated with ouabain at the next higher concentration. This compound caused a progressive, concentration-dependent increase in basal tone of 255% of the initially recorded level at 100 μM; the level of tone reached with the subsequent addition of carbachol was not influenced by the presence of ouabain. This agent inhibited the relaxations induced by K⁺ (n = 6) in a concentration-dependent fashion (Fig. 5), as verified with the Spearman rank correlation coefficient test (ρ = −0.398 at a K⁺ concentration of 20.8 mM,P = .013; ρ = −0.607 at a K⁺concentration of 50.8 mM, P = .0002).

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Figure 5

Concentration-effect relationship for the inhibitory action of ouabain on the immediate relaxation induced by K⁺on precontracted colonic segments. Data are expressed as percent of control. ▪, K⁺ = 20.8 mM (n = 6; ρ = −0.398, P = .013). ○, K⁺ = 50.8 mM (n = 6; ρ = −0.607, P = .0002; for statistics, seeMaterials and Methods).

Effect of Inhibitors of First- or Second Messenger Mechanisms on Relaxations to K⁺ on Precontracted Preparations

First Messenger Inhibitors.

Apamin (0.5 μM;n = 6) elicited an increase in phasic activity (cf.Börjesson et al., 1997) but did not significantly influence basal tone or tone induced by carbachol. K⁺ induced relaxations were significantly reduced by apamin (Fig. 4, A and B), possibly indicating an involvement of ATP in the response.

We investigated the effect of the P_2y antagonist reactive blue 2 (50 μM; n = 7), to further corroborate an involvement of ATP in the K⁺-induced relaxation. This compound caused a minor increase in the phasic activity, as previously reported (Börjesson et al., 1997). Basal tone was lowered to 72 ± 5% (P < .05), but tone induced by carbachol was unchanged. Relaxations induced by K⁺ were significantly reduced by reactive blue 2 (Fig. 4, A and B).

A possible contribution of NO in the nonadrenergic relaxation elicited by K⁺ was analyzed with the use of L-NNA (100 μM; n = 8). This compound elicited a marked increase in phasic activity, whereas basal tone and carbachol-induced tone were unaffected (cf. Börjesson et al., 1997). The relaxation in response to K⁺ was significantly reduced (Fig. 4, A and B). Conversely, the biologically inactived-enantiomer D-NNA (100 μM;n = 7) affected neither phasic contractile activity, basal tone, induced tone, nor relaxations induced by K⁺ (as above; Fig. 4, A and B).

When both reactive blue 2 (50 μM) and L-NNA (100 μM) were added to the tissue (n = 7), there was a marked increase in phasic activity, whereas basal tone and tone induced by carbachol were unchanged (cf. Börjesson et al., 1997). The relaxations in response to K⁺ were significantly diminished (Fig. 4, A and B). The combination of the two blockers caused an inhibition of the relaxation in response to K⁺(20.8 mM) that was significantly greater than the blockade caused by reactive blue 2 or L-NNA when administered alone (P < .01 and P < .05, respectively).

Finally, when we combined reactive blue 2 (50 μM) and L-NNA (100 μM) with VIP_10–28 (3 μM; n = 2), there was a similar change as above with regard to basal tone and phasic activity while carbachol-induced tone was unchanged. Relaxations in response to K⁺ were abolished (data not shown).

Second Messenger Inhibitors.

Methylene blue (10 μM;n = 6) did not change the phasic activity of the tissue. Basal tone was lowered by ∼35% (P < .05), but tone induced by carbachol was unchanged. The relaxations in response to K⁺ were unaffected by methylene blue (Table1).

ODQ (10 μM; n = 6) affected neither the phasic activity of the tissue, basal tone, nor tone induced by carbachol. The relaxations in response to K⁺ were significantly diminished (Table 1).

H-89 (1 μM; n = 6) affected neither the phasic activity of the tissue, basal tone, nor tone induced by carbachol. The relaxations in response to K⁺ (at 20.8 mM) were significantly diminished, whereas the response to 50.8 mM was unchanged (Table 1).

Effect of VIP or SNP and of Putative Inhibitors of These Compounds on Carbachol-Precontracted Preparations.

A single concentration of VIP (0.1 μM; n = 17) or the NO donor SNP (100 μM; n = 16; cf. Feelish and Noack, 1987) was administered. In a few experiments, after washouts, the tissues were left undisturbed for 30 min, whereupon carbachol, followed by VIP or SNP, was again added to the chambers to investigate the reproducibility of these latter compounds. VIP induced a slow relaxation to 80.9 ± 2.3% of tone recorded before the administration of this compound. This effect was seemingly reproducible (n = 3). The response to SNP mimicked the effect to VIP and relaxed the tissue to ∼65% of tone recorded immediately before SNP administration; this effect was reproducible (n = 6).

