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(Circulation Research. 1999;84:921-928.)
© 1999 American Heart Association, Inc.

Original Contribution

Acid-Evoked Currents in Cardiac Sensory Neurons

A Possible Mediator of Myocardial Ischemic Sensation

Christopher J. Benson, Stephani P. Eckert, Edwin W. McCleskey

From the Vollum Institute (C.J.B., S.P.E, E.W.M.) and Division of Cardiology (C.J.B.), Oregon Health Sciences University, Portland, Ore.

Correspondence to Christopher J. Benson, Division of Cardiology, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd, UHN-62, Portland, OR 97201-3098. E-mail bensonc{at}ohsu.edu

	Abstract

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Abstract—Sensory neurons that innervate the heart sense ischemia and mediate angina. To use patch-clamp methods to study ion channels on these cells, we fluorescently labeled cardiac sensory neurons (CSNs) in rats so that they could later be identified in dissociated primary culture of either nodose or dorsal root ganglia (DRG). Currents evoked by a variety of different agonists imply the importance of lowered pH (7.0) in signaling ischemia. Acidic pH evoked extremely large depolarizing current in almost all cardiac afferent neurons from the DRG (CDRGNs). In contrast, only about half of the unlabeled DRG neurons responded to acid, and their current amplitudes were much less than that in CDRGNs. In all respects tested—kinetics, selectivity, and pharmacology—the acid-evoked current was similar to that of previously described native and cloned acid-sensing ion channels. Cardiac afferents from the nodose ganglia differed from CDRGNs in having smaller acid-evoked currents but clearly larger currents evoked by ATP. Serotonin, acetylcholine, bradykinin, and adenosine elicited currents in fewer CSNs than did ATP or lowered pH, and the currents were relatively small. Capsaicin, an activator of small nociceptive sensory neurons that innervate skin, evoked only small and rare currents in CDRGNs. The extremely large amplitude and prevalent expression of acid-evoked current in CSNs imply a critical role for acidity in sensation associated with myocardial ischemia.

Key Words: myocardial ischemia • cardiac sensory neuron • proton • whole-cell patch clamp

	Introduction

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Although much is understood about the effect of autonomic nervous system input to the heart, the cardiac sensory, or afferent, system and its role in physiological and pathological conditions are less well understood. Historically, the major impetus for research on the cardiac sensory system has been to find the source of cardiac pain, or angina. During the first half of this century, the neuroanatomical pathways of the cardiac sensory system were defined by clinical reports of surgical attempts to relieve angina and by experimental studies.^{1 2} These studies revealed that the cardiac sensory neurons (CSNs) follow the sympathetic and vagal nerve tracts en route to the central nervous system (Figure 1). The cell bodies of those sensory axons following the sympathetic tracts are found in the upper thoracic dorsal root ganglia (DRG); those following the vagal tracts are located in the nodose ganglia. The sensory innervation of the fibrous and serous parietal pericardium, separate from that of the heart and epicardium, follows the phrenic nerves to the cervical DRG (C₃-C₅).^{3 4}

View larger version (40K): [in this window] [in a new window]   Figure 1. Illustration of the cardiac and pericardial sensory pathways. Cardiac sensory signals travel from terminals throughout the heart through both the sympathetic and the vagal nerve tracts en route to the central nervous system. Axons of some CSNs exit the heart via the sympathetic cardiac nerves and course through sympathetic ganglia (middle [MCG] and inferior cervical and upper thoracic) en route to their cell bodies, located in the lower cervical and upper thoracic DRG. Axons of other CSNs travel within the vagal nerve to their cell bodies in the nodose ganglia. Afferent neurons from the pericardium follow the phrenic nerves to their cell bodies in the DRG of the upper cervical region (C3-C5).

It has long been understood that cardiac pain is associated with myocardial ischemia, which causes oxygen supply/demand insufficiency.⁵ In various whole-animal preparations, occlusion of a coronary artery activates the cardiac afferent nerve fibers in the sympathetic^{6 7 8} and vagal tracts.^{9 10} Various substances released during myocardial ischemia have been implicated as chemical mediators of myocardial ischemic sensation. Several of these substances have been shown to activate CSNs: ATP,^{8 10} serotonin (5HT),¹¹ bradykinin (BK),^{12 13} and adenosine.^{8 10} In turn, stimulation of sensory fibers elicits reflexes specifically mediated by the sympathetic tract¹⁴ and by the vagal tract.¹⁵ Still, the precise stimuli that are sensed during myocardial ischemia are incompletely understood (see Reference 16¹⁶ for review).

