INTRODUCTION

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THE CONTRACTILE RESPONSE of the guinea pig ileum to stimulants, such as ANG II or ACh, depends on membrane depolarization, which leads to an increase of the intracellular free Ca²⁺ concentration (4). This increase is due to an enhanced Ca²⁺ influx, mainly through nonselective cation channels (NSCC) and/or voltage-dependent Ca²⁺ channels rather than the release of Ca²⁺ from intracellular pools (18, 27). ANG II, in addition to its potent vasoconstrictor action and other physiological effects, has a direct action on the guinea pig ileum (19, 21). Its contractile response presents two components: a fast and transient increase of the tonus, the phasic component, followed by a partial relaxation of the tissue to a sustained tonus, the tonic component. Prolonged treatment of this tissue with ANG II promotes a gradual decay of this component to near basal levels usually within 15 min (20). This response, named desensitization, does not occur when the guinea pig ileum is stimulated with ACh, since the tonic component remains sustained (1).

The molecular mechanisms underlying desensitization of AT₁ receptor-mediated responses in vascular smooth muscles appear to involve receptor phosphorylation, downregulation, and internalization (29). In guinea pig ileum, it has been proposed that desensitization of the contractile response to ANG II may be due to a negative-feedback mechanism mediated by protein kinase C (PKC) affecting a step in the stimulus-response chain after phospholipase C activation (24). On the other hand, over the last years, the reports converge to the concept that K⁺ channels may be an important cell strategy for controlling smooth muscle function (6). The relationship between increased K⁺ channel activity and smooth muscle relaxation was explored in many tissues and K⁺ channel types, such as ATP-dependent K⁺ channels in intestinal smooth muscles (8), small-conductance Ca²⁺-dependent K⁺ channels in vascular smooth muscle (25), and high-conductance Ca²⁺-dependent K⁺ (maxi-K⁺) channels in vascular (9) and intestinal smooth muscles (28).

Maxi-K⁺ channels are ubiquitously distributed among tissues, and it has been suggested that they contribute to the resting potential (11, 26) and the repolarization of the action potential (3). In addition to its sensitivity to intracellular Ca²⁺ concentration and membrane potential (15), it has been reported that it is modulated by a wide variety of agents, including hormones, lipids, cyclic nucleotides, neurotransmitters, and PKC (for a review, see Ref. 2), thus providing a link between the metabolic and electric state of the cells. In a previous paper (23), we demonstrated that the pharmacomechanical coupling of ANG II to the AT₁ receptor is well preserved in high-K⁺ depolarized longitudinal myocytes of the guinea pig ileum by indirect and persistent activation of maxi-K⁺ channels. This long-lasting ANG II effect contrasts with the suppression of Ca²⁺-dependent K⁺ currents by ACh in other tissues (7, 13), so that it raises a major possibility that there is a close relationship between ANG II desensitization and maxi-K⁺ channel activity.

In this study, we explored the role of maxi-K⁺ channel on the desensitization mechanism of the guinea pig ileum to ANG II. We compared the maxi-K⁺ channel activity resulting from prolonged treatment of the cells with ANG II and ACh and studied the contractile response to these agonists when the membrane potential was altered, by either blocking maxi-K⁺ channel activity or adding KCl in the physiological solution. We conclude that desensitization of the tonic component of the contractile response to ANG II is largely due to the sustained opening of maxi-K⁺ channel population, leading to repolarization of the membrane potential, thus closing the voltage-dependent Ca²⁺ channels responsible for the Ca²⁺ influx that sustains the tonic component in this smooth muscle.

	METHODS

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Animals. Either male or female albino guinea pigs, weighing between 200 and 250 g, were used in this study.

Cell preparation. Guinea pig ileum smooth muscle cells were isolated according to the method described by Romero et al. (23). Briefly, 2.5-cm segments of the longitudinal muscle layer of the guinea pig ileum were washed in 5 ml of Ca²⁺-free solution and exposed to Ca²⁺-free solution containing 0.5 mg/ml collagenase, 0.3 mg/ml pronase (from Streptomyces griseus), and 2.0 mg/ml BSA for 7 min at room temperature. The enzymatic digestion was interrupted by washing the tissue fragments in high-Ca²⁺ solution containing 2.0 mg/ml BSA and 0.1% trypsin inhibitor. The digested fragments were rinsed in Ca²⁺-free solution, and the cells were released by successively drowning the tissue fragments in and out of a blunt glass pipette. Cells were collected by centrifugation at 700 g for 30 s, and the cell pellet was resuspended in Ca²⁺-free solution, seeded on circular coverslips, and kept at 4°C for 1 h. At the time of the experiment, one coverslip was washed with the appropriate saline solution and transferred to the stage of a microscope for electrophysiological measurements.

