The isolated posterior midgut of the tobacco hornworm maintains a vigorous transepithelial K+ transport from the hemolymphal side to the lumen side at a rate accurately measured by its short-circuit current. Previous studies using the K+ channel blocker Ba2+ suggested that partial inhibition of the short-circuit current by hemolymphal Ba2+ was due to blockage of one of at least two parallel transbasal entry routes for K+ into the intracellular transport pool. The present studies show that the local anesthetic lidocaine, at a concentration of 5 mmol I-1 on the hemolymphal side, partly inhibits net transepithelial K+ transport. The inhibition is accompanied by hyperpolarization of the basal membrane and an increase in transbasal resistance, suggestive of a block of transbasal K+ conductance. The effects of lidocaine and Ba2+ are additive, suggesting that the inhibitors distinguish separate, parallel K+ uptake processes.
The posterior midgut of lepidopteran larvae consists of two major cell types, columnar and goblet, which show restricted coupling with intracellular Lucifer Yellow dye but are apparently well coupled with respect to small ions and electrical current (Moffett et al. 1982; Moffett and Koch, 1988a). The isolated, short-circuited tissue transports K+ from hemolymph to lumen at a rate as high as 1 μmolcm-2min-l. Active K+ transport involves two transmembrane steps: entry across the basal membrane followed by extrusion across the apical membrane. Studies using X-ray microanalysis (Dow et al. 1984) and K+-selective microelectrodes (Moffett and Koch, 1988b) show that K+ is actively transported across the goblet cell apical membrane against a large electrochemical gradient. Under typical experimental conditions the transbasal electrochemical gradient is favorable for K+ entry (Moffett et al. 1982). However, the transbasal gradient becomes unfavorable when extracellular K+ concentration is low and under hypoxia (Chao et al. 1990). The continuation of some net K+ transport under these conditions is evidence that transbasal K+ movement involves an active component that is masked under typical conditions in vitro.
Evidence for multiple parallel pathways for K+ entry across the basal membrane was provided by the effects of Ba2+. Although generally regarded as a competitive inhibitor of K+ passage through membrane channels, Ba2+ partly inhibits net K+ transport by the midgut in standard saline, but stimulates it in high-K+ saline. This finding was interpreted as evidence that Ba2+ inhibits one route of transbasal K+ entry while stimulating another; about one-third of transbasal K+ influx appears to be Ba2+-insensitive (Moffett and Koch, 1985). In standard saline, in which the inhibitory effect dominates, application of Ba2+ results in hyperpolarization of the basal membrane and an increase in transbasal resistance (Moffett and Koch, 1988a), effects that are consistent with blockage of a current-carrying process.
The present studies characterize the effect of lidocaine on net K+ transport by the posterior midgut. Lidocaine exerts its local anesthetic effect by blocking the voltage-gated Na+ and Ca2+ channels of excitable cells, but it has also been reported to block K+ channels in the basolateral membrane of amphibian urinary bladder (Van Driessche, 1986) and turtle colon (Dawson et al. 1988). In the midgut, lidocaine, like Ba2+, inhibits the short-circuit current (Isc) and increases basal membrane resistance. However, the effect of each inhibitor is almost unaffected by the presence of the other, and the two inhibitors show different forms of dependence on extracellular K+ concentration. These results suggest that lidocaine and barium distinguish separate routes of transbasal K+ entry.
A preliminary account of these studies has been presented (Koch and Moffett, 1990).
Materials and methods
Larvae of Manduca sexta were reared on an artificial diet (Yamamoto, 1969). The morphologically distinct posterior midgut (Cioffi, 1979) was removed from cold-anesthetized fifth-instar larvae and immediately transferred to oxygenated ice-cold bathing solution. The Malpighian tubules and adhering fat body were removed and the tissue was mounted as described below.