A likely contributor is acid. The heart is an organ of high metabolic activity and is susceptible to drops in pH during ischemia or hypoxia. It has been demonstrated that pH is lowered intracellularly¹⁷ and extracellularly^{18 19} in ischemic heart models and clinically in patients with coronary artery disease.²⁰ In dogs, lowered pH stimulates afferent cardiac sympathetic nerve fibers.²¹ In another organ system, rat skin, acid plays a dominant role in exciting sensory neurons when compared with other potential chemical mediators of inflammation.²²

Acid evokes depolarizing currents in sensory neurons studied in primary dissociated culture,^{23 24 25 26} and a variety of different components are distinguished by kinetic criteria.²⁷ Most components activate somewhere between pH 7 and pH 6 and desensitize in response to a maintained stimulus. These desensitizing currents all have the unusual property of selectively passing Na⁺ over K⁺ about as effectively as voltage-gated Na⁺ channels. In addition to the desensitizing, Na⁺-selective currents in DRG neurons, there is a sustained, nonselective current that is evoked by pH below 6.0.²⁶ The channels underlying these currents are believed to be the recently cloned acid-sensing ion channels (ASICs), which are members of the amiloride-sensitive Na⁺ channel/degenerin family of cation channels.²⁸

These acid-evoked currents may play a role in mediating the pain of cardiac and skeletal muscle ischemia and perhaps also of inflammation. It is difficult to explore this possibility in culture, because the sensory modality and the site of innervation of individual neurons are not known. The first goal of the present study was to fluorescently label CSNs in the rat so that they can later be distinguished from other sensory neurons in dissociated culture; we accomplished this using a retrogradely transported dye placed in the pericardial space. We found that acid evoked extraordinarily large currents in the cardiac afferent neurons from the DRG (CDRGNs) compared with other, unlabeled DRG neurons (UDRGNs). The very high expression of these currents in cells thought to be specialized for sensing ischemia suggests an important role of acid in mediating cardiac pain.

	Materials and Methods

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In Vivo Labeling of CSNs
Surgical Preparation
Sprague-Dawley rats (200 to 300 g) were anesthetized by intramuscular injection of 1 mL/kg rat anesthetic (in mg/mL, ketamine 55, xylazine 5.5, and acepromazine 1.1). Each animal was intubated and respiration maintained with a rodent ventilator (Harvard model 683). The heart and thymus were exposed through a left lateral thoracotomy at the fifth intercostal space. The thymus, with the anterior superior portion of the pericardium adherent to its undersurface, was gently retracted cephalad to better delineate the pericardium and pericardial space. Twenty-five microliters of a suspension of 17 mg/mL of 1,1'-dioctadecyl-3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI; Molecular Probes) in saline solution was injected into the pericardial space. The rat pericardial membrane is thin and contains microscopic pores²⁹ ; thus, a suspension rather than solution of the lipophilic DiI was used to decrease the potential of leakage of dye from the pericardial space. After injection, the ribs were approximated, the thoracic cavity was evacuated, and the incision was closed in layers. The animals were cared for in accordance with the current Guide for the Care and Use of Laboratory Animals (US Public Health Service, Department of Health and Human Services) and guidelines of the Institutional Animal Care and Use Committee of Oregon Health Sciences University.

Tissue Culture and Identification of Labeled CSNs
During the 2- to 4-week postoperative period, DiI was carried through retrograde transport back to the cell bodies of the CSNs. The rats were then sacrificed, and the right and left dorsal root (C₈-T₃) and nodose ganglia were collected. The ganglia were dissociated and cultured as previously described, except that the Percoll spin was omitted.³⁰ In brief, the ganglia underwent enzymatic dissociation successively in papain and collagenase/dispase solutions; this was followed by trituration in Hanks solution. The cells were then plated on polylysine- and laminin-coated plastic in F12 medium plus nerve growth factor (50 ng/mL) at 37°C in 5% CO₂. After several hours, the medium was changed to L15 plus nerve growth factor, and the cells were maintained at 22°C in air. CSNs were identified by fluorescence microscopy (Figure 2C and 2D). From the animals that underwent the pericardial space injection preparation, the following 3 populations of sensory neurons were obtained for study: (1) labeled CDRGNs, (2) UDRGNs, and (3) labeled neurons from the nodose ganglia (CNodNs).

View larger version (151K): [in this window] [in a new window]   Figure 2. Fluorescent labeling of CSNs. A and B, Corresponding phase (A) and fluorescence micrograph (B) of an apical slice of myocardium 3 weeks after surgical injection of fluorescent dye into the pericardial space. C and D, Phase (C) and fluorescence micrograph (D) of 2 CSNs in primary dissociated culture of DRG neurons. E and F, Phase (E) and fluorescence micrograph (F) of a myocardial slice after an intramural injection of dye. Scale bars represent 0.5 mm for panels A, B, E, and F and 0.25 mm for panels C and D.

Control Experiments
To check for dye leakage from the pericardium and to explore the effects of labeling at different injection sites, we performed several control procedures. First, after a pericardial space injection, the heart and lungs were sectioned at the time of euthanization and viewed under a fluorescence microscope. Whereas the heart consistently displayed confluent fluorescence over the epicardium (Figure 2A and 2B), the surface of the lungs contained only an occasional isolated crystal of dye. Next, to test whether dye leakage from the pericardial space would cause significant contamination, we intentionally injected dye in the following sites in separate animals: the left pleural space, the left ventricular chamber, or the right ventricular chamber. As expected, sectioning of the lungs after injecting into the right ventricular chamber revealed confluent pulmonary vascular embolization of the dye. Next, in an effort to label sensory nerve terminals deeper within the myocardium, we stabilized the heart with sutures and made several intramural injections into the left ventricular myocardium (Figure 2E and 2F).