Recording techniques. Single-channel currents were recorded using either the inside-out or cell-attached modes of the patch-clamp technique (10). Patch electrodes were made of borosilicate glass (Garner Glass, Claremont, CA) through a two-stage puller (model PP-83; Narishige, Japan) and fire-polished (model MF-83 forge; Narishige) to a final pipette tip resistance of 5-10 M. A 1 M KCl-agar bridge connecting the Ag-AgCl reference electrode was used to ground the bath solution. The cells and the electrode were visualized with an inverted microscope (model Diaphot, Nikon, Japan). Single-channel currents were captured and amplified through a patch-clamp amplifier (EPC7; List Electronics, Darmstadt, Germany) and were stored on videotape (model PVC-6000; Philco-Hitachi, São Paulo, Brazil) through an analog-to-digital converter (model DR-384; Neuro-Corder, Neuro Data Instruments, New York, NY). Data were displayed on-line or from the videotape to a physiograph (model RS 3200; Gould, Cleveland, OH) and to an oscilloscope (model MO 1221; Minipa, São Paulo, Brazil) via a low-pass filter (8-pole Bessel filter; Frequency Devices, Haverhill, MA) at 3 kHz. All experiments were done at room temperature and at 40 mV membrane potential.

Data acquisition and analysis. Currents were acquired through a 16-bit analog-to-digital converter (TL-1 DMA interface; Axon Instruments, Foster City, CA) controlled by the Fetchex software (pClamp 5.1, Axon). Records were analyzed using the computer program Transit (version 1.0, kindly offered by R. Latorre, Universidad de Chile). The duration and amplitude of each current level were determined using idealized records from the original data, constructed through the recognition of the transitions between distinct levels. Transitions were detected any time dI/dt (where I is current amplitude) was higher than the slope threshold criterion, usually set at ±3 of the mean baseline noise. NP_o values, where N is the number of channels in the patch available to open and P_o is the open probability of the channel, were calculated by the ratio of the mean current to the unitary single-channel current. The mean current was obtained from the amplitude current distribution histogram, using the following expression: I_mean = A₁f₁ + A₂f₂ + ...... A_nf_n, where A₁, A₂, and A_n represent the area under the Gaussian curve for each current level (f₁, f₂, f_n) present in the patch.

Tension measurements. Guinea pig ileum longitudinal smooth muscle strips were prepared as previously described (22). Segments of the longitudinal muscle ileum (3-3.5 cm) were suspended in a 5-ml chamber containing Tyrode solution at 37°C and bubbled with air. The isometric tension was recorded through a Narco Bio-System model F-60 force transducer connected to an ECB model 102-B potentiometric recorder. The agonist concentrations used were maximal, the time contact of the tissues with the agonist was 15 min, and a control response was obtained before any experimental protocol and between two successive agonist challenges after a 30-min resting period to fully recover the initial contractile response (21).

Solutions and drugs. The following solutions were used for isolation of the cells. Ca²⁺-free solution contained (in mM) 132.4 NaCl, 5.9 KCl, 1.2 MgCl₂ · 6H₂O, 11.5 glucose, and 10 HEPES, pH 7.4 (1 M NaOH), in the presence of 100 U/ml penicillin and 100 µg/ml streptomycin. For high-Ca²⁺ solution plus albumin, 2.0 mg/ml BSA and 2.5 mM CaCl₂ · 2H₂O were added to Ca²⁺-free solution. For patch-clamp experiments, the composition of both the bath and pipette solutions was (in mM) 150 KCl, 1 MgCl₂ · 6H₂O, and 10 HEPES, pH 7.4 (1 M KOH), referred to as high-K⁺ solution. In cell-attached experiments, all chemicals were added to the cells by perfusion of homogeneous solutions, and data were obtained after 3 min, so that the complete substitution of the volume of the chamber was guaranteed. The composition of the Tyrode solution was (in mM) 137 NaCl, 2.68 KCl, 1.36 CaCl₂ · 2H₂O, 0.49 MgCl₂ · 6H₂O, 12 NaHCO₃, 0.36 NaH₂PO₄, and 5.5 D-glucose.

