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Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  • 1
    Previous studies indicate that prostacyclin (PGI2) increases the activity of baroreceptor afferent fibres. The purpose of this study was to test the hypothesis that PGI2 inhibits Ca2+-activated K+ current (IK(Ca)) in isolated baroreceptor neurones in culture.
  • 2
    Rat aortic baroreceptor neurones in the nodose ganglia were labelled in vivo by applying a fluorescent dye (DiI) to the aortic arch 1–2 weeks before dissociation of the neurones. Outward K+ currents in baroreceptor neurones evoked by depolarizing voltage steps from a holding potential of −40 mV were recorded using the whole-cell patch-clamp technique.
  • 3
    Exposure of baroreceptor neurones to the stable PGI2 analogue carbacyclin significantly inhibited the steady-state K+ current in a dose-dependent and reversible manner. The inhibition of K+ current was not caused indirectly by changes in cytosolic Ca2+ concentration. The Ca2+-activated K+ channel blocker charybdotoxin (ChTX, 10−7m) also inhibited the K+ current. In the presence of ChTX or in the absence of Ca2+, carbacyclin failed to inhibit the residual K+ current. Furthermore, in the presence of high concentrations of carbacyclin, ChTX did not cause further reduction of K+ current.
  • 4
    Carbacyclin-induced inhibition of IK(Ca) was mimicked by 8-bromo-cAMP and by activation of G-protein with GTPγS. The inhibitory effect of carbacyclin on IK(Ca) was abolished by GDPβS, which blocks G-protein activation, and by a selective inhibitor of cAMP-dependent protein kinase, PKI5–24.
  • 5
    The results demonstrate that carbacyclin inhibits ChTX-sensitive IK(Ca) in isolated aortic baroreceptor neurones by a G-protein-coupled activation of cAMP-dependent protein kinase. This mechanism may contribute to the PGI2-induced increase in baroreceptor activity demonstrated previously.

Arterial baroreceptors are mechanosensitive nerve terminals located in the adventitia of the aortic arch and carotid sinuses that are activated by vascular stretch during increases in arterial blood pressure (Kirchheim, 1976; Brown, 1980). Previous studies in our laboratory have shown that prostacyclin (PGI2), the major prostanoid produced by the vascular endothelium, increases the activity of arterial baro-receptors (McDowell, Axtelle, Chapleau & Abboud, 1989; Chen, Chapleau, McDowell & Abboud, 1990). For instance, inhibition of PGI2 formation in the carotid sinus with inhibitors of cyclo-oxygenase or by endothelial denudation decreased baroreceptor activity, whereas injection of exogenous PGI2 into the isolated carotid sinus increased baroreceptor activity without a change in the vessel distensibility (Chen et al. 1990). It has also been shown that PGI2 increases the sensitivity of nociceptors (Taiwo & Levine, 1990), cardiopulmonary receptors and other visceral sensory receptors connected to vagal and sympathetic afferents (Staszewska-Barczak, 1983; Hintze & Kaley, 1984). The underlying mechanisms by which PGI2 increases the sensitivity of various sensory nerves are not known.

A particular difficulty often encountered in the investigation of peripheral sensory nerve function and chemical modulation is the small size of sensory nerve terminals and their complex arrangement within the surrounding tissues (Kirchheim, 1976; Krauhs, 1979; Brown, 1980). In this study we applied a fluorescent dye, DiI (Honig & Hume, 1989), to the site of baroreceptor innervation in the aortic arch to retrogradely label the cell soma of aortic baroreceptor neurones in the nodose ganglia. We then studied the dissociated baroreceptor neurones maintained in culture and examined the effects of the stable PGI2 analogue carbacyclin (Whittle & Moncada, 1985) on the steady-state outward K+ currents using the whole-cell patch-clamp technique. With the use of charybdotoxin (ChTX), and by altering Ca2+ availability in the recording solutions, we specifically tested the hypothesis that PGI2 inhibits the ChTX-sensitive IK(Ca) in baroreceptor neurones.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

Labelling of aortic baroreceptor neurones in rats

The fact that the rat aortic depressor nerve innervating the aortic arch contains only baroreceptor afferent fibres and no chemoreceptor afferents (Sapru, Gonzalez & Krieger, 1981) allowed us to selectively label the aortic baroreceptor neurones in the nodose ganglia using a retrogradely transported fluorescent dye, DiI (1,1′-dioleyl-3,3,3′,3′-tetramethylindocarbocyanine methanesulphonate; Molecular Probes, Inc.) (Fig. 1). The lipophilic dye incorporates into nerve endings and diffuses throughout the entire cell membrane with no transfer to adjacent cells (Honig & Hume, 1989). Briefly, adult Sprague–Dawley rats weighing 250–300 g were anaesthetized with ketamine (100 mg kg−1, i.p.) and acepromazine (10 mg kg−1, i.p.) (Midwest Veterinary Supplies, Desmoines, IA, USA) and mechanically ventilated. A right thoracotomy was performed under sterile conditions. We injected 1–2 μl DiI (25 mg DiI in 0.5 ml methanol) onto the adventitia of the aortic arch with a fine-tipped glass pipette. After DiI application, the surgical incision was closed, negative intrathoracic pressure was re-established, and the animals were allowed to recover. Care and use of the animals conformed to the standards established by the United States Department of Agriculture and by the National Institutes of Health. All protocols were approved by the University of Iowa Institutional Animal Care and Use Committee.

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Figure 1. DiI-labelled baroreceptor neurones

Shown is a microphotograph of rat nodose neurones maintained in culture for ∼6 h after cell dissociation. Cells in the same field were viewed under Hoffman contrast optics (× 600) in regular light (left) and fluorescent light (right). A DiI-labelled baroreceptor neurone showing red-orange colour was identified under fluorescent light with an excitation wavelength of 546 ± 10 nm, a dichroic mirror of 580 nm and an emission barrier filter of 580 nm. Scale bar, 50 μm.

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Cell dissociation and culture

After the surgery for DiI labelling, 1–2 weeks were allowed for the dye to diffuse to the neuronal cell soma of the aortic baroreceptor neurones in the nodose ganglia. Then the rats were anaesthetized in a sealed desiccator chamber containing gauze saturated with 100% halothane (Halocarbon Lab., River Edge, NJ, USA). The deeply anaesthetized rats were unresponsive to noxious stimuli before decapitation. The nodose ganglia were quickly removed, stripped of the connective tissue capsule, transferred into ice cold culture media, and minced with micro scissors. The nodose tissue was digested in modified L-15 Leibovitz culture medium containing collagenase (Type Ia, 1 mg ml−1), trypsin (Type III, 1 mg ml−1) and DNase (type IV, 0.1 mg ml−1) at 37 °C for 50 min. The chemical digestion was terminated by adding soybean trypsin inhibitor (2 mg ml−1) and bovine serum albumin (1 mg ml−1). The digested tissue fragments were gently triturated with a siliconized sterile Pasteur pipette and centrifuged at 800 r.p.m. for 5 min. The neurones were resuspended in the modified L-15 medium supplemented with 3.7% (v/v) rat serum and 1.5% (v/v) chick embryo extract and plated onto poly-l-lysine-coated glass cover-slips. Neurones in the modified L-15 culture media were kept at 37 °C in an incubator saturated with 5% CO2 and water vapour. The neurones were studied within 24 h of dissociation. During this period, neurones were attached onto the poly-l-lysine-coated coverslips but devoid of extensive neurites, ensuring an excellent voltage-clamp of the whole cell membrane. Figure 1 shows micro-photographs of the nodose neurones (left) viewed under Hoffman contrast optics (× 600) in regular light and a DiI-labelled baroreceptor neurone (right) in the same field, which was identified by a red-orange colour under fluorescent light with appropriate fluorescent filters (Nikon G-1A combination with excitation filter, 546 ± 10 nm, dichroic mirror, 580 nm; and emission barrier filter, 580 nm). Only DiI-labelled neurones were selected for study. Carbocyanine dyes such as DiI do not cause any adverse effects on cell viability or membrane properties when using brief periods of illumination for cell identification (Honig & Hume, 1989).

Solutions and chemicals

The pipette solution contained (mm): potassium aspartate, 116; KCl, 10; NaCl, 5; CaCl2, 2.3; MgCl2, 4.8; EGTA, 10; Hepes, 10; ATP (magnesium salt), 4.0; and GTP (Tris salt), 0.5. The pH was adjusted to 7.2 with NaOH. The free [Ca2+] in the pipette solution was estimated to be about 10−7m according to the Chelator program (Schoenmakers, Visser, Flik & Theuvenet, 1992). For the experiments including an inhibitor of cAMP-dependent protein kinase, PKI5–24, in the pipette solution, the pipette was first tipfilled for < 1mm with the normal pipette solution and then backfilled with PKI5–24containing solution to avoid potential interference of PKI5–24 (mol wt, 2223) with seal formation. The bath solution typically contained (mm): NaCl, 140; KCl, 5.4; CaCl2, 2.0; MgCl2, 1.0; Hepes, 10; d-glucose, 5.5; CdCl2, 0.2; tetrodotoxin (TTX), 0.001; and the pH was adjusted to 7.4. Unless indicated otherwise, CdCl2 was present in the extracellular (bath) solution to block Ca2+ current. When Ca2+-free solutions were required, Ca2+ was substituted by equimolar Mg2+ in the pipette solution and by Mg2+ plus 1 mm EGTA in the bath solution to minimize the effects of surface charge changes. TTX was present in the bath solution to block Na+ current. Osmolarity of the solutions was measured with a freezing-point depression osmometer (Osmette, Precision Systems Inc., Natick, MA, USA) and adjusted to 295–300 mosmol l−1 with mannitol if necessary. Stable recording of whole-cell outward K+ currents could be obtained for more than 30 min with these solutions. The bath solution in the recording chamber was exchanged when desired with a DC-driven dual-channel pump (Model 700, Instech Laboratory Inc., Plymouth, PA, USA). The perfusion rate was set at 1 ml min−1, which enabled a complete exchange of the chamber solution within 2–3 min. Carbacyclin, 6–keto-PGF, 8-bromo-cAMP, PKI5–24 and RO20–1724 were obtained from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA, USA). TTX, tetraethylammonium (TEA), ChTX, apamin and other chemicals were purchased from Sigma. All experiments were performed at room temperature (21–23 °C).

Whole-cell current recordings and data analysis

The outward K+ currents in the DiI-labelled baroreceptor neurones were studied using the standard whole-cell patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Neurones attached onto the coverslips were transferred into a 0.5 ml recording chamber on the stage of an inverted microscope (Nikon, Diaphot) and superfused with the bath solution. Whole-cell currents were acquired using an Axopatch 200A amplifier (Axon Instruments, Inc.). Patch pipettes were pulled from thin-walled borosilicate glass (TW150-4, World Precision Instruments Inc., Sarasota, FL, USA) and had tip resistances of 1–3 MΩ when filled with the pipette solution. A reference electrode was connected through a Ag–AgCl pellet to the bath solution via a 3 M KCl–agar bridge. The off set potential between the pipette and bath solution was zeroed prior to seal formation, and the liquid junction potential measured between the pipette and the bath solutions was less than 3 mV and not corrected for. Gigaseals (2–20 GΩ) were formed between the pipette electrode and the cell membrane by applying suction to the pipette. The pipette capacitance was compensated and additional suction applied for membrane rupture to establish the whole-cell recording configuration. More than 5 min were usually allowed for the cell membrane conductance to stabilize before the application of any agent. The whole-cell current was filtered at 2 kHz with a 4-pole Bessel filter and digitized using a 12-bit analog-to-digital interface (Digidata 1200, Axon Instruments, Inc.). Voltage protocols, data acquisition and analysis were accomplished using pCLAMP software (versions 5.7.1 and 6.0.2, Axon Instruments, Inc.) and data were stored in a 486DX Gateway computer for later analysis. The Axopatch 200A amplifier allowed us to achieve compensation of the series resistance to ∼90% and the resistance and compensation were checked for stability during the experiments and were corrected accordingly. Whole-cell outward K+ current was elicited by a series of 200 ms voltage steps in 10 mV increments from a holding potential of −40 mV, and the steady-state current at the end of 200 ms voltage steps was measured. The leak and residual capacitative currents were digitally subtracted online using the P/n pulse protocol built into the pCLAMP software. All data were expressed as means ±s.e.m. (n), where n refers to the number of cells tested. The Student's t test was used to compare the data obtained before and after intervention, and one-way ANOVA followed by contrast testing was used to compare the data from multiple groups. Statistical significance was determined at P < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

Outward K+ currents in DiI-labelled baroreceptor neurons

Figure 2 shows that voltage steps from a holding potential of −40 mV to various test potentials of 200 ms duration elicited outward currents. In the presence of Ca2+ without Cd2+ added to the bath solution, the corresponding current and voltage exhibits an ‘N-shaped’ relationship (Fig. 2A and D), suggesting the presence of a Ca2+-dependent K+ current component secondary to Ca2+ influx during membrane depolarization. Addition to the bath solution of 0.2 mm Cd2+, which effectively blocked all inward Ca2+ current (data not shown), reduced the outward current and changed the N-shaped current–voltage curve to a simple rising function of membrane depolarization (Fig. 2B and D). The outward current began to activate at approximately −20 mV and increased in response to stronger depolarization without significant inactivation. To avoid the influence of Ca2+ influx through Ca2+ channels on the outward current we included 0.2 mm Cd2+ in the bath solution except when stated otherwise. Application of 10−7M ChTX, a potent blocker of the large conductance Ca2+-activated K+ channel (Miller, Moczydlowski, Latorre & Phillips, 1985), caused a further reduction of the outward current (Fig. 2C and D). Exposure of the DiI-labelled neurones to 40 μm apamin, a selective blocker of the small conductance Ca2+-activated K+ channel (Hugues, Romey, Duval, Vincent & Lazdunski, 1982), did not cause a noticeable reduction of the K+ current (n= 3, data not shown). These results indicate that the total outward current in baroreceptor neurones consists of both ChTX-sensitive and ChTX-insensitive components.

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Figure 2. Representative recordings of the outward K+ currents in a baroreceptor neurone (A, B and C) and the corresponding current–voltage (I–V) curves (D)

Whole-cell currents were elicited by voltage steps from −40 mV to various test potentials in 10 mV increments shown in the top traces. The currents were recorded during control with 2 mm Ca2+ in the bath solution (A; see Methods), after addition of 0.2 mm Cd2+ (B), and after addition of 10−7 M ChTX in the presence of 0.2 mm Cd2+ (C). The corresponding I–V curves were constructed from the currents measured at 200 ms of depolarization. ○, control; □, Cd2+; ▵, Cd2++ ChTX. Note that Cd2+ eliminated the ‘N-shaped’ outward K+ currents and ChTX further reduced the K+ currents.

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The outward current was sensitive to blockade by TEA (IC50= 3.2 mm, Fig. 3A). When the extracellular (bath) [K+] was changed, the reversal potential of tail currents shifted close to the values predicted by the Nernst equation for a pure K+-selective electrode (the K+-Nernst equation) (Fig. 3B). Fitting the data of the shifted reversal potentials with the Goldman–Hodgkin–Katz voltage equation yields the permeability ratio, PNa/PK, of 0.0171. These results indicate that K+ is the major ionic carrier of the outward current.

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Figure 3. Dose-dependent inhibition of outward K+ current by TEA (A) and the reversal potentials of tail currents as a function of extracellular K+ concentration (B)

A, K+ current was evoked by voltage step from −40 to 20 mV and measured at 200 ms of depolarization. The currents were recorded with 0.2 mm Cd2+ present in the bath solution. The current amplitude at different TEA concentrations was normalized to the control. Addition of TEA was accompanied by equimolar reduction of Na+ in the bath solution to maintain the same osmolality. The data were fitted to the equation:inline imagewhere a1 is the current in the control and is equal to 1, a2 is the derived current at maximal inhibition and is equal to 0.03, IC50 is the [TEA] at half-maximal inhibition of the current and is equal to 3.2 mm, and H is the Hill coefficient and is equal to 0.61. B, the relationship between the reversal potential of the tail currents and the extracellular (bath) [K+] which was varied by equimolar substitution of KCl for NaCl in the bath solution. Tail currents were elicited upon repolarization to membrane potentials ranging from −100 to 0 mV from 200 ms depolarization at 40 mV. Holding potential =−40 mV. The data of reversal potentials were fitted by the Goldman–Hodgkin–Katz voltage equation with a permeability ratio of PNa/PK= 0.0171 (the continuous line). The Nernst equation predicted for a pure K+-selective electrode is shown by the dashed line with a slope of 58.3 mV for a 10-fold change in extracellular [K+].

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Effect of the PGI2 analogue carbacyclin on the outward K+ currents

Figure 4A and B shows an example of the K+ currents recorded from a DiI-labelled baroreceptor neurone before, during, and after exposure to the stable PGI2 analogue carbacyclin. In all of the neurones tested, carbacyclin significantly inhibited the outward K+ current and the inhibitory effect reached a maximum in 5–10 min. The inhibitory effect was rapidly reversible upon removal of carbacyclin from the bath solution (Fig. 4B). As a control, the inactive metabolite of prostacyclin, 6-keto-PGF (Pace-Asciak, 1976) dissolved in the same vehicle (ethanol) as was used for carbacyclin, did not alter the K+ currents (n= 4, Fig. 4C).

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Figure 4. Reversible inhibition of outward K+ current by carbacyclin (A), time course of the inhibition and recovery (B), and lack of effect of 6-keto-PGF in vehicle ethanol (C)

A, representative recordings of outward K+ currents in control, during exposure to carbacyclin dissolved in vehicle ethanol, and after washout of carbacyclin. Top traces show the voltage protocols. The bath solution contained 0.2 mm Cd2+ in each condition. B, the time course of carbacyclin-induced inhibition of the K+ current and the reversibility of the inhibition after washout. The currents were measured at 200 ms of depolarization to 20 mV in the same cell as shown in A. C, lack of effect of 6-keto-PGF on the outward K+ current. 6-Keto-PGF was dissolved in the same vehicle (0.1% ethanol) as used for carbacyclin.

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Inhibition of the K+ current by carbacyclin was dose dependent (IC50= 2.59 × 10−5 M, Fig. 5A). The current was restored to 94.5 ± 2.3% of the control in about 10 min after washout of carbacyclin from the bath solution (n= 11). To determine if the ChTX-sensitive K+ current or the ChTX-insensitive K+ current was inhibited by carbacyclin, we exposed the cells to ChTX after the K+ current was inhibited by a high concentration (3 × 10−4 M) of carbacyclin. ChTX did not cause any further reduction of the residual current after carbacyclin (Fig. 5A, inset), suggesting that carbacyclin inhibited essentially all of the ChTX-sensitive component of the K+ current. Inhibition of the K+ current by carbacyclin was not associated with a change in the voltage-dependent activation of K+ current, as indicated by the overlapping activation curves (Fig. 5B). Fitting the activation curves to the Boltzmann equation yielded mean half-activation potentials (V1/2) of 10.5 ± 1.0 and 9.7 ± 1.1 mV, and average slope factors (k) of 15.2 ± 0.8 and 14.5 ± 0.7 mV per e-fold change in conductance in the control condition and during exposure to 10−4m carbacyclin, respectively (n= 5, P > 0.05).

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Figure 5. Dose-dependent inhibition of the total outward K+ currents by carbacyclin (A) without a change in the voltage-dependent activation curve of the K+ currents (B)

A, dose–response curve for carbacyclin-induced inhibition of the total outward K+ currents which were elicited by depolarization at 60 mV with 0.2 mm Cd2+ in the bath solution. The currents were measured after the inhibitory effects reached a stable level at each concentration and normalized to the current magnitude during control. Numbers in parentheses indicate the number of cells tested at each concentration. The continuous line shows the best fit by the Hill equation with IC50 of 25.94 × 10−6 M. The inset bar graph shows the responses to 10−4 M and 3 × 10−4 M carbacyclin (Carb), and then to 10−7 M ChTX in the same cells (n= 5). * indicates statistically significant difference at P < 0.05 compared with the control, and n. s. indicates no statistically significant difference as analysed by one-way ANOVA followed by contrast testing. Note that ChTX did not cause further inhibition of the K+ current after a high concentration (3 × 10−4 M) of carbacyclin. B shows the voltage-dependent activation curves of the outward K+ currents constructed from the tail currents at −40 mV following 200 ms step depolarization at different potentials (n= 5). The tail currents were curve-fitted with a single exponential to derive the tail amplitude at time 0 (I) and normalized to the maximum tail current (Imax). The normalized tail currents (I/Imax) at the corresponding depolarization (Vm) were fitted to the Boltzmann equation: I/Imax= 1/[1 + exp(V1/2Vm)/k], where V1/2 is the voltage for half-maximal activation of the steady-state whole-cell conductance and k is the slope factor of the activation curve. The V1/2 and k values were not different during control (○) and after exposure of the cells to 10−4 M carbacyclin (□; n= 5).

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Type of K+ current inhibited by carbacyclin

The lack of further current reduction by ChTX after high doses of carbacyclin (Fig. 5A) suggested that only the ChTX-sensitive Ca2+-activated K+ current, IK(Ca), was inhibited by carbacyclin. We also examined the effect of carbacyclin on the residual K+ current after the IK(Ca) was blocked by ChTX (Fig. 6). After reduction of the outward K+ current by ChTX (10−7 M), carbacyclin (10−4 M) did not cause further inhibition of the residual K+ current (n= 6), thus confirming that it is the ChTX-sensitive IK(Ca) that was selectively inhibited by carbacyclin.

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Figure 6. Original recordings and the group data showing inhibition of K+ current by ChTX and lack of further inhibition by carbacyclin in the presence of ChTX

The currents were activated by depolarization at 20 mV during control, after exposure to 10−7 M ChTX, and after ChTX plus 10−4 M carbacyclin (Carb). The K+ currents were all recorded with 0.2 mm Cd2+ in the bath solution. The inset shows group data and the K+ currents were normalized to the control (n= 6). * indicates P < 0.05 compared with control, and there was no significant difference between the currents after ChTX and the currents after ChTX plus carbacyclin.

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We further examined the inhibitory effect of carbacyclin on K+ currents in the presence and absence of Ca2+ (Fig. 7). In this protocol, the pipette solution did not contain Ca2+ and the bath solution contained 2 mm Ca2+. Cd2+ was not contained in the bath solutions so that Ca2+ influx through voltage-activated Ca2+ channels would be the only source of Ca2+ for activating IK(Ca). When the Ca2+ in the bath solution was replaced with equimolar Mg2+ plus 1 mm EGTA, the total outward K+ current was significantly reduced and the residual K+ current was not inhibited by carbacyclin (n= 4, Fig. 7). When Ca2+ was returned to the bath solution, the total K+ current was restored and was again inhibited by carbacyclin. Addition of ChTX reduced the total K+ current to a level equivalent to that seen after removal of Ca2+ (Fig. 7).

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Figure 7. Inhibition of the ChTX-sensitive K+ current by carbacyclin is dependent on the presence of free Ca2+

Shown is the time course of the outward K+ currents at 10 mV elicited once every 20 s. In contrast to the data shown previously, Ca2+ was removed from the pipette solution and no Cd2+ was added to the bath solutions which contained 2 mm CaCl2. To remove the bath Ca2+, a Ca2+-free bath solution was prepared by replacing the 2 mm CaCl2 in the original bath solution with equimolar MgCl2 plus 1 mm EGTA. After removal of Ca2+, the outward K+ current decreased significantly and the residual Ca2+-independent K+ current was not changed by carbacyclin (Carb). Restoration of the bath Ca2+ restored the total K+ current and the inhibitory effect of carbacyclin. Note that ChTX (10−7 M) reduced the total outward K+ current to a level similar to that seen after removal of Ca2+.

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Signal transduction pathway

PGI2 is known to increase cAMP production through G-protein activation in various tissues (Kerins, Murray & Fitzgerald, 1991). To determine the signalling mechanism of carbacyclin on the K+ current, we first examined the role of G-protein by replacing GTP in the pipette solution with either GDPβS, which blocks G-proteins, or with GTPγS, which activates G-proteins (Gilman, 1987). Equimolar (0.5 mm) substitution of GDPβS for intracellular GTP prevented the carbacyclin-induced inhibition of the K+ current (n= 4, Fig. 8A). In contrast, when GTPγS was substituted for GTP, total outward K+ current decreased progressively over a 10 min period and the addition of carbacyclin caused a further inhibition of the current (n= 4, Fig. 8B). However, washout of carbacyclin did not restore the K+ current, as was seen in the absence of GTPγS (Fig. 4B). The sustained inhibition of the current may reflect the irreversible activation of G-proteins by the non-hydrolysable GTPγS (Gilman, 1987). These results indicate that G-protein activation is required for carbacyclin-induced inhibition of the K+ current.

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Figure 8. G-protein-dependent inhibition of the K+ current by carbacyclin

A, substitution of GDPβS for GTP (0.5 mm) in the pipette solution prevented inhibition of the K+ current by carbacyclin (Carb). The outward K+ current evoked at 20 mV was recorded once every 20 s in the presence of 0.2 mm Cd2+ in bath solution. The inset bar graph shows the magnitude of the K+ current normalized to the control value (n= 4). B, equimolar substitution of GTPγS for GTP in the pipette solution inhibited the K+ current by itself after establishing the whole-cell recording configuration. The inset bar graph shows the group data (n= 4) and *indicates P < 0.05 compared with the current immediately after establishing whole-cell recording (Control). Addition of 10−4 M carbacyclin caused further inhibition of the current and the inhibition was not reversible after washing carbacyclin out of the bath solution.

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Next, we examined whether a membrane-permeable cAMP analogue mimics the carbacyclin-induced inhibition of K+ current. Exposure of the neurones to 8-bromo-cAMP (1–5 mm) significantly suppressed the outward K+ current (n= 6, Fig. 9). In addition, exposure to a selective cAMP-phosphodiesterase inhibitor, RO20–1724 (Bang, Ericsen & Aarbakke, 1994) also reduced the K+ current by ∼10% (n= 2, data not shown). These results suggest that cAMP formation may mediate the inhibition of K+ current by carbacyclin.

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Figure 9. The membrane-permeable cAMP analogue 8-bromo-cAMP inhibits K+ current

A shows outward K+ currents recorded over time during exposure of baroreceptor neurones to 8-bromo-cAMP. The K+ currents were elicited once every 20 s in the presence of 0.2 mm Cd2+. B shows the group data for maximum inhibition of the K+ currents by 1–5 mm 8-bromo-cAMP. * indicates P < 0.05 compared with control.

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Finally, we examined the role of cAMP-dependent protein kinase (PKA) using the peptide PKI5–24, a pseudosubstrate inhibitor highly selective for PKA (Kemp, Cheng & Walsh, 1988; Walsh & Glass, 1991). When PKI5–24 (50 μM) was included in the pipette solution, the amplitude of the outward K+ current increased progressively after establishing the whole-cell recording configuration, and carbacyclin now failed to inhibit K+ current (n= 6, Fig. 10).

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Figure 10. Carbacyclin-induced inhibition of the K+ current is abolished by PKI5–24

The K+ currents were elicited by voltage steps from -40 to 20 mV once every 20 s in the presence of 0.2 mm Cd2+. PKI5–24 (50 μM), the peptide inhibitor specific for PKA, was included in the pipette solution. The dashed line represents the gradual delivery of PKI5–24 to the interior of the neurone by diffusion. The inset bar graph shows group data of the currents measured before and ∼10 min after administration of 10−4 M carbacyclin (Carb). Note that the K+ current spontaneously increased after establishing the whole-cell recording configuration with the PKA inhibitor PKI5–24 in the pipette solution, and that carbacyclin failed to inhibit the current in the presence of PKI5–24.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

The major findings of this study are as follows. (1) The PGI2 analogue carbacyclin inhibits outward K+ current in aortic baroreceptor neurones without an associated change in the voltage-dependent activation of the current. (2) Carbacyclin does not inhibit the residual K+ current after the removal of Ca2+ or in the presence of ChTX. (3) The inhibitory effect of carbacyclin on K+ current was mimicked by 8-bromo-cAMP or intracellular application of the G-protein activator GTPγS, and it was abolished by the G-protein blocker GDPβS or the peptide inhibitor (PKI5–24) of PKA. These results indicate that carbacyclin selectively inhibits ChTX-sensitive IK(Ca) in baroreceptor neurones through G-protein-coupled activation of the cAMP-dependent protein kinase.

Inhibition of K+ current by carbacyclin

Several types of K+ currents have been described in rat nodose neurones, including Ca2+-activated K+ current (Hay & Kunze, 1994a, b; Schild, Clark, Hay, Mendelowitz, Andresen & Kunze, 1994), transiently activated K+ current and delayed rectifier K+ current (Cooper & Shrier, 1989; McFarlane & Cooper, 1991; Schild et al. 1994). The transiently activated K+ currents are inactivated by depolarizing membrane holding potential, and the delayed rectifier K+ current and the Ca2+-activated K+ channels exhibit little or no inactivation (Cooper & Shrier, 1989; McFarlane & Cooper, 1991; Hay & Kunze, 1994b; Schild et al. 1994). In the present study we recorded the steady-state outward K+ current elicited from a holding potential of −40 mV. K+ is the major carrier of the outward current elicited in the presence of Cd2+ and TTX as indicated by the sensitivity of the current to TEA and by the shift of reversal potentials of tail currents as predicted by the K+-Nernst equation (Fig. 3). The lack of current reduction by ChTX after the K+ current was inhibited by high doses of carbacyclin (Fig. 5A), the lack of further inhibition of the residual K+ current by carbacyclin after ChTX (Fig. 6), and the observation that carbacyclin inhibited K+ current in the presence but not in the absence of Ca2+ (Fig. 7) together indicate that it is the ChTX-sensitive IK(Ca) that was selectively inhibited by carbacyclin.

It is important to consider the possibility that inhibition of IK(Ca) by carbacyclin may be secondary to a reduction of intracellular [Ca2+]. This is unlikely in the current study since Ca2+ entry through voltage-gated Ca2+ channels was effectively blocked by Cd2+ in the bath solution and the intracellular free Ca2+ was clamped by Ca2+ and EGTA in the pipette solution. In fact, increased cAMP and activation of PKA increases rather than decreases Ca2+ current in rat nodose neurones (Gross, Moises, Uhler & MacDonald, 1990; Gross, Uhler & MacDonald, 1990). An increased Ca2+ current might have even opposed the carbacyclin-induced direct inhibition of IK(Ca) in the protocol that did not utilize Cd2+ (Fig. 7).

Intracellular signal transduction pathway

PGI2 has been shown to stimulate G-protein-coupled increases of intracellular cAMP and/or IP3 formation and intracellular Ca2+ release in various tissues and cell systems (Kerins et al. 1991; Vassaux, Gaillard, Ailhaud & Negrel, 1992; Namba et al. 1994). In addition, G-protein subunits, cAMP, and protein kinases may each gate or modulate ion channels independently (Clapham 1994; Goulding, Tibbs & Siegelbaum, 1994; Kume, Hall, Washabau, Takagi & Kotlikoff, 1994). We originally considered that the inhibition of IK(Ca) by carbacyclin might occur through multiple pathways. Our results indicate that G-protein-coupled PKA activation mediates the carbacyclin-induced inhibition of the K+ current in baroreceptor neurones. The evidence for this conclusion includes the following. First, the inactive PGI2 metabolite 6-keto-PGF dissolved in the vehicle (ethanol) as a control for the PGI2 analogue carbacyclin did not alter the K+ current (Fig. 4C). Second, blocking the G-protein activation by replacement of GTP in the pipette solution with GDPβS prevented the inhibition of K+ current by carbacyclin (Fig. 8A). Conversely, activating G-protein by replacement of GTP with GTPγS caused a progressive inhibition of the K+ current, which was facilitated by the addition of carbacyclin (Fig. 8B). The inhibitory effect of GTPγS on K+ current was sustained after the washout of carbacyclin, presumably because of its non-hydrolysable nature (Gilman, 1987). Third, the effect of carbacyclin was mimicked by the membrane-permeable cAMP analogue 8-bromo-cAMP (Fig. 9) and by the cAMP-elevating agent RO20–1724, a selective inhibitor of cAMP-phosphodiesterase. Fourth and most importantly, inhibition of K+ current by carbacyclin was abolished by PKI5–24 (Fig. 10), the specific inhibitor of PKA, suggesting that PKA activation is essential in mediating the carbacyclin-induced inhibition of the K+ current while the other messengers (e. g. G-protein subunits and cAMP) apparently do not directly inhibit the K+ current. In addition, the observation that K+ current increased as PKI5–24 diffused intracellularly (Fig. 10) and that inhibition of the K+ current by carbacyclin was readily reversed upon removal of carbacyclin (Figs 4B and 7) suggest intrinsic activities of PKA and protein phosphatase, both of which may tonically modulate the function of Ca2+-activated K+ channels. Indeed, endogenous PKA and phosphatase activities have been shown to be closely associated with Ca2+-activated K+ channels from the rat brain (Chung, Reinhart, Martin, Brautigan & Levitan, 1991; Reinhart, Chung, Martin, Brautigan & Levitan, 1991).

It is interesting to note that phosphorylation by PKA of the Ca2+-activated K+ channels from various types of smooth muscle cells uniformly increases channel opening leading to muscle relaxation (Sadoshima, Akaike, Kanaide & Nakamura, 1988; Minami, Fukuzawa, Nakaya, Zeng & Inoue, 1993). Ca2+-activated K+ channels from rat brain may be either up-or downregulated by PKA (Sikdar, McIntosh & Mason, 1989; Reinhart et al. 1991; Kume et al. 1994). Single channel recordings in neonatal rat nodose neurones revealed a large conductance (220 pS) Ca2+-activated K+ channel which is highly sensitive to ChTX, and a smaller conductance (60 pS) Ca+-activated K+ channel which is present at lower density and insensitive to either ChTX or apamin (Hay & Kunze, 1994a, b). It is not known whether the ChTX-insensitive Ca2+-activated K+ channel is present in baroreceptor neurones or whether it is modulated by PKA.

Experimental approach and physiological significance

We propose that the results of the present study may be relevant to the actions of PGI2 on baroreceptor afferent endings in vivo. PGI2 is released from the blood vessel wall during vascular stretch. It is known to increase the action potential firing frequency of various types of sensory afferent fibres including baroreceptors (Chen et al. 1990), nociceptors (Taiwo & Levine, 1990) and visceral receptors (Staszewska-Barczak, 1983; Hintze & Kaley, 1984). Numerous studies have shown that ligand-binding receptors and ion channels present on nerve terminals are often also located on the membrane of the cell soma (Higashi, 1986; Harper, 1991; Matsumura, Watanabe, Onoe & Watanabe, 1995). A recent autoradiographic study of the rat nervous system has demonstrated that a high density of binding sites for iloprost (a PGI2 analogue) exist in the afferent fibres of vagal sensory neurones, in the central terminals of these neurones in the nucleus tractus solitarii, as well as in the cell soma of nodose neurones (Matsumura et al. 1995). Comparisons of the electrophysiological properties of acutely dissociated nodose neurones and nodose neurones in situ have shown that the electrophysiological and chemoreceptive properties of acutely dissociated nodose neurones are very similar to those observed for neurones in the intact ganglia (Ikeda, Schofield & Weight, 1986; Leal-Cardoso, Koschorke, Taylor & Weinreich, 1993). Therefore, we believe that study of the cell soma can provide insight into the function of the nerve terminals. In a preliminary study we have observed that carbacyclin causes significant depolarization and increases the spike firing frequency in cultured aortic baro-receptor neurones (Li, Lee, Bielefeldt, Abboud & Chapleau, 1995), which is consistent with the PGI2-induced increase in spike frequency recorded from the baroreceptor afferent fibres (Chen et al. 1990).

Modulation of Ca2+-activated K+ channels is a common effector mechanism affecting action potential duration, spike frequency adaptation and possibly resting membrane potential (Blatz & Magleby, 1987; Schild et al. 1994; Hay & Kunze, 1994b). The presence of PGI2 receptors in both the peripheral and central terminals of the vagal afferent fibres as well as in the cell soma in the nodose ganglia (Matsumura et al. 1995) implies that changes of membrane conductance by PGI2 may not only contribute to the PGI2-induced increase in afferent baroreceptor activity (Chen et al. 1990) but may also increase the efficacy of synaptic transmission at the central afferent terminals in the nucleus tractus solitarii.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

We thank Laurie J. Waite and Carol A. Whiteis for help in the DiI labelling and culturing of the nodose neurones, and Drs Toshinori Hoshi, Brett A. Adams and Erwin F. Shibata for valuable suggestions. We also thank Dr Ruth E. Wachtel for helpful comments on the manuscript. The work was supported by research funds from the National Heart, Lung, and Blood Institute Grant HL14388 and from the Department of Veterans Affairs.