The numbers of labeled sensory neurons obtained in culture of the DRG after the various injection experiments are listed in the Table. The pericardial space injection resulted in a significantly greater number of labeled neurons in the DRG than the various control injection site experiments. Thus, despite the potential for dye leakage from the pericardial space, it would cause little contamination because relatively few cells were labeled with intentional injection into the pleural space. Because of the low number of neurons labeled by the intramural injections, we abandoned this experimental preparation. The number of nodose ganglion neurons labeled by the pericardial space injection was not quantified; however, there appeared to be a higher fraction of labeled cells in the nodose ganglion (10%) than in the DRG (1%) cultures.

View this table: [in this window] [in a new window]   Table 1. Number of Labeled DRG Neurons Per Dye Injection Site

Whole-Cell Patch-Clamp Recording
Whole-cell currents were recorded with an EPC-9 amplifier (HEKA Elektronik). For most experiments, pipettes of 2- to 4-M resistance were filled with KCl internal solution containing (in mmol/L) KCl 100, EGTA 10, HEPES 40, MgCl₂ 5, Na₂ATP 2, and Na₃GTP 0.3, adjusted to pH 7.4 with KOH unless otherwise stated. For the monovalent permeability experiments, NaCl replaced KCl in the internal solution, and pH was adjusted with NaOH; high internal Na⁺ eliminated contamination by large, outward K⁺ currents. For the Ca²⁺ permeability experiments, the internal solution consisted of (in mmol/L) N-methyl glucamine 90 (titrated with HCl), NaCl 10, EGTA 10, HEPES 40, MgCl₂ 5, Na₂ATP 2, and Na₃GTP 0.3, pH adjusted with tetramethylammonium (TMA)-OH. Our strategy was to measure relative Na⁺ and Ca²⁺ permeability by using similar Na⁺ and Ca²⁺ activities inside and outside the cell, respectively. Standard extracellular solutions contained (in mmol/L) NaCl 130, KCl 5, CaCl₂ 2, MgCl₂ 1, HEPES 10, and MES 10, pH adjusted with TMA-OH. TMA-Cl was added to the various pH solutions to equalize the concentration of TMA. For the monovalent permeability experiments, the extracellular solutions consisted of (in mmol/L) NaCl (or KCl, NaCH₃SO₃, or CsCl) 130, CaCl₂ 2, MgCl₂ 1, HEPES 10, and MES 10; pH was adjusted with NaOH, KOH, or CsOH. For the Ca²⁺ permeability experiments, external solutions consisted of (in mmol/L) N-methyl glucamine 120 (titrated with HCl), HEPES 10, MES 10, and CaCl₂ 10 or 30. For experiments on Ca²⁺ block, external solutions consisted of (in mmol/L) NaCl 130; HEPES 10; MES 10; and CaCl₂ 1, 2, or 10. The series resistance ranged from 3 to 7 M, and it was compensated by 50%.

All dose-response curves were made by random-order application of various concentrations at 30-second intervals. Solutions were applied through an array of 1- or 10-µL pipes positioned 50 µm from the cell under 40 cm of water pressure. Rapid solution exchanges were controlled via computer-driven solenoid valves and were accomplished within 5 ms as measured by an osmotically induced change in current (Figure 5A). Cells were held at –70 mV unless otherwise stated. Experiments were performed at room temperature (22°C). We studied most cells after 1 to 2 days in culture; however, some experiments were done on cells cultured up to 7 days. We saw no obvious difference in the responses of cells cultured for longer times.

View larger version (20K): [in this window] [in a new window]   Figure 5. Kinetics of acid-evoked transient currents in CDRGNs. A, Typical transient current evoked by pH 6.8. Trace on the right is a shorter pH application to the same cell, displayed on a faster time scale to demonstrate the fast activation. Top right trace shows the measured time course of pH application (see Materials and Methods). B, In a small subpopulation of CDRGNs, acid evoked a different transient current with slower activation and desensitization. Left and right scale bars correspond to left and right traces for both panels A and B. C, Activation, desensitization, and recovery from desensitization are each faster in the fast transient () than the slow transient () current. Time constants for activation and desensitization at various pH solutions are from exponential fits to the rising and falling phase of currents, as in panels A and B. Recovery times are at pH 7.4. Horizontal axis indicates interval at pH 7.4 spent between 2 pulses to pH 6.8. The first pulse completely desensitizes the current, and the second tests the extent of recovery. Vertical axis is the relative amplitude of currents evoked by the second and first pulses. Time constants of the exponential fits are 0.44 and 6.8 seconds for the fast and slow currents, respectively.

Data Analysis
The equation I(H⁺)=1/{1+(K_0.5/[H⁺])ⁿ}, where pH at half-maximal response is –log K_0.5, and I is the current at a given proton concentration, [H⁺], was best-fit to the dose-response data using the program NFIT (University of Texas Medical Branch, Galveston, TX), a least-squares algorithm. PulseFit (HEKA Elektronik) was used to determine the time constants of current activation and desensitization, fit to a single exponential. Igor software (WaveMetrics, Inc) was used to curve-fit the time of recovery from desensitization. Permeability ratios were calculated from reversal potentials using the Goldman-Hodgkin-Katz equation.³¹ P_Na:P_K was calculated from the change in reversal potential (E_rev) when K⁺ replaced Na⁺ in the external solution: E_rev=(RT/F)ln{P_Na⁺[Na⁺]_o/P_K⁺[K⁺]_o}, where T is absolute temperature, R and F are gas and Faraday constants, respectively, and brackets indicate concentrations. P_Na:P_Ca was calculated from the absolute reversal potential with Na⁺ and Ca²⁺ as the only current carriers inside and outside the cell, respectively, using the following equation: E_rev=(RT/2F)ln{4P_Ca2+[Ca²⁺]_o/P_Na+[Na⁺]_i}. Data are reported as mean±SEM. Statistical analysis was performed with an unpaired t test. A value of P<0.01 was considered statistically significant.