Chemicals. All chemicals were of analytical grade. Penicillin, streptomycin, HEPES, trypsin inhibitor, BSA, iberiotoxin, and ACh were purchased from Sigma (St. Louis, MO). Collagenase I (217 U/ml) was from Worthington Biochemical (Freehold, NJ), and pronase was from Boehringer (Mannheim, Germany). ANG II and [2-lysine]ANG II (Lys²-ANG II) were purified peptides routinely synthesized in our laboratory. Stock solutions (1 mg/ml) were prepared in water and kept at 0°C, and an appropriate dilution was made at the moment of the experiment. Other salts and D-glucose were from Merck (Darmstadt, Germany).

	RESULTS

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Indirect effect of ANG II and ACh on maxi-K⁺ channel activity. To test the possibility that desensitization of the contractile response might be related to repolarization of the membrane potential through activation of K⁺ channels, we compared the effect of prolonged exposure of the guinea pig intestinal myocytes, bathed in high-K⁺ solution, to maximal concentration of 10⁷ M ANG II and 10⁶ M ACh on maxi-K⁺ currents recorded through cell-attached configuration of the patch-clamp technique at 40 mV membrane potential. ANG II invariably enhanced maxi-K⁺ channel activity by keeping NP_o values elevated as long as the peptide was maintained in the bath solution, as already reported by Romero et al. (23). It usually caused simultaneous channel openings as illustrated by the multiple current levels in Fig. 1. Contrasting with ANG II, prolonged exposure to maximal ACh concentration caused quite variable effects on maxi-K⁺ channel activity: a transient activation returning to the control activity within 12 min even in the presence of ACh in the bath solution (Fig. 1; in 4 of 8 experiments), no effect at all (data not shown; in 2 of 8 experiments), or a sustained increase, which was reversible upon washout (data not shown; in 2 of 8 experiments). In this latter group, the ACh activation was lower than that caused by ANG II challenged at the same patches. The observed sustained P_o induced by ANG II strongly suggests that relaxation of the whole tissue might be related to the repolarization of the cell.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Indirect effect of ANG II and ACh on maxi-K+ channel activity. Continuous K+ current records were obtained from cell-attached patches of different freshly dispersed myocytes from longitudinal layer of guinea pig ileum, in presence of high-K+ solution in bath and pipette. Agonists, 107 M ANG II or 106 M ACh, were added to bath solution. Patches were submitted to 40 mV membrane potential. NPo values were 0.019 and 1.044 for control condition and ANG II-induced maxi-K+ channel activation, respectively, and 0.007, 0.053, and 0.007 for control condition, 2-min and 12-min ACh-induced channel activation, respectively. Arrows at right indicate zero-current level, and inward currents are displayed as downward deflections. Experiments were performed at room temperature. Data were sampled at 125 Hz. Individual records were from different patches and are representative of 10 experiments for ANG II and 4 of 8 for ACh.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Kinetic parameters of maxi-K+ channel in ANG II-stimulated myocytes. Data were obtained from a cell-attached patch containing just 1 channel, at 40 mV membrane potential. Total recording time was 30 s, and data sampling was at 10 kHz. Histograms and fitted sums (solid line) of probability density function for each component (pdf, dot lines) are traced for open and closed dwell times, in absence (A) and after a 2-min stimulation period with 107 M ANG II added to bath solution (B). Time constant values correspond to peak of pdf, and area under the curve of respective component is contribution to mean dwell time.

Desensitization and membrane potential. If a sustained activation of maxi-K⁺ channels by ANG II would repolarize the cell, leading to tissue relaxation, it would be expected that maxi-K⁺ channel blockers would interfere with the desensitization of its contractile response. Thus we investigated the effects of iberiotoxin, a specific maxi-K⁺ channel blocker (5), on the contractile response of the muscle tissue to ANG II, its synthetic analog Lys²-ANG II, or ACh. Because there were no previous reports that the maxi-K⁺ channels present in our experimental model are sensitive to iberiotoxin, we first tested its effect at single-channel level, in inside-out patches of longitudinal layer myocytes bathed in symmetrical high-K⁺ solutions. Iberiotoxin (100 nM) in the pipette solution inhibited the channel activity in a similar pattern as it is usually described for this toxin blockade in other tissues (5). The channel presented prolonged silent periods lasting several seconds alternated with bursts of activity (data not shown), a quite different behavior as compared with the usual activity pattern recorded in the absence of the blocker (23). In whole tissue experiments, 100 nM iberiotoxin increased the fluctuation of basal tension as well as ANG II and ACh-induced tonic contractions. Furthermore, this blocker completely prevented the desensitization of the tonic contraction to 10⁷ M Lys²-ANG II (or 10⁶ M ANG II) applied 5 min after the toxin, while the steady amplitude of the ACh tonic component was not altered (Fig. 3). For all agonists, the contractile waveform did not fully recover within 30 min, as assessed by the delayed onset for dissipation of the Lys²-ANG II tonic component and slower increase of the tonus for ACh (Fig. 3).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effect of iberiotoxin (IbTx), a specific maxi-K+ channel blocker, on contractile response of longitudinal layer of guinea pig ileum induced by Lys2-ANG II or ACh. Isometric contractions, elicited by 106 M Lys2-ANG II or 106 M ACh, were recorded in absence (control) and presence of 100 nM IbTx added 5 min before agonists as well as after a 30-min washout period (Rev). Muscle strips were bathed in Tyrode solution at 37°C. Experiments with ANG II and ACh were carried out on distinct muscle strips, and records are representative of 10 experiments for each agonist.