The standard bathing solution, designated ‘32KS’, contained, in mmoll-1: 32 KC1, 5 CaCl2, 5 MgCl2, 5 Tris-HCl (pH8.0), 166 sucrose. In some experiments, the concentration of KC1 was varied as indicated by the solution designation; for example ‘70KS’ contains 70 mmol I-1 KC1. In other experiments the solutions were also made nominally divalent-cation-free by omission of CaCl2 and MgCl2. Except for the experiments on side specificity, all experiments were conducted with identical solutions on both sides of the tissue. Unless indicated otherwise, bathing solutions were oxygenated with 100 % O2.
Pure lidocaine (Sigma) and its quaternary amine congeners QX-222 and QX-314 (kindly provided by Dr Bertil Takman of Astra Pharmaceutical) were added to bathing solutions to give the concentrations indicated. At the pH of the bathing solutions used in this study (8.0) the maximum concentration of lidocaine that can be attained is 5 mmoll-1. Barium chloride was added to the half-chambers as a 1 moll-1 solution in distilled water to give the final concentrations indicated.
Short-circuit current studies
For studies of inhibitor effects on Isc, the tissue was mounted in a modified Ussing chamber as in previous reports (Moffett and Koch, 1985). Except for the experiments on side specificity, all experiments were conducted with identical solutions on both sides of the tissue. Tissues were continuously short-circuited and Isc was recorded using a strip chart recorder. Solution resistance was compensated using predetermined values.
The results of inhibitor experiments are expressed in terms of the ratio of Iscafter inhibition to the control value of Isc measured just before addition of inhibitor (I/Io). Recovery of Isc is expressed as a percentage of the current recorded immediately before inhibitor was added. Although Isc decays spontaneously with time, with our present techniques the rate of decay is less than 10% h-1 after initial equilibration. Since the effects of Ba2+ and lidocaine are rapid and exposures to the inhibitors were brief, no corrections for timedependent decay of Isc were made.
Microelectrodes were pulled from microcapillary glass (Fredrich Haer 1011) in a vertical puller (David Kopf) and filled with 1 moll-1 KC1. Useful electrodes had tip resistances of 25–30MΩ when measured in 32KS. The midgut was mounted in a chamber modified from the design of Thompson et al. (1982) and penetrated from the hemolymphal side with electrical reference to the hemolymphal solution. All experiments were conducted under short-circuit conditions with identical solutions on both sides. The microelectrode voltage was amplified by a Keithley electrometer and recorded on one channel of a Gould Brush four-channel pen recorder, with Isc being simultaneously recorded on another channel.
The microelectrode was advanced at right angles to the tissue by a hydraulic drive (Friedrich Haer). Penetration of a cell was indicated by a sharp negative deflection of between –20 and –40 mV (Moffett et al. 1982); this is the transbasal potential (Vb). The change in transbasal resistance was measured as the change in the magnitude of deflection of Vb in response to the passage of a pulse of constant current. This is a relative measure; the absolute value of transbasal resistance cannot be measured readily by this method because of the large series resistance component presented by the bathing solution.
All data are given ±the standard errors of their means. For each tissue in the microelectrode experiments, several impalements were made and the averages of the values obtained were taken as best estimates for that tissue. The means presented are the averages of these best estimates.
Lidocaine inhibition of Ivc
Bilateral addition of lidocaine partly inhibits ISC (Fig. 1). In six tissues I/Io and the percentage reversibility of inhibition were measured as a function of the duration of exposure to lidocaine for durations of 5, 10, 20 and 40 min. In three of the tissues the order of exposure durations was ascending; in the other three the order was descending. Results from the two series were not distinguishable, and pooled results are shown in Fig. 2. The inset to Fig. 2 shows the correlation between I/I0 and fractional recovery.
On the basis of time course and reversibility, two phases of lidocaine inhibition can be distinguished in these results. The initial phase consists of a rapid drop in Iscduring the first 10min. The inhibition that occurs during this phase is almost completely reversible (Fig. 2). If exposure to lidocaine continues beyond 10 min, a slower drop in Isc is seen. This phase takes up to l h to complete and is accompanied by a progressive decrease in reversibility (Fig. 2). To maximize reversibility, data were collected at 7.5 min after addition of lidocaine in all subsequent experiments.