	Results

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Response of CSNs to Chemical Stimuli
To study the response of CSNs to potential chemical mediators of ischemia, the following chemicals were each dissolved in an external solution of pH 7.4: in µmol/L, ATP 30,³² 5HT 30,³³ capsaicin 1,³⁴ acetylcholine (ACh) 200,³⁵ and adenosine 200,³⁷ as well as 500 nmol/L BK.³⁶ The concentrations chosen for each compound produced maximal responses in our experiments and in the references cited. The largest measured currents (8.58±1.44 nA) were consistently evoked (93%) by acid applied to CDRGNs (Figure 3). A smaller percentage of UDRGNs (54%) and CNodNs (74%) responded to acid, and the cells that responded displayed significantly smaller currents (3.48±1.10 nA in UDRGNs and 1.15±0.23 nA in CNodNs; P<0.01 versus pH-evoked current in CDRGNs) (Figure 3B and 3C). These differences between cell populations refer to the amplitude of transient acid-evoked current (see below); the sustained component evoked by very low pH was seen in virtually every neuron and did not distinguish different cell populations.

View larger version (25K): [in this window] [in a new window]   Figure 3. CSNs respond to a variety of chemical activators. A, Representative currents evoked by application of various chemicals to CDRGNs. Note the different scale bars and application times. Each of the test solutions was applied to cells, in random order, for a minimum of 3 seconds; a longer application was made if needed to see the peak current amplitude. Control solution flowed onto the cells for 30 seconds between chemical applications. B, Percentages of CDRGNs, UDRGNs, and CNodNs that responded to the following: pH 5.0; (in µmol/L) ATP 30, 5HT 30, capsaicin 1, ACh 200, and adenosine 200; and 500 nmol/L BK. A positive response was defined as an evoked current >50 pA. For pH response, the number of CDRGNs studied was 29; UDRGNs, 22; and CNodNs, 19. Not all of the cells studied were tested for all chemicals; each bar represents at least 12 cells. C, Mean current amplitudes of the responding neurons. Data are mean±SEM. *P<0.01 vs pH-evoked current in CDRGNs. Cap indicates capsaicin; Aden, adenosine.

Another consistent and large response (4.87±0.59 nA) was evoked by ATP acting on ion channels called P2X receptors in CNodNs. The current was slow activating and only partially desensitized (data not shown), which is indicative of the heteromeric combination of P2X2 and P2X3 receptor subtypes previously described in nodose neurons.³⁸ In contrast, the ATP-evoked currents in CDRGNs and UDRGNs were substantially smaller and consisted primarily of a fast-activating and fast-desensitizing current (Figure 3A), which suggests either the P2X1 or P2X3 receptor subtypes.³⁹ CSNs sometimes responded to the other chemicals indicated in Figure 3; however, they did so inconsistently, and the amplitude of evoked currents in the responders was much smaller than with either pH or ATP (Figure 3B and 3C).

Biophysical and Pharmacological Properties of Acid-Evoked Currents in CDRGNs
The exceptionally large amplitude and prevalence of the transient acid-evoked current in CDRGNs suggests its significance in sensing cardiac ischemia. Therefore, we characterized its biophysical and pharmacological properties to see how these compared with the variety of acid-evoked currents seen in sensory neurons.^{23 24 25 26} In short, we found no properties unexpected from those described in the literature.

A drop in pH to 7.0 reproducibly evoked a transient, rapidly activating and rapidly desensitizing current in CDRGNs (Figure 4A and 4C). This transient current was half-activated by a pH step to 6.6 and half-desensitized by preincubation at pH 7.2 (Figure 4C). The Hill coefficient of the activation curve was 2.5. A smaller, nondesensitizing current was evoked by more extreme decreases in pH (6). The activation curve for this current is fitted with a pH_0.5 of 3.7. The combination of transient and sustained components at low pH has previously been seen in unlabeled rat sensory neurons.^{26 27}

View larger version (21K): [in this window] [in a new window]   Figure 4. Activation and desensitization of acid-evoked currents in CDRGNs. A, Typical currents evoked by applying various pH solutions to a CDRGN. Note the transient, fast-activating, and fast-desensitizing current that is evoked by relatively low proton concentrations. At higher proton concentrations, a nondesensitizing, sustained current is evoked. Resting pH=8.0 at –70 mV. B, Superimposed currents evoked by pH 5.0 from varying resting pH values. The resting pH was applied for at least 3 seconds. C, Dose-response data for acid-evoked transient (, n=6) and sustained (, n=6) currents. indicates data obtained using the desensitization protocol shown in panel B (n=7). Transient responses are normalized to the peak current obtained from application of pH 5.0 from a resting pH of 8.0. The sustained responses are normalized to the saturation level of the curve fit. Curves are best fits of the Hill equation, I(H+)=1/{1+[K0.5/(H+)]n} (activation), or I(H+)=1/{1+[(H+)/K0.5]n} (desensitization). Half-activation values were pH 6.6 (transient) and pH 3.7 (sustained); half-desensitization was pH 7.2. Hill coefficients (n) were 2.5 (transient activation), 1.2 (sustained activation), and 2.6 (transient desensitization). Boxed inset magnifies the region where the transient activation and desensitization curves overlap. Points represent mean±SEM.