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View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effect of distinct membrane depolarization levels on contraction of guinea pig ileum in response to ANG II. Shown are isometric contractile responses of longitudinal muscle strips induced by 107 M ANG II (control) and in presence of indicated KCl concentrations in external medium, applied 2 min after exposure of tissues to 107 M ANG II. Muscle tissues were bathed in Tyrode solution at 37°C. Individual traces were from different experiments and are representative of 10 experiments.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Effect of membrane depolarization on contraction of guinea pig ileum in response to ACh. Shown are isometric contractile responses of longitudinal muscle strip induced by 106 M ACh and in presence of 8 mM KCl in external medium, applied 2 min after exposure of tissues to 106 M ACh. Inset: isometric contractile response of longitudinal muscle strip induced by 106 M ACh in 20% of experiments. Horizontal bar, 1 min; vertical bar, 0.5 g. Muscle tissues were bathed in Tyrode solution at 37°C. Record is representative of 10 experiments.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effect of same membrane depolarization level on Lys2-ANG II tonic component. Shown are isometric contractile responses of muscle strips to 106 M Lys2-ANG II (control) and in presence of 8 mM KCl added in external medium at 7, 4, and 0.5 min of tonic contraction time course. Muscle tissues were bathed in Tyrode solution, at 37°C, and allowed to rest for 30 min between preparation washout and next challenge with peptide. Individual traces were from different experiments and are representative of 5 experiments.

	DISCUSSION

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In this study we provide evidence that desensitization of the contractile response induced by ANG II in the longitudinal layer of the guinea pig ileum is intimately related to the membrane potential. This conclusion was supported by the fact that ANG II activated maxi-K⁺ channels in cell-attached patches and the desensitization of the contractile response was abolished by iberiotoxin and elevation of extracellular K⁺ concentration.

In intestinal smooth muscles, ACh and ANG II share some common pathways of the transduction signaling. Both activate a NSCC population (12, 17) promoting Ca²⁺ and/or Na⁺ influx, depolarization, activation of L-type Ca²⁺ channels, rise in intracellular Ca²⁺ concentration, and thus contraction. In guinea pig ileum, the NSCC channels activated by ACh and ANG II present different characteristics. Although both are permeable to Ca²⁺, the first is very sensitive to intracellular Ca²⁺ (12), whereas the second is not, being a Na⁺- and voltage-sensitive channel (17). Both receptors, namely, the AT₁ and M₃ muscarinic receptors, are coupled to the G_q/G₁₁ protein class (16, 30). However, the effector response differs in the maintenance of the tonic component during prolonged contact of the tissue with ANG II or ACh. ANG II invariably promotes dissipation of the tonic component to basal level (desensitization), whereas ACh does not (1; Fig. 5).