The limited solubility of lidocaine in bathing solutions at pH 8.0 appears to preclude the maximal effect of the inhibitor. At a concentration of 5 mmol l-1, the inhibitory effect was almost exactly twice that at 2.5 mmol l-1 (Fig. 3).
Sidedness of lidocaine inhibition
The rapid effect of lidocaine is on the hemolymphal side (Fig. 4). In four experiments, 75±3.1% of the inhibition in effect by 7.5 min after bilateral application was attributable to the hemolymphal lidocaine, regardless of which side was exposed first.
The effects of lidocaine on transbasal voltage (Vb) and transbasal resistance (Rb) were measured in microelectrode experiments. Transbasal penetrations were made in tissues superfused with 32KS oxygenated with 100% O2. The mean Vbwas –36.5±0.84 mV in seven experiments. The superfusate was then changed to oxygenated 32KS with 5 mmol l-1 lidocaine and changes in Vb, Rb and Isc were recorded. Fig. 5A shows typical results. The Vb hyperpolarized synchronously with the decrease in ISC; the mean change in Vb was –6.35±1.63 mV. The increase in the deflection of Vb in response to the transepithelial constant-current pulse indicates increased Rb; the mean change in basal resistance was 22±6%. The effects of lidocaine on Vb, Rb and Isc are comparable to those of 6 mmol l-1 Ba2+ (Fig. 5B). As in previous studies (Moffett and Koch, 1988a), Ba2+ hyperpolarized the basal membrane with a concomitant increase in Rb and decrease in Isc.
Although in previous studies we were usually able to maintain impalements through a number of solution changes, in these experiments re-introduction of lidocaine-free solution usually caused the microelectrode to be dislodged. This observation could have been due to contraction of the intestinal muscularis following washout of lidocaine.
Non-effectiveness of charged lidocaine analogues
Two quaternary amine analogues of lidocaine, QX-222 and QX-314, were used to determine whether lidocaine must permeate the cell membrane to be effective. The pKa of lidocaine is 8.1, so at pH8.0 about half of the dissolved lidocaine is in the uncharged, lipid-soluble form. In contrast, the analogues are charged in solution and thus only weakly lipid-soluble. In two trials, neither analogue had a measurable effect on Isc after 30 min of exposure to 10 mmol I-1, the highest concentration tested.
Effect of bathing solution K+ concentration
These experiments determined the effect of lidocaine on the relationship between bathing solution K+ concentration ([K+]o) and Isc. Each tissue was presented with stepwise changes in concentration over the series 10, 20, 32, 50 and 70 mmol l-1 K+. In three of the six experiments, the tissues were equilibrated in 10KS and presented with ascending [K+]o; the other three were equilibrated in 70KS and presented with descending [K+]o. The timing of the experiments was similar to that shown in Fig. 3 of Moffett and Koch (1985), except that in the present experiments percentage recovery was also measured. Briefly, tissues were equilibrated in the initial solution until ISC became stable (usually about 45 min). Then the corresponding lidocaine-containing solution was substituted. After 7.5 min of lidocaine exposure, the original solution was restored for measurement of recovery, which typically required 10–15 min. The bathing solution was then replaced with the next solution in the series, and the sequence of lidocaine exposure and recovery repeated.
The mean values of Isc for each [K+]o differed between the two protocols (Table 1), and I/I0 was consistently lower for the tissues equilibrated at high [K+]o than for those equilibrated at low [K+]o. Nevertheless, within each series the lidocaine-sensitive fraction of the total current was almost constant. The reversibility of the inhibition was independent of the K+ concentration of the equilibration solution and test solutions. The mean reversibility for the series was 0.72±0.03.