Varying the pH before a test stimulus of pH 5 revealed that a significant fraction of the transient current is desensitized at a resting pH of 7.4 (Figure 4B). This is consistent with the previous demonstration that acid-evoked channels need not open to desensitize.²⁵ The steady-state desensitization and activation curves show clear overlap in the vicinity of pH 7 (Figure 7C and inset), suggesting that the channel can generate a standing current at pH 7.

View larger version (21K): [in this window] [in a new window]   Figure 7. Fast transient and sustained acid-evoked currents in CDRGNs differ in ion selectivity and in amiloride sensitivity. A, Superimposed acid-evoked currents in NaCl and NaCH3SO3 (left). Both the transient and sustained acid-evoked currents were unchanged when extracellular Cl– was replaced with the impermeable anion CH3SO3-. Thus, neither current is carried by anions. Right, Currents evoked in extracellular solutions containing either NaCl or CsCl. Transient current is more permeable to Na+ than Cs+, whereas the sustained current is more permeable to Cs+ than Na+. B, Left, currents evoked by pH 5.0 and by pH 5.0 plus 100 µmol/L amiloride (*). The transient current was significantly reduced by amiloride, whereas the sustained current was unchanged. Right, dose-response curve for amiloride on current evoked by a pH change from 7.4 to 6.8. IC50=9.2 µmol/L (n=5).

A closer look at the time constants of activation and desensitization of the transient currents in CDRGNs revealed multiple transient components (Figure 5), as noted by Krishtal and Pidoplichko.²⁷ Most cells exhibited a rapidly activating and desensitizing current, but some had a current that was 10-fold slower. The fast and slow transient currents were not exclusive; a few neurons displayed both currents, which was evident as biphasic activation and/or desensitization. These cells were not included in the analysis of time constants. The rate of recovery from desensitization differed for the fast and slow transient currents in CDRGNs (Figure 5C). In a control solution of pH 7.4, the fast transient current recovered with =0.44 seconds, and the slow transient current recovered with =6.8 seconds.

The fast transient current is Na⁺ selective but Ca²⁺ permeable (Figure 6). With the usual internal (high K⁺) and external (high Na⁺) solutions, the reversal potential was in the vicinity of +50 mV (data not shown). To quantify the evident preference for Na⁺ over K⁺, we measured reversal potentials with Na⁺ as the internal ion (see Materials and Methods). The reversal potential shifted –50±1.2 mV when K⁺ replaced Na⁺, corresponding to a P_Na/P_K of 6.8.

View larger version (27K): [in this window] [in a new window]   Figure 6. Fast transient acid-evoked currents in CDRGNs are Na+ selective. A, Transient currents evoked by applying pH 5.0 during steps to various membrane potentials in an extracellular solution of NaCl (left) or KCl (right). Internal solution was NaCl. B, Current vs voltage curves from the data in panel A. Data points indicate differences between currents at pH 7.4 and 5.0. The mean shift in reversal potential was –50±1.2 mV (n=3); thus, PNa/Pk=6.8. C, Currents evoked by pH 6.0 at the indicated potentials in 10 Ca2+ (external) and 15 Na+ (internal). Mean reversal potential was –47.2±2.3 mV (n=7); thus, PNa/PCa=105.

Current-voltage relationships measured in 10 mmol/L extracellular Ca²⁺ with 15 mmol/L intracellular Na⁺ displayed an average reversal potential of –47.2±1.2 mV (n=7), corresponding to a P_Na/P_Ca of 105 (Figure 6C). In all cells, current amplitude increased when [Ca²⁺] was increased from 10 to 30 mmol/L (n=7; amplitude increase varied from 28% to 725%; data not shown). Thus, as previously shown,⁴⁰ the current can be carried by Ca²⁺. To determine whether the acid-evoked currents are blocked by millimolar extracellular Ca²⁺, as seen in some native^{24 25} and cloned⁴¹ channels, we measured Na⁺ currents elicited by pH 6.0 in 1, 2, and 10 mmol/L Ca²⁺. There was no change in amplitude (n=5; data not shown); therefore, the current was not blocked by millimolar Ca²⁺ concentrations.

The fast transient and sustained current components differ in 2 respects: ion selectivity (Figure 7A) and pharmacology (Figure 7B). Both components are cation selective, because they were unchanged when extracellular Cl^– was replaced with the impermeant anion CH₃SO₃^-. Replacement of extracellular Na⁺ with Cs⁺ revealed that the transient current does not readily pass Cs⁺, whereas the sustained current does. Thus, as previously described in UDRGNs,²⁶ the transient current is selective for Na⁺, whereas the sustained current is a nonselective cationic current.

