Resting Membrane Properties of Locust Muscle and Their Modulation II. Actions of the Biogenic Amine Octopamine

Christian Walther and Klaus E. Zittlau

Physiological Institute, Neuroendocrinology Working Group, University of Marburg, 35037 Marburg, Germany

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Walther, Christian and Klaus E. Zittlau. Resting membrane properties of locust muscle and their modulation. II. Actions of the biogenic amine octopamine. J. Neurophysiol. 80: 785-797, 1998. Ionic currents in the resting membrane of locust jumping muscle and their modulation by the biogenic amine octopamine were investigated using the two-electrode voltage clamp. A Cl- conductance, GCl,H, which slowly activates on hyperpolarization, can be induced by raising the intracellular Cl- concentration via diffusion of Cl- ions from the recording electrode. The instantaneous I-V characteristic of the current, ICl,H, is linear and reverses at the same potential as the gamma -aminobutyric acid (GABA)-mediated Cl- current. Elevation of [Cl-]i increases the maximal steady state GCl,H (Gmax) and shifts the activation curve of GCl,H to more positive potentials. Octopamine enhances GCl,H, mainly by increasing Gmax. Octopamine also lowers the resting K+ conductance (GK,r). It reduces a hyperpolarization-activated component (GK,H) of GK,r, mainly by decreasing Gmax. Octopamine also transiently stimulates the Na+/K+ pump although this effect was not always seen. The effects of octopamine on the Cl- and K+ conductances are mimicked by membrane permeant cyclic nucleotides. The modulation of GK,r, but not that of GCl,H, seems to be mediated by protein kinase A (PKA). PKA seems to be constitutively activated as indicated by the pronounced increase in GK,r induced by a PKA inhibitor, H89. The properties of GCl,H and related Cl- conductances in invertebrate and vertebrate neurons are compared. GCl,H probably supports efflux of Cl- ions accumulating in the fibers during synaptic inhibition. Octopamine's multiple modulation at the level of the muscle cell membrane, in conjunction with previously established effects on synaptic transmission and excitation-contraction coupling, are suited to support strong and rapid muscle contractions.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The biogenic amine octopamine is an important neuromodulator in insects and other arthropods (Agricola et al. 1988; David and Coulon 1985; Evans 1985a; Evans and Myers 1986; Nässel 1996). In insect ventral nerve cord, it is produced by many unpaired dorsal median (DUM) neurons (Nässel 1996; Stevenson and Spörhase-Eichmann 1995). Octopamine is released both within the CNS and into the hemolymph (e.g., Evans 1985a; Orchard 1982), partly from neurosecretory endings closely associated with certain skeletal (Hoyle 1974) or visceral (Orchard and Lange 1987) muscles. Specific subsets of DUM neurons are active, i.e., release octopamine, when specific movements are initiated (Burrows and Pflüger 1995). However, octopamine also exerts multiple actions in "fight and flight responses" (Adamo et al. 1995; Evans 1985a) or "arousal syndrome" (Corbet 1991), and its complete absence in a Drosophila mutant leads to reduced viability under unfavorable environmental conditions (Monastirioti et al. 1996). A wealth of knowledge has accumulated with respect to octopamine's receptor pharmacology (Osborne 1996; Robb et al. 1994; Roeder et al. 1995) and its physiological actions, including a role in memory (Hammer and Menzel 1995). Little, however, is known about its effects on ionic currents in insect tissues (e.g., Achenbach et al. 1997), although in many cases it is quite clear that modulation of ionic current(s) is involved in the particular actions of octopamine.

In the well-studied jumping muscle of the locust (Hoyle 1978), which is innervated by the neurosecretory octopaminergic neuron DUMETi, octopamine increases the amplitude of neurally evoked contractions and speeds up their relaxations (Evans and Myers 1986; O'Shea and Evans 1979). The first effect is due, at least partly, to potentiation of excitatory transmitter release (e.g., Walther and Schiebe 1987), whereas the second effect is not yet understood. Relaxation, in part of the fibers, also is accelerated on activity of an inhibitory neuron (e.g., Burns and Usherwood 1979; Wolf 1990), which causes Cl--mediated inhibitory junctional potentials (Usherwood and Grundfest 1965). The postsynaptic inhibition is important for rapid relaxation of tonic and intermediate fibers (cf. Hoyle 1978), which otherwise would oppose rapid movements (Burns and Usherwood 1979; Wolf 1990).

In the present study, we wanted to clarify whether the actions of octopamine in the jumping muscle also involve modulation of muscle membrane resting currents. Up to now, this did not seem very likely because the previously observed effects were not conspicuous and sometimes inconsistent. On intracellular recording some reduction of the resting conductance (Walther and Schiebe 1987) and a slight hyperpolarization (e.g., May et al. 1979; O'Shea and Evans 1979) were noticed. However, as we have discovered on using the voltage clamp, octopamine in fact induces profound changes of several resting currents. Here, we analyze these effects in detail and test whether they are mediated by octopamine's ability to stimulate adenylate cyclase in this muscle (Evans 1984a,b).

We have found a Cl- current that activates on hyperpolarization or when [Cl-]i is increased. This current may prevent [Cl-]i from rising to the extent that postsynaptic inhibition is impaired. Octopamine potentiates this Cl- current but in addition reduces the resting K+ conductance and enhances the activity of the Na+/K+ pump. These latter effects appear suitable to raise electrical excitability of the muscle fibers. The multiple actions of octopamine partly cancel out on the level of the membrane potential, and their net effect can greatly differ depending on electrode filling solutions. This readily explains why the previous membrane potential measurements did not lead to major insights. Some of the results have been presented in abstract form (e.g., Walther 1996; Walther and Zittlau 1991; Zittlau and Walther 1992).


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FIG. 1. Loading a muscle fiber with Cl-- ions induces a Cl- current. Fiber was impaled at time 0 with a 3 M KCl-recording electrode and a 2 M K+ citrate-current electrode. Holding potential was set at -75 mV, the approximate EK. IK,H was blocked by 2 mM Ba2+. Hyperpolarizing voltage jumps (20 mV × 40 ms) were performed continuously at a rate of 0.16 Hz. Groups of the resulting current pulses, monitored by a chart recorder, are shown. Zero current level is indicated by the position of the time axis. Note that the current pulse amplitude and the inward holding current began to increase after ca. 60 min. Inset: currents elicited by voltage jumps to -109 mV for 2.6 s, performed at the times indicated by the letters. For clarity of presentation, the traces were aligned by subtraction of the holding currents so that they start from the same level. Note slowly activating inward current followed by an inward tail current. At b these current-relaxations are just apparent; subsequently they greatly increase parallel to the above changes. This is due to intracellular accumulation of Cl- ions, diffusing from the KCl electrode; this leads to the development of a Cl- conductance, which slowly activates at voltages more negative than ECl (cf. RESULTS). From the data of this figure, it is calculated that, at -75 mV, this conductance is of the order of 0.02 µS at b and increases to ~0.25 µS at e. [Cl-]i raises to ~20 mM at b and to ~60 mM at e according to the calculated reversal potentials of the tail currents.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Preparation, solutions and electrophysiological techniques

Experiments were performed in the distal part of the jumping muscle (M. Extensor tibiae, region f) (Hoyle 1978, his Fig. 2) of the desert locust Schistocerca gregaria at 30°C (for details of preparation, saline and experimental setup, see Walther et al. 1998). This part of the muscle is particularly responsive to octopamine (Evans 1985b). The bundles investigated, i.e., the second and the third (counted distally to proximally), usually lack inhibitory innervation yet exhibit extrajunctional receptors for gamma -aminobutyric acidic (GABA), whereas the fibers of the first bundle (i.e., the most distal), which occasionally also were used, are consistently innervated by one common inhibitory neuron, termed CI1 (Cull-Candy 1984; Cull-Candy and Miledi 1981; Hale and Burrows 1985). Data from >200 muscles were evaluated.