In an additional series of experiments, after washouts, the preparations that had been challenged with VIP were incubated for 30 min with one of the following compounds: VIP_10–28 (8 μM; n = 5), H-89 (1 μM; n = 7), or ODQ (10 μM; n = 6). These tissues were then rechallenged with carbachol followed by VIP, as above. (Because of the cost of the compound, any further analyses using VIP_10–28 were not undertaken in the current study.) Conversely, the preparations that had been challenged with SNP were incubated for 30 min with either of the following compounds: methylene blue (10 μM; n = 8), ODQ, (10 μM; n = 6), or H-89 (10 μM;n = 4). The preparations were then rechallenged with carbachol followed by SNP, as above.

VIP_10–28 affected neither basal tone, phasic activity, nor tone induced by carbachol. The effect to VIP was reduced to 23.9 ± 11.5% of the control response. H-89 or ODQ significantly reduced the relaxation in response to VIP (Table 1). The relaxation in response to SNP was increased after methylene blue pretreatment, whereas ODQ abolished relaxations in response to SNP. Conversely, H-89 (at a concentration 10 times greater than that used elsewhere throughout the study) did not affect relaxations induced by SNP (Table 1); therefore, the results obtained strongly indicate that methylene blue is an unreliable tool to investigate the cGMP pathway.

In all experiments evaluating the effect of inhibitors of first or second messengers (above), papaverine (cf. Huddart and Saad, 1980; 45 μM) was administered at the end of the experiment (the gut segment was still precontracted). This agent was able to relax the tissues consistently to a level even below the initially recorded basal tone.

Effect of K⁺ and Papaverine on Tone Induced by Lidocaine.

The local anesthetic lidocaine serves not only as a nerve-blocking agent but also as a spasmogen (cf. Börjesson et al., 1997). This agent was administered on basal tone, resulting in a stable increase in tone, that was 39.2 ± 7.4 mN after 15 min of equilibration. Then, K⁺ (20.8 and 50.8 mM, as above;n = 6) was administered. In these experiments, K⁺ caused no relaxation, but qualitatively and quantitatively contractions were seemingly not different from those observed on carbachol-induced tone. The addition of papaverine (n = 6) relaxed the segments to or slightly below basal tone.

Discussion

Neurogenic Relaxation in Response to K⁺.

In the current study, we extended previous observations concerning the inhibitory effect of K⁺ on various parts of gut muscle (seeTøttrup et al., 1993, for references). Thus, in the longitudinal muscle layer of rat distal colon, the addition of K⁺elicited nonadrenergic relaxations of the tissue, the initial component of which was almost abolished by TTX or entirely by lidocaine (see below). By using two concentrations of K⁺ (20.8 and 50.8 mM), we pursued the pharmacological analysis of such a neurogenic response.

Ouabain.

Relaxation of smooth muscle may be induced by an augmentation of the extracellular K⁺ as a direct effect (Thomas, 1972; Anderson, 1976); the neurogenic component of the response to K⁺ was maximally antagonized by ouabain (when used instead of TTX) by ∼40% of control. The net effects of ouabain on the muscle are, however, complex because this compound may via several mechanisms interfere with functional properties of the motoneurons and/or muscle excitability and thereby affect the resulting effect to K⁺ administration (Thomas, 1972; Sandoval, 1980;Adam-Vizi, 1992; Broadley, 1996).

Hexamethonium.

The relaxation in response to K⁺was unchanged by the nicotinic receptor antagonist hexamethonium, suggesting it is due to the activation of postganglionic motoneurons.

Purinergic Neurotransmission.

The evidence for the involvement of ATP in NANC inhibitory neurotransmission in the gastrointestinal tract and, in particular, rat colon have been previously summarized byBörjesson et al. (1997). When using either of the two compounds apamin or reactive blue 2, we found that the relaxations induced by the two different concentrations of K⁺ were antagonized by ∼75 and ∼45%, respectively. Therefore, we propose a considerable purinergic component of this response.

Nitrergic Neurotransmission.

The relaxation in response to K⁺ was inhibited by ∼60% by L-NNA. NO is considered to mediate smooth muscle relaxation by increasing cGMP, which in turn causes the activation of cGMP-dependent protein kinase (Hobbs and Ignarro, 1996). In the current study, although the cAMP blocker H-89 did not affect the response to the NO donor SNP, this effect was abolished by the cGMP inhibitor ODQ. This finding strongly indicates that SNP relaxes gut muscle singularly via cGMP activation (cf. Murthy and Makhlouf, 1995). Moreover, ODQ diminished the inhibitory effect of K⁺ by ∼60%, in concert with the view that the relaxation in response to K⁺ is greatly dependent on NO.

Our findings are, however, in conflict with those of Suthamnatpong et al. (1993) in an investigation of the longitudinal muscle layer of rat proximal, middle, and distal colon via electrical field stimulation at spontaneous tone (i.e., without precontraction). According to these authors, NO is responsible for the evoked relaxation in only the proximal colon, whereas another transmitter (presumably VIP) is responsible in the distal part. The discrepancy between our results and those of Suthamnatpong et al. (1993) may, at least in part, be explained by the difference in mode of activation of the enteric nerves and by the fact that we used a precontracted preparation (cf.Börjesson et al., 1997).