Amiloride, a K⁺-sparing diuretic that has been shown to block proton-activated currents in mouse neuroblastoma cells,⁴² blocked the fast transient current (IC₅₀=9.2 µmol/L) but not the sustained current (Figure 7B). This difference is consistent with one of the ASICs.⁴³ The slow transient current was similarly inhibited by amiloride (data not shown). The amiloride derivative ethylisopropylamiloride inhibited the fast transient current with an IC₅₀ of 37.6 µmol/L (data not shown). These blocking concentrations are 100-fold greater than those needed to block the epithelial amiloride-sensitive Na⁺ channel⁴⁴ and are high enough to block unrelated ion channels.⁴²

	Discussion

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There are 4 key findings in this study, as follows. (1) We describe a method to distinguish CSNs from neurons of other sensory modalities in primary dissociated tissue culture. (2) Compared with other sensory neurons, CDRGNs have extremely large currents evoked when pH drops to 7.0 or below. (3) CNodNs have smaller acid-evoked currents, but larger ATP-evoked currents than CDRGNs. (4) Other than its large amplitude, the acid-evoked current in CSNs has no biophysical or pharmacological properties that are not predicted by previous studies of acid-evoked currents in sensory neurons. The large amplitude indicates the importance of acid in mediating pain due to cardiac ischemia, but it certainly does not imply that other potential mediators are unimportant.

Isolation of CSNs
As described in Materials and Methods, considerable effort was made to validate this preparation. Each of the control injection experiments resulted in significantly fewer labeled neurons compared with the pericardial space injections; this lends support to our assertion that we have specifically labeled and isolated CSNs. Finally, the different responses between the labeled DRG neurons and UDRGNs in our whole-cell patch-clamp experiments provide further evidence that we have isolated a distinct subgroup of sensory neurons from the DRG population at large.

Although neuroanatomy was not the primary focus of this study, some anatomical information can be gleaned from our data. The number of labeled neurons we obtained was consistent with previous neuroanatomical studies in the rat in which fluorescent tracers were injected into the pericardial space.^{4 45} In contrast to the large number of labeled neurons appearing after pericardial space injections, we found relatively few labeled neurons after intramural injections. This may reflect either less tissue exposure to dye compared with the pericardial space injection or a true paucity of nerve terminals within the rat intramural myocardium. The endocardial layers were not labeled in our study. Interestingly, in a dog preparation, myocardial ischemia caused cardiac sympathetic afferent firing only if the ischemia was transmural and involved the superficial epicardial layers.¹⁰

Chemical Activation of CSNs
Several insights arise from a comparison of responses to different agonists in the different cell populations that we isolated. Most importantly, modest decreases in extracellular pH (ie, to pH 7.0 or below) evoke exceptionally large currents in almost all epicardial CDRGNs. In contrast, and as reported previously,^{26 27} only 50% of UDRGNs respond to acid; those that did respond had much smaller average currents than CDRGNs.⁴⁶

Cardiac afferents with cell bodies in the DRG differed from those in the nodose ganglia. CNodNs had significantly smaller acid-evoked currents and larger ATP-evoked currents than CDRGNs. This raises the possibility that the 2 cell populations sense different chemical signals during cardiac ischemia.

There are 2 classes of molecules that are proposed to sense changes in extracellular pH in sensory neurons: ASICs and vanilloid (capsaicin) receptors. Vanilloid receptors are activated by noxious heat and by capsaicin (the compound in pepper that tastes "hot"); also, current through vanilloid receptors is strongly increased by acidic pH.^{34 47} Therefore, it is considered that vanilloid receptors, in addition to sensing heat, may mediate sensory responses to acidity caused by inflammation and ischemia. The neurons we isolated detect cardiac ischemia, yet only a small fraction exhibit capsaicin-activated current, and those that do respond have small currents compared with UDRGNs. In contrast, almost all exhibit grossly large currents through ASICs. These results argue that ASICs are more important than vanilloid receptors for sensing myocardial ischemia.

5HT evoked currents that were substantial in cardiac afferents, but they were still smaller than the acid- or ATP-evoked currents. Various cells that contribute to the immune response to tissue damage release 5HT, so these currents may provide a means of communication between immune cells and CSNs.

Of the chemicals tested, protons, ATP, 5HT, and capsaicin activate ion channels (the ASICs,²⁸ P2X receptors,^{38 39} 5HT3 receptor,³³ and vanilloid receptors,³⁴ respectively) that are presumed to serve as sensory transducers in sensory neurons. No current was ever evoked by ACh, but currents were occasionally seen in response to BK or adenosine. We do not infer anything from the relatively rare and small currents evoked by these compounds, because they do not directly gate ion channels in sensory neurons; in fact, adenosine inhibits a Ca²⁺-activated K⁺ channel.³⁷ The current we saw presumably arose from modulation of a channel by an intracellular signaling cascade; the importance of this may be understated by simply comparing the amplitude with those of channels directly gated by protons, ATP, or 5HT.

Physiological and Pathophysiological Significance
CSNs respond to lowered pH (in the range produced by myocardial ischemia) with consistent and robust depolarizing currents; this suggests that acid is a potential mediator of myocardial ischemic sensation.