Effect of hypoxia
The effect of decreased O2 tension on lidocaine inhibition of ISC was measured in 10 experiments in which the tissues were equilibrated in 32KS gassed with 100 % O2, then switched to 32KS gassed with air (21 % O2) and then to 32KS gassed with 5 % O2. A 7.5 min exposure to 5 mmol l-1 lidocaine was interposed during each of the three O2 tensions.
In contrast to the constancy of I/I0 when Isc was altered by changing [K+]o, reduction of O2 tension greatly enhanced the inhibitory effect of lidocaine (Table 2). However, decreased O2 tension reduced the reversibility of the inhibition. In 21 % O2 the recovery was only 0.60±0.03 as compared to 0.73 in 100 % O2, and currents were so low in the 5 % O2 experiments that percentage recovery was not computed.
Non-dependence on alkaline earth ions
The experiments reported above on the dose-response relationship, the effect of varying [K+]o and the effect of low O2 tension were also carried out in nominally divalent-cation-free solutions. As previously reported (Moffett and Koch, 1985), initial values of ISC were higher in these solutions; the mean ISC in 32K was 543±μAcm-2 for four experiments compared to the combined mean of 376μAcm-2 for the six experiments in the present report (Table 1). The relationship between ISC and [K+]o became quasi-saturating, as expected from previous studies (Moffett and Koch, 1985). The dose-response curve for lidocaine in divalent-cation-free solutions was similar to that in standard solution: on average, 2.5 mmol l-1 lidocaine inhibited 19% of the current while 5 mmol l-1 lidocaine inhibited 44%. As in standard solutions, I/I0 for 5 mmol I-1 lidocaine was constant when [K+]o was varied: the overall mean for two experiments using ascending [K+]o was 0.68±0.02 and for two with descending [K+]o it was 0.45±5.1. Enhancement of lidocaine inhibition by decreased O2 tension in divalent-cation-free solution was similar to that observed in standard solutions; in two experiments, I/Io in 5 mmol l-1 lidocaine was 0.67±0.03 in 100% O2 but 0.06±0.02 in 5% O2.
Combined effects of Ba2+ and lidocaine
The extent of competition between Ba2+ and lidocaine for inhibition of Isc was determined by comparing the I/I0 of each inhibitor given singly to the 1/IO of that inhibitor in the presence of the other. In nine of eighteen experiments 6 mmol I-1 Ba2+ was given first, followed by 5 mmol I-1 lidocaine; in the other nine experiments the order of inhibitors was reversed. Fig. 6 shows the average results in the form of two diagrams of changes in I/Io, one for experiments in which Ba2+ was given first and one for those in which lidocaine was given first. When given first, Ba2+ inhibited 57% of the current (I/I was 0.43), but when given in the presence of lidocaine, Ba2+ inhibited 69 % of the remaining current. Similarly, when given first, lidocaine inhibited 44% of the current, but it inhibited 68% of the current remaining after Ba2+ blockade.
Previous studies suggested that the transbasal electrical potential of midgut consists of at least two components; a Nernstian component generated mainly by the transmembrane gradient of K+, and a contribution due to the current of net K+ entry through conductive pathways (Chao et al. 1990). Cation channels in the basal membrane detected by patch-clamp (Moffett and Lewis, 1990) and noise analysis (Zeiske et al. 1986) are one possible route of conductive K+ entry. These would be expected to mediate passive K+ entry in tissues bathed in well-oxygenated solution containing normal to high K+ concentrations, conditions under which the transbasal K+ electrochemical gradient favors K+ entry (Moffett and Koch, 1988a; Chao et al. 1990). An active component of K+ uptake was inferred from observations that a short-circuit current may be maintained under low extracellular [K+] and/or hypoxia, while under these conditions the electrochemical gradient is unfavorable for K+ entry (Chao et al. 1990). If it were electrogenic, an active pathway would also contribute to the entry-related component of the transbasal potential.