The combined treatment of reactive blue 2 and L-NNA caused a significantly greater blockade of the K⁺ induced relaxations (at 20.8 mM) than either of the two compounds alone, as noted by others as well (Maggi and Giuliani, 1993; Börjesson et al., 1997). These findings could suggest that in the rat distal colon, ATP and NO may act in parallel and independent of each other. The coexistence of ATP and NO in the rat intestine has previously been proposed in functional studies of the pyloric sphincter, small intestine, and distal colon (see Börjesson et al., 1997, for references) and may also be supported by some histochemical investigations (Belai and Burnstock, 1994).

VIP-ergic Neurotransmission.

There are previous immunohistochemical (Browning and Lees, 1994) and pharmacological (Suthamnatpong et al., 1993) data that may support a role for VIP in the K⁺-induced relaxation. The intracellular mechanisms via which VIP induces gut muscle relaxation is complex; both the cAMP (Bitar and Makhlouf, 1982; Jin et al., 1993) and cGMP (Jin et al., 1993; Murthy and Makhlouf, 1995) pathways may be involved. The view that neurally released VIP in turn causes the release of NO (see Jin et al., 1993, for older references; Dick and Lefebvre, 1998) is, however, controversial (Sanders et al., 1992). In the current study, VIP was found to relax the precontracted tissue, a response antagonized by the VIP receptor antagonist VIP_10–28 by 75%. Such relaxations could be reduced by H-89 (by 50%) or ODQ (by 60%). Furthermore, only the relaxation induced by 20.8 mM K⁺ (but not 50.8 mM) was significantly reduced by H-89 (by 40%), an observation that could suggest VIP has only a minor role in this effect.

After pretreatment with the combination of reactive blue 2 and L-NNA, a residual relaxation in response to K⁺ was still evident at 50.8 mM (suggesting the involvement of a third neurotransmitter), but at 20.8 mM, the combined treatment with reactive blue 2 and L-NNA was as effective as TTX. Thus, the third component was evident only at concentrations of K⁺ of >20.8 mM. The combination of reactive blue 2, L-NNA, and VIP_10–28 abolished the K⁺-induced relaxation (50.8 mM). Therefore, we propose that ATP, NO, and VIP all serve as inhibitory neurotransmitters in the rat distal colon; that they may act in parallel; and that each mediator seemingly contributes with an additive effect to the total response.

Comparisons with Previous Findings.

The relaxation in response to 20.8 and 50.8 mM K⁺ on the longitudinal muscle of rat distal colon, as obtained in the current study, differs from that elicited by 10 mM K⁺ on longitudinal muscle of canine duodenum (Toda et al., 1992) in the following respects.

In the rat, ouabain was found to partially inhibit the relaxation in response to K⁺ and apamin inhibited it by a substantial degree, whereas either compound was inefficient in the dog. Moreover, L-NNA was far more efficient in the dog (and in the feline lower esophageal sphincter, in response to isotonic 124 mM K⁺ as found by Tøttrup et al., 1993) than in the rat, strongly suggesting that different mediators participate in the effect in the different locations as well as species. This view may be strengthened by the results of Okamura et al. (1998) on the longitudinal muscle of canine proximal colon (seeIntroduction), which are similar to ours with regard to the partial sensitivity to ouabain or NOS antagonism. Interestingly, these authors could not confirm with the use of electrical field stimulation of the tissue the presence of either purinergic or VIP-ergic inhibitory nerves to the muscle, despite the good evidence for the existence of such nerves along the gastrointestinal tract, albeit in an heterogeneous fashion (see Bennett, 1997; Börjesson et al., 1997, for references). Indeed, it should be pointed out that when investigating the longitudinal muscle of rat proximal colon (without prior precontraction), Suthamnatpong et al. (1993) found that antagonism to NO, but not VIP, markedly reduced the relaxation in response to a wide range of stimulation frequencies (but a possible purinergic component was not investigated in that study). When dismissing the significance of purinergic nerves in proximal colon,Okamura et al. (1998) use only one stimulation frequency (5 Hz), which may not be optimal for purinergic responses (cf. Lundberg, 1996). Using aminophylline, these authors block the relaxation in response to exogenously administered ATP but not to nerve stimulation. It is likely, however, that ATP is rapidly degraded to adenosine in the colon tissue (Bailey and Hourani, 1992), and interestingly, aminophylline is an antagonist at extracellular receptors for adenosine rather than P₂ purinoceptors (Burnstock, 1978). Therefore, in our view, Okamura et al. (1998) have not unequivocally ruled out a transmitter role for ATP in their preparation.

To conclude, we demonstrated in carbachol-precontracted segments from rat distal colon that K⁺ induced neurogenic, nonadrenergic relaxation. This was unchanged with hexamethonium, suggesting it is a consequence of the direct activation of inhibitory, postganglionic motoneurons. The relaxation was markedly inhibited by either of two compounds known to antagonize purinergic neurotransmission and also by an NOS blocker but only to a minor extent by a VIP antagonist. When all three hypothesized mediators were interfered with, the response to K⁺ was abolished. Our results indicate that ATP, NO, and VIP may be inhibitory neurotransmitters in the rat distal colon.

The analysis of the intracellular mediators involved in the neurogenic relaxation in response to K⁺ indicates that this response involves both cGMP and cAMP.