Substance P Regulates Ih via a NK-1 Receptor in Vagal Sensory Neurons of the Ferret

M. Samir Jafri and Daniel Weinreich

Department of Pharmacology and Experimental Therapeutics, University of Maryland, School of Medicine, Baltimore, Maryland, 21201-1559

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Jafri, M. Samir and Daniel Weinreich. Substance P regulates Ih via a NK-1 receptor in vagal sensory neurons of the ferret. J. Neurophysiol. 79: 769-777, 1998. Substance P (SP) hyperpolarizes ~80% of ferret vagal sensory neurons (nodose ganglion neurons) via NK-1 receptor-mediated activation of a potassium current (IK). A depolarizing current activated by membrane hyperpolarization could minimize the SP-induced hyperpolarization. Such a current exists in 65% of the nodose neurons (n = 264). In this study, we examine this current and how it can interact with SP-induced membrane hyperpolarizations. This slowly developing, noninactivating inward current, designated Ih, was activated maximally at about -120 mV and had a reversal potential value of -23 ± 4.4 mV (n = 4). The time course of activation followed voltage-dependent, monoexponential kinetics. Steady-state activation curves derived from tail current analysis were well fit by a Boltzmann equation yielding a half-activation potential (V1/2) of-77 ± 1.5 mV and a ks value of 18 ± 0.5 (n = 8). In the presence of 1 mM cesium, the current was completely abolished. These parameters are consistent with those derived for Ih in other neurons. Substance P (200 nM) reduced the magnitude of Ih elicited by membrane hyperpolarizations to about -110 mV but did not affect the magnitude of Ih elicited by hyperpolarizations to more negative potentials. Tail current analysis revealed that this effect was the result of a SP-induced shift of the Ih activation curve to more negative membrane potentials. The V1/2 value for Ih was shifted by -20 ± 1.4 mV in the presence of SP with no change in ks (18 ± 0.7; n = 5). The SP effect on Ih, like its effect on IK, was blocked reversibly by 10 nM CP99,994, a NK-1 antagonist, and was mimicked by the NK-1 agonist Ac-[Arg6, Sar9, Met(O2)11]SP(6-11) (ASMSP; 200 nM). Ih was not affected by NK-2 or NK-3 selective agonists (n = 4 for each) nor was the effect of SP on Ih reduced by an NK-2 antagonist (n = 4). These results show that SP activates a NK-1 receptor coupled to the Ih channel. Thus NK-1 receptor activation in ferret vagal afferents not only leads to membrane hyperpolarization but it also can enhance synergistically this inhibitory effect by decreasing Ih.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

A hyperpolarization-activated, nonspecific cation current (If) was first described in cells of the sinoatrial node (Brown and DiFrancesco 1980; Yanagihara and Irisawa 1980). Subsequently, this current was identified in a variety of mammalian neurons (reviewed by Pape 1996) and called Ih or IQ.

Ih plays a role in the generation of spontaneous action potentials (DiFrancesco and Tortora 1991; McCormick and Pape 1990b; Noble et al. 1992), and modulation of Ih results in the regulation of firing frequencies (Banks et al. 1993; Denyer and Brown 1990; DiFrancesco et al. 1989; McCormick and Pape 1990a). Ih is modulated by neurotransmitters (serotonin or acetylcholine), inflammatory mediators (prostaglandin E2), and even neuropeptides (opioids) (Bobker and Williams 1989; DiFrancesco and Tromba 1988; DiFrancesco et al. 1986; Ingram and Williams 1994; McCormick and Pape 1990a; Tokimasa and Akasu 1990).

Substance P (SP) is contained in (Helke and Hill 1988; Katz and Karten 1980; Kummer et al. 1992) and released from (Saria et al. 1988) some vagal primary afferent neurons and acts as an inflammatory neuropeptide (reviewed by Otsuka and Yoshioka 1993) or as a neurotransmitter (De Koninck and Henry 1991). We have demonstrated that the vagal afferent somata of ferret nodose ganglion possess tachykinin NK-1 receptors (Jafri and Weinreich 1996). Activation of these receptors induces a membrane hyperpolarization that could potentially activate Ih. Many nodose neurons possess Ih (Ingram and Williams 1994; Undem and Weinreich 1993), including nodose neurons from the ferret (unpublished observations). The inward Ih-current could mitigate the inhibitory effect of the SP-induced hyperpolarization. In this study, we have demonstrated that SP, also via the NK-1 receptor, inhibits the activation of Ih by shifting the voltage dependence of Ih to more negative potentials, thereby synergistically enhancing the SP-induced inhibitory effect.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Preparation of tissue

