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¹ Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE 68198-5850, USA

	Abstract

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Angiotensin II (Ang II) plays an important role in the enhanced chemoreflex function that occurs in congestive heart failure (CHF), but the mechanism of this effect within the carotid body (CB) is not known. We investigated the sensitivity of Ca²⁺-independent, voltage-gated K⁺ (Kv) channels to hypoxia in CB glomus cells from CHF rabbits, and whether endogenous angiotensin II (Ang II) modulates this action. Using the conventional whole-cell patch clamp technique, we found that Kv currents (I_K) under normoxic conditions were blunted in the CB glomus cells from CHF rabbits compared with sham rabbits. In addition, the inhibition of I_K and the decrease of resting membrane potential (RMP) induced by hypoxia were greater in CHF versus sham glomus cells. Ang II, at 100 pM, had no direct effect on I_K at constant normoxic P_O2, but increased the sensitivity of I_K and RMP to hypoxia in sham glomus cells. In CHF glomus cells, an AT1 receptor (AT₁R) antagonist, L-158 809 (1 µM), alone did not affect I_K at normoxia, but it decreased the sensitivity of I_K and RMP to hypoxia. At higher concentrations, Ang II dose dependently (0.1–100 nM) reduced I_K under constant normoxic conditions in sham and CHF glomus cells, with threshold concentrations of about 900 and 600 pM, respectively. Immunocytochemical and Western blot assessments demonstrated the down-expression of Kv3.4 but not Kv4.3 channels in CHF glomus cells. These results indicate that: (1) Ang II/AT₁R signalling increases the sensitivity of Kv channels to hypoxia in CB glomus cells from CHF rabbits; (2) high concentrations of Ang II (> 1 nM) directly inhibit I_K in CB glomus cells from sham and CHF rabbits; (3) changes in Kv channel protein expression (Kv3.4 versus Kv4.3) in the CB glomus cell may contribute to the suppression of I_K and enhanced sensitivity of I_K to hypoxia in CHF.

(Received 30 March 2006; accepted after revision 11 June 2006; first published online 15 June 2006)
Corresponding author H. D. Schultz: Department of Cellular and Integrative Physiology, University of, Nebraska Medical Center, Omaha, NE 68198-5850, USA.  Email: hschultz{at}unmc.edu

	Introduction

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The carotid body (CB) is the primary arterial (peripheral) chemoreceptor, which plays an important role on respiratory and cardiovascular control. The CB is composed of neurotransmitter-enriched glomus cells and glial-like sustentacular cells among other neuronal structures (Gonzalez et al. 1994). Although there are still some discrepancies about the detailed steps of the chemo-neurotransduction cascade in the CB, it is believed that the glomus cells, which lie in synaptic apposition with afferent axons, are the initial sites of chemotransduction. The glomus cell membrane contains several types of ion channels. Of these channels, the hypoxia-inactivated outward K⁺ channels are proposed to play a key role for the initial depolarization, with subsequent activation of voltage-gated Ca²⁺ channels, release of neurotransmitter, and increases of sensory discharge in the carotid sinus nerve (Gonzalez et al. 1994; Prabhakar, 1994).

An enhancement of peripheral chemoreflex sensitivity occurs in pacing-induced CHF rabbits (Sun et al. 1999a). Furthermore, an augmented afferent input from the CB chemoreceptors is involved in the enhancement of peripheral chemoreflex function in the CHF rabbits (Sun et al. 1999b). Although the cellular and molecular mechanisms involved in enhancing CB chemoreceptor sensitivity during CHF still remain unclear, evidence shows that angiotensin II (Ang II) is an important modulator of sympathetic outflow in the central nervous system and peripheral neural sites (Reid, 1992; Brooks & Osborn, 1995; Brooks, 1997; Li et al. 2006). Plasma and tissue Ang II levels are increased during CHF patients and animals (Liu et al. 2000; Roig et al. 2000; Cardin et al. 2003; van de et al. 2006). A locally generated Ang II system has been confirmed in the rat CB (Lam & Leung, 2002), and Ang II has been shown to increase the CB chemoreceptor activity via the AT₁ receptor (AT₁R) (Allen, 1998). Furthermore, our recent study has shown that endogenous Ang II concentration and AT₁R expression are increased in the CBs of CHF rabbits, and that CB Ang II mediates the enhanced CB chemoreceptor sensitivity to hypoxia in these animals (Li et al. 2006).

A blunted Ca²⁺-dependent K⁺ current (K_Ca) in CB glomus cells contributes to the enhancement of CB chemoreceptor activity in the normoxic state in CHF rabbits (Li et al. 2004b). However, the hypoxia-sensitive K⁺ channels in adult rabbit CB glomus cells are voltage-gated, Ca²⁺-insensitive K⁺ (Kv) channels (Ganfornina & López-Barneo, 1992), which encompass Kv4.1 and Kv4.3 subunits (Sanchez et al. 2002). Since Ang II enhances CB chemoreceptor sensitivity to hypoxia (Li et al. 2006), we reasoned that endogenous Ang II blunts the Kv currents and affects the sensitivity of Kv channels to hypoxia in the CB glomus cells in the CHF state. In the present study, therefore, we assessed the effect of Ang II and the AT₁R antagonist L-158,809 on the outward Kv currents (I_K) in the primary cultured CB glomus cells from sham and CHF rabbits. We also measured the effect of hypoxia on I_K before and after treatment with Ang II or L-158,809. In other experiments, we measured protein expression of Kv3.4 and Kv4.3 channel subunits in the CB glomus cells from sham and CHF rabbits using immunocytochemical and Western blot analysis.