As previously reported (Moffett and Koch, 1988a), hemolymphal Ba2+ hyperpolarizes the basal membrane and increases transbasal resistance. In view of the reputation of Ba2+ as a competitive inhibitor of K+ channels, this effect was interpreted as the consequence of blockage of one route of K+ entry, with diversion of some K+ entry current to a second conductive pathway (Moffett and Koch, 1985). According to this interpretation, the increase in resistance to the K+ entry current caused by blockage of the Ba2+-sensitive pathway increases the magnitude of the entry-related component of Vb, hyperpolarizing the basal membrane. The decrease in Isc that occurs at low and normal K+ concentrations can be regarded as the result of the increased transapical potential against which the apical pump must work (Moffett and Koch, 1988a). In contrast, direct inhibition of the apical pump by hypoxia decreases the entry-related voltage. component, depolarizing the basal membrane (Moffett and Koch, 1988a; Chao et al. 1990).
The present studies show that the effects of lidocaine on Vb and Rb are similar to those of Ba2+, arguing that both inhibitors affect the entry-related component of Vb. The fractional inhibition of either inhibitor, defined in terms of the current remaining after the first inhibitor has taken effect, is increased in the presence of the other (Fig. 6). This is evidence against competition between the two inhibitors, for such competition would have decreased the fractional inhibition of lidocaine after Ba2+, rather than increasing it. However, the absolute decrease in current due to either of the inhibitors is less when it is added second than when it is added first (Fig. 6). The decrease in the amount of current abolished by either inhibitor when it is second in order of addition is consistent with an overlap between the two inhibitors of about 18 % of the total transbasal current. That is to say, if the basal membrane contained a number of channels all of equal conductance, about 18% of the channels would be susceptible to blockage by either inhibitor. The 17 % of ISC that remains in the presence of both inhibitors may be carried by an uptake pathway insensitive to either inhibitor, but could well be due to the fact that, because of its low solubility in 32KS solution, the concentration of lidocaine was suboptimal.
The concentrations of lidocaine used in these studies (2.5–5 mmol l-1) are an order of magnitude higher than the range of about 200–800 μmol l-1 in which effects on Na+ and K+ channels are detectable and in which the drug is effective as a local anesthetic. The possibility that such a high dose might still be suboptimal could be explained by the likelihood that in this system, as in others, the binding of lidocaine to its active site is reversible. To achieve maximal inhibition of Isc, the inhibitor must be concentrated enough to block the susceptible channels essentially all the time. In contrast, blockage of neuronal conduction requires only that some critical fraction of the voltage-gated channels be blocked at any instant.
The relationship between Isc and [K+]o has been analyzed as the sum of a saturating and a linear component (Moffett and Koch, 1983, 1985). These components are believed to reflect different concentration dependences of parallel transbasal uptake processes. According to this analysis, under standard conditions about two-thirds of the transported K+ enters the cells through the saturating component. The relative contribution of the saturating component to total K+ uptake is greatest at low [K+]o and decreases as [K+]o increases.
The control values of Isc measured in the experiments in which a series of [K+]o was presented (Table 1) are not comparable to results obtained in our previous studies of the K+ concentration dependence of Isc (Moffett and Koch, 1985), which used a similar protocol. The difference is assumed to reflect the accumulation of irreversible lidocaine block over the course of the repeated exposures to lidocaine in the present experiments, making control values of Isc measured later in each experiment progressively lower than would be expected from the previous studies. Nevertheless, the fractional inhibition of the remaining ISC by lidocaine was remarkably constant over the range of extracellular K+ concentrations, regardless of whether the concentrations were presented in ascending or descending order (Table 1). This result indicates that, unlike the situation for the divalent-cation-sensitive uptake process, the relative contribution of the lidocaine-sensitive uptake process to total uptake is independent of [K+]o.
