Introduction

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Chemical activation of the nerve supply of a preparation of smooth muscle may be more selective than electrical stimulation for the analysis of neurotransmission mechanisms. Thus, different compounds may excite different subpopulations of neurons, as investigated mainly in afferent, but also to some extent efferent, nerves (Maggi, 1991; Toda et al., 1992; Broadley, 1996; Hong et al., 1996; Okamura et al., 1998). With reference to the gut, K⁺ may be of particular interest. Gabella (1978) noted that (isotonic) K⁺ solutions (concentrations, 36-127 mM), when administered to isolated preparations of guinea pig gastrointestinal tract, elicited triphasic responses (relaxation followed by contraction and a secondary relaxation). The first component could be abolished by the nerve-blocking agent tetrodotoxin (TTX) but was not affected by the noradrenergic nerve blocker guanethidine. These findings suggest that the TTX sensitive component be due to the activation of inhibitory, nonadrenergic motoneurons to the muscle (Gabella, 1978).

Furthermore, Toda et al. (1992) reported that selectively nonadrenergic, neurogenic relaxations of the longitudinal muscle of canine duodenum could be elicited by K⁺. Such relaxations were abolished by interference with NO synthase (NOS) but were unchanged by the inhibitor of Na⁺,K⁺- ATPase, ouabain. In a recent follow-up study on the longitudinal muscle of canine proximal colon, these authors noted that K⁺ elicited neurogenic relaxations that could be reduced (but not abolished) by either ouabain or an NOS antagonist (Okamura et al., 1998).

In the current study, which was undertaken with a preparation of the longitudinal muscle of rat distal colon, we investigated the effect of the administration of K⁺ to the tissue with the following, specific aims: Could neurogenic relaxations be elicited by K⁺? If so, what similarities or differences are there with regard to the action of K⁺ on other tissues (cf. Toda et al., 1992; Tøttrup et al., 1993; Okamura et al., 1998) when investigated with a variety of nerve-blocking or neurotransmitter-blocking agents? Moreover, which transmitter candidates could mediate this response, with a specific focus on ATP, NO, and vasoactive intestinal peptide (VIP; for a review, see Bennett, 1997)? Finally, are the second messengers cAMP and cGMP involved in such an effect to K⁺?

	Materials and Methods

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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. (See Börjesson et al., 1997, for the rationale for the use of "closed" instead of the conventional, "open" colon segments.)

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 inactive D-enantiomer N^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 (see Ward 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.

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

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

View larger version (7K): [in this window] [in a new window]   Fig. 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.

View larger version (14K): [in this window] [in a new window]   Fig. 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).

View larger version (5K): [in this window] [in a new window]   Fig. 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).

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).

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).

View larger version (32K): [in this window] [in a new window]   Fig. 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); VIP10-28 (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).

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 ( = 0.398 at a K⁺ concentration of 20.8 mM, P = .013;  = 0.607 at a K⁺ concentration of 50.8 mM, P = .0002).

View larger version (10K): [in this window] [in a new window]   Fig. 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;  = 0.398, P = .013). , K+ = 50.8 mM (n = 6;  = 0.607, P = .0002; for statistics, see Materials 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.

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 (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).

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

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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 (see Tøttrup 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 by Börjesson 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.

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.

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.