Nodose ganglia were isolated from adult male ferrets (1-2 kg) purchased from Marshall Farms USA (North Rose, NY). After the animals were killed by CO2 asphyxiation followed by exsanguination, the ganglia were removed and placed in 4°C Locke's solution of the following composition (in mM): 136 NaCl, 5.6 KCl, 14.3 NaHCO3, 1.2 NaH2PO4, 2.2 CaCl2, 1.2 MgCl2, and 10 dextrose, equilibrated continuously with 95% O2-5% CO2 (pH 7.2-7.4). Ganglia were dissociated enzymatically on the day of dissection using the procedure described by Christian, Togo, Naper, Koschorke, Taylor, and Weinreich (1993). Briefly, the ganglion was desheathed, cut into three to four pieces and placed in Ca2+-free, Mg2+-free Hanks balanced salt solution (CMFH) of the following composition (in mM): 138 NaCl, 5.0 KCl, 4.0 NaHCO3, 0.3 Na2HPO4, 0.3 KH2PO4, 5 dextrose, and 0.03 phenol red. All incubations were done at 37°C. The pieces of tissue were incubated for 7 min in 10 ml CMFH containing papain (100 µl, 0.1 mg/ml, Boehringer Mannheim, Indianapolis, IN), which was activated by L-cysteine (0.2 mg/ml, Sigma, St. Louis, MO). The tissue then was washed twice with CMFH and incubated for 10 min in 4 ml CMFH containing dispase, grade II (2 mg/ml, Boehringer Mannheim) and collagenase, type 1A (1 mg/ml, Sigma). During the incubation, the tissue was triturated with a fire-polished Pasteur pipette at 8 and 10 min. After washing twice with Lebovitz L-15 medium (GIBCO, Grand Island, NY) containing 10% fetal bovine serum (FBS, vol/vol, JRH Biosciences, Lexena, KS), cells were resuspended in 150 µl L-15/10% FBS per coverslip. A 24-well culture plate was prepared by placing a 15-mm poly-D-lysine-coated coverslip in the bottom of each well. After resuspension, 150 µl of the cell suspension was added to each well. Neurons were allowed to settle and attach overnight at 37°C before use. Recordings were performed on neurons within 48 h of plating. The Institutional Animal Care and Use Committee approved all methodology used in these experiments.

Electrophysiological studies

Intracellular glass micropipettes were fabricated from aluminosilicate capillary glass (1.0 mm OD, 0.68 mm ID, Sutter Instruments, San Francisco, CA) on a Brown and Flaming puller (Sutter) and back-filled with a solution of 3 M KCl. Micropipettes had DC resistances ranging from 20 to 40 MOmega . The micropipettes were connected to an Axoclamp-2A amplifier (Axon Instruments, Foster City, CA). The discontinuous current injection mode (switching frequency 5-6 kHz) of the amplifier was used for both current- and voltage-clamp applications; the headstage voltage was monitored continuously. Current and voltage outputs were viewed on-line on an oscilloscope and chart recorder and digitized with a Neurocorder (Neurodata Instruments, Delaware Water Gap, PA) for storage on videocassette tapes for off-line analysis. Data acquisition and voltage-clamp protocols were controlled using pCLAMP5 software (Axon Instruments).

Neurons were superfused with oxygenated Locke solution (3-4 ml/min) at room temperature (20-22°C). The superfusate level was lowered to ~50 µm above the surface of the neurons to minimize electrode stray capacitance. A neuron was judged acceptable for study only if its resting membrane potential (Vm; < -50 mV) and action potential overshoot (>0 mV) remained stable for 5 min after impalement. Stable (<10% change) Vm and membrane input resistance (Ri) values typically could be achieved for >2 h. Current recordings from voltage-clamp experiments are the average of three trials except where noted (e.g., in single pulse pharmacology experiments; Fig. 6). Voltage traces from current-clamp experiments are not averaged.