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FIG. 2. Current-voltage relationships of chloride currents activated either by hyperpolarization or by gamma -aminobutyric acid (GABA). Recordings, all obtained from a sequence of measurements in one fiber, demonstrate that the reversal potentials of both currents are similar and can be shifted to a similar extent by lowering [Cl-]i. Zero current levels are indicated by the positions of the voltage axes. Holding potential: -75 mV. Ba2+ (2 mM), to suppress the hyperpolarization-activated K+ current, IK,H, and 1 mM glutamate (cf. METHODS) were present throughout the experiment. In addition, in conditions 1 and 2, 10 µM octopamine and 100 µM 3-isobutyl-1-methylxanthine (IBMX) were present to enhance the hyperpolarization-activated Cl- current, ICl,H. (cf. RESULTS). Recording electrode contained 2 M K+acetate and 1.5 M KCl. A: amplitudes of tail currents recorded on jumping to the various test potentials after an activating prepulse from -75 to -110 mV for 3 s. 1 and 2: before and after lowering [Cl-]i by prolonged GABA-application (cf. B); 3: 10 min after octopamine and IBMX had been washed off. Inset: tail currents shown were recorded in condition 1. Although inward IK,H was blocked completely due to the presence of Ba2+, block of outward IK,H was incomplete, e.g., only ~75% at -55 mV. Therefore, small residual outward currents, recorded with the same voltage jump protocol at an early stage of the experiment when ICl,H had not yet developed, were subtracted from the currents obtained by jumping to -65, -55, and -45 mV. B: currents obtained with linear voltage ramps sweeping through the complete range within 310 ms. Traces, digitally smoothed, represent the difference between the currents flowing in the presence and absence of 1 mM GABA. In condition 1, [Cl-]i was elevated due to prolonged (140 min) diffusion of Cl- from the recording electrode into the cell. In condition 2, [Cl-]i was reduced as a consequence of 8 min application of 1 mM GABA, which caused an average inward current (i.e., Cl- efflux) of -25 nA at the holding potential.

Chloride-free saline consisted of Ca-propionate and Mg-gluconate and either K- and Na-sulfate or K- and Na-gluconate. Ca2+-free saline contained 1 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid. The equipment and methods for voltage clamping as well as experimental protocols were essentially as described before (Walther et al. 1998). For reasons given there, the range of voltages over which the currents could be investigated was limited to about -130 to -45 mV. Current electrodes contained 2 M K citrate plus 10 mM KCl. Recording electrodes were filled with 3 M KCl or 2 M K acetate or a mixture of both. When the mixture was used the hyperpolarization-activated Cl- current, ICl,H never became so large that space clamp was impaired. We also had the impression that drift of ICl,H amplitude was less serious than when a KCl electrode was used.

Application of octopamine to the muscle in situ can cause a reduction in muscle tone (O'Shea and Evans 1979) and thus might introduce mechanical artifacts. We did not find this effect, however, in our completely isolated preparation.

Determination of Cl- equilibrium potential

GABA in insect skeletal muscle fibers opens channels selective for Cl- ions (Usherwood and Grundfest 1965). Determination of the reversal potential of a GABA-induced current therefore offers a convenient way of estimating ECl. GABA often induced an inward current, i.e., Cl- efflux, at holding potentials around -70 mV because Cl- ions, due to diffusion from a KCl-filled recording electrode, had accumulated in the fiber. Thus to minimize the resultant fall of [Cl-]i, it was necessary to keep the GABA application as short as possible (<= 0.5 min) and to avoid currents >10 nA. The reversal potential of the GABA-induced current, ICl,GABA, depended on whether glutamate was present or not. In the absence of glutamate, the reversal potential was 7 ± 3 mV (mean ±SD; n = 11) more positive than that of the hyperpolarization-activated Cl- current, ICl,H. The discrepancy, however, disappeared when the experiments were performed in the continuous presence of 1 mM glutamate (see RESULTS and Fig. 2) so that junctional and extrajunctional glutamate receptors were largely desensitized (Cull-Candy 1984). A conclusion from these somewhat unexpected findings is that GABA seems to have a weak affinity for glutamate receptors, which, in neuromuscular junctions, mediate excitatory transmission (e.g., Usherwood and Cull-Candy 1975). Thus, at the high GABA concentrations required here, the chloride current is contaminated by an inward current when glutamate receptors are not desensitized.

Drugs

The following substances were obtained from Sigma: 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), 8-bromoadenosine 3':5'-cyclic monophosphate (8-br-cAMP) and 8-bromoguanosine 3':5'-cyclic monophosphate (8-br-cGMP), anthracene-9-carboxylic acid, DL-octopamine (DL-p-hydroxyphenylethanolamine), ouabain, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS). N-[2-bromocinnamyl(amino)ethyl]-5-isoquinolinesulfonamide (H-89) was obtained from Biomol, Hamburg; L-glutamic acid from Research Biochemicals; GABA and 3-isobutyl-1-methylxanthine (IBMX) came from Serva; cantharidin and okadaic acid came from Calbiochem; and dihydro-ouabain from Zyma SA, Nyon, Switzerland. 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was kindly supplied by Dr. R. Greger, Freiburg, FRG.

Evaluation of data

Digitized recordings were evaluated by means of routines supplied by Krenz Electronics. Fits to mathematical expressions were performed using software from Wavemetrics (Igor). Means are given ±SD; n = number of experiments. Statistical significance was determined using Student's t-test for pair differences.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

ICl,H, a Cl- current that activates slowly on hyperpolarization

Electrophysiological properties of the muscle fibers of the locust jumping muscle already have been characterized partially (Walther et al. 1998). Briefly, there is a K+ current that increases on hyperpolarization (Zittlau and Walther 1991). The underlying conductance, GK,H, is partially activated at the K+ equilibrium potential (EK). It thus contributes significantly to the resting K+ conductance, GK,r, part of which, however, seems to be voltage independent (Walther et al. 1998). We now describe a second conductance that is also activated by hyperpolarization and that is modulated by octopamine. As in the accompanying investigation, one of our standard procedures was to clamp muscle fibers at EK (determined from the relaxations of IK,H) (cf. Fig. 2 of Walther et al. 1998) and to apply short hyperpolarizing commands to monitor the membrane resting conductance (e.g., Figs. 1 and 4). In this condition, any shift of the holding current (Ihold) will give information concerning the type of conductance that is changing.


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FIG. 4. Effects of octopamine on input conductance and holding current. Recordings are from 2 fibers. Holding potentials corresponded approximately to EK. Repetitive 20 mV × 40 ms hyperpolarizing commands were given at 6-s intervals. Top: holding current and miniature excitatory currents that render the lower edges of the traces noisy. Bottom: inward current levels attained during the short command pulses; the fast down- and upstrokes of the current pulses are not seen. Traces in A and B from 1 fiber loaded with Cl- ions diffusing from the recording electrode; the Cl- conductance, GCl,H (cf. Fig. 1), was therefore partially activated. A: 2 effects of octopamine overlapped, i.e., a reduction of the K+ resting conductance (arrowhead; see also C) and a more slowly developing enhancement of GCl,H. B: due to the presence of 2 mM BaCl2, the resting K+ conductance was strongly reduced; therefore only the effect of octopamine on GCl,H is seen, i.e., an increase in pulse amplitude and an inward shift of the holding current. Recording electrode at A and B was filled with 1 M K acetate plus 2 M KCl. C: from another fiber; the recording electrode was filled with 2 M K acetate so that GCl,H was not activated. Therefore only the reduction of the resting K+ conductance by octopamine is seen, i.e., a decrease in pulse amplitudes without a change of holding current. Effect of octopamine in this example did not recover completely on subsequent washing. Zero current levels are indicated (right). Horizontal bars mark the presence of 10-5 M octopamine, which, in each case, had been applied once before; hence no noticeable effects on the Na+/K+ pump occurred (c.f. Fig. 8).