	Methods

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Pacemaker implant and production of CHF

All experiments were carried out on male New Zealand White rabbits weighing 2.5–3.5 kg. Experiments were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee and were carried out in accordance with the National Institutes of Health and the American Physiological Society's Guides for the Care and Use of Laboratory Animals. Rabbits were assigned to sham-operated and CHF groups. They were housed in individual cages under controlled temperature and humidity and a 12: 12 h dark–light cycle, and fed standard rabbit chow with water available ad libitum.

Rabbits were anaesthetized with a cocktail consisting of 1.2 mg acepromazine, 5.9 mg xylazine and 58.8 mg ketamine, given as an I.M. injection. Using sterile technique, a left thoracotomy was performed as previously described (Sun et al. 1999a). Briefly, a pin electrode was attached to the left ventricle for pacing. Two sonomicrometer crystals (Sonometrics Corp., London, ON, Canada) were attached to opposing walls of lateral left ventricle for measuring external diameter. The chest was closed. Rabbits were placed on an antibiotic regimen consisting of 5 mg kg^–1 Baytril I.M. for 5 days. After 2 weeks, baseline left-ventricular end-systolic and end-diastolic external diameter (D), fractional shortening, and shortening velocity (dD/dt_max) were measured by sonomicrometry (Triton Technology Inc., San Diego, CA, USA). The pacing was started at 320 beats min^–1, held for 7 days, and then the rate was gradually increased to 380 beats min^–1, with an increment of 20 beats min^–1 each week. Sonograms and experimental procedures were performed with the pacemaker turned off for at least 60 min before the recordings were started. Rabbits with > 40% reductions in dD/dt_max and shortening fraction are considered in CHF (generally after 3–4 weeks). Sham-operated animals underwent a similar period of sonographic measurements with the pacemaker turned off. Any rabbit exhibiting abnormal arterial blood gases (P_a,O2 < 85 Torr; 45 Torr < P_a,CO2 < 30 Torr) were excluded from the study (Sun et al. 1999a).

Isolation and identification of CB glomus cell

The isolation and identification of CB glomus cells were performed as described in our previous study (Li et al. 2004b). Briefly, the carotid bifurcations on both sides were removed surgically from sham or CHF rabbits anaesthetized with the cocktail described above. The rabbits were then killed with an I.V. injection of 150 mg kg^–1 sodium pentobarbital. The removed tissue was subjected to a two-step enzymatic digestion protocol. Each step lasted for 30 min at 37°C. The enzymatic solution for the first step of digestion contained trypsin (type II, 2 mg ml^–1) and collagenase (type IV, 2 mg ml^–1); and that for the second step contained collagenase (4 mg ml^–1) and bovine serum albumin (BSA, 5 mg ml^–1). The isolated cells including glomus cells, glial cells and vascular cells were co-cultured at 37°C in a humidified atmosphere of 95% air–5% CO₂ and studied within 24 h of dissociation.

Culture cells were superfused with an extracellular solution containing Na⁺. Those cells that exhibited Na⁺ current were considered to be glomus cells (Overholt & Prabhakar, 1997). Once the presence of Na⁺ current was confirmed, the extracellular solution was changed to solution containing 0.5 µM TTX and 50 µM CdCl₂ to record I_K (see below).

Recording of whole-cell I_K and resting membrane potential

The hypoxia-sensitive K⁺ channels in adult rabbit glomus cells are voltage-gated, Ca²⁺-insensitive K⁺ (Kv) channels (Ganfornina & López-Barneo, 1992). Our previous study has shown that K_Ca currents but not Kv currents run-down in glomus cells in conventional whole-cell recordings due to intracellular dialysis of Ca²⁺ (Li et al. 2004b). In addition, we also found 100 nM iberioxin (a selective K_Ca blocker) did not alter the effect of hypoxia on the Kv currents under perforated-patch recording (data not shown). In the present study, I_K was measured in the conventional whole-cell configuration (Hamill et al. 1981) derived from Kv channels (demonstrated to be 4-amino-pyridine sensitive, Li et al. 2004b). I_K and resting membrane potentials (RMP) were monitored with a patch-clamp amplifier (Warner Instrument Corp., Hamden, CT, USA), which allowed the same cell to be recorded in either the voltage-clamp or current-clamp mode.

Patch pipettes had resistances of 4–6 M when filled with (mM): 105 potassium aspartate, 20 KCl, 1 MgCl₂, 10 EGTA, 5 Mg-ATP, 10 Hepes and 25 glucose, pH 7.2. The extracellular solution had the following composition (mM): 140 NaCl, 5.4 KCl, 1 MgCl₂, 5.5 Hepes, 11 glucose, 10 sucrose, and pH 7.4. Na⁺ and Ca²⁺ channels were blocked by 0.5 µM TTX and 50 µM CdCl₂.

Current traces were sampled at 10 kHz and filtered at 5 kHz. Holding potential was –80 mV. Current–voltage (I–V) relations were elicited by 400 ms test pulses from –80 mV to +80 mV applied in 10 mV increments (5 s between steps). Peak currents were measured for each test potential and were plotted against the corresponding test potential.