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FIG. 6. Substance P (SP) decreases Ih through tachykinin NK-1 receptor activation. A: substance P decreases the amplitude of Ih measured as the difference between the instantaneous (Iinst) and the steady state (Iss) current during a hyperpolarizing voltage command step (hold, -60 mV; step, -90 mV; 3-s duration; see Fig. 1 for protocol). These traces were not averaged. B: amplitude of Ih is reversibly decreased during superfusion of SP (200 nM) or a tachykinin NK-1 receptor specific agonist (100 nM ASMSP). Tachykinin NK-2 or NK-3 receptor agonists [1 µM neurokinin A (NKA) or 1 µM Senk, respectively] did not affect Ih. Agonist application periods shown by bars. Pulses were applied every 15 s. Arrows (a, b, c) indicate traces shown in A. C: bar graph summarizing effects of tachykinin receptor agonists and antagonists as a percent block of Ih (n = 4 for each treatment). SP (200 nM) and the NK-1 agonist ASMSP (200 nM) were almost equally effective at blocking Ih. NKA and senktide, NK-2 and NK-3 agonists, respectively, were ineffective at blocking Ih at concentrations ranging from 200 nM to 1 µM. The NK-1 antagonist CP99,994 abolished the effect of 200 nM SP while the NK-2 antagonist SR48968 had no effect on the SP-induced block.

Preparation and delivery of solutions

Solutions were prepared daily for experiments from stock aliquots, which were stored at -20°C. Reservoirs containing solutions of various drugs were connected to the inflow line of the recording chamber by three-way valves, which could divert rapidly the source of superfusion from the main reservoir. This means of drug delivery introduced a 15-s delay from the activation of the valve to arrival of the drug solution in the chamber.

Substance P was obtained from Sigma. CP99,994 kindly was provided by Dr. James Heym, Pfizer.

Data analysis

Data are expressed as means ± SEM. Statistical significance was assessed using two-tailed, unpaired Student's t-test at theP < 0.05 level of significance. Figure construction and fitting of mathematical functions to data were accomplished with SigmaPlot software (Jandel Scientific, San Rafael, CA).

The analysis and interpretation of the voltage-clamp data obtained in these experiments were performed as outlined in the appendices of Banks et al. (1993) and will be summarized here. The difference current (Idiff) was derived using the equation Idiff = Iss - Iinst (Fig. 1) where Iss is the steady state current recorded during the test voltage command (Vc) and Iinst is the instantaneous current determined by fitting the current during Vc with a single exponential and extrapolating to time 0 of Vc.


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FIG. 1. Voltage-clamp recording of Ih identifying variables used for analysis. Sample voltage clamp recording of Ih in an isolated nodose ganglion neuron of the ferret identifying measurements made the variable names used in the calculations.- - -, monoexponential fits of Ih activation and tail current. Inset: voltage protocol.

The steady state activation (ninf) at command voltage V was obtained using the equation
<IT>n</IT><SUB>inf</SUB>(<IT>V</IT>) = <FR><NU><IT>I</IT><SUB>tail,diff</SUB>(<IT>V</IT>) − <IT>I</IT><SUB>tail,diff</SUB>(<IT>V</IT><SUB>min</SUB>)</NU><DE><IT>I</IT><SUB>tail,diff</SUB>(<IT>V</IT><SUB>max</SUB>) − <IT>I</IT><SUB>tail,diff</SUB>(<IT>V</IT><SUB>min</SUB>)</DE></FR> (1)
where Itail,diff = Itail,inst - Itail,ss (Fig. 1) and Vmax and Vmin are the voltages corresponding to maximum and zero activation of Ih, respectively. Substitution of Ohm's Law
<IT>I</IT><SUB>h</SUB>(<IT>V</IT>) = <IT>g</IT><SUB>h</SUB>(<IT>V</IT>)*(<IT>V − E</IT><SUB>h</SUB>)
into Eq. 1 simplifies to ninf(V) = gh(V)/gh,max where gh(V) is the conductance of Ih at voltage V and gh,max is the maximal conductance of Ih. Thus ninf is the fraction of maximal Ih conductance activated at a given command voltage. The activation curves were fit using a Boltzmann equation of the following form
<IT>n</IT><SUB>inf</SUB>(<IT>V</IT>) = <FR><NU>1</NU><DE>1 + exp[(<IT>V − V</IT><SUB>1/2</SUB>)/<IT>k</IT><SUB>s</SUB>]</DE></FR>
where V is the membrane potential, V1/2 is the half-activation voltage, and ks describes the slope of the curve.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

General characteristics of ferret nodose neurons

Dissociated ferret nodose neurons used in these experiments appeared round or ovoid in shape and usually are devoid of visible processes. Some dissociated cells did reveal small tufted processes reminiscent of periglomerular and pericellular arborizations associated with some nodose neurons (Cajal 1904; Lieberman 1976). The average resting membrane potential of the neurons used in this study was -60 ± 1.3 mV (n = 30; range -42 to -72 mV), and their input resistance at resting membrane potential was 26 ± 2.7 MOmega (n = 30; range 5-70 MOmega ). These values are in agreement with values previously reported for ferret nodose neurons recorded in intact nodose ganglia or in isolation (Jafri and Weinreich 1996).