In cell-attached membrane patches, prolonged treatment of the myocytes with ANG II peptides promoted a sustained increase of the maxi-K⁺ channel activity (23; Fig. 1). Because the pharmacomechanical coupling of ANG II to its receptor was preserved in this preparation (23), it was attractive to verify whether there is a relationship between the enhanced maxi-K⁺ channel activity that would lead to the repolarization of the membrane potential and the dissipation of the ANG II tonic contractile response (Fig. 3). ACh was used as a counterproof in the electrophysiological studies as well as in the whole tissue contraction experiments. In contrast to the homogeneity of the sustained increase of maxi-K⁺ channel activity in long-term ANG II-stimulated myocytes (Fig. 1), ACh caused variable responses. Most of the cells tested presented transient (50% of the cells) or no activation (25%) of maxi-K⁺ channels, and just in 25% of the ACh-stimulated cells there was a sustained maxi-K⁺ channel activation, although lower than the ANG II effect. Similarly, ACh-induced contractile response of the guinea pig ileum also presented some variability concerning the amplitude of the steady tonic component, which however never attains basal level in the presence of this agonist. The most usual ACh waveform presents a tonic component already sustained at its onset (Fig. 5), and in a few cases, there is a partial relaxation before a steady level is attained (Fig. 5, inset). This parallelism between ACh-induced maxi-K⁺ channel activity and ACh contraction suggests that the major contribution to the most usual ACh contractile response results from myocytes that presented transient or no activation of maxi-K⁺ channels. This would lead to a sustained depolarization of the cell membrane, favoring Ca²⁺ currents through L channels, and thus contraction. On the other hand, the observations that iberiotoxin increased the fluctuation of basal and stimulated tension in ACh contraction (Fig. 3) and the partial relaxation of the tonic component in 25% of the cases (Fig. 5, inset) indicate an active involvement of maxi-K⁺ channels throughout the contraction induced by this agent. However, the eventual repolarization of the membrane potential, as a consequence of maxi-K⁺ channel activity, would not be enough to overcome the initial depolarization induced by ACh, which involves activation of Ca²⁺- sensitive NSCC (12) sustaining the depolarization level of the membrane. In the case of ANG II, on the contrary, the cell balance does converge to the repolarization of the cell membrane and thus relaxation of the tissue. Indeed, repolarization due to sustained maxi-K⁺ channel activation would exert a negative-feedback modulation on L-type Ca²⁺ channels. The nondesensitization in the presence of iberiotoxin (Fig. 3) reinforces the close dependency of maxi-K⁺ channel activity and modulation of the ANG II tonic component. When this channel population is blocked, the contractile response to ANG II is similar to the ACh contraction (Fig. 3), as sustained ANG II depolarization should occur, probably by maintaining elevated the P_o of voltage-dependent channels, mainly NSCC and L channels, thus keeping the tissue contracted. If this interpretation is correct, then any maneuver causing membrane depolarization should prevent this phenomenon. Indeed, exposure of the tissue to KCl concentrations, which did not cause a sustained response per se, prevented the fade of the ANG II tonic component (Fig. 4) and did not alter the time course of the most usual ACh response (Fig. 5). Moreover, it appears that the membrane potential must attain a threshold to counteract the repolarization induced by maxi-K⁺ opening (Fig. 4), but once it is attained, there is no time dependency along the tonic component because the desensitization process was shut off by addition of KCl at any moment of the tonic response time course (Fig. 6). This prompt response suggests that the balance of the voltage-dependent channels recruited by ANG II (maxi-K⁺, NSCC, and L channels) is a very effective regulatory mechanism for the contractility of the guinea pig ileum. However, our data also suggest that this is probably not the only mechanism underlying desensitization, since it is not clear how ACh and ANG II, which initiate their response through the same G protein (16, 30), may present differential channel type coupling. Our data convey the notion that the two receptors must differentially activate distinct G proteins and hence balance the types of channels activated and the responses achieved. Nevertheless, some caution is required for this straightforward conclusion because no data are yet available focusing this intriguing point, and alternatively, the signaling transduction of one or both agonists may involve other pathways. It has been shown that tyrosine kinase activity is of great functional importance in regulating muscarinic NSCC in guinea pig ileum (for a review, see Ref. 14) or that muscarinic activation enhanced production of arachidonic acid, thereby increasing Ca²⁺ influx into the cell (14). Another point liable to speculation concerns the molecular mechanism for the gating properties of the channels recruited by ACh and ANG II. It has been proposed that the concentration of active G protein -subunits might critically determine the state of voltage dependence of the channel (14). Furthermore, one must also consider that different ,-subunits can also regulate a number of cellular effectors, including ionic channels (14). Finally, we cannot rule out the involvement of PKC or some other unknown intracellular mechanisms that transduce cell-surface signals to the channel proteins (14), which might be different according to the agonist used. Indeed, it has been proposed that desensitization to ANG II in guinea pig ileum may be due to a negative-feedback mechanism exerted by PKC on a step of the stimulus-response chain after phospholipase C activation (24).

In conclusion, ANG II desensitization in guinea pig ileum is likely to rely on membrane repolarization, assigning a relevant role to maxi-K⁺ channel population to the modulation of this phenomenon, although the precise type and contribution of G protein other than G_q/G₁₁ remains unclear.