The values of I/Io for the ascending series of [K+]o are significantly greater than those of the descending series (Table 1). Although no definite conclusion can be drawn, the difference could reflect an effect of the equilibration [K+]o on intracellular [K+]. Previous studies gave values of about 70 mmol I-1 for tissues superfused in 10KS, but greater than 100 mmol l-1 for tissues superfused in 70KS (Moffett et al. 1982). Although any difference in the cytoplasmic K+ concentration ([K+]i of the tissues at the start of the two experiments undoubtedly diminished as the experiments progressed, the greater degree of inhibition occurred in the descending concentration series in which tissues could be expected to have higher [K+]i values. The result implies that the fraction of total uptake that is lidocaine-inhibitable is more sensitive to [K+]i than to [K+]o. Although higher [K+]i is associated with more negative values of Vb, voltage-sensitivity of lidocaine block can be ruled out because I / I0 is constant over a range of [K+]o previously shown to result in large changes in Vb (Moffett and Koch, 1988a).
Deletion of Ca2+ and Mg2+ from bathing solution favors the saturating component of K+ uptake, while addition of Ba2+ favors the linear component at the expense of the saturating component, particularly if the bathing solutions are also Ca2+-and Mg2+-free (Moffett and Koch, 1983, 1985). In Ca2+,Mg2+-free solution, Ba2+ stimulates Isc when [K+]o is 50 mmol l-1 or higher. Lidocaine inhibition, unlike that of Ba2+, was not affected by removal of Ca2+ and Mg2+, and lidocaine inhibited ISC at high as well as normal and low [K+]o. The insensitivity of the lidocaine effect to [K+]o and to deletion of alkaline earth ions is additional evidence that lidocaine affects a component of transbasal K+ uptake insensitive both to Ba2+ and to the presence or absence of Ca2+ and Mg2+, unlike the pathway identified by Moffett and Koch (1985).
Both Ba2+ and lidocaine are regarded as blockers of membrane channels. Such blockers of passive movement would be expected to inhibit ISC only under conditions in which the transbasal K+ electrochemical gradient favors K+ entry, and to have no (or perhaps a stimulatory) effect when the transbasal gradient is unfavorable for K+ entry. In 32KS gassed with 5% O2, the transbasal K+ electrochemical gradient rapidly becomes unfavorable for K+ entry. Under these conditions the inhibitory effect of Ba2+ actually increased. This result was taken to suggest that the entry process inhibited by Ba2+ is thermodynamically active (Chao et al. 1990). The similar increase in effectiveness of lidocaine in hypoxia found in the present studies suggests that lidocaine, like Ba2+, inhibits a thermodynamically active K+ uptake process.
Quaternary analogues of lidocaine are effective when applied to the cytoplasmic side of axons but not when applied to the outside (Frazier et al. 1970). The failure of extracellular quaternary analogues to inhibit the ISC of midgut suggests that lidocaine must dissolve in the membrane lipid to reach its site of action. Lidocaine competes favorably with K+ for detection by resin-based K+-selective microelectrodes, and was detected in the cytoplasm with such microelectrodes after application to the hemolymphal side (K. F. Moffett and A. Koch, unpublished observations). However, the block of Isc probably does not come primarily from free intracellular lidocaine, since the drug is markedly more effective when applied to the hemolymphal side of the tissue, although lidocaine would be expected to enter the cytoplasm with equal ease from either side of the tissue. Thus, lidocaine may reach its blocking site from within the lipid component of the basal cell membrane, as has been suggested for lidocaine block of Na+ channels in frog muscle (Schwarz et al. 1977).
The effects of lidocaine on transepithelial K+ transport described here add to the evidence that K+ enters the intracellular transport pool of posterior midgut by multiple pathways, rather than by a single readily characterizable mechanism. The substantial effect on ISC of partial inhibition of K+ uptake by Ba2+ and lidocaine suggests that regulation of the uptake pathways could be a potent mechanism for physiological control of transport of K+.
These studies were supported by NSF DCB 8315739. We thank Robin Woods and Anna-Lise Dragoy for technical assistance.