Characterization of Ih

Many ferret nodose neurons are hyperpolarized by SP (Jafri and Weinreich 1996) and also possess Ih. To minimize interpretational complexity, we specifically have chosen to analyze the effects of SP on Ih in neurons that are not hyperpolarized by SP. A sample voltage-clamp recording of Ih identifying the variables used in our analysis is shown in Fig. 1 (see METHODS). In current-clamp experiments, Ih appeared as a time- and voltage-dependent relaxation of the membrane potential hyperpolarization in response to hyperpolarizing current steps (Fig. 2A). Under voltage clamp, hyperpolarizing voltage steps from holding potentials of -50 to -60 mV elicited a time- and voltage-dependent inward current associated with an increase in membrane conductance (Fig. 2B) in 65% (n = 264) of the neurons. In some neurons, Ih was active between -50 and -60 mV; however, in most neurons it was not. The change in conductance associated with this inward current was determined by comparing the instantaneous conductance (Gi) with the steady state conductance (Gss). At -70 mV, Gi was 38 ± 5.9 nS (n = 10) and Gss was 45 ± 1.4 nS (n = 10). At -120 mV, these values were 33 ± 3.4 (n = 10) and 55 ± 6.2 (n = 10), respectively. Therefore, the conductance increase associated with Ih averaged 7 nS at -70 mV and 22 nS at -120 mV. The inward current increased in amplitude with greater hyperpolarizing steps until a maximum was reached at about -120 mV. The activation kinetics of Ih in these neurons was best described by a single exponential, and the kinetics were voltage sensitive. There was no difference in the activation kinetics of Ih in response to hyperpolarizing stimulus pulses ranging from 400 ms to 8 s.


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FIG. 2. Current- and voltage-clamp recordings of Ih from a single isolated ferret nodose neuron. A: voltage traces recorded under current clamp in response to incremental 200-pA hyperpolarizing current pulses. open circle  and bullet , times at which measurements were taken for current-voltage (I-V) plots in B. Resting membrane potential was -65 mV. B: current-clamp I-V relationship derived from responses in A. open circle , transient voltage response; bullet , steady state voltage amplitude after activation of Ih. C: current traces recorded from the same cell as in A and B under voltage clamp in response to incremental 10-mV hyperpolarizing voltage command steps to -120 mV. After each test pulse, the neuron was held at -70 mV. Transient currents shown during this period are the tail currents reflecting the conductance activated by the test voltage steps. open circle  and bullet , times at which measurements were taken for current-voltage (I-V) plots in D. Holding potential was -50 mV. D: voltage-clamp I-V relationship derived from responses in C; open circle , instantaneous current amplitude; bullet , steady state current amplitude after activation of Ih.

H current is a mixed sodium potassium current. Its reversal potential (Erev) value has been reported to range between -20 and -50 mV (reviewed by Pape 1996). We have estimated the Erev value for the hyperpolarization-activated current in ferret nodose neurons by examining the instantaneous current-voltage (I-V) relationship in a single neuron generated from different holding potentials. A different number of Ih channels will be open at each holding potential. Because current at any given test potential will be based on the number of channels open (conductance) and the driving force, the only condition where the current will be equal at any holding potential is when the driving force is zero. The driving force for an ionic current is zero at its reversal potential. Thus the point at which these I-V curves intersect is the Erev value. The Erev value for this hyperpolarization-activated current was -20 mV; the population average was -23 ± 4.4 mV (n = 4; range, -20 to -29 mV), consistent with the Erev for neuronal Ih.

Extracellular cesium in millimolar concentrations is known to block Ih (Pape 1996). In ferret nodose neurons, rectification was reversibly blocked by 1 mM external cesium (100% block in 5 of 5 neurons). In voltage-clamp experiments, 1 mM external cesium also reversibly blocked Ih (100% in 5 of 5 neurons). Taken together these data indicate that the hyperpolarization-activated current in ferret nodose neurons is Ih.

Effect of substance P on Ih

The magnitude of Ih elicited by hyperpolarizing voltage commands was diminished in the presence of 200 nM SP (Fig. 3B) compared with control (Fig. 3A). The instantaneous (Iinst) and steady state (Iss) I-V relationships from these experiments show a decrease in the induced current at all test potentials (Fig. 3C). The difference between Iss and Iinst is the magnitude of Ih. A plot of this difference current shows that Ih is decreased in the presence of 200 nM SP (Fig. 3D).


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FIG. 3. Modulation of Ih by 200 nM substance P (SP). Current traces recorded under voltage clamp in response to hyperpolarizing voltage command pulses given at 10-mV increments in control Locke solution (A) or in Locke solution containing 200 nM SP (B). Symbols above traces indicate times at which measurements of instantaneous (circles) or steady state (squares) currents were taken for current-voltage (I-V) plots in C. Holding potential was -55 mV (A) or -60 mV (B). C: voltage-clamp I-V relationship derived from responses in control Locke solution (open symbols; data from A) or in the presence of 200 nM SP (filled symbols; data from B). Circles indicate the instantaneous current amplitude; squares indicate the steady state current amplitude after activation of Ih. D: difference current (Iss - Iinst) under control conditions (open circle ) or in the presence of 200 nM SP (bullet ) of data shown in previous panels.

The voltage dependence of Ih is modulated by a variety of physiological stimuli (reviewed by Pape 1996). Determination of the SP effect on the voltage dependence of Ih activation was accomplished by constructing steady state activation curves using tail-current analysis (Fig. 4) in control Locke solution and in the presence of 200 nM SP. Using the voltage-clamp protocol shown in Fig. 2, tail currents were elicited (Fig. 4A, top). These tails were fit by single exponentials to estimate the instantaneous current at the time of repolarization (Fig. 4A, bottom). Plotted as a ratio of the maximum Ih, these activation curves could be described by the Boltzmann equation (Fig. 4B, solid lines). Under control conditions, the half-activation potential for Ih in these neurons was -77 ± 1.5 mV (n = 8). The decrease in the current amplitude in response to SP application was a result of a leftward shift in the V1/2 of 20 ± 1.4 mV (n = 5; range, 16-24 mV). The ks value did not change in the presence of 200 nM SP (18 ± 0.7; n = 5) as compared with control (18 ± 0.5; n = 5).


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FIG. 4. Substance P (SP) shifts the activation curve of Ih. A, top: current recordings in response to hyperpolarizing voltage commands given in 10-mV increments from a holding potential of -70 mV. After each test pulse, the neuron was held at -90 mV for 375 ms before returning to the holding potential of -70 mV. Bottom: expanded view of tail currents shown above with single exponential fits superimposed over the data traces. B: steady state activation curve derived from tail currents in control Locke solution (bullet ; data shown in A) or in the presence of 200 nM SP (black-triangle). Solid lines, Boltzmann fits of the data. V1/2 value shifts from -77 mV under control conditions to -98 mV in the presence of SP. ninf value represents the fraction of channels open (see METHODS for derivation).

Functionally the effect of this decrease in Ih can be seen in current-clamp experiments. The membrane hyperpolarization in response to hyperpolarizing current stimuli showed substantial rectification in control (Fig. 5A). Using the same stimulus protocol, this rectification was diminished in the presence of 200 nM SP (Fig. 5B). The net depolarization resulting from activation of Ih in response to strong stimuli is halved in the presence of SP (Fig. 5C). The increase in resistance seen in the presence of SP at the smallest current injections (Fig. 5B) may be due to block of Ih that is active at rest or it may be an effect on a distinct channel. This effect was seen in 2 of 12 current-clamped neurons and may reflect the heterogeneity of the cell population. By decreasing the depolarizing effect of Ih, SP maintains a more hyperpolarized membrane potential, thus making the neuron less excitable.


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FIG. 5. Effect of 200 nM substance P (SP) on current clamp I-V relationship in a single isolated ferret nodose neuron. Voltage traces recorded under current clamp in response to hyperpolarizing current pulses in control Locke solution (A) or in Locke solution containing 200 nM SP (B). Resting membrane potential was -50 mV. C: voltage change associated with activation of Ih (peak Vm - Vss from A or B) was diminished in the presence of 200 nM SP (bullet ) compared with control (open circle ).

Pharmacology of substance P effect on Ih

Endogenous tachykinins [SP, neurokinin A (NKA), and neurokinin B] activate the three known tachykinin receptor subtypes, designated NK-1, NK-2, and NK-3 (reviewed by Otsuka and Yoshioka 1993). To assess which tachykinin receptor subtype might subserve the SP-mediated effect on Ih, we applied agonists and antagonists selective for different tachykinin receptor subtypes. Agonists were applied at 100 times greater than their dissociation constant (Kd). To measure the effects of these agents on Ih, a voltage-clamped neuron was stepped from a holding potential of -60 to -100 mV for 3 s (Fig. 6B). The resulting Ih was measured by subtracting the instantaneous current (Iinst) at the start of the step from the steady state current (Iss) during the step. The command step hyperpolarization was applied every 15 s under control conditions and in the presence of tachykinin receptor subtype specific agonists and antagonists.

Application of 200 nM SP decreased the amplitude of Ih by 85 ± 2.1% (n = 4; range, 80-90%; Fig. 6, A and B). The NK-1 receptor subtype specific agonist, ASMSP, was as effective as SP, diminishing the amplitude of Ih by96 ± 1.4% (200 nM; n = 4; range, 93-99%; Fig. 6B). At concentrations as high as 1 µM, neither [beta -ala8]NKA nor senktide, selective NK-2 and NK-3 receptor agonists, respectively, affected the amplitude of Ih (no effect in 4 of 4 neurons for each agonist; Fig. 6B). In all cases, neurons that did not respond to these latter two agonists did respond to SP or ASMSP.

CP99,994 and SR48968 are specific nonpeptide antagonists for the NK-1 (McLean et al. 1993) and NK-2 (Emonds-Alt et al. 1993) receptors, respectively. At concentrations as low as 1 nM CP99,994 completely abolished the effect of 200 nM SP on Ih (100% prevention in 4 of 4 neurons). In the presence of 1 µM SR48968, the SP effect was reduced by only 4.5 ± 1.8% (n = 4; range, 1-9%). The cumulative pharmacological evidence indicate the SP effect on Ih is mediated by a tachykinin NK-1 receptor.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

The major observations of this work are that nanomolar concentrations of SP decrease the amplitude of Ih in ferret nodose neurons, that this inhibition is accomplished through a shift of the voltage activation curve to more negative potentials, and that this effect is mediated via activation of a tachykinin NK-1 receptor subtype.

Characterization of Ih

In ferret nodose neurons, the activation kinetics of Ih were voltage dependent and were best described by a single exponential. The time constant of activation, tau , ranged from ~300 ms at -80 mV to 100 ms at -130 mV. This is in agreement with Ih recorded in other neurons where the activation kinetics exhibit voltage dependence and are described by a single exponential (Kamondi and Reiner 1991; Kiehn and Harris-Warrick 1992; McCormick and Pape 1990b; Tokimasa and Akasu 1990). In cat cortical neurons and in neurons of the rat median nucleus of the trapezoid body, Ih displays biexponential activation kinetics (Banks et al. 1993; Spain et al. 1987). The kinetic characteristics of Ih in vagal afferents have not been studied; however, in mouse dorsal root ganglion neurons the kinetics appear more complicated (Mayer and Westbrook 1983). The variations in the kinetic characteristics in different neurons may reflect differentially modulated Ih channels in each cell type or may indicate the presence of multiple subtypes of this channel.

Analysis of the steady state activation of Ih in ferret vagal afferents revealed a half-activation voltage of -77, which is consistent with that obtained in other primary afferent neurons. The half-activation potential in rat dorsal root ganglion neurons was -81 mV (Mayer and Westbrook 1983) and in guinea pig nodose neurons was -70 to -79 mV (Ingram and Williams 1994, 1996). This range is also in agreement with a variety of other neuronal cell types including guinea pig enteric neurons (-70 mV) (Galligan et al. 1990), thalamic relay neurons (-75) (McCormick and Pape 1990b) and cat sensory motor cortex neurons (-82) (Spain et al. 1987). The values in other neuronal cell types vary widely ranging from -106 mV in crustacean motor neurons (Kiehn and Harris-Warrick 1992) to -67 mV in tiger salamander rod photoreceptors (Hestrin 1987). This variability may reflect different functional roles the Ih plays in these neurons. The level of activation of Ih at resting membrane potential and the contribution of Ih to neuronal function strongly depends on individual neuronal characteristics, particularly the membrane potential and input resistance. Additional evidence supporting the identification of this current as Ih is the Erev value of -23 mV in ferret nodose neurons; this is comparable with that for Ih measured in other neurons (reviewed by Pape 1996) and clearly distinguishes this current from the fast inwardly rectifying potassium current identified in guinea pig olfactory cortex neurons (Constanti and Galvan 1983). Thus the characteristics of the hyperpolarization-activated inward current in ferret nodose neurons indicate that this current is Ih.

Modulation of Ih

Modulation of Ih by a variety of neurotransmitters has been described in both neuronal and nonneuronal systems. Most frequently, the activation curve is shifted to more positive potentials. Less common are reports of receptors that cause a shift of the activation curve for Ih to more negative potentials. This type of response has been identified in primary afferent neurons from guinea pig nodose ganglia in response to µ-opioid receptor activation (Ingram and Williams 1994). However, opioids had no effect on Ih alone or in the presence of adenosine 3',5'-cyclic monophosphate analogues but reversed the effects of forskolin on Ih, implying that these compounds exert their effect through a decrease in the activity of adenylyl cyclase. In the CNS, activation of adenosine A1 receptors in thalamic (Pape 1992) and mesopontine (Rainnie et al. 1994), neurons causes anegative shift of the Ih activation curve without prior activation of adenylyl cyclase, implying that these compounds act through a decrease in the basal activity of adenylyl cyclase. In both these cases, the shift in the half-activation potential was -5 to -10 mV.

Substance P receptor activation in ferret nodose neurons shifts the half-activation potential of Ih by about -20 mV. This shift occurs without prior addition of adenylyl cyclase activators. All known tachykinin receptors are coupled to G proteins and have been associated with plethora of second messenger systems, including inhibition of adenylyl cyclase (reviewed by Otsuka and Yoshioka 1993). The coupling of these receptors in ferret nodose neurons remains to be determined.

The endogenous tachykinins SP, NKA, and neurokinin B show preference for the NK-1, NK-2, and NK-3 tachykinin receptor subtypes, respectively (reviewed by Otsuka and Yoshioka 1993). The SP response characterized in this study is mediated by the NK-1 receptor based on the following results. It is mimicked by a NK-1 receptor specific agonist ASMSP (100 nM) but not by agonists specific for the NK-2 or NK-3 receptors. Furthermore, the high-specificity NK-1 receptor antagonist CP99,994 (Ki = 250 pM) (McLean et al. 1993) at 1 nM abolishes the SP effect on Ih while SR48,968, a NK-2 receptor specific antagonist, did not alter the action of SP. Thus our cumulative pharmacological data with tachykinin agonists and antagonists indicate that the SP effect on Ih is mediated specifically through a NK-1 subtype of tachykinin receptor.

Functional implications

Substance P has been localized to primary afferent neurons of the dorsal root (Dalsgaard et al. 1982; Lindh et al. 1983), trigeminal root (McCarthy and Lawson 1989), jugular (Katz and Karten 1980), and nodose (Helke and Hill 1988; Katz and Karten 1980; Kummer et al. 1992) ganglia. Activation of these neurons elicits release of mediators, including tachykinins such as SP, from the sensory nerve terminals in the CNS and in the periphery (Saria et al. 1988). At peripheral endings, released SP participates in neurogenic inflammation (reviewed by Otsuka and Yoshioka 1993), whereas at the central nerve terminals, SP functions as an excitatory neurotransmitter (De Koninck and Henry 1991). The release of SP can be modulated by inhibitory autoreceptors for SP that have been identified on ferret nodose neurons (Jafri and Weinreich 1996). Activation of these receptors results in a membrane hyperpolarization of ~10 mV from the average resting membrane potential of -62 mV via activation of a calcium-dependent potassium channel (Jafri and Weinreich 1996). From this resting membrane potential, the hyperpolarization is of sufficient magnitude to activate Ih, an inward current that would reduce the inhibitory effect of the outward hyperpolarizing SP-induced current. The substantial shift of the activation curve of Ih to more negative potentials by SP, as measured by the -20-mV shift of its half-activation potential, is sufficient to prevent the activation of Ih by the SP-induced hyperpolarization. If these channels and receptors also exist on the nerve terminal membranes, these two actions may inhibit synergistically the further release of SP.

The SP-induced IK has been shown to be mediated by activation of tachykinin NK-1 receptors (Jafri and Weinreich 1996); the same tachykinin receptor underlying the SP-induced shift in the activation curve of Ih characterized in this study. Modulation of two distinct currents by activation of a single autacoid receptor subtype appears to be a common motif used by ferret vagal primary afferents. Histamine increases the excitability of ferret nodose neurons both by eliciting a membrane depolarization through block of a potassium current active at rest [IK(rest)] and by blocking a slow postspike afterhyperpolarization (IAHP). It is interesting to note that the modulation of these two currents by histamine is mediated through one histamine receptor subtype, the H1 subtype (Jafri et al. 1997).

In contrast to the SP-induced membrane hyperpolarization observed in ferret nodose (Jafri and Weinreich 1996) and rabbit superior vagal (jugular) neurons (unpublished observations), studies in other primary afferent neurons have shown exclusively depolarizing effects produced by SP (Dray and Pinnock 1982; Spigelman and Puil 1990; Akasu et al. 1996). In most of these studies, relatively high concentrations of SP were used: 2-3 µM in guinea pig trigeminal root ganglia (Spigelman and Puil 1990), 1 mM pressure ejection in rat dorsal root ganglia (Dray and Pinnock 1982); however, the EC50 value for SP-induced inward currents in bullfrog dorsal root ganglion neurons was estimated at 7 nM (Akasu et al. 1996). Membrane hyperpolarizations, by contrast, were elicited by nM concentrations of SP, and even at concentrations <= 5 µM, SP still produces a membrane hyperpolarization in nodose neurons.

Given that NK-1 receptor activation can modulate both IK (Jafri and Weinreich 1996) and Ih (current work), is it possible that SP also may activate a depolarizing current that is masked by the hyperpolarization? We believe this is unlikely for several reasons. First, when the SP-induced hyperpolarization is blocked by potassium channel blockers, in nominally zero extracellular calcium, or pharmacologically with NK-1 receptor antagonists, SP-induced depolarization is not observed. Second, concentrations of SP as high as 5 µM consistently hyperpolarized the membrane potential of ferret nodose neurons. The micromolar SP concentrations used to demonstrate SP-induced membrane depolarization in other primary afferents may reflect the activation of a low affinity receptor or may be due to a nonreceptor mediated effect (Mousli et al. 1990) on the membrane potential in those neurons. Finally, the reversal potential value for the SP-induced membrane hyperpolarization and its Nernstian dependance on the concentration of extracellular potassium ion indicates that this SP effect is mediated a potassium conductance. It is unlikely that this result would occur if a depolarizing component were mixed in, unless it were due solely to the block of a resting potassium conductance.

Although the data support SP modulation of Ih in a majority of these neurons, some neurons may have other potential effector targets for SP. In 2 of 12 neurons (see Fig. 2), small hyperpolarizing stimuli appear to increase the resistance of the neuron, whereas stronger stimuli reflect a block Ih similar to that seen in most cells. Anomalous results such as these may reflect the heterogenous population of neurons in the nodose ganglion. While >90% of the somata of nodose ganglion neurons are associated with C-fiber afferents (Stansfeld and Wallis 1985), these somata represent a heterogenous population of sensory modalities and innervate a variety of target tissues. This diversity also may explain why 1 of 10 neurons studied showed a decrease in resistance in response to small stimuli in 1 mM cesium. Elaboration on these points awaits future studies on modality- and tissue-identified nodose neurons.

Studies on axons of rat vagal afferents and human sural nerves have implicated Ih in maintenance of conduction velocity during repetitive activation and reduction of conduction failure at sites of reduced safety factor, such as branch points. Reduction of Ih then, can decrease primary afferent excitability by decreasing their conduction velocity and increasing the chance of conduction failure at branch points. Two areas where primary afferent axonal branching may exert effects on action potential conduction are at the peripheral arborization and at the T-junction near the soma where the stem process meets the axon (Ducreux et al. 1993). Both these locations are particularly susceptible to exposure to SP. If the responses in the axons are similar to those described here in the soma, SP released from the peripheral nerve terminals (Saria et al. 1988) may increase the chance of conduction failure at branch points in the peripheral arborization by depressing Ih activation. In the same way, SP released from the somata of primary afferents in the sensory ganglia (Amir and Devor 1996; Huang and Neher 1996) may affect conduction at the T-junction, also present in the ganglia.

In summary, vagal afferents are known to possess SP inhibitory autoreceptors of the tachykinin NK-1 subtype. We have shown that SP, also acting through an NK-1 receptor, shifts the activation curve of Ih to more negative potentials. If these receptors also exist on the axonal membranes, diminution of Ih may alter action potential conduction properties. In this manner, these two SP-mediated mechanisms can produce potentially synergistic effects on impulse traffic and on neurosecretion in these vagal afferent C fibers.

    ACKNOWLEDGEMENTS

  The authors thank Drs. Robert W. Greene and Brad Undem for constructive suggestions on an earlier draft of this manuscript. The expert technical assistance of G. Taylor is greatly appreciated.

  This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-22069.

    FOOTNOTES

  Address for reprint requests: D. Weinreich, Dept. of Pharmacology and Experimental Therapeutics, University of Maryland, School of Medicine, Room 542 Health Sciences Facility, 685 W. Baltimore St., Baltimore, MD 21201-1559.

  Received 17 July 1997; accepted in final form 13 October 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

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