When the recording electrode contained 3 M KCl, 30-60 min after impalement, the membrane resting conductance (Gr) usually started to rise. At the same time, the inward holding current at EK increased (Fig. 1). Both effects reached a maximum some 2-3 h after impalement and then started to decline. In parallel with these changes, an inward current developed that slowly activated during a hyperpolarizing command; it was followed by an inward tail current on jumping back to EK (Fig. 1, inset; cf. also Fig. 3C). This current resembled the K+ current, IK,H (cf. Fig. 6; suppressed by Ba2+ in the experiment shown in Fig. 1), which, however, has somewhat faster on- and off-relaxations. Because the current, as will be demonstrated, is carried by chloride, it will be subsequently referred to as ICl,H (or the underlying conductance as GCl,H). ICl,H also was observed in the distal-most bundle of the muscle region used here where the fibers are regularly innervated by the common inhibitory neuron. It is not restricted to the jumping muscle because its presence also was demonstrated in the mandibular closer muscle (Walther, unpublished observations).


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FIG. 3. Steady-state activation of the chloride conductance, GCl,H under various conditions. After activating 5-s prepulses to the voltages (Vpre) indicated on the abscissae the amplitudes of tail currents were measured on jumping to the approximate EK as shown for 1 example in C. Currents were converted to conductance, gtail, by dividing them by the driving force for Cl- ions. Cl- equilibrium potential was determined from the reversal potential of GABA-induced current (cf. Fig. 2). Hyperpolarization-activated K+ current, IK,H, was blocked due to the presence of 2 mM Ba2+. down-arrow , voltages at which activation is half-maximal. A: activation depends on [Cl-]i. open circle , [Cl-]i approx  28 mM due to accumulation of Cl- ions diffusing from the recording electrode; bullet , same fiber, [Cl-]i approx  16 mM after application of 1 mM GABA for 15 min, which led to efflux of Cl- ions. Figures of [Cl-]i were derived from the indicated Nernst potentials (ECl). Octopamine (10 µM) was present throughout the experiment. B: octopamine (10 mM) potentiates GCl,H. C: currents recorded during and after 5-s jumps from -73 mV to the voltages indicated. Note the inward tail currents. Octopamine present (10 µM); the data at B (black-square) were derived from these records. Traces were smoothed digitally. Recording electrode contained 3 M KCl at A and 2 M K+acetate plus 1.5 M KCl at B and C. Saline contained 1 mM glutamate both at A and at B (cf. METHODS).


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FIG. 6. Octopamine reduces the hyperpolarization-activated K+ current, IK,H. All panels show currents recorded from the same fiber during and after a long hyperpolarizing voltage jump to -115 mV. Thereafter, as denoted at A, the voltage was stepped either to -75 mV, i.e., the approximate EK, or to -55 mV. A: note the slow on-relaxation (indicated by the rectangular bracket to the left) and the outward tail current (off-relaxation) at -55 mV which are due to activation and deactivation of IK,H, respectively. B: 3 min after application of 10 µM octopamine the on- and off- relaxations of IK,H are both reduced by 30%; the instantaneous current due to the jump to -115 mV also is reduced, indicating a decrease of input conductance. C: IK,H is blocked by 2 mM Ba2+; there is a small inward relaxation at -115 mV, possibly due to an anion current. D: octopamine, in the presence of Ba2+, does not produce any change of current. Each trace represents the digitally smoothed average of 2 records. ···, 0 current levels; [, amplitudes of current relaxations. Both the current and the recording electrode were filled with K+ citrate.

When the recording electrode did not contain Cl-, but acetate or citrate instead, ICl,H did not develop. Occasionally during hyperpolarization a small inward current with a rather slow time course was noticed. This current, which was not affected by octopamine (Fig. 6; in contrast to ICl,H; cf. further), was not further investigated. ICl,H was not observed if preparations had been kept for >= 30 min in solutions where chloride was substituted for by an impermeant anion (gluconate or sulfate). Thus as first shown for a similar Cl- current in molluscan neurons (Chesnoy-Marchais,1983), ICl,H becomes measurable only if [Cl-]i is raised above the level in resting, unimpaled fibers (cf. further). The amplitude of the current, unfortunately, varied during prolonged recording, probably because of its dual dependence on [Cl-]i, i.e., via both driving force and activation.

CURRENT-VOLTAGE CHARACTERISTICS OF ICL,H. We performed tail current measurements to establish the I-V characteristics of the activated current and to determine its reversal potential. The tail currents were measured in the presence of 2-5 mM Ba2+ to block IK,H. As shown by the example in Fig. 2A, and found in six other experiments, the I-V characteristic of ICl,H is practically linear for the investigated range. Thus the flow of Cl- current through the activated conductance is more complex than permeation of ions through a simple pore in which case the Goldman equation predicts a slight outward rectification. The conductance does not rectify at ECl because with sufficiently positive potentials outward going ICl,H tail currents could be demonstrated (cf. Fig. 2A, inset). The reversal potentials, mostly determined by linear extrapolation of the measured I-V relations, ranged from -72 to -45 mV. The mean was -57 ± 9 (n = 10), and the average [Cl-]i, according to the Nernst equation and [Cl-]o = 188 mM, was 21 mM.

If ICl,H is carried solely by Cl- ions, its reversal potential should be the same as that of the GABA-induced Cl- current (ICl, GABA). The latter can be activated via synaptic and nonsynaptic receptors in this preparation (the significance of the latter is not clear) (Cull-Candy and Miledi 1981; Usherwood and Grundfest 1965). It differs from ICl,H by its pronounced outward rectification (Cull-Candy 1984) (Fig. 2B). The reversal potential of ICl,GABA was obtained by means of voltage ramps performed in the presence and absence of 0.5-1 mM GABA and subtraction of the induced currents. These experiments---which required certain precautions (see METHODS)---indeed indicated that ICl,GABA and ICl,H reversed at nearly the same potential (within ~2 mV; n = 3; Fig. 2; compare curves 1 in A and B). This was also true after [Cl-]i had been lowered (Fig. 2, curves 2) by prolonged application of 1 mM GABA, which produced a pronounced Cl- efflux. The parallel shifts of the reversal potentials of ICl,H and ICl,GABA were confirmed in four other experiments.

Inhibitory junction potentials (ijps), under appropriate conditions, are hyperpolarizing (Usherwood 1968; Usherwood and Grundfest 1965). Thus although in our investigations [Cl-]i was artificially raised, ECl in undisturbed fibers should be more negative than the resting membrane potential, Vr (presumably due to some sort of chloride transport) (e.g., Aickin et al. 1984). This was confirmed by a series of measurements. Using acetate in the recording electrode, we determined ECl 5-10 min after impalement of fibers. To obtain the likely resting membrane potential the voltage measured on impalement was corrected for leak-induced depolarization (cf. METHODS in Walther et al. 1998). Seven of 10 measurements performed in the presence of glutamate indicated that ECl was ~10 mV (10.9 ± 4.4 mV) more negative than Vr, i.e., -65.8 ± 4.8, and [Cl-]i thus 10 mM. In the remaining three fibers, the measured difference was only -3 mV or less.