In humans, the only conscious sensation from the heart is pain or angina, which most commonly occurs during myocardial ischemia. However, objective measurements of myocardial ischemia often do not correlate with the presence or severity of chest pain. In fact, ambulatory electrocardiographic monitoring in patients with myocardial ischemia has revealed that the majority of ischemic episodes are not reported as painful.⁴⁸ Attempts to model ischemic cardiac pain in animals have produced variable results. The pseudoaffective measures of pain in these behavioral studies correlate poorly with sensory neuronal activation.^{13 16 49} Thus, it is reasonable to conclude that activation of CSNs with acid does not equate with nociception. For example, in patients with "silent" ischemia (defined as objective myocardial ischemia that is painless), there is evidence of sensory activation to the level of the thalamus, whereas patients with typical angina have additional activation of the cerebral cortex.⁵⁰ Thus, the conscious perception of chest pain most certainly involves complex central processing and integration at multiple levels, and activation of CSNs is probably necessary but not sufficient to produce pain. Regardless, ischemia- or acid-induced activation of CSNs, whether painful or not, may be an important initiator of cardiovascular reflexes in pathological cardiac conditions.

A broad range of cardiovascular disease processes, including myocardial ischemia,⁵¹ congestive heart failure,⁵² and arrhythmias,⁵³ are precipitated or worsened by perturbations in the autonomic nervous system. Much of the current pharmacological therapies are directed toward blocking the compensatory, but often deleterious, neurohormonal systems that are activated in these diseases. In human skeletal muscle, ischemia-induced acidic pH is coupled with sympathetic efferent nerve discharge.⁵⁴ Also, abdominal visceral ischemia leads to profound cardiovascular reflex changes, the degree of which appears related to the level of the resulting acidosis.⁵⁵ A similar acid-evoked reflex loop may exist in the heart and contribute to the detrimental effect of sympathetic activation in myocardial ischemic conditions. Specific blockade of acid-evoked activation of CSNs presents a potential new therapeutic management strategy in the treatment of ischemic heart disease.

	Acknowledgments

 
This study was supported by the following NIH grants: RO1DAO7415, Cardiovascular Signaling Training Grant T32-HL07817 (to C.J.B.), and Neuronal Signal Transduction Training Grant NS07381 (to S.P.E.). We thank G. Giraud and K. Thornburg for their comments on the manuscript and V.C. Dang for technical assistance.

Received August 12, 1998; accepted February 10, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Cutler EC. Summary of experiences up-to-date in the surgical treatment of angina pectoris. Am J Med Sci. 1927;173:613–624.

2. White JC. Cardiac pain: anatomic pathways and physiologic mechanisms. Circulation. 1957;16:644–655.[Medline] [Order article via Infotrieve]

3. Berry M, Bannister LH, Standering SM, eds. Gray's Anatomy. New York, NY: Churchill Livingstone; 1995;901–1397.

4. McNeill DL, Burden HW. Convergence of sensory processes from the heart and left ulnar nerve onto a single afferent perikaryon: a neuroanatomical study in the rat employing fluorescent tracers. Anat Rec. 1986;214:441–444.[Medline] [Order article via Infotrieve]

5. Lewis T. Pain in muscular ischemia: its relation to anginal pain. Arch Int Med. 1932;49:713–727.[Abstract/Free Full Text]

6. Brown AM. Excitation of afferent cardiac sympathetic nerve fibres during myocardial ischemia. J Physiol (Lond). 1967;190:35–53.[Abstract/Free Full Text]

7. Minisi AJ, Thames MD. Activation of cardiac sympathetic afferents during coronary occlusion: evidence for reflex activation of sympathetic nervous system during transmural myocardial ischemia in the dog. Circulation. 1991;84:357–367.[Abstract/Free Full Text]

8. Huang MH, Horackova M, Negoescu RM, Wolf S, Armour JA. Polysensory response characteristics of dorsal root ganglion neurones that may serve sensory functions during myocardial ischaemia. Cardiovasc Res. 1996;32:503–515.[Medline] [Order article via Infotrieve]

9. Thames MD, Klopfenstein HS, Abboud FM, Allyn LM, Walker JL. Preferential distribution of inhibitory cardiac receptors with vagal afferents to the inferoposterior wall of the left ventricle activated during coronary occlusion in the dog. Circ Res. 1978;43:512–519.[Abstract/Free Full Text]

10. Armour JA, Huang MH, Pelleg A, Sylven C. Responsiveness of in situ canine nodose ganglion afferent neurones to epicardial mechanical or chemical stimuli. Cardiovasc Res. 1994;28:1218–1225.[Abstract/Free Full Text]

11. James TN. A cardiogenic hypertensive chemoreflex. Anesth Analg. 1989;69:633–646.[Abstract/Free Full Text]

12. Baker DG, Coleridge HM, Coleridge JCG, Nerdrum T. Search for a cardiac nociceptor: stimulation by bradykinin of sympathetic afferent nerve endings in the heart of the cat. J Physiol (Lond). 1980;306:519–536.[Abstract/Free Full Text]

13. Euchner-Wamser I, Meller ST, Gebhart GF. A model of cardiac nociception in chronically instrumented rats: behavioral and electrophysiological effects of pericardial administration of algogenic substances. Pain. 1994;58:117–128.[Medline] [Order article via Infotrieve]

14. Malliani A, Schwartz PJ, Zanchetti A. A sympathetic reflex elicited by experimental coronary occlusion. Am J Physiol. 1969;217:703–709.