To measure the resting membrane potentials (RMP), the amplifier was switched from voltage-clamp mode to current-clamp mode with zero current being delivered. pCLAMP 8.1 programs (Axon Instruments, Inc., Union City, CA, USA) were used for data acquisition and analysis. All experiments were done at 22°C. In order to measure the effect of each treatment, 10 glomus cells from five to seven rabbits were used in each group and the effects of one or two treatments were measured in one cell.

The concentration–response relation for the inhibition of Kv current by Ang II was drawn by a Hill equation with the use of the least-squares method:

where [C] is the concentration of Ang II, IC₅₀ is the Ang II concentration at the half-maximum inhibition of Kv current, and h is the Hill coefficient.

Immunofluorescence for Kv3.4, Kv4.3 and tyrosine hydroxylase (TH) detection

Both CBs in each rabbit were rapidly removed and placed in ice-cold Ca²⁺/Mg²⁺-free solution (mM: NaCl 140, KCl 5, Hepes 10, glucose 5, pH 7.2). The CBs were minced with microscissors and then subjected to the enzymatic digestion solution (2 mg ml^–1 type II trypsin, 2 mg ml^–1 type IV collagenase, and 5 mg ml^–1 BSA) for 50 min at 37°C. The digested tissue fragments were gently triturated for 1 min every 10 min during the process of digestion. CB cells were obtained after centrifugation of the digested tissue at 150 g for 5 min. The isolated cells were plated onto poly-L-lysine-coated coverslips with 2 ml culture medium (50/50 mixture of Delbecco's modified Eagle's medium (DMEM) and Ham's F12 medium supplemented with antibiotics and 10% fetal bovine serum), and maintained at 37°C in a humidified atmosphere of 95% air–5% CO₂ within 24 h.

CB cells plated onto coverslips were fixed with 50/50 mixture of ethanol and methanol for 20 min at –20°C, washed with PBS-Triton solution (phosphate-buffered-saline + 0.1% Triton X-100), and blocked with 10% normal goat serum for 1 h at room temperature. Primary anti-Kv3.4, Kv4.3 (Alomone Laboratories, Israel), and anti-TH antibodies (Santa Cruz, CA, USA) were incubated with the CB cells overnight at 4°C. Then the CB cells were incubated with appropriate secondary antibodies (Invitrogen, CA, USA) for 60 min at room temperature. After washing in PBS, the coverslips were mounted on pre-cleaned microscope slides. The CB cells were observed under a Leica fluorescent microscope with appropriate excitation/emission filters. Pictures were captured by a digital camera system. Kv3.4 and Kv4.3 immunofluorescent images were quantified using Openlab 4.0.2 (Improvision, Lexington, MA, USA). For each animal, 20 TH-positive cells from three to five sections were scored (n = 5 rabbits for each group). No staining was seen when the procedure described above was used but PBS was used instead of the primary antibody.

Western blot analysis for Kv3.4 and Kv4.3

CBs were rapidly removed and immediately frozen in dry ice and stored at –80°C until analysed. CBs of two rabbits were pooled and homogenized with the lysing buffer (10 mM Tris, 1 mM EDTA, 1% SDS, pH 7.4) plus protease inhibitor cocktail (100 µl ml^–1). Following centrifugation at 12 000 g for 20 min at 4°C, the protein concentration in the supernatant was determined using a BCA protein assay kit (Pierce Chemical, Rockford, IL, USA). The protein sample was mixed with loading buffer containing ß-mercaptoethanol and heated at 100°C for 5 min. Protein (20 µg) was loaded. Protein was fractionated in a 10% polyacrylamide gel along with molecular weight standards and transferred to PVDF membrane. The membrane was probed with rabbit primary Kv3.4 and Kv4.3 antibodies (Alomone Laboratories, Israel) and a peroxidase-conjugated goat anti-rabbit IgG. The signal was detected using enhanced chemiluminescence substrate (Pierce Chemical, Rockford, IL, USA) and the bands were analysed using UVP BioImaging Systems. Protein loading was controlled by probing all Western blots with ß-tubulin antibody (Santa Cruz) and normalizing Kv3.4 or Kv4.3 protein intensity to that of ß-tubulin. Four samples (8 rabbits) were assayed in each group.

Data analysis

All data are presented as means ± S.E.M. Statistical significance was determined by Student's paired and unpaired t test for haemodynamic parameters. A two-way ANOVA, with a Bonferroni procedure for post hoc was used in comparisons of Kv currents and RMP. Statistical significance was accepted when P < 0.05.

	Results

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Induction of CHF

Rapid left ventricular pacing induced CHF by the 3rd or 4th week of pacing. LV dD/dt_max and LV shortening fraction were reduced by 60.4 ± 1.8 and 60.7 ± 2.0%, respectively, after 3 or 4 weeks of pacing compared with prepaced baselines (P < 0.05, Table 1). There was no significant change in the LV dD/dt_max and LV shortening fraction between the baseline and the 3rd or 4th week in sham rabbits (Table 1).

Under resting, normoxic conditions (P_O2 = 104 ± 1.4 Torr) I_K was significantly blunted in glomus cells from CHF rabbits compared with those from sham rabbits (P < 0.05, sham-control versus CHF-control in Fig. 1A, B and C). There was no difference in membrane capacitance in glomus cells from sham (2.73 ± 0.22 pF) and CHF (3.16 ± 0.29 pF) rabbits. These results are consistent with those observed in our previous study (Li et al. 2004b).

View larger version (36K): [in this window] [in a new window]   Figure 1.  Effect of Ang II on IK in CB glomus cells from sham and CHF rabbits under normoxic conditions (PO2 = 104 ± 1.4 Torr) IK was evoked by 400 ms depolarizing pulses from –80 to +80 mV, 10 mV steps). A and B, IK from a sham and CHF glomus cell, respectively, before (control) and after treatment with 100 nM Ang II in the extracellular medium. C, peak I–V relationships (n = 10 cells from 7 rabbits in each group) from sham and CHF glomus cells obtained before and after treatment with 100 nM Ang II. Data are means ± S.E.M. *P < 0.05 versus sham-control; #P < 0.05 versus CHF-control. D, dose–response curves of extracellular Ang II concentration versus percentage reduction of peak IK, in sham and CHF glomus cells. Data are means ± S.E.M.n = 10 cells from 7 rabbits for each point. *P < 0.05 versus sham. E, peak IK of sham and CHF glomus cells before (control) and after exposure to either 100 nM Ang II, 1 µM L-158,809 (AT1R antagonist), or 100 nM Ang II + 1 µM L-158,809. Data are means ± S.E.M.n = 10 cells from 7 rabbits in each group. *P < 0.05 versus control; #P < 0.05 versus Ang II; P < 0.05 versus sham). Peak IK in D and E measured in response to a test pulse from –80 to +70 mV.

Effects of Ang II and L-158,809 on the sensitivity of I_K to hypoxia in CB glomus cells from sham rabbits

Hypoxia (P_O2 = 41.7 ± 2.2 Torr) significantly blunted I_K in glomus cells from sham rabbits (Fig. 2A). This effect is consistent with the well-documented response of Kv channels to hypoxia in rabbit glomus cells (Kaab et al. 2005). It was not possible to test the effects of high concentrations of Ang II on the sensitivity of I_K to hypoxia because high concentrations of Ang II (> 1 nM) itself directly decreased the I_K at normoxia (see above). Nevertheless, 100 pM Ang II, a dose that did not affect I_K at normoxia (P_O2 = 103 ± 1.8 Torr), markedly increased the sensitivity of I_K to hypoxia (Fig. 2) in glomus cells from sham rabbits. Average data are illustrated in Fig. 2D. Ang II significantly augmented the inhibition of I_K in response to hypoxia (22.5 ± 3.9% inhibition before versus 46.2 ± 4.1% inhibition after treatment with 100 pM Ang II, P < 0.05, Fig. 2D). L-158,809 (1 µM) alone did not affect the sensitivity of I_K to hypoxia (22.5 ± 3.9% inhibition before versus 21.7 ± 3.4% inhibition after L-158,809 treatment), but it inhibited the effect of Ang II on the sensitivity of I_K to hypoxia (46.2 ± 4.1% inhibition after treatment with Ang II versus 24.2 ± 3.7% inhibition after treatment with L-158,809 plus Ang II, P < 0.05, Fig. 2D).

View larger version (46K): [in this window] [in a new window]   Figure 2.  Effects of Ang II and L-158,809 on the sensitivity of IK to hypoxia (PO2 = 41.7 ± 2.2 Torr) in CB glomus cells from sham rabbits IK was elicited as in Fig. 1. A and B, effects of hypoxia on IK before (A) and after (B) administration of 100 pM Ang II to the extracellular medium. C, peak I–V relationships in 10 cells from 7 sham rabbits for data illustrated in A and B. Data are means ± S.E.M. *P < 0.05 versus control; #P < 0.05 versus hypoxia before Ang II. D, percentage change of IK by hypoxia (Icontrol – Ihypoxia)/Icontrol) in sham glomus cells before (hypoxia alone) and after exposure to either 100 pM Ang II, 1 µM L-158,809 (AT1R antagonist), or 100 pM Ang II + 1 µM L-158,809. Data are means ± S.E.M.n = 10 cells from 7 rabbits in each condition. *P < 0.05 versus hypoxia alone; #P < 0.05 versus Ang II (100 pM) + hypoxia).

Hypoxia (P_O2 = 42.2 ± 1.3 Torr) also significantly reduced I_K in glomus cells from CHF rabbits (Fig. 3). Furthermore, the effect of hypoxia on I_K in CHF rabbits (53.5 ± 4.4% inhibition) was greater than that in sham rabbits (22.5 ± 3.9% inhibition, P < 0.05, Figs 2A and 3A). After administration of L-158,809 alone (1 µM), the sensitivity of I_K to hypoxia in glomus cells from CHF rabbits was markedly decreased (27.8 ± 3.8% inhibition, P < 0.05, Fig. 3D), to a level similar to that normally observed in the glomus cells from sham rabbits (see above). However, Ang II alone (100 pM) neither affected I_K at normoxia, nor altered the sensitivity of I_K to hypoxia in glomus cells from CHF rabbits (55.8 ± 3.9% inhibition, Fig. 3D).

View larger version (40K): [in this window] [in a new window]   Figure 3.  Effects of Ang II and L-158,809 on the sensitivity of IK to hypoxia (PO2 = 42.2 ± 1.3 Torr) in CB glomus cells from CHF rabbits IK was elicited as in Fig. 1. A and B, effects of hypoxia on IK before (A) and after (B) administration of 100 pM Ang II to the extracellular medium. C, peak I–V relationships in 10 cells from 7 CHF rabbits for data illustrated in A and B. Data are means ± S.E.M. *P < 0.05 versus control; #P < 0.05 versus hypoxia before L-158,809). D, percentage change of IK by hypoxia (Icontrol – Ihypoxia)/Icontrol) in CHF glomus cells before (hypoxia alone) and after exposure to either 100 pM Ang II, 1 µM L-158,809 (AT1R antagonist), or 100 pM Ang II + 1 µM L-158,809. Data are means ± S.E.M.n = 10 cells from 7 rabbits in each condition. *P < 0.05 versus hypoxia alone; #P < 0.05 versus Ang II (100 pM) + hypoxia).

In CHF rabbits, RMP of the glomus cells was depolarized, compared with that in sham rabbits (43.2 ± 2.3 mV in sham rabbits versus 32.8 ± 2.0 mV in CHF rabbits; P < 0.05, Fig. 4). Hypoxia (41.9 ± 2.1 Torr) induced depolarization of RMP in glomus cells from both sham and CHF rabbits (Fig. 4A), but the effect of hypoxia on RMP in CHF rabbits was greater than that in sham rabbits (35.9 ± 1.8% decrease in RMP in CHF rabbits and 21.4 ± 1.4% in sham rabbits, P < 0.05; Fig. 4B). Ang II (100 pM) significantly facilitated the effect of hypoxia to decrease RMP in glomus cells from sham rabbits but not CHF rabbits, whereas L-158,809 alone (1 µM) markedly inhibited the effect of hypoxia to decrease RMP in CHF rabbits but not sham rabbits (Fig. 4B).

View larger version (38K): [in this window] [in a new window]   Figure 4.  Effects of Hyproxia, Ang II, and L-158,809 on RMP in CB glomus cells from sham and CHF rabbits A, effect of hypoxia (41.9 ± 2.1 Torr) on resting membrane potential (RMP) at different conditions in CB glomus cells from sham and CHF rabbits. Data are means ± S.E.M.n = 8 cells from 5 rabbits in each condition. *P < 0.05 versus sham-normoxia or CHF-normoxia; #P < 0.05 versus sham-normoxia. B, percentage reduction of RMP by hypoxia (Icontrol – Ihypoxia)/Icontrol) in sham and CHF glomus cells before (hypoxia alone) and after exposure to either 100 pM Ang II, 1 µM L-158,809 (AT1R antagonist), or 100 pM Ang II + 1 µM L-158,809. Data are means ± S.E.M.n = 8 cells from 5 rabbits in each condition. *P < 0.05 versus hypoxia alone; #P < 0.05 versus Ang II (100 pM) + hypoxia).

Figure 5A illustrates typical images of isolated glomus cell clusters with immunofluorescent double-labelling for TH and Kv3.4 in CB glomus cells from sham and CHF rabbits. Kv3.4 clearly appeared in the CB glomus cells from sham rabbits (Fig. 5A), validated by the presence of TH as an immunohistochemical marker for CB glomus cells (Kameda et al. 1990). Immunoflourescent labelling of Kv3.4 was markedly less in the CB glomus cells from CHF rabbits, compared with that from sham rabbits (Fig. 5B). However, there was no measurable difference in the Kv4.3 protein labelling in the CB glomus cells between sham and CHF rabbits (Fig. 6, P > 0.05). Similarly, Western blot data showed that protein expression of Kv3.4 but not Kv4.3 was decreased in the CB tissue from CHF rabbits versus sham rabbits (Fig. 7).

View larger version (40K): [in this window] [in a new window]   Figure 5.  Co-localization of tyrosine hydroxylase (TH) and Kv3.4 in CB glomus cells from sham and CHF rabbits A, CB from a sham rabbit (a–c) and CHF rabbit (d–f). Green immunofluorescent image for TH in a and d, red immunofluorescent image for Kv3.4 showed in b and e, and the merged image for overlap of TH and Kv3.4 in c and f. B, normalized fluorescence intensity for Kv3.4 in CB glomus cells from sham and CHF rabbits (see detail in Methods). Data are means ± S.E.M.n = 5 rabbits for each group. *P < 0.05 versus sham.

View larger version (42K): [in this window] [in a new window]   Figure 6.  Co-localization of tyrosine hydroxylase (TH) and Kv4.3 in CB glomus cells from sham and CHF rabbits A, CB from a sham (a–c) and CHF (d–f) rabbit. Green immunofluorescent image for TH in a and d, red immunofluorescent image for Kv4.3 in b and e, and the merged image for overlap of TH and Kv4.3 in c and f. B, normalized fluorescence intensity for Kv4.3 in CB glomus cells from sham and CHF rabbits (see detail in Methods). Data are means ± S.E.M.n = 5 rabbits for each group. *P < 0.05 versus sham.

View larger version (31K): [in this window] [in a new window]   Figure 7.  Protein expression of Kv3.4 and Kv4.3 The representative (A) and summary (B) data for protein expression of Kv3.4 and Kv4.3 in the CBs from sham and CHF rabbits. Data are means ± S.E.M.n = 4 samples in each group. *P < 0.05 versus sham.

	Discussion

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Role of Ang II in the enhanced sensitivity of Kv channels to hypoxia in CHF glomus cells

The hypoxia-sensitive K⁺ channels in adult rabbit CB glomus cells are calcium-insensitive, voltage-gated K⁺ (Kv) channels (Ganfornina & López-Barneo, 1992), which encompass those with Kv4.1, and Kv4.3 subunits (Sanchez et al. 2002). Data (Gonzalez et al. 1994; Prabhakar, 1994) regarding O₂-sensing mechanisms have shown that I_K inhibition induced by hypoxia causes membrane depolarization in glomus cells, inducing an intracellular Ca²⁺ increase through voltage-gated Ca²⁺ channel opening, and leading to release of Ca²⁺-dependent neurotransmitter(s) that act on apposing afferent nerve terminals to enhance sensory discharge in the carotid sinus nerve. Our results suggest that this is a likely mechanism by which CB chemoreceptor activation to graded hypoxia is enhanced in CHF rabbits (Sun et al. 1999b). The present study confirms that hypoxia decreases I_K and induces depolarization of RMP in rabbit glomus cells, and that these hypoxic responses are enhanced in the CHF state.

This is the first study to demonstrate that the sensitivity of Kv channels to hypoxia is enhanced in isolated glomus cells from CHF rabbits and that blockade of the AT₁R (L-158,809) alone is capable of normalizing this enhanced hypoxic sensitivity. In addition, exposing normal CB glomus cells to Ang II can mimic this effect of CHF on Kv channel function. These observations have two important implications: first, Ang II is capable of enhancing the O₂ sensitivity of Kv channels via a AT₁R-mediated pathway; and second, this cellular Ang II–AT₁R signalling mechanism is operating in isolated glomus cells from CHF but not normal rabbits. This notion is supported by previous studies that have identified components of a local Ang II system in CB tissue (Lam & Leung, 2002) and have found chronic hypoxia activates a local Ang-generating system and up-regulates the expression and function of AT₁R in rat CB (Leung et al. 2000; Lam & Leung, 2003; Lam et al. 2004), and by our recent study that revealed that Ang II concentration and AT₁R expression are elevated in CB tissue from CHF rabbits (Li et al. 2006).

This effect of cellular Ang II to enhance the hypoxic sensitivity of Kv channels appears to be functioning maximally in glomus cells from our CHF animals since exposure of these cells to exogenous Ang II (100 pM) had no further effect, whereas the same concentration of Ang II markedly enhanced O₂ sensitivity in glomus cells from normal rabbits. Conversely, the fact that AT₁R blockade (L-158,809) alone did not affect the O₂ sensitivity of Kv channels in glomus cells from sham rabbits, but had marked effects in CHF glomus cells, would suggest that ability of Ang II to sensitize Kv channels to hypoxic inhibition is not functioning (or at least not demonstrable in our isolated experimental conditions) in the CB of normal rabbits.

We recently established that perfusion of the CB with 100 pM Ang II enhances CB chemoreceptor responses to hypoxia in normal rabbits (Li et al. 2006). Furthermore, AT₁R blockade normalizes the enhanced CB chemoreceptor responses to hypoxia in CHF rabbits (Li et al. 2006). In the present study, we found that 100 pM Ang II enhanced the sensitivity of I_K to hypoxia and facilitated the effect of hypoxia on RMP in sham rabbits, whereas L-158,809 attenuated the sensitivity of I_K to hypoxia and markedly decreased the effect of hypoxia on RMP in CHF rabbits. In addition, both Ang II and AT₁R levels are increased in the CB of CHF rabbits (Li et al. 2006). These results suggest that up-regulation of the local Ang II system in the CB contributes to the enhanced CB responsiveness to hypoxia characterized in CHF (Sun et al. 1999b) as a result of the ability of the peptide to enhance the O₂ sensitivity of Kv channels.

The concentration of exogenously administered Ang II (100 pM) we used to invoke this effect in normal glomus cells approaches that of maximal physiological circulating levels. We have previously reported that plasma levels of Ang II in CHF rabbits at 3–4 weeks of pacing are near 50 pg ml^–1 (Li et al. 2006). In preliminary experiments, however, we found that 50 pM Ang II had little effect on the O₂ sensitivity of Kv channels in sham glomus cells. It is possible that this disparity may be an experimental artifact: dispersion and isolation of CB cells required for patch clamping may render the glomus cells less sensitive to Ang II. We think it is more likely that these results suggest that intracellular rather than extracellular Ang II is important in modulating Kv channel function in the glomus cells. Thus high levels of Ang II are needed in the extracellular medium to allow internalization of sufficient amounts of the peptide to have a physiological effect in normal glomus cells. In the CHF glomus cells, an enhanced intracellular production of Ang II can explain its effects on Kv channel function independent of extracellular Ang II.

Although our results suggest that elevation of local tissue Ang II in the CB plays an important role on the hypersensitivity of I_K to hypoxia in CHF, it is not clear how Ang II within an isolated glomus cell interacts with AT₁R to affect the sensitivity of Kv channels. Recent studies have shown that intracellular administration of Ang II increases the peak inward calcium current density and decreases the junctional conductance via intracellular Ang II receptors in cardiac myocytes (De Mello, 2003; De Mello & Monterrubio, 2004), and intracellular treatment of losartan (a selective AT₁R antagonist) abolishes the effect of intracellular Ang II (De Mello, 2003; De Mello & Monterrubio, 2004). Based on these studies, we speculate that the enhanced sensitivity of I_K to hypoxia in CHF may be due to elevation of intracellular Ang II in CB glomus cells binding to intracellular AT₁R. This notion is confirmed by our observation (data not shown) that intracellular administration of L-158,809 (added to the recording pipette solution) blunted the sensitivity of I_K to hypoxia in the CB glomus cells from CHF rabbits.

The process that occurs in the course of the development of CHF that causes an up-regulation of this local Ang II–AT₁R signalling in CB glomus cells is not evident from the present study. This phenomenon, however, is not unique to the CB. Previous studies have documented that Ang II effects are enhanced in several tissues in CHF, including the heart (Schulz & Heusch, 2005; de Resende et al. 2006), brainstem (Ferguson & Latchford, 2000; DiBona, 2001), kidney (Schunkert et al. 1992; de Resende et al. 2006), vascular smooth muscle (Crespo, 1999), and skeletal muscle (Jones & Woods, 2003). It is interesting to note that the changes in Kv channel function we describe in glomus cells in CHF rabbits are similar in most respects to those described in rabbit glomus cells exposed to chronic hypoxia (Kaab et al. 2005). This observation raises the question whether the chronically decreased cardiac output of CHF may be sufficient to produce a decrease in O₂ delivery to the carotid body (akin to chronic hypoxia) that leads to the functional changes that occur in glomus cells. This issue warrants further study.

Chronic exposure of rabbit CB glomus cells to hypoxia induces down-regulation of Kv3.4 channels, an O₂-insensitive component of the Kv current, but not the hypoxia-sensitive Kv4.3 channels in rabbit CB glomus cells (Kaab et al. 2005). This shift in balance of Kv channels results in a more predominant role of the hypoxia-sensitive Kv4.3 to the total outward K⁺ current, with a subsequent relative increase in the magnitude of acute hypoxia-induced inhibition of I_K. We found that in CHF, there is a similar decrease in the expression of Kv3.4 but not Kv4.3 in rabbit CB glomus cells. It is possible that the increased proportion of Kv4.3 channels to the total outward K⁺ current contributes to the enhanced sensitivity of I_K to hypoxia in glomus cells in CHF. Nevertheless, the marked effect of AT₁R blockade to reverse the exaggerated hypoxic sensitivity of I_K observed in our CHF glomus cells (Fig. 3D), points to an important regulatory role for Ang II on post-translational Kv activity in the glomus cell in the CHF state.

The mechanism by which Ang II–AT₁R activation enhances the sensitivity of I_K to hypoxia in CB glomus cells is not known. One possible candidate is the protein kinase C (PKC) signalling pathway. AT₁R activation results in a rapid, phospholipase C-dependent sustained release of diacylglycerol, which leads to activation of PKC (Ushio-Fukai et al. 1998). PKC was shown to cause inhibition of K_Ca currents in CB glomus cells of rats, but PKC activation could not account for inhibition of these channels by acute hypoxia (Peers & Carpenter, 1998). The effect of PKC on the O₂ sensitivity of Kv channels in rabbit CB glomus cells has not yet been addressed. Alternatively, Ang II recently has been implicated in activation of ROS, specifically superoxide anion, in CHF (Tojo et al. 2002; Korantzopoulos et al. 2003; Lindley et al. 2004). AT₁R activation promotes activation of NADPH oxidase (Touyz & Berry, 2002). Our preliminary experiments have shown that mRNA and protein expressions of NADPH oxidase subunits are enhanced in CBs from CHF rabbits, and a NADPH oxidase inhibitor (apocynin) and superoxide dismutase mimetic (tempol) inhibit the effect of Ang II on the sensitivity of I_K to acute hypoxia (Li et al. 2004a).

Effect of Ang II on Kv channel activity during normoxia in glomus cells

The glomus cell membrane in rabbits possesses a calcium-dependent, voltage-gated K⁺ (K_Ca or maxi K) channel in addition to Kv channels that contributes to I_K. Previously, we observed that I_K carried by K_Ca channels is blunted under normoxic conditions in CB glomus cells from CHF rabbits, which contributes to the decrease of total outward K⁺ currents observed in these cells. We also previously established that a decrease in the availability of endogenous nitric oxide (NO) in the CB mediates the suppression of K_Ca channels in CB glomus cells in CHF (Li et al. 2004b). This mechanism is consistent with the observation that NO donors inhibit the elevated baseline (normoxic) discharge of CB chemoreceptor afferents in CHF rabbits, and that NO synthase inhibitors increase the baseline discharge of CB chemoreceptors in sham rabbits (Sun et al. 1999b). In the present study, the contribution from K_Ca channels was excluded in the experimental design (see detail in Methods). By doing so, we observed that the I_K carried by remaining Kv channels is also blunted (28.3 ± 2.4% decrease) at normoxia in glomus cells from CHF rabbits compared with sham, but to a lesser extent than that of K_Ca (41.6 ± 2.8% decrease; Li et al. 2004b).

The present results indicate that neither extracellular Ang II at a dose within the high physiological range (100 pM), nor AT₁R blockade itself have effects on Kv channel I_K at normoxia in either sham or CHF glomus cells (see Figs 2B and C, and 3B and C). These results are consistent with our recent observation that exposure of the isolated CB to a similar level of Ang II or to L-158,809 had no effect on the activity of chemoreceptor discharge at normoxia in either sham or CHF rabbits (Li et al. 2006). Thus endogenous Ang II cannot account for the suppression of Kv channel activity in glomus cells (Fig. 1) or the enhanced chemoreceptor activity observed in CHF rabbits (Sun et al. 1999b; Li et al. 2006) in normoxic (control) conditions. The possible role of NO depletion on the suppression of K_V channel activity in glomus cells of CHF rabbits during normoxia was not addressed in the present study, but is worthy of further study. It is also likely that the down-regulation of Kv3.4 that occurred in CHF glomus cells (Fig. 5) would contribute to the blunted I_K we observed in CHF glomus cells under normoxic conditions.

A confounding revelation from our experiments that seems at odds with the discussion above, is that, at very high concentrations (> 1 nM), exogenous Ang II directly inhibits Kv channels under constant P_O2 (normoxic) conditions in both sham and CHF glomus cells. This effect also is mediated via AT₁R activation. This effect of high levels of Ang II to directly inhibit Kv channels differs from its effect (at lower levels) to only enhance oxygen sensitivity of the channels. First, endogenous Ang II, even in the elevated CHF state, is not capable of generating this effect to directly inhibit I_K from Kv channels in glomus cells at normoxia. In addition, the threshold concentration needed to evoke this direct inhibitory affect is at least 6–10 times greater than which produced maximal effects on O₂ sensitivity (100 pM). Another significant observation is that glomus cells from CHF rabbits were more sensitive than sham cells to the direct inhibitory effect of Ang II on I_K at normoxic P_O2. It would appear therefore that the total Ang II concentration (endogenous + exogenous) contributes to this response. Due to the very high levels of exogenous Ang II required to evoke this direct inhibitory effect on Kv channel activity, even in the CHF state, the physiological significance of this response remains uncertain. Nevertheless, it is conceivable that under certain pathophysiological conditions, such as severe heart failure, the total Ang II content of the CB may be sufficiently elevated to mediate this inhibitory effect on Kv channel function under normoxic conditions.

A recent study may shed some light on a possible mechanism by which Ang II inhibits Kv channels. In HEK 293 cells co-transfected with AT₁R and Kv4.3 expression vectors, Doronin et al. (2004) found that the AT₁R forms a stable complex with Kv4.3 on the cell surface. In the presence of Ang II (1 µM), Kv4.3 protein was internalized and co-localized with AT₁R in intracellular compartments. This mechanism is thought to be involved in the reduction of I_K in the presence of Ang II observed in these transfected cells (Doronin et al. 2004), and can also explain our present results in which Ang II at the high concentration (> 1 nM) attenuated I_K in glomus cells.

Role of other K⁺ channels in glomus cells

It was evident from our experiments that steady state K⁺ currents (I_Kss), as well as the peak transient outward currents, were similarly influenced by CHF, hypoxia, and Ang II (see current traces in Figs 1–3). Kv3.4 and Kv4.3 channels that we describe in this study, and K_Ca that we described in a previous study (Li et al. 2004b) are known to be principle conduits of the peak transient outward K⁺ current in rabbit glomus cells (Ganfornina & López-Barneo, 1992;. Sanchez et al. 2002; Li et al. 2004b). The family of K⁺ channels that comprise the I_Kss in the CB glomus cells from rabbit are not well described, nor extensively studied. HERG-like K⁺ channel (I_Kr), one component of delayed rectifier K⁺ channels, has been identified in the CB glomus cells of the rabbit (Overholt et al. 2000) but its sensitivity to the conditions we describe in this study have yet to be studied. Other components of delayed rectifier K⁺ channels (such as rapidly activating, slowly inactivating delayed rectifier K⁺ channel and slowly activated delayed rectifier K⁺ channels) are not reported in the rabbit CB glomus cells. Background K⁺ channels (such as TASK channels) that are O₂ sensitive in rat CB glomus cells (Buckler et al. 2000), also have not been described in rabbit CB glomus cells. It also is possible that changes in inactivation kinetics of Kv channels could influence the magnitude of the I_Kss. Therefore, further study is needed to explore the underlying mechanisms of alterations of I_Kss induced by CHF, hypoxia, and Ang II in rabbit glomus cells.

In conclusion, this study indicates that Kv channel currents are suppressed in CB glomus cells from CHF rabbits, and these channels exhibit an enhanced sensitivity to hypoxic inhibition. This study also reveals that a cellular Ang II–AT₁R mechanism contributes to the enhanced sensitivity of Kv channels to hypoxia in CB glomus cells in the CHF state. The down-expression of Kv3.4 channels in glomus cells during CHF may contribute to the reduction of I_K. Exposure of glomus cells to high levels of Ang II directly inhibits Kv channel currents, but the functional relevance of this effect is less clear. Our study establishes an autocrine role of Ang II in the carotid body to modulate chemosensitivity to hypoxia via effects on O₂-sensitive K⁺ channels in glomus cells. Up-regulation of this tissue angiotensin system contributes to the enhanced sensitivity of the carotid body chemoreflex in heart failure.

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	Acknowledgements

 
The authors wish to thank Kaye Talbitzer and Kristin J. Bohling for their technical assistance, and Dr Kurtis Cornish for his surgical assistance. This study was supported by a Program Project Grant from the Heart, Lung and Blood Institute of the NIH (PO1-HL62222).

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