ACTIVATION OF GCL,H. The different slopes of the I-V curves 1 and 2 in Fig. 2A indicate that the conductance GCl,H decreases if [Cl-]i is reduced (n = 5). To further investigate this [Cl-]i dependence and to establish a basic characterization of GCl,H, its steady-state activation was established in the absence of the K+ current, IK,H. For this purpose, activating jumps to increasingly negative potentials were performed and the tail currents measured which occurred on jumping back to a fixed potential (~EK; Fig. 3C). ECl was derived, as above, from a short application of GABA. The amplitudes of the tail currents were converted to conductance. Data were fitted (Fig. 3A) according to the Boltzmann relation
<IT>G = G</IT><SUB>max</SUB>(1/{1 − exp[(<IT>V − V</IT><SUB>0.5</SUB>)/<IT>S</IT>]}) (1)
where Gmax is the maximal conductance, V0.5 the voltage at which activation is half-maximal, and S the slope factor. From 16 Cl--loaded muscle fibers, we obtained the following mean activation parameters: Gmax = 0.84 ± 0.46 µS; V0.5 = -103.5 ± 4.5 mV; and S = 8.9 ± 1.51 mV. The average ECl in these fibers was -54.8 ± 4.5 mV. This corresponds to a mean [Cl-]i of 23.0 mM ([Cl-]o = 188 mM). In this situation activation of GCl,H became measurable only at voltages some 10 mV negative from ECl. Activation was half-maximal at a voltage ~50 mV more negative than ECl, and it was still incomplete at -125 mV (Fig. 3, A and B).

 
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TABLE 1. GABA-induced reduction of [Cl-]i alters activation of GCl,H

When [Cl-]i was lowered by prolonged GABA-application, Gmax was considerably reduced. In addition, the activation curve was shifted on the voltage axis in the negative direction (Fig. 3A; Table 1). The extent of this shift was similar to that of ECl. Thus the internal Cl- concentration somehow affects the mechanism of activation. That Gmax for inward Cl- current (i.e., Cl- ions leaving the cell) is reduced as [Cl-]i is lowered seems to be conceivable, yet there is no simple model from which the extent of this reduction can be predicted. As seen from Table 1, the percent reduction of Gmax was at least of the same order of magnitude as the percent reduction of [Cl-]i. These measurements, in addition, demonstrate that at the membrane resting potential, which under our conditions was at EK (cf. Walther et al. 1998) and thus between -70 to -75 mV, ICl,H becomes just about measurable if [Cl-]i is raised to ~25 mM (cf. control in Fig. 3B).

The time course of ICl,H was not systematically investigated because ICl,H often was not sufficiently large and stationary for carrying out a detailed study. Usually deactivation of ICl,H could be fitted by a single exponential (data not shown), whereas for activation, such fits slightly but systematically deviated from the measurements. The time constant of deactivation at V approximately equal to -75 mV ranged from 0.3 to 0.6 s (mean: 0.42 ± 0.14 s; n = 6). Experiments using -75 and -85 mV as test voltages indicated that activation and deactivation, at least at these voltages, follow a similar time course. Activation at -95 mV was not as fast as deactivation at -75 mV, i.e., the shorter of the two time constants (tau 1) obtained from a double exponential fit was 0.76 ± 0.19 s (same 6 fibers as above; the other, rather variable time constant, tau 2, was generally of the order of 10 s). Thus kinetics of ICl,H seem to become more rapid at more positive voltages (cf. also inset of Fig. 2A). After reduction of [Cl-]i, in three of the four experiments summarized in Table 1, the time course of ICl,H was slightly slower, i.e., tau 1 was increased by 15 ± 4%. This tendency corresponds to the dependence of kinetics on [Cl-]i previously established for of a related Cl- current in Aplysia neurons (Chesnoy-Marchais 1983).


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FIG. 5. Octopamine induces the hyperpolarization-activated Cl- current, ICl,H. All panels show currents recorded from the same fiber during and after a long hyperpolarizing voltage jump denoted at A. A: hyperpolarization causes as small slow inward current relaxation due to activation of IK,H; there is no tail current on jumping back to -75 mV, the approximate EK. B: after 12 min application of 20 µM octopamine, the hyperpolarizing pulse produces a much larger slow inward current than in A; now there is also an inward tail current. C: IK,H is blocked by 2 mM Ba2+, and there are no slow current relaxations. D: octopamine has induced the Cl- current, ICl,H, the slow relaxations of which are unmasked due to the presence of Ba2+. Because the I-V characteristic of ICl,H is linear (cf. Fig. 2), the approximate ECl can be calculated from the magnitude of the on- and the off-relaxation (Ion and Ioff), marked by the rectangular brackets at D, according to: (75 × Ion - 115 × Ioff)/(Ioff - Ion) = -45 mV. Each trace represents the digitally smoothed average of 2 records. ···, 0 current levels. Recording electrode was filled with KCl. Measurements started 45 min after impalement of the fiber.

 
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TABLE 2. Effect of octopamine (10-5 M) on activation parameters of GCl,H

PHARMACOLOGICAL CHARACTERIZATION OF ICL,H. A small selection of known chloride conductance blockers was tested by investigating their effects on ICl,H tail currents recorded at EK. Approximately 50% reduction was achieved by 1 mM SITS, by 0.1 mM DIDS, or with 10 mM SCN-. NPPB (10-4 M) (Greger 1990) produced ~20% block (10-5 M had no effect). Anthracene-9-carboxylic acid (0.1 mM) had no effect. Ni2+ (1 mM) and 0.1 mM Zn2+ effectively blocked ICl,H, whereas 2 mM Ba2+ (routinely used to suppress K+ currents) or 2 mM Cd2+ reduced ICl,H at most by ~5%. Most of the agents that did block ICl,H also had effects on the resting K+ conductance.

Modulatory effects of octopamine

To get some first indications how octopamine might affect the resting membrane currents of the muscle, fibers were clamped at EK and repetitive hyperpolarizing commands applied. The effects of octopamine on the membrane resting conductance, Gr, and the holding current were variable, particularly if a KCl-electrode was used for recording (Fig. 4A). However, applying octopamine under conditions in which either IK,H or ICl,H were eliminated led to more consistent results, indicating that GCl,H was enhanced (Fig. 4B) and the resting K+ conductance, GK,r was reduced (Fig. 4C). These responses to octopamine overlapped (Fig. 4A), yet the change of GK,r was faster than that of GCl,H (compare Fig. 4, B and C). The time to 50% effect was 25 ± 13 s for GK,r and 196 ± 42 s for GCl,H; on washing, the time to 50% decline was 2.5-3 min for GK,r and 5-10 min for GCl,H (figures based each on sets of 8 measurements; 10 µM octopamine was applied for 5-10 min under conditions where GCl,H or GK,r, respectively, were eliminated). These effects of octopamine also were observed in Ca2+-free saline.

On a first application of octopamine, there was often a slight outward shift of Ihold, which could not be attributed to modulation of either current. Obviously yet another current must have been affected, which, as will be shown, is caused by the electrogenic Na+/K+ pump. It should be pointed out that octopamine, for unknown reasons, in approx 20% of the trials was practically ineffective.

EFFECTS ON CL- CONDUCTANCE. When a fiber had been loaded with Cl- ions, application of octopamine induced or potentiated ICl,H (Fig. 5; also seen from comparison of measurements 2 and 3 in Fig. 2A). This often led to an inward shift of Ihold and an increase in Gr (Fig. 4B). The increase in GCl,H, judged by the amplitudes of tail currents at EK, varied considerably. Often it was more than threefold when the electrodes had been in the fiber for periods of 1-2 h before octopamine was applied. Octopamine did not cause a consistent change of ECl (Table 2), although in some cases, a small negative shift occurred if octopamine induced a particularly large increase in ICl,H.

The effects of octopamine on GCl,H were further characterized by activation measurements (Fig. 3B, Table 2). Octopamine (10 µM) led to an increase in the extrapolated maximal conductance by 40-140% (mean approx 100%). In addition it shifted the midpoint of the activation curve (V0.5) in the positive direction although only to a small, variable extent. The mean difference between V0.5 and ECl was reduced slightly, on average by 8.0 ± 4.4 mV (statistically significant: P < 0.01). Thus in the presence of octopamine a lower intracellular chloride concentration and a smaller hyperpolarization should suffice to induce a sizeable ICl,H and at ECl, there may be already a slight activation. The change of the activation curve roughly predicted the observed inward shift of Ihold at about -75 mV and the increase in the amplitudes of the current pulses routinely elicited by short negative going voltage jumps (as in Fig. 4B).

Activation and deactivation of ICl,H were accelerated by octopamine; the corresponding time constants, at -95 and -75 mV, respectively (cf. earlier text), were reduced by 33 ± 18% and 17 ± 10%, respectively (n = 6).

EFFECTS ON K+ CONDUCTANCE. In the absence of ICl,H, 10 µM octopamine reduced the resting conductance, Gr (Fig. 4C), by 25.0 ± 12% (n = 15). Ramp measurements indicated that a conductance was suppressed that had a slightly outward rectifying I-V characteristic (not shown; similar to Fig. 1D of Walther et al. 1998). The current flowing through this conductance reversed close to EK provided there was no effect on the electrogenic Na+/K+-pump current (cf. further). The reversal potential changed by ~17 mV when [K+]o was increased from 10 to 20 mM (n = 2; the Nernst equation predicts 18 mV at 30°C), indicating that octopamine reduced the resting K+ conductance, GK,r. It also reduced IK,H (or GK,H), i.e., the K+ current (or conductance) that slowly activates on hyperpolarization (Fig. 6). Octopamine (10 µM) reduced IK,H by 43 ± 17% (n = 11; based on 2.7 s test pulses going 20 mV negative from EK). In the presence of 2 mM Ba2+, which blocks GK,H and GK,r (Walther et al. 1998), these effects of octopamine were occluded. After prolonged (>10 min) applications of octopamine, there was often only a partial recovery of GK,r and GK,H on 30-60 min washing, but even after short applications recovery was not always complete (e.g., Fig. 4C).

To characterize how octopamine affects IK,H, tail current measurements were performed as detailed previously (Walther et al. 1998). Briefly, activating jumps to potentials between -65 and -125 mV were performed to activate the current. The amplitudes of the outward going tails on jumping back to a fixed test potential were taken as a measure of the activation attained during the prepulses. Data were fitted according to
<IT>I = I</IT><SUB>max</SUB><IT>×</IT>(1/{1 − exp[(<IT>V − V</IT><SUB>0.5</SUB>)/<IT>S</IT>]}
− 1/{1 − exp[(<IT>V</IT><SUB>test</SUB><IT>− V</IT><SUB>0.5</SUB>)/<IT>S</IT>]}) (2)
where Imax is the maximal current, V0.5 the voltage at which activation is half-maximal, S the slope factor, and Vtest the potential at which the tail currents were measured. The second term accounts for the finding that IK,H at the test potential (around -55 mV; actually the holding potential) is usually not completely deactivated (Walther et al. 1998).

Figure 7 gives an example and Table 3 summarizes the findings. It should be pointed out that these figures are not representative for the average fiber because for these measurements, fibers with fairly large resting conductances were selected and because the experiments were aborted if octopamine did not produce a sizeable effect. Octopamine always decreased the maximal current. It also shifted the activation curve in the negative direction, but this effect was rather variable (7-21 mV shift in 3 fibers with an initial V0.5 of approximately equal to ±87 mV; practically no shift in 2 fibers with an initial V0.5 of approximately equal to ±100 mV). The time course of IK,H during a jump to EK - 20 mV was somewhat slower in the presence of octopamine, a point not further investigated here.


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FIG. 7. Octopamine modifies the activation of the hyperpolarization-activated K current, IK,H. Amplitudes of outward-going tail currents at -55 mV (cf. Fig. 6A), recorded after 2 s jumps to the voltages indicated on the abscissa, are shown for 1 cell. Data points are averages from 2 successive runs. Fits were calculated according to Eq. 2 (cf. RESULTS). Inset: complete theoretical steady-state activation curves calculated by means of the Boltzmann relation I = Imax × (1/{1 - exp[(V - V0.5)/S]}). Fit parameters were for control: Imax = 32.4 nA, V0.5 = -103 mV, and S = 23 mV; and for octopamine: Imax= 18.6 nA, V0.5 = -101 mV, and S = 21 mV. Circles mark midpoints of curves. Octopamine in this cell practically did not shift the activation curve on the voltage axis but reduced the maximal current. Recording electrode filled with 2 M K acetate.

 
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TABLE 3. Effect of octopamine (10-5 M) on activation parameters of IK,H and on resting membrane conductance, Gr

From the changes of the activation parameters, one can estimate the reduction of the resting K+ conductance, GK,r, which should result if octopamine acted solely on GK,H. By comparing activation curves constructed with the mean fit parameters obtained before and after application of octopamine (same fibers as in Table 3), one arrives at a figure of 0.28 µS. This has to be corrected both for incomplete activation and for Goldman rectification (cf. Walther et al. 1998), yielding ~0.25 µS. This figure is nearly 50% less than the mean decrease of 0.44 µS measured for the resting conductance (cf. Table 3). Even though the accuracy of such calculations may not be high the discrepancy suggests that octopamine, in addition to GK,H, also reduces the voltage-independent component of GK,r.

CONCENTRATION DEPENDENCE OF OCTOPAMINE EFFECTS ON CL- AND K+ CONDUCTANCES. The effects of octopamine on GK,r and GCl,H did not appear to desensitize. The threshold for modulation of GCl,H was ~10-9 M and the saturating concentration probably somewhat higher than 10-5 M (n = 3). The concentration dependence of the effect on GK,r seemed to be similar. This compares well to the concentration range over which octopamine, in this muscle, raises the level of cyclic AMP (Evans 1984a). cAMP seems to be involved in the modulation of both conductances (see further).

EFFECTS ON THE ELECTROGENIC NA+/K+ PUMP. Under resting conditions there is a steady outward current of 1-2 nA, which is blocked reversibly by dihydro-ouabaine (DHOu; Fig. 8B). It thus seems to be produced by the Na+/K+ pump. Octopamine enhances this current according to the following observations: on its application often (in more than half of the experiments), a transient outward shift of Ihold by 1-3 nA was noticed (Fig. 8A) if the fiber was clamped at the approximate EK. The shift attained a maximum after 0.5 min and then largely declined within 2 min. There were usually no significant concomitant conductance changes. Generally a second application of octopamine, performed 20-40 min after the first one, was practically ineffective (Fig. 8A, right).


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FIG. 8. Octopamine stimulates the electrogenic sodium pump. Recordings from 2 fibers voltage-clamped to ~EK in the presence of 2 mM Ba2+. Recording electrodes in both experiments were filled with 2 M K acetate. Under these conditions GK and GCl,H were eliminated and could not account for changes of the holding current. A: 1st application of 10-5 M octopamine led to a transient outward shift of the holding current (initial level = -3 nA). Second application, after 15 min of washing (part of the record is not shown), had a much smaller effect. Repetitive inward current pulses, resulting form 20 mV × 40 ms hyperpolarizing commands, were not affected by octopamine (i.e., the input conductance remained constant). B: fiber from another preparation. First application of 5 × 10-4 M dihydro-ouabaine (DHOu) led to a reversible inward shift of the holding current (initial level = -3.5 nA). Second application of DHOu, now in the presence of 2 × 10-5 M octopamine, had a more pronounced effect. Due to the longer application of DHOu and the presence of octopamine, withdrawal of DHOu led to a rebound outward shift of the holding current that was accompanied by a slight increase of the input conductance (cf. RESULTS). After the first application of DHOu, such effects were barley noticeable. Holding potential was -70 mV at A and -73 mV at B.

The shift of Ihold cannot be due to an effect on a K+ conductance (because Vhold approx  EK). Accordingly this shift also was noticed when octopamine was applied in the presence of Ba2+ (like in the example of Fig. 8A), i.e., when GK,r was strongly reduced. It cannot be due to a change of a Cl- conductance either because these experiments were performed with acetate in the recording electrode. Thus ICl,H was absent and ECl should have been ~10 mV negative from Vhold (cf. preceding text). If the outward current were due to an increase of some other Cl- conductance, the conductance increase should have been large enough to be recognized from the short current pulses in Fig. 8A (or larger than the increases in Fig. 8B; for these as well as for the slight pulse reductions induced by DHOu we presently have no explanation).

That octopamine stimulates the Na+/K+ pump also is supported by experiments in which octopamine was applied in the presence of DHOu (Fig. 8B). DHOu (5 × 10-4 M; a nearly saturating concentration) produced a considerably greater effect if applied soon after octopamine, i.e., when the outward shift of Ihold induced by the latter had not yet declined (Fig. 8B). After washing off DHOu, there appeared to be a rebound outward shift of Ihold (Fig. 8B). This may indicate an enhanced pump rate due to an increased [Na+]i.

Intracellular mediation of the octopamine effects

CYCLIC NUCLEOTIDES AND A PHOSPHODIESTERASE-BLOCKER MIMIC THE EFFECTS OF OCTOPAMINE ON K+ AND CL- CONDUCTANCES. It has been well established that octopamine raises the concentration of cyclic AMP in locust jumping muscle, particularly in the distal part used for our present study (Evans 1984a, 1985b). We therefore tested whether membrane permeant cAMP analogues produce effects similar to those described above for octopamine. As demonstrated by Fig. 9, A and B, this in fact was observed with 8-bromo-cAMP (n = 15) but also with 8-bromo-cGMP (n = 14). This nucleotide acted at least as potently as the former. At 5 × 10-4 M both nucleotides reduced GK,r as indicated by the initial reduction of pulse amplitudes (Fig. 9, A and B) and the amplitude of IK,H (not shown). They also enhanced GCl,H as indicated by the slow increase in pulse amplitudes and the increased amplitudes of ICl,H tail currents (Fig. 9, A and B, left and right, respectively). The respective dibutyryl analogues acted similarly but were much less effective than the 8-bromo-compounds.


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FIG. 9. Cyclic nucleotides and a phosphodiesterase-blocker mimic the actions of octopamine on K + and Cl- conductance. Left: chart recordings from 3 voltage-clamped fibers showing the holding current at Vhold approx  EK and the repetitive inward current pulses caused by 20 mV × 80 ms hyperpolarizing commands given at 6-s intervals. Calibration marks (10 nA) and 0 current levels are shown (right). Right: digitized recordings of currents elicited by a 44 mV × 2.7 s hyperpolarizing command schematically depicted underneath A and applied at the times indicated at the chart recordings by a and b. For clarity of presentation, the pairs of traces were aligned (by subtraction of the holding currents) so that they start from the same level. Inward current relaxations result from activation of a K+ and a Cl- current, IK,H and ICl,H. Inward tail currents that flow on stepping back to Vhold are solely due to deactivation of ICl,H (because Vhold approx  EK). Calibration bars: 40 nA. A: 5 × 10-4 M 8-bromoadenosine 3':5'-cyclic monophosphate (8-bromo-cAMP); B: 5 × 10-4 M 8-bromoguanosine 3':5'-cyclic monophosphate (8-bromo-cGMP); and C: 1 × 10-4 M IBMX. Drugs essentially produce the same 2 overlapping effects. 1) Current pulses initially become smaller (arrowheads) while there is little change of the holding current; this indicates a reduction of the resting K+ conductance. 2) With a slower time course, the current pulses re-increase and the holding current shifts inwardly. This results from increased activation of the hyperpolarization-activated Cl- current, ICl,H as indicated by the increased inward current on hyperpolarization and the comcomitant increase in the inward tail current (right). Response pattern at A probably indicates that Vhold was slightly more negative than EK. Recording electrodes were filled with 3 M KCl. For cyclic nucleotides (or IBMX), rough average figures from 9 to 15 experiments were 13% (13%) initial reduction of Gr (cf. arrowheads); after 12-15 min, which was the approximate time until steady state was reached, 15% (12%) net increase in Gr, -5 nA (-4 nA) inward shift of holding current, and 200% (150%) increase in ICl,H-tail current amplitude. On washing, the effects subsided within 20-30 min.

The unspecific inhibitor of phosphodiesterases, IBMX (10-4 M) produced much the same effects as the cyclic nucleotides (Fig. 9C; n = 9). It has to be pointed out that the different patterns and magnitudes of the examples shown in Fig. 9 are not related to differences between the applied drugs but rather reflect variation typically seen with each of the drugs (see legend of Fig. 9 for quantitation of effects). Any of these drugs occasionally was practically ineffective.

Although the effects of cyclic nucleotides and IBMX developed more slowly than those of octopamine, there was a similarity with respect to their relative time courses of actions. The change of GK,r developed and reversed (on washing) more quickly than that of GCl,H (cf. Fig. 9, left). We tried to find out whether cyclic nucleotides or IBMX also had an effect on the Na+/K+ ATPase but failed to obtain conclusive results.

EFFECTS OF PROTEIN KINASE AND PHOSPHATASE INHIBITORS. The different time courses observed for the modulation of GK,H and GCl,H suggest that the steps leading, after the rise of [cAMP] (or [cGMP]), to a conductance change are not the same for GK,H and GCl,H. We therefore tested whether H89, a highly specific inhibitor of protein kinase A (e.g., Geilen et al. 1992), suppresses the octopamine effects on GK,H or GCl,H. H89 (5 × 10-5 M), in fibers that had not previously been exposed to octopamine (cf. further), had rather marked effects on its own (Fig. 10). It increased the resting conductance by <= 250%. Ihold remained nearly constant; the slight changes usually seen probably occurred because Vhold was not precisely at EK. Thus H89 specifically affects GK,r. This is supported by the fact that in the presence of 2 mM Ba2+ the effects of H89 were almost entirely occluded. This effect of H89 is complementary to the above observation that IBMX led to a reduction of GK,r. Both findings can be explained by a basal activity level of adenylyl cyclase and, therefore, of protein kinase A (PKA), restricting steady-state activation of GK,r.


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FIG. 10. Effects of H89, a blocker of protein kinase A, on the membrane resting conductance and the hyperpolarization-activated K+ current, IK,H. Holding potential, -72 mV. Repetitive 20 mV × 80 ms hyperpolarizing commands were given at 6-s intervals. Top: holding current and miniature excitatory currents, which render the lower edges of the trace noisy. Bottom: inward current levels attained during the short command pulses; the fast down- and upstrokes of the current pulses are not seen. Increased amplitude of these current pulses in the presence of H89 indicates an increase in resting K+ conductance because there was only a small concomitant shift of the holding current; probably EK was slighlty more positive than the holding potential. Before and during application of H89 single prolonged (2.7 s × 20 mV) voltage jumps were performed that elicited the currents (*) shown on an expanded time scale. Rectangular brackets indicate the amplitudes of IK,H relaxations which were clearly reduced after 12 min application of H89. Recording electrode contained 2 M K acetate.

H89 did not increase the amplitudes of IK,H relaxations elicited by negative going voltage jumps from EK; on the contrary, it always reduced them (cf. single pulses in Fig. 10). A reduction of IK,H amplitudes at voltages below EK would be expected if H89 shifted the activation curve of this current to more positive potentials. In that case, activation already would approach saturation at EK, and steps to more negative potentials would cause only little further activation. We attempted to investigate, by means of tail current measurements, whether H89 in fact has such an effect on activation. Although three experiments with 2 × 10-5 M H89 (which increased the resting conductance by only ~35%) seemed to support this hypothesis (H89 leading to a shift of V0.5 from -97 ± 5 to -86 ± 1 mV), this finding can only be regarded as preliminary. The problem is, that, particularly at the higher concentration of H89, the increased inward (or outward) K+ currents obviously led to pronounced K+ depletion (or accumulation) in the transverse tubules, which under normal conditions occurs only on a small scale (Walther et al. 1998). This explains various anomalies (e.g., unusual time courses of IK,H) observed in the presence of H89 and renders activation measurements in most cases impossible.

Interestingly, the effect of H89 on GK,r depended on whether a preparation was first treated with octopamine or not. In untreated preparations, H89 (20 of 21 experiments) led to the described conductance increase (by 75 ± 50%; figures referring to total resting conductance). However, if applied 15-30 min after a challenge with octopamine (10-5 M for ~10 min), H89 produced a much weaker conductance increase (by 32 ± 14%; n = 6) or even a slight reduction (by 11 ± 7%; n = 5). Presently we have no explanation for these aftereffects of octopamine.

H89 (5 × 10-5 M) inhibited the action of octopamine on GK,r. If octopamine was applied when H89 already was present for 10 min, it had no significant effect or caused a slight increase in GK,r (8 of 9 experiments). Likewise, when H89 was added 10-20 min after octopamine already had been applied (and still was present), GK,r re-increased up to or beyond control level (n = 6). H89 did, however, not clearly affect the modulation of GCl,H by octopamine. In four of five experiments, it did not block or reduce the octopamine-induced potentiation (in 1 there was a slight reduction). It also did not block the increase in GCl,H-amplitude induced by 8-bromo cyclic nucleotides. H89 on its own had no consistent and significant effects on GCl,H (n = 8). Thus PKA seems to be involved in the octopamine-dependent modulation of the K+ conductance but not of the Cl- conductance.

We investigated the effects of two inhibitors of type 1 and 2A protein phosphatases to further test this hypothesis, but the results were not conclusive. There should be a basal rate of dephosphorylation, compensating for phosphorylation, and on inhibition of phosphatases phosphorylation should become more effective. Thus one might expect that GK,r, but not GCl,H, will be reduced on application of okadaic acid (2.5 µM) or cantharidin (250 µM). The effects of these inhibitors (n >=  2 experiments for each combination) were similar: both of them slowly reduced GK,r as well as GCl,H. The reductions induced by okadaic acid amounted roughly to -75% within 20 min (longer applications were not tested) and completely reversed within 1 h of subsequent washing. Cantharidin produced somewhat smaller effects within 15-20 min. During 1 h of subsequent washing, however, GK,r and GCl,H further decreased, to <= 10% of control. 2 h of further washing led only to ~50% recovery. These effects may indicate that cantharidin both enters and leaves the cells more slowly than okadaic acid. One explanation for the findings with these phosphatase inhibitors would be that the channels (or some protein(s) controlling them) bear multiple phosphorylation sites and that phosphorylation of some of them is essential for the channels to be capable of opening.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

We have demonstrated that at voltages around the resting membrane potential a hyperpolarization-activated Cl- conductance, GCl,H, becomes activated if [Cl-]i rises above normal resting levels. Octopamine increases GCl,H, but it reduces the resting K+ conductance. These effects are mediated by the rise of [cAMP], which is induced by octopamine in this muscle (Evans 19841,b). It remains to be shown whether a third octopamine effect, i.e., stimulation of the electrogenic sodium pump, is also mediated by cAMP or by a different signal. The effects of octopamine presented here, including potentiation of a voltage-dependent Ca2+ current (Walther, unpublished observations), add to the previously established octopamine-mediated potentiation of transmitter release and modification of excitation-contraction coupling (Evans and Myers 1986; O'Shea and Evans 1979; Walther and Schiebe 1987). Presently it is difficult to assess how the various effects may act in concert and, therefore, we will regard them separately.

HYPERPOLARIZATION-ACTIVATED CL- CONDUCTANCE, GCL,H, AND ITS MODULATION BY OCTOPAMINE. Cl- conductances activating on hyperpolarization exist in various genera of invertebrates, e.g., mollusks (Brezina et al. 1994; Chesnoy-Marchais 1983) and probably crayfish (e.g., Reuben et al. 1962), as well as vertebrates (e.g., Thiemann et al. 1992). They occur in many tissues (Jentsch et al. 1995; see also Bretag 1987 for chloride conductances in skeletal muscles). Common features of these conductances are their slow activation and deactivation (both taking seconds), the lack of inactivation, the dependence of activation on the difference between membrane potential and ECl, and, in several examples, a nearly linear instantaneous I-V relationship. For GCl,H of locust muscle and for the Cl- conductance in Aplysia neurons, the voltage for half-maximal activation (V0.5) is considerably more negative than ECl, i.e., by ~45 mV in locust (Table 1) and by ~35 mV in Aplysia (estimated from Fig. 2 of Chesnoy-Marchais 1983). Furthermore, in both preparations the steady state maximum conductance increased and decreased with [Cl-]i. In these respects both Cl- conductances differ from ClC2 (Jentsch et al. 1995), a Cl- conductance that is expressed for example in certain vertebrate neurons (Smith et al. 1995). V0.5 of ClC2 is only 15 mV more negative then ECl and its open channel conductance depends on [Cl-]i in a manner predicted by the Goldman equation (Staley 1994).

In all these examples, activation starts off only at voltages more negative than ECl. In resting muscle cells of the locust, according to our measurements, ECl was slightly more negative than the membrane potential (approx EK). The situation in vivo seems to be similar (Usherwood 1968), probably due to some outward transport of Cl- ions. Hence GCl,H should not contribute to the resting conductance and should activate only when ECl becomes more positive than the resting potential. Perhaps this occurs on dehydration of the animal, i.e., if the cells lose water, leading to an increased internal ion concentration and, accordingly, some hyperpolarization. Thus GCl,H may be relevant to regulation of cell volume as suggested for related Cl- conductances in vertebrates (e.g., Thieman et al. 1992).

GCl,H may, however, also play a role in other membrane events. For example, under some circumstances GCl,H or an interaction of GCl,H and GK,H might lead to slow oscillations of membrane potential. In the jumping muscle, slow oscillations occur in a specialized bundle capable of rhythmical spontaneous contractions (e.g., Burns and Usherwood 1978). One also wonders whether GCl,H may support GABA-mediated postsynaptic inhibition of the muscle cells, particularly because GCl,H is potentiated by octopamine. In mammalian hippocampus, those neurons that exhibit GABAA-receptor-mediated postsynaptic inhibition rather than paradoxical excitation express the Cl- conductance, ClC2 (Smith et al. 1995). ClC2 prevents Cl- from accumulating in the cells during intense synaptic activity (Staley 1994; cf. also Staley et al. 1996). In locust jumping muscle, such a possibility also may be envisaged even though one could argue that only a fraction of them is innervated by an inhibitory neuron, whereas GCl,H probably occurs in all its fibers.

Information about the firing patterns of the common inhibitory neuron CI1 (Burns and Usherwood 1979; Wolf 1990), and the inhibitory synaptic currents elicited by the CI1 neuron (Cull-Candy 1984) allow us to estimate the order of magnitude for synaptic Cl- influx into a muscle fiber during walking. Importantly, CI1 neuron and the slow excitatory neuron fire largely during the same phase of a step cycle. Thus because of excitatory synaptic depolarization, Cl- flux should mostly be inward, even if ECl becomes more positive than the resting potential. At a moderate walking speed (2 steps/s), the mean firing frequency of CI1 is ~20 Hz (Wolf 1990; Wolf personal communication). In the absence of compensating outward flow or transport of Cl- ions, this would lead, within 1 min, to a rise of [Cl-]i by ~1 mM (assumptions: fiber of 100 µm diameter and 1.5 mm length; cytosol amounting to roughly 50% of fiber volume; average driving force for Cl- ~20 mV; average charge per inhibitory junctional potential ~0.25 nC). This is only ~10% of resting [Cl-]i in vivo (assumptions: physiological resting membrane potential ~10 mV positive from EK approximately equal to -75 mV and initial ECl ~10 mV negative from resting potential, thus ECl around -75 mV and [Cl-]i ~ 10 mM). Considerably greater increases are to be expected on fast walking when the firing frequency of the CI1 neuron is elevated. An increase of only 4 mM would suffice to shift ECl (from -75 mV) by 10 mV and thus to reduce the efficiency of postsynaptic inhibition significantly.

This suggests that a mechanism to avoid Cl- loading during a high rate of synaptic inhibition would be advantageous. However, will [Cl-]i rise high enough during the periods of activity and will the membrane potential, during the intervals, become sufficiently negative to warrant activation of GCl,H? This is difficult to decide without more quantitative knowledge of various parameters under in vivo conditions including the efficiency of the Cl- transporter postulated above. It is tempting to speculate that GCl,H at least will come into the play when octopamine shifts its voltage dependence slightly positive and greatly potentiates its amplitude. A rise in octopamine concentration during a high firing rate of the slow excitatory motoneuron is likely to occur because the octopaminergic neuron innervating the jumping muscle (DUMETi) is active in this condition (Pflüger, personal communication). In addition some of the octopamine released elsewhere into the hemolymph (e.g., at neurohemal organs) (Evans 1985a) may circulate into the leg.

Modulation of Cl- conductances resembling GCl,H has been demonstrated previously both in vertebrates (hippocampal CA1 neurons: increase with norepinephrine, decrease with phorbol esters) (Madison et al. 1986; Staley 1994) and invertebrates (Aplysia neurons: increase with the peptide FMRFamide, reduction with serotonin) (Lotshaw and Levithan 1987; Thompson and Ruben 1988). Octopamine most probably potentiates GCl,H via the increase of cAMP (cf. Fig. 9A), and some as yet unidentified modulator might affect GCl,H via cGMP (cf. Fig. 9B). The subsequent steps do not seem to involve activation of PKA as on modulation of GK,r (cf. further) because the cyclic nucleotides did not act with the same time course on K+ and Cl- conductance and an inhibitor of PKA did not prevent the potentiation of GCl,H by octopamine. Perhaps the cyclic nucleotides affect the Cl- channels directly by some binding step as has been demonstrated for a variety of channels (Finn et al. 1996).

MODULATION OF THE RESTING K+ CONDUCTANCE BY OCTOPAMINE. Octopamine, like the peptides (YGG)FMRFamide and proctolin, reduced GK,r. All three substances consistently affected the activation curve of the voltage dependent component, GK,H (for the relationship between GK,H and GK,r, cf. Walther et al. 1998), by reducing Gmax and, to a variable extent, by a positive shift on the voltage axis. As in the case of GCl,H, octopamine's effects on GK,r seem to result from the rise of [cAMP]. However, the results obtained with an inhibitor of PKA strongly suggest that the subsequent steps involve phosphorylation via PKA. That both cAMP and cGMP reduce GK,r may be due to a susceptibility of PKA to both nucleotides (like in honeybee) (Altfelder and Müller 1991). There is a substantial tonic downmodulation of GK,r even in the absence of octopamine because inhibitors of phosphodiesterase or PKA on their own caused pronounced effects on GK,r (decrease and increase, respectively; Figs. 9C and 10). This opens the interesting possibility that some modulator(s) may enhance GK,r e.g., through activation of a phosphatase (like in certain Ca2+ activated K+ channels) (White et al. 1991).

MODULATION OF THE ELECTROGENIC SODIUM PUMP BY OCTOPAMINE. Neither the increase in GCl,H nor the reduction in GK,H can account for the spurious hyperpolarizations previously noted on application of octopamine in this (e.g., O'Shea and Evans 1979; Walther and Schiebe 1987) and other insect muscle preparations (e.g., Fitch and Kammer 1986). Most probably they result from the enhanced activity of the Na+/K+ pump. That this octopamine effect usually fades within a few minutes might be due to negative feed back, i.e., a fall of [Na+]i, or/and receptor desensitization. A stimulatory effect of octopamine on the sodium pump was previously noted in an insect mechanoreceptor (Zhang et al. 1992).

During a high level of phasic muscle activity, the concomitant release of octopamine from the neurosecretory endings of DUMETi may lead, among other consequences, to more effective coping of the muscle cells with massive ion fluxes. Na+ influx due to excitatory synaptic currents, Ca2+ influx during action potentials (cf. Pichon and Ashcroft 1985; Washio 1972) and K+ efflux due to delayed rectifier and Ca2+ activated K+ currents (Walther, unpublished observations) certainly put increased demand on the Na+/K+ pump, assuming that extrusion of Ca2+ occurs at least partly via Na+ dependent transport. Presently it is not clear whether the transient polarizing effect of enhanced pumping is of relevance. It might support the activation of the hyperpolarization-activated currents and possibly potentiate low threshold Ca2+ current(s) by reducing inactivation.

    ACKNOWLEDGEMENTS

  We thank Drs. J. E. Brayden, C. Erxleben, and D. Wicher for commenting on the manuscript and G. Karger for technical assistance. We also thank Dr. H. Achenbach for providing some data from experiments performed during a research student training program in our laboratory.

  This work was supported by the Deutsche Forschungsgemeinschaft (Grant Wa 223/4-1,2,3 to C. Walther and a Postoctoral Fellowship to K. E. Zittlau).

    FOOTNOTES

  Address for reprint requests: C. Walther, Physiological Institute, University of Marburg, Deutschhausstrabeta e 2, 35037 Marburg, Germany.

  Received 26 August 1997; accepted in final form 25 January 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society