15. Sleight P. A cardiovascular depressor reflex from the epicardium of the left ventricle in the dog. J Physiol (Lond). 1964;173:321–343.

16. Meller ST, Gebhart GF. A critical review of the afferent pathways and the potential chemical mediators involved in cardiac pain. Neuroscience. 1992;48:501–524.[Medline] [Order article via Infotrieve]

17. Jacobus WE, Taylor GT IV, Hollis DP, Nunnally RL. Phosphorus nuclear magnetic resonance of perfused working rat hearts. Nature. 1977;265:756–758.[Medline] [Order article via Infotrieve]

18. Cobbe SM, Poole-Wilson PA. The time of onset and severity of acidosis in myocardial ischemia. J Mol Cell Cardiol. 1980;12:745–760.[Medline] [Order article via Infotrieve]

19. Hirsch HJ, Franz CHR, Bos L, Bissig R, Lang R, Schramm M. Myocardial extracellular K⁺ and H⁺ increase and noradrenaline release as possible cause of early arrhythmias following acute coronary artery occlusion in pigs. J Mol Cell Cardiol. 1980;12:579–593.[Medline] [Order article via Infotrieve]

20. Cobbe SM, Poole-Wilson PA. Continuous coronary sinus and arterial pH monitoring during pacing-induced ischaemia in coronary artery disease. Br Heart J. 1982;47:369–374.[Abstract/Free Full Text]

21. Uchida Y, Murao S. Acid-induced excitation of afferent cardiac sympathetic nerve fibers. Am J Physiol. 1975;228:27–33.

22. Steen KH, Steen AE, Reeh PW. A dominant role of acid pH in inflammatory excitation and sensitization of nociceptors in rat skin, in vitro. J Neurosci. 1995;15:3982–3989.[Abstract]

23. Krishtal OA, Pidoplichko VI. A receptor for protons in the nerve cell membrane. Neuroscience. 1980;5:2325–2327.[Medline] [Order article via Infotrieve]

24. Konnerth A, Lux HD, Morad M. Proton-induced transformation of calcium channel in chick dorsal root ganglion cells. J Physiol (Lond). 1987;386:603–633.[Abstract/Free Full Text]

25. Davies NW, Lux HD, Morad M. Site and mechanism of activation of proton-induced sodium current in chick dorsal root ganglion neurons. J Physiol (Lond). 1988;400:159–187.[Abstract/Free Full Text]

26. Bevan S, Yeats J. Protons activate a cation conductance in a sub-population of rat dorsal root ganglion neurones. J Physiol (Lond). 1991;433:145–161.[Abstract/Free Full Text]

27. Krishtal OA, Pidoplichko VI. A receptor for protons in the membrane of sensory neurons may participate in nociception. Neuroscience. 1981;6:2599–2601.[Medline] [Order article via Infotrieve]

28. Waldmann R, Lazdunski M. H⁺-gated cation channels: neuronal acid sensors in the NaC/DEG family of ion channels. Curr Opin Neurobiol. 1998;8:418–424.[Medline] [Order article via Infotrieve]

29. Nakatani T, Shinohara H, Fukuo Y, Morisawa S, Matsuda T. Pericardium of rodents: pores connect the pericardial and pleural cavities. Anat Rec. 1988;220:132–137.[Medline] [Order article via Infotrieve]

30. Eckert SP, Taddese A, McCleskey EW. Isolation and culture of rat sensory neurons having distinct sensory modalities. J Neurosci Methods. 1997;77:183–190.[Medline] [Order article via Infotrieve]

31. Hille B. Selective permeability: independence. In: Ion Channels of Excitable Membranes. Sunderland, Mass: Sinauer Associates; 1992:344–347.

32. Cook SP, Vulchanova L, Hargreaves KM, Elde R, McCleskey EW. Distinct ATP receptors on pain-sensing and stretch-sensing neurons. Nature. 1997;387:505–508.[Medline] [Order article via Infotrieve]

33. Maricq AV, Peterson AS, Brake AJ, Myers RM, Julius D. Primary structure and functional expression of the 5HT₃ receptor, a serotonin-gated ion channel. Science. 1991;254:432–436.[Abstract/Free Full Text]

34. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature. 1997;389:816–824.[Medline] [Order article via Infotrieve]

35. Sucher NJ, Cheng TPO, Lipton SA. Neural nicotinic acetylcholine responses in sensory neurons from postnatal rat. Brain Res. 1990;533:248–254.[Medline] [Order article via Infotrieve]

36. Nicol GD, Cui M. Enhancement by prostaglandin E₂ of bradykinin activation of embryonic rat sensory neurones. J Physiol (Lond). 1994;480:485–492.[Abstract/Free Full Text]

37. Allen TGJ, Burnstock G. The actions of adenosine 5'-triphosphate on guinea-pig intracardiac neurones in culture. Br J Pharmacol. 1990;100:269–276.[Medline] [Order article via Infotrieve]

38. Lewis C, Neidhart S, Holy C, North RA, Buell G, Suprenant A. Coexpression of P2X₂ and P2X₃ receptor subunits can account for ATP-gