Nitric Oxide Inhibits L-Type Ca2+ Current in Glomus Cells of the Rabbit Carotid Body Via a cGMP-Independent Mechanism

Beth A. Summers, Jeffrey L. Overholt, and Nanduri R. Prabhakar

Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4970


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Summers, Beth A., Jeffrey L. Overholt, and Nanduri R. Prabhakar. Nitric oxide inhibits L-type Ca2+ current in glomus cells of the rabbit carotid body via a cGMP-independent mechanism. Previous studies have shown that nitric oxide (NO) inhibits carotid body sensory activity. To begin to understand the cellular mechanisms associated with the actions of NO in the carotid body, we monitored the effects of NO donors on the macroscopic Ca2+ current in glomus cells isolated from rabbit carotid bodies. Experiments were performed on freshly dissociated glomus cells from adult rabbit carotid bodies using the whole cell configuration of the patch-clamp technique. The NO donors sodium nitroprusside (SNP; 600 µM, n = 7) and spermine nitric oxide (SNO; 100 µM, n = 7) inhibited the Ca2+ current in glomus cells in a voltage-independent manner. These effects of NO donors were rapid in onset and peaked within 1 or 2 min. In contrast, the outward K+ current was unaffected by SNP (600 µM, n = 6), indicating that the inhibition by SNP was not a nonspecific membrane effect. 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (carboxy-PTIO; 500 µM), an NO scavenger, prevented inhibition of the Ca2+ current by SNP (n = 7), whereas neither superoxide dismutase (SOD; 2,000 U/ml, n = 4), a superoxide scavenger, nor sodium hydrosulfite (SHS; 1 mM, n = 7), a reducing agent, prevented inhibition of the Ca2+ current by SNP. However, SNP inhibition of the Ca2+ current was reversible in the presence of either SOD or SHS. These results suggest that NO itself inhibits Ca2+ current in a reversible manner and that subsequent formation of peroxynitrites results in irreversible inhibition. SNP inhibition of the Ca2+ current was not affected by 30 µM LY 83,583 (n = 7) nor was it mimicked by 600 µM 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP; n = 6), suggesting that the effects of NO on the Ca2+ current are mediated, in part, via a cGMP-independent mechanism. N-ethylmaleimide (NEM; 2.5 mM, n = 6) prevented the inhibition of the Ca2+ current by SNP, indicating that SNP is acting via a modification of sulfhydryl groups on Ca2+ channel proteins. Norepinephrine (NE; 10 µM) further inhibited the Ca2+ current in the presence of NEM (n = 7), implying that NEM did not nonspecifically eliminate Ca2+ current modulation. Nisoldipine, an L-type Ca2+ channel blocker (2 µM, n = 6), prevented the inhibition of Ca2+ current by SNP, whereas omega -conotoxin GVIA, an N-type Ca2+ channel blocker (1 µM, n = 9), did not prevent the inhibition of Ca2+ current by SNP. These results demonstrate that NO inhibits L-type Ca2+ channels in adult rabbit glomus cells, in part, due to a modification of calcium channel proteins. The inhibition might provide one plausible mechanism for efferent inhibition of carotid body activity by NO.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The carotid bodies are sensory organs that detect changes in arterial oxygen. Hypoxia augments the sensory discharge of the carotid bodies, and the resulting reflexes are crucial for maintaining homeostasis during hypoxemia (see Fitzgerald and Lahiri 1986 for references). The chemoreceptor tissue is composed of neurotransmitter-enriched glomus (type I) cells, which are of neural crest origin, and sustentacular (type II) cells that are glial-like. Currently it is believed that glomus cells, which lie in synaptic apposition with sensory nerve endings, are the initial sites of sensory transduction. Several hypotheses have been proposed for the transduction of the hypoxic stimulus at the carotid bodies (Gonzalez et al. 1992). Whatever the mechanism of transduction, much evidence indicates that neurotransmitters play important roles in sensory transmission at the carotid body (Fidone and Gonzalez 1986; Gonzalez et al. 1992; Prabhakar 1994). Thus it is of considerable importance to understand the cellular mechanisms associated with the actions of neurotransmitters at the carotid body.

Nitric oxide (NO) is a gas molecule with free radical properties that functions as a transmitter molecule in both the peripheral and central nervous systems. It is synthesized during the catalytic conversion of arginine to citrulline by the enzyme nitric oxide synthase (NOS) (Snyder 1992). NO is an unusual neurotransmitter in that it diffuses toward its target cells instead of being stored and released from synaptic vesicles. Recent immunocytochemical studies have shown that NOS is present in both nerve fibers (Prabhakar et al. 1993; Wang et al. 1993) and blood vessels within the carotid body (Grimes et al. 1995; Wang et al. 1993). Inhibitors of NOS augment (Chugh et al. 1994; Prabhakar et al. 1993; Wang et al. 1995), whereas NO donors inhibit, the sensory discharge of the carotid bodies (Wang et al. 1994). Recently, Kline et al. (1998) examined ventilatory responses to hypoxia in neuronal NOS knockout mice. These authors found that respiratory responses to hypoxia are augmented selectively in nNOS knockout mice, in part due to exaggerated peripheral chemoreceptor sensitivity, suggesting that NO generated by neuronal NOS is an important physiological regulator of carotid body activity. Furthermore our previous studies have shown that hypoxia inhibits NOS activity in the carotid body, indicating that NO generation is modulated by oxygen (Prabhakar et al. 1993). Taken together, these studies suggest that NO is an important modulator of carotid body activity and that NO production can be regulated by hypoxia. However, very little is known about the cellular mechanisms associated with the actions of NO in the carotid body.

There are a number of pathways through which NO can exert its effects. In many systems, NO acts through the activation of heme containing guanylate cyclase and subsequent elevation of cellular levels of cyclic GMP (cGMP) (Snyder 1992). NO signaling also can encompass the actions of other naturally occurring NO derivatives, such as S-nitrosothiols (RSNOs), the actions of which depend on the redox environment of the cell (Campbell et al. 1996; Stamler et al. 1992). Cellular targets of cGMP and/or S-nitrosothiols include channel proteins that regulate ionic conductances. In addition, NO has been shown to directly affect ion channel activity by nitrosylation of sulfhydryl groups on the channel protein (Bolotina et al. 1994; Campbell et al. 1996; Hu et al. 1997). Several studies have shown that NO affects Ca2+ currents in a variety of cells. Regardless of the pathway, reported effects of NO on Ca2+ current are complex and include both inhibition and augmentation. For example, in vascular smooth muscle cells (A7r5 cells), Ca2+ currents are inhibited by NO (Blatter and Wier 1994). On the other hand, NO donors augment Ca2+ currents in neurons of the superior cervical ganglion (Chen and Schofield 1995).

Ca2+ channels play an important role in carotid body chemoreception. As in other systems, Ca2+ channels are involved in the release of neurotransmitters from glomus cells (Gonzalez et al. 1992). This Ca2+-dependent neurotransmitter release from glomus cells is believed to be an obligatory step in the chemotransduction process (Biscoe and Duchen 1990; Fidone and Gonzalez 1986; Prabhakar 1992; Shirahata and Fitzgerald 1991). Previous studies have shown that both rat and rabbit glomus cells exhibit high-voltage-activated (HVA), but not low voltage-activated, (LVA) Ca2+ current (e Silva and Lewis 1995; Urena et al. 1989). The HVA Ca2+ current has been shown to be sensitive to dihydropyridines in both rabbit (Obeso et al. 1992) and rat (e Silva and Lewis 1995; Fieber and McCleskey 1993; Peers et al. 1996), suggesting that they express L-type channels. In addition, we recently have reported that the Ca2+ current in rabbit glomus cells is conducted by L, N, P/Q and resistant channel types (Overholt and Prabhakar 1997).

NO, once released from the nerve endings, affects carotid body sensory activity by changing carotid body blood flow via its action on vascular smooth muscle cells and/or by direct action on the glomus cell acting as a retrograde messenger as it does elsewhere in the nervous system (Snyder 1992). Because several lines of evidence indicate that glomus cells are important for sensory transduction at the carotid body, we investigated the cellular action(s) of NO in this cell type. Our hypothesis is that NO affects one or more of the HVA Ca2+ channel types in glomus cells. To test this possibility, we monitored the effects of NO donors on the macroscopic Ca2+ current in glomus cells isolated from rabbit carotid bodies. Our results demonstrate that NO inhibits voltage-gated Ca2+ channels, an effect that primarily is confined to L-type Ca2+ channels. Further the effects of NO on Ca2+ current in glomus cells appears to be a direct action on the channel protein and/or an associated channel protein rather than through a cGMP-dependent mechanism.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General procedures

Experiments were performed on glomus cells freshly isolated from the carotid bodies of adult rabbits killed with CO2. Individual glomus cells were dissociated enzymatically as described previously (Overholt and Prabhakar 1997). Briefly, carotid bodies were incubated at 37°C in a solution that contained trypsin (type II, 2 mg/ml, Sigma) and collagenase (type IV, 2 mg/ml, Sigma). The composition of the incubation medium was (in mM) 140 NaCl, 5 KCl, 10 HEPES, and 5 glucose, pH 7.2. The carotid body tissue was triturated mechanically with a fire-polished glass pasteur pipette every 10 min. After 30 min of incubation, cells were pelleted after centrifugation at 1,800 g for 5 min. Dissociated cells were resuspended in a 50/50 mixture of Dulbecco's minimum essential medium (DMEM) and HAM F12 supplemented with penicillin-streptomycin (GIBCO-BRL), insulin, transferrin, selenium (ITS, Sigma), and 10% heat-inactivated fetal bovine serum. Cells were maintained at 37°C in a CO2 incubator and were used within 36 h. All experiments were performed at room temperature.

Isolation of Ca2+ current

Ca2+ current was monitored using the whole cell configuration of the patch-clamp technique (Hamill et al. 1981). Pipettes coated with silicone elastomer (Sylgard, Dow Corning) were made from borosilicate glass capillary tubing and had resistances of 4-5 MOmega . Currents were recorded using an Axopatch 200A voltage-clamp amplifier, filtered at 5 kHz and sampled at a frequency of 28.6 kHz using an IBM-compatible computer with a Digidata 1200 interface and pCLAMP software (Axon Instruments). Currents were not leak subtracted. Current-voltage (I-V) relations were elicited from a holding potential of -80 mV using 25-ms steps (5 s between steps) to test potentials over a range of -50 to +70 mV in 10-mV increments. Current at each potential was measured as the average over a 2.5-ms span at the end of the 25-ms step.

Ca2+ current was isolated by using K+- and Na+-free intra- and extracellular solutions. The intracellular solution had the following composition (in mM): 115 CsCl, 20 TEA-Cl, 5 MgATP, 0.1 TrisGTP, 5 EGTA, 10 phosphocreatine, and 5 HEPES, and the pH was adjusted to 7.2 with CsOH. The extracellular solution contained (in mM) 140 NMGCl, 5.4 CsCl, 10 BaCl2, 10 HEPES, and 11 glucose, and the pH was adjusted to 7.4 with CsOH. The extracellular solution was changed using a fast-flow apparatus consisting of a linear array of borosilicate glass tubes (Overholt and Prabhakar 1997). In these experiments, Ba2+ was the charge carrier. For simplicity, Ba2+ current conducted by Ca2+ channels will be referred to as Ca2+ current. To observe Na+ current to identify a glomus cell, cells first were superfused with an extracellular solution containing Na+ (see solutions for recording K+ current for composition).

Rundown of Ca2+ current and the effects of drugs were monitored using a wash protocol (25 ms step to 0 mV, 10 s between steps). The effects of NO agents were compensated for rundown using a linear regression of the current decrease during the wash protocol in the absence of test compounds. Rundown was negligible compared with drug effects over the same time period (e.g., 1.0 ± 0.3%/min, mean ± SD; n = 4). Cells in which rundown was excessive or did not appear linear were excluded from the analysis. For comparison of I-V relations, Ca2+ current at each potential was normalized to the maximum value recorded during the control I-V relation in individual cells (usually 0 mV).

Isolation of K+ current

K+ current was monitored using Na+- and K+-containing solutions. The intracellular solution had the following composition (in mM): 120 K-glutamate, 20 KCl, 5 MgATP, 0.1 TrisGTP, 5 EGTA, and 5 HEPES, and pH was adjusted to 7.2 with KOH. The extracellular solution contained (in mM) 140 NaCl, 5.4 KCl, 2.5 CaCl2, 0.5 MgCl2, 5.5 HEPES, and 11 glucose, and pH was adjusted to 7.4 with NaOH. The effects of drugs were monitored using a wash protocol that consisted of a 100-ms step to +10 mV from a holding potential of -80 mV (with 10 s between steps). These currents did not need to be corrected for rundown. I-V relations were elicited from a holding potential of -80 mV using 75-ms steps (5 s between steps) to test potentials over a range of -50 to +50 mV in 10-mV increments. Current at each potential was measured as the average over a 9-ms span at the end of the 100-ms step.

Drugs

Stock solutions of sodium nitroprusside (SNP, Sigma), spermine-NO (Research Biochemicals International, RBI), 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium (carboxy-PTIO, RBI), superoxide dismutase (SOD, Alexis), and 8-bromoguanosine 3':5'-cyclic monophosphate sodium salt (8-bromo-cGMP, Sigma) were prepared fresh in extracellular solutions just before each experiment. All NO donor solutions were protected from direct light. LY 83,583 (RBI) and N-ethylmaleimide (NEM, Sigma) were prepared as stock solutions in ethanol. The final concentration of ethanol was 0.1%. In control experiments (n = 4), the ethanol vehicle alone (i.e., without LY or NEM) did not effect the Ca2+ current. Nisoldipine (Miles Laboratories) was prepared as a stock solution in polyethylene glycol (MW = 400, Sigma). omega -Conotoxin GVIA and norepinephrine were prepared in an aqueous and a 50 mM ascorbic acid stock solution, respectively (RBI). Sodium hydrosulfite (SHS, i.e., sodium dithionite, Sigma) was added to the extracellular solution to reach a final concentration of 1 mM. This solution then was bubbled under an air-tight flask with nitrogen gas for 30 min. The pH was readjusted to 7.4 with NaOH and the pO2 was measured with a blood gas analyzer (Laboratory Instruments). The pO2 was monitored routinely and found to be between 35-40 mmHg.

Data analysis

All values are presented as means ± SE. Statistical significance was determined by a paired t-test or a one-way ANOVA where appropriate. P values < 0.05 were considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of glomus cells

Freshly dissociated cells from carotid bodies contain several cell types, including glomus and type II cells. Previous studies have shown that rabbit glomus cells are excitable cells and they exhibit Ca2+, K+, and Na+ currents. Whereas, type II cells are nonexcitable and exhibit only a small outward K+ current but not Na+ or Ca2+ currents (Urena et al. 1989). At the beginning of each experiment, cells were exposed to an extracellular solution containing Na+ and K+. Once the presence of Na+ current was confirmed, the extracellular solution was switched to a Na+/K+-free solution to isolate Ca2+ current (see METHODS for solutions). Those cells that exhibited Na+ and Ca2+ currents were considered to be glomus cells.

Sodium nitroprusside inhibits Ca2+ currents in glomus cells

NO in the carotid bodies is produced primarily in the nerve fibers that innervate the glomus tissue (Chugh et al. 1994; Prabhakar et al. 1993; Wang et al. 1994). However, primary cell cultures from the carotid body do not contain nerve fibers. Therefore it is necessary to administer NO exogenously to study its effect on Ca2+ currents in isolated glomus cells. Nitrosyl compounds such as SNP release NO in physiological solutions and are potential tools to study the biological actions of NO (Lipton et al. 1993). An example illustrating the effects of SNP (600 µM, 5 min application) on Ca2+ current recorded from a glomus cell is shown in Fig. 1. Examples of whole cell Ca2+ current traces before and during exposure to SNP are shown in Fig. 1A. It is obvious from these traces that SNP decreased the amplitude of the Ca2+ current. Figure 1A, inset, depicts the same current traces, but the current in the presence of SNP is scaled up to match the current at the end of the step during control to show that SNP had no effect on the rate of activation. The time course for changes in Ca2+ current elicited at 0 mV in Fig. 1B shows that the effect of SNP began within tens of seconds and reached a plateau within a minute. The current did not return to control values within 5 min after washing out SNP. To assess whether SNP affected the current-voltage (I-V) relationship of the Ca2+ current, the effects of SNP (600 µM) were tested over a broad range of membrane potentials. As shown in Fig. 1C, SNP reduced the magnitude of the current equally over the entire range of membrane potentials tested, suggesting that the effect of SNP is voltage independent. Figure 1D summarizes the effects of varying concentrations of SNP on the magnitude of the Ca2+ current recorded at 0 mV (n = 7). There was a progressive inhibition of the Ca2+ current as the concentration of SNP in the extracellular solution was increased.



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Fig. 1. Sodium nitroprusside (SNP), a nitric oxide (NO) donor, inhibits the Ca2+ current in rabbit glomus cells. A: raw, whole cell Ca2+ current elicited by a 25-ms step from -80 to 0 mV before and after a 5-min exposure to 600 µM SNP. Inset: same raw current traces but the current in the presence of SNP is scaled up to match the current at the end of the step during control. B: time course for changes in Ca2+ current elicited at 0 mV as the extracellular solution is changed to and from one containing 600 µM SNP (same cell as in A). C: average (n = 7), normalized I-V relations recorded before and after a 5-min exposure to 600 µM SNP in the extracellular solution. Currents are corrected for rundown and normalized to the maximum (usually 0 mV) inward control current recorded in individual cells. D: percentage inhibition of control current at 0 mV by 0.1, 0.3, 0.6, and 1.0 mM SNP (n = 7). Percentage inhibition by SNP of the control current was calculated with the following formula: percentage inhibition= (1 - Idrug/Icontrol)100. *Significance with P < 0.05 from control.

SNP does not affect K+ currents in glomus cells

To test whether the effect of SNP is a nonspecific membrane effect on all ionic currents in glomus cells, we tested its effects on the outward K+ current. We chose this current because it is the most widely studied current in glomus cells and is believed to be sensitive to hypoxia (i.e., Lopez Barneo et al. 1988). An example illustrating the lack of an effect of SNP on the on K+ current is shown in Fig. 2. Figure 2A shows the current traces elicited by a step to +10 mV before and during exposure to 600 µM SNP. Figure 2B shows the time course for changes in K+ current at +10 mV, and Fig. 2C shows the average (n = 6), normalized I-V relations in the presence and absence of SNP. In contrast to the Ca2+ current, the amplitude of the outward K+ current was unaffected by SNP in any of the six cells tested. These results demonstrate that SNP does not affect the outward K+ current in glomus cells and suggest that the effect of SNP is not a nonspecific membrane effect.



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Fig. 2. NO does not affect the outward K+ current in glomus cells. A: raw, whole cell K+ currents elicited by a 100-ms step from -80 to +10 mV from a freshly isolated glomus cell using K+ and Na+ containing solutions (see METHODS) in the presence (SNP) and absence (Con) of 600 µM SNP. B: time course for changes in K+ current elicited by a step to +10 mV under the conditions shown in A. C: average (n = 6), normalized I-V relations recorded before and after a 5-min exposure to 600 µM SNP in the extracellular solution. Currents are normalized to the maximum (+10 mV) outward control current recorded in individual cells.

Inhibition of Ca2+ current by SNP is not due to cyanide

In addition to NO, SNP also can generate cyanide anions. (CN) (Wink et al. 1996). Biscoe and Duchen (1989) reported that CN- ions inhibit Ca2+ current in glomus cells. To establish whether or not the effects of SNP are due to CN- ions, we examined the effect of spermine-NO (SNO; 100 µM), an NO donor that does not generate CN- ions in solution, on the Ca2+ current. As shown in the current traces in Fig. 3A, like SNP, SNO decreased the amplitude of the Ca2+ current without altering the rate of activation (see inset). Figure 3B shows that the time course of the response to SNO also resembled that elicited by SNP. Furthermore, as with SNP, Ca2+ current did not return to control levels within 5 min after washing out SNO. The I-V relation in Fig. 3C shows that, like SNP, SNO inhibited the Ca2+ current equally over the range of potentials tested, suggesting the effects are voltage independent. The effects of SNO were seen consistently in all seven cells tested. On average, the Ca2+ current was inhibited 21 ± 5% at 0 mV by 100 µM SNO (P < 0.01). Routine use of SNO is difficult because of its short half-life (i.e., 40 min). Therefore SNP was used as the NO donor for the remaining experiments.



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Fig. 3. Spermine nitric oxide (SNO), another NO donor, inhibits the Ca2+ current in rabbit glomus cells. A: raw, whole cell Ca2+ current recorded before and after a 5-min exposure to an extracellular solution containing 100 µM SNO. Inset: same raw current traces but the current in the presence of SNO is scaled up to match the current at the end of the step during control. B: time course for changes in Ca2+ current elicited at 0 mV as the extracellular solution is changed to and from one containing 100 µM SNO (same cell as in A). C: average (n = 7), normalized I-V relations recorded before and after a 5-min exposure to 100 µM SNO in the extracellular solution.

To further establish that the effects of SNP are due to generation of NO, we monitored its effects on Ca2+ current in the presence of carboxy-PTIO (CPTIO, 500 µM), a potent scavenger of NO (n = 7). It is evident from the current trace shown in Fig. 4A as well as from the time course shown in Fig. 4B that SNP did not inhibit the Ca2+ current in the presence of CPTIO. SNP did not have a significant effect on Ca2+ current in the presence of CPTIO in any of the seven cells tested. Figure 4B also shows that CPTIO by itself had no effect on Ca2+ current. On average, in the presence of CPTIO, SNP inhibited Ca2+ current only by 7 ± 5%, which was not significant compared with baseline values (P > 0.05, paired t-test, n = 7; Fig. 4C).



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Fig. 4. The effects of 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium (CPTIO), an NO scavenger, on the SNP-induced inhibition of the Ca2+ current. A: raw, whole cell Ca2+ currents elicited by a step to 0 mV before and after a 5-min exposure to an extracellular solution containing 500 µM CPTIO and 600 µM SNP. B: time course for changes in the macroscopic Ca2+ current at 0 mV as the extracellular solution is changed to one containing 500 µM CPTIO alone and then to one containing 500 µM CPTIO and 600 µM SNP (same cell as in A). C: comparison of the average percentage inhibition of Ca2+ current at 0 mV by SNP alone and in the presence of CPTIO (n = 7). *Significance with P < 0.05.

Reversibility of SNP-induced inhibition of the Ca2+ current in the presence of SOD and SHS

In the experiments described in the preceding text, calcium currents did not return to controls within 5 min after washing out NO donors (either SNP or SNO). NO, however, is known to generate peroxynitrite in the presence of oxygen by binding to superoxide ions (Stamler 1994). The following experiments were conducted to test whether peroxynitrites contribute to the lack of reversibility of the response. We examined the effect of SNP on Ca2+ current in the presence of superoxide dismutase (SOD 2000 U/ml), a scavenger of superoxide ions (n = 4). We reasoned that, in the presence of SOD, peroxynitrite formation should be minimal and consequently the effects of SNP on Ca2+ currents should be reversible. As shown in the currents in Fig. 5A and the time course in Fig. 5B, SNP inhibited the Ca2+ current in the presence of SOD and Ca2+ currents returned to the control values within 5 min after washout of SNP in three of the four cells tested. Moreover, the response to SNP in the presence of SOD was qualitatively and quantitatively similar to that in the absence of SOD (compare with Fig. 1, A and B). The average results shown in Fig. 5E show that in the presence of SOD, SNP inhibited the Ca2+ current by 25 ± 6%, which is comparable with that seen without SOD (30 ± 6%; P > 0.05; unpaired t-test).



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Fig. 5. Effects of SOD and sodium hydrosulfite (SHS) on the SNP-induced inhibition of the Ca2+ current. A: raw, whole cell Ca2+ currents elicited by a step to 0 mV before, during, and after exposure to an extracellular solution containing 2,000 U/ml SOD and 600 µM SNP. B: time course for changes in the macroscopic Ca2+ current at 0 mV under the conditions shown in A. C: raw, whole cell Ca2+ currents elicited by a step to 0 mV before, during, and after exposure to an extracellular solution containing 1 mM SHS and 600 µM SNP in a glomus cell. D: time course for changes in the macroscopic Ca2+ current at 0 mV under the conditions shown in C. E: comparison of the average percentage inhibition of Ca2+ current at 0 mV by SNP alone and in the presence of SOD (n = 4) or SHS (n = 7). n.s., no significant difference (P > 0.05).

Peroxynitrite formation is minimal in the presence of reducing agents (Gaston et al. 1993). Therefore, we examined the effect of SNP on the Ca2+ current in the presence of 1 mM SHS (i.e., sodium dithionite, pO2 35-40 mmHg), a reducing agent. As shown in the current traces in Fig. 5C and the time course in Fig. 5D, SNP inhibited the current in the presence of SHS and the effects of SNP were reversible in all seven cells tested. The average results presented in Fig. 5E show that in the presence of SHS, SNP inhibited the Ca2+ current by 25 ± 4%, which is comparable with that seen without SHS (n = 7, 30 ± 6%, P > 0.05, unpaired t-test). These observations indicate that peroxynitrites contribute to the lack of the reversibility of Ca2+ current inhibition by SNP.

Inhibition of the Ca2+ current by NO: evidence for nitrosylation

To test whether SNP inhibition of the Ca2+ current is associated with activation of guanylate cyclase, we monitored the effects of SNP on the Ca2+ currents in the presence of LY 83,583 (LY), a guanylate cyclase inhibitor. As evidenced by the current traces in Fig. 6A and the time course in Fig. 6B, LY by itself had no effect on the Ca2+ current. More importantly, these figures show that LY did not significantly affect the magnitude of the inhibition or alter the time course of the response to SNP. On average, SNP reduced Ca2+ current by 20 ± 6% (n = 7) and 30 ± 6% (n = 7) in the presence and absence of LY, respectively. Although the magnitude of the response tended to be less in the presence of LY, the difference was not statistically significant (P > 0.05; unpaired t-test).



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Fig. 6. Nitric oxide does not inhibit the Ca2+ current in glomus cells via a cyclic monophosphate sodium (cGMP)-dependent mechanism. A: raw, whole cell Ca2+ currents elicited by a step to 0 mV before and during exposure to an extracellular solution containing either 30 µM LY 83,583 (a guanylate cyclase inhibitor) or LY and 600 µM SNP. B: time course for changes in Ca2+ current elicited at 0 mV under the conditions shown in A (n = 7). C: raw, whole cell Ca2+ currents elicited by a step to 0 mV before and during exposure to an extracellular solution containing either 600 µM 8 Br-cGMP or 8-Br-cGMP and 600 µM SNP. D: time course for changes in Ca2+ current elicited at 0 mV under the conditions in C (n = 6).

We reasoned that if the effects of SNP are mediated by cGMP, then 8-Br-cGMP should mimic the effects of SNP on the Ca2+ current. This possibility was tested in six additional cells. Applications of 8-Br-cGMP (600 µM) for as long as 10 min had no effect on the Ca2+ current (data not shown). An example of the raw whole cell Ca2+ current traces and the time course of the effects of 8-Br-cGMP on glomus cell Ca2+ current are shown in Fig. 6, C and D, respectively. It can be seen that 8-Br-cGMP alone had no effect on Ca2+ current even though subsequent application of SNP (600 µM) in the presence of 8-Br-cGMP caused a prompt inhibition of the Ca2+ current. Further, the magnitude of SNP-induced inhibition was the same with and without 8- Br-cGMP (29 ± 3% and 30 ± 6%, respectively; P > 0.05, unpaired t-test). Under the present experimental conditions, these results further support the idea that SNP inhibits Ca2+ current via a cGMP-independent mechanism(s).

To test whether the NO-induced inhibition of the Ca2+ current is due to a modification of sulfhydryl groups on Ca2+ channels, we examined the effect of SNP in the presence of NEM, which is known to covalently modify sulfhydryl groups making them incapable of nitrosylation (Bolotina et al. 1994). Figure 7A shows the effects of NEM (2.5 mM) on raw Ca2+ current traces and the response to SNP in the presence of NEM. As can be seen, NEM by itself inhibited the Ca2+ current. Subsequent addition of SNP (600 µM) during NEM application resulted in no further inhibition of the Ca2+ current. On average, NEM inhibited the Ca2+ current by 36 ± 9% (n = 6, data not shown). In the presence of NEM, there was no significant inhibition of the Ca2+ current by SNP (6 ± 6%, unpaired t-test, P > 0.05; n = 6, Fig. 7D).



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Fig. 7. N-ethylmaleimide (NEM) inhibits the Ca2+ current in glomus cells and blocks the inhibitory effect of SNP. A: raw, whole cell Ca2+ currents elicited by a step to 0 mV before and during exposure to an extracellular solution containing either 2.5 mM NEM or NEM and 600 µM SNP. B: raw, whole cell Ca2+ currents elicited at 0 mV before and during exposure to an extracellular solution containing either 2.5 mM NEM or NEM and 10 µM norepinephrine (NE). Inset: same raw current traces, but the current in the presence of NE is scaled up to match the current at the end of the step during control. C: time course for changes in Ca2+ current elicited at 0 mV under the conditions shown in B. D: comparison of the average percentage inhibition of Ca2+ current at 0 mV by either SNP (n = 6) or NE (n = 7) alone and in the presence of NEM. *Significance P < 0.05; n.s., no significant difference P > 0.05.

To test whether Ca2+ channel proteins were nonspecifically damaged by NEM, we examined the effects of norepinephrine (NE, 10 µM) on the Ca2+ current in six additional cells. We chose NE because it inhibits Ca2+ current in glomus cells of the rabbit carotid bodies (Almaraz et al. 1997; Overholt and Prabhakar 1998), via a G-protein-dependent mechanism (Overholt and Prabhakar 1998). As exemplified in the current traces shown in Fig. 7B and the time course in Fig. 7C, NE reversibly inhibited the magnitude and slowed the rate of activation (see inset) of the Ca2+ current in the presence of NEM. These results are summarized in Fig. 7D, which compares the average percentage of the Ca2+ current inhibited by NE (21 ± 6%, n = 7) alone with that inhibited by NE (15 ± 5%, n = 7) in the presence of NEM. These results suggest that NEM at concentrations of 2.5 mM did not nonspecifically affect modulation of Ca2+ channel proteins and that NO modulates Ca2+ channel activity, in part, via a covalent modification of sulfhydryl group(s).

SNP-induced inhibition of the Ca2+ current is mediated by effects on L-type calcium channels

NO has been shown to primarily affect L-type Ca2+ channels in other cells (Campbell et al. 1996; Hu et al. 1997). To test whether the effects of NO are selectively coupled to L-type Ca2+ channels in glomus cells, we monitored the effects of SNP on Ca2+ current in the presence of 2 µM nisoldipine, a specific L-type Ca2+ channel blocker (n = 6). Parallel experiments with 1 µM omega -conotoxin GVIA (CONO), a selective blocker of N-type Ca2+ channels, served as controls (n = 9). Figure 8A shows current traces elicited by a step to 0 mV before and during application of either nisoldipine alone or nisoldipine and SNP in the extracellular solution. As expected, nisoldipine by itself blocked a portion of the Ca2+ current (37 ± 4%). However, in the presence of nisoldipine, SNP had little effect on the Ca2+ current as can be seen in the raw currents shown in Fig. 8A and in a time course shown in Fig. 8B. In contrast, SNP further inhibited Ca2+ current in the presence of CONO (Fig. 8, C and D), which by itself reduced the basal Ca2+ current by 18 ± 2% (n = 9). These results are summarized in Fig. 8E, which compares the average percentage of the Ca2+ current inhibited by SNP alone with that inhibited by SNP in the presence of NISO or CONO. SNP inhibited the remaining Ca2+ current in the presence of nisoldipine by only 8 ± 6%, and this effect was not significant compared with baseline controls (unpaired t-test, P >0.05; n = 6). On the other hand, SNP inhibited Ca2+ current in the presence of CONO by 29 ± 3% (unpaired t-test, P < 0.001; n = 9), a response comparable with that seen without CONO. These observations suggest that NO primarily affects L-type Ca2+ channels in glomus cells.



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Fig. 8. NO inhibits L-type Ca2+ current in rabbit glomus cells. A: raw, whole cell Ca2+ currents elicited by a step to 0 mV before and during exposure to an extracellular solution containing either 2 µM nisoldipine (NISO) or NISO and 600 µM SNP. B: time course for changes in Ca2+ current elicited at 0 mV under the conditions described in A. C: raw, whole cell Ca2+ currents elicited by a step to 0 mV before and during exposure to an extracellular solution containing either 1 µM omega -conotoxin GVIA (CONO) or CONO and 600 µM SNP in the extracellular solution. D: time course for changes in Ca2+ current elicited at 0 mV under the conditions described in C. E: comparison of the percentage of the Ca2+ current inhibited by SNP alone (n = 7), and in the presence of NISO (n = 6), or CONO (n = 9). *Significance P < 0.05; n.s., no significant difference P > 0.05.


    DISCUSSION
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Previous studies have shown that NO inhibits carotid body sensory activity (see INTRODUCTION for references). To begin to understand the cellular mechanisms associated with the actions of NO in the carotid body, we monitored the effects of NO donors on the macroscopic Ca2+ current in glomus cells isolated from rabbit carotid bodies. Our results provide evidence that NO inhibits Ca2+ current and that this effect is confined primarily to L-type Ca2+ channels. Furthermore the effects of NO on Ca2+ current appears to be a direct action on the channel protein and/or an associated channel protein, rather than through a cGMP-dependent mechanism.

Inhibition of the calcium currents in glomus cells by NO donors is due to nitric oxide

It is clear from our results that NO donors inhibit Ca2+ current in glomus cells of rabbit carotid bodies. The onset of the effects were rapid, occurring within seconds after the application of NO donors (i.e., Fig. 1B). This pattern of response to NO donors is consistent with those reported elsewhere in the nervous system (Snyder 1992). The present results, however, are at variance with those reported by others who found no effect of NO donors on Ca2+ currents in rat glomus cells (Hatton and Peers 1996). One possibility for this discrepancy is that previous studies employed SNAP as the NO donor, whereas we used SNP or SNO in the present study.

SNP also may generate CN- ions in solution (Wink et al. 1996). Given that CN- inhibits Ca2+ channel activity in glomus cells (Biscoe and Duchen 1989), it could be argued that the SNP-induced inhibition of the Ca2+ current is due to CN- and not due to NO. Such a possibility, however, seems unlikely because SNO, a NO donor that does not produce CN-, also inhibited Ca2+ current. In a different series of experiments, we monitored NO production from SNP by microvoltammeteric technique using a NO microelectrode (WPI). Using both differential pulse voltammetry (DPV) and amperometry, we found that NO indeed is generated from SNP (N. R. Prabhakar and M. Gratzl, unpublished observations). These observations, taken together with the observations that SNP no longer inhibited the Ca2+ current in the presence of an NO scavenger (carboxy PTIO), support the idea that the effects of SNP are indeed due to release of NO. We also do not believe that the effects of NO donors are nonselective as reported for other pharmacological substances (e.g., cytochrome P-450 inhibitors) (Hatton and Peers 1996), because conotoxin-sensitive Ca2+ channels (i.e., N type) as well as the outward K+ current were unaffected by SNP. These observations thus demonstrate that NO donors inhibit Ca2+ current in glomus cells from rabbit carotid bodies and that this effect is due to NO itself not from byproducts such as CN-.

A striking finding of the present study is that the effects of NO donors (both SNP and SNO) on Ca2+ current are not reversible even after 5 min of terminating the challenges. These observations give an impression that the effects of NO are irreversible. NO generates peroxynitrites in the presence of superoxide ions (Stamler 1994) that could potentially affect cell membranes. We believe that the lack of reversibility of the response to NO is due to peroxynitrite formation for the following reasons. SNP responses were reversible in the presence of SOD, a potent scavenger of superoxide ions and SHS, a strong reducing agent. Both these substances prevent peroxynitrite formation. SOD is a cytosolic enzyme, and it is likely that the effective levels of SOD are expected to be low in our cells due to dialysis of the cell under the whole cell configuration of the patch-clamp technique. Therefore under our experimental conditions, low levels of endogenous SOD would not be able to effectively scavenge superoxide anions, thereby preventing consequent generation of peroxynitrite. As expected if this were the case, exogenous addition of SOD would effectively reverse SNP-induced inhibition of calcium current. Further, isolated cells were exposed to atmospheric oxygen (~pO2 = 149 mmHg), wherein generation of superoxide ions is expected to be high compared with in vivo carotid body where tissue oxygen levels are expected to be much lower. Thus in intact carotid bodies, the effects of NO are expected to be reversible because of high levels of SOD and relatively low tissue oxygen levels resulting in minimal levels of peroxynitrites. It is important to note that the magnitude of the inhibition of calcium currents was unaffected by either SOD or SHS, indicating that peroxynitrites do not contribute to the suppression of the Ca2+ currents by SNP.

Mechanisms of calcium channel inhibition by NO in glomus cells

Considerable evidence indicates that NO exerts many of its effects through the activation of guanylate cyclase and increased levels of cGMP (Snyder 1992). Consistent with such a notion, we (Prabhakar et al. 1993) as well as others (Wang et al. 1989) have reported previously that NO stimulates cGMP levels in the carotid body. On the basis of these observations, we performed several experiments to test whether guanylate cyclase and cGMP are involved in the NO-mediated inhibition of the Ca2+ current in glomus cells. Under the conditions of the whole cell patch configuration, results from these experiments indicate that the effects of NO on calcium currents may, in part, be due to a mechanism independent of cGMP. First, a guanylate cyclase inhibitor, LY 83,583, failed to prevent the SNP-induced inhibition of the Ca2+ current (Fig. 6, A and B). Second, a membrane permeant analogue of cGMP, 8-Br-cGMP, did not mimic the SNP-induced inhibition (Fig. 6, C and D). Third, when SNP was added to glomus cells in the presence of the cGMP analogue, it still caused inhibition of calcium current. Our present observations are in accord with those reported by Hatton and Peers (1996), who also found that both 8-Br-cGMP and PET-cGMP, analogues of cGMP, had no affect on the whole cell Ca2+ current in rat glomus cells. However, the possible involvement of cGMP and the subsequent activation of protein kinase G, cannot be ruled out using the whole cell configuration of the patch-clamp technique because the cell has been dialyzed, allowing escape of small molecules and cytosolic enzymes. Nonetheless, it is possible that NO might directly affect the channel protein or an associated channel protein. Several studies have shown that NO can directly activate or inhibit channel activity through S-nitrosylation. Bolotina et al. (1994) found that NEM, an agent that prevents S-nitrosylation of proteins, prevented activation of Ca2+-activated K+ current by NO in vascular smooth muscle cells. Likewise, after NEM treatment, NO donors no longer activated L-type Ca2+current in ferret ventricular myocytes (Campbell et al. 1996). Hu et al. (1997) reported that specific cysteine modifying agents also could prevent inhibition of the L-type Ca2+ current by NO donors. These observations by other investigators prompted us to test if S-nitrosylation is involved in NO-mediated inhibition of the Ca2+ current in glomus cells. Our results provide evidence that NO is acting through S-nitrosylation in glomus cells. In the presence of NEM, a reagent that prevents S-nitrosylation, SNP did not affect Ca2+ current in glomus cells. We believe that this is not a nonspecific effect of NEM on Ca2+ channel function, because much Ca2+ current remained in the presence of NEM and NE was able to suppress this Ca2+ current remaining. This suggests that the signaling pathway mediating the actions of NE in glomus cells is unaffected by NEM. Taken together, these observations under whole cell conditions provide evidence that inhibition of Ca2+ current by NO in glomus cells involves S-nitrosylation of channel proteins.

NO inhibition is primarily confined to L-type Ca2+ channels

It is evident from recent studies that glomus cells express multiple calcium channels (e Silva et al. 1995; Overholt and Prabhakar 1997; Peers et al. 1996). It is well known that neurotransmitters can preferentially affect a specific type of Ca2+ channel within a given cell while sparing others. For instance, we recently have found that NE selectively inhibits a toxin-resistant non-L, N, P/Q type channel in glomus cells of the rabbit carotid body (Overholt and Prabhakar 1998). Therefore, we examined whether the effects of NO are confined preferentially to one type of Ca2+ channel in glomus cells. Our data indicate that the effects of NO are confined to L-type Ca2+ channels in glomus cells. Inhibition of L-type Ca2+ current by NO seen in glomus cells resembles that reported for L-type current in cardiac myocytes (Campbell et al. 1996). Furthermore, NO donors (both SNP and SNO) inhibited the Ca2+ current in every rabbit glomus cell tested, suggesting that the effects of NO are uniform among individual glomus cells.

What might be the physiological importance of NO-induced inhibition of calcium channels in the glomus cells? Several lines of evidence indicate that NO inhibits carotid body activity (Chugh et al. 1994, Kline et al. 1998; Prabhakar et al. 1993; Wang et al. 1995). NOS is expressed in the sinus nerve (Chugh et al. 1994; Prabhakar et al. 1993; Wang et al. 1993) as well as the nerve fibers that originate from the petrosal ganglion (Wang et al. 1993). Stimulation of the sinus nerve inhibits the sensory activity (efferent inhibition) (see Wang et al. 1995 for references). It has been suggested that NO is a mediator of efferent inhibition in the carotid body (Prabhakar et al. 1993; Wang et al. 1995). Previous studies have documented that L-type calcium channels are associated with the release of neurotransmitter(s) in the carotid body (Obeso et al. 1992). Perhaps one of the mechanisms by which NO mediates efferent inhibition is by inhibiting L-type Ca2+ channels in glomus cells and subsequent neurotransmitter release. However, under the conditions of the present experiments, we do not know how much NO is released by NO donors nor do we know the levels of NO released in the carotid body. Therefore further studies are necessary to demonstrate the effects of NO on calcium channels during efferent inhibition of sensory activity.

In summary, the present results demonstrate that NO inhibits L-type voltage-gated Ca2+ channels in adult rabbit glomus cells, in part, via a direct effect on calcium channel protein. It is suggested that inhibition of L-type Ca2+ current in glomus cells would constitute one of the cellular mechanism associated with actions of NO associated with efferent inhibition of carotid body activity.


    ACKNOWLEDGMENTS

The authors thank Drs. S. W. Jones and R. D. Harvey for constructive suggestions during this study. We also thank Dr. Harvey for providing us with nisoldipine for our experiments.

This work was supported by National Institute of Heart, Lung, and Blood Grant HL-25830; J. L. Overholt is a Parker B. Francis Fellow in Pulmonary Research, and Beth Summers was supported by NHLBI Training Grant T32HL-07653.


    FOOTNOTES

Address for reprint requests: N. R. Prabhakar, Dept. of Physiology and Biophysics, School of Medicine, 10900 Euclid Ave., Case Western Reserve University, Cleveland, OH 44106-4970.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 3 September 1998; accepted in final form 16 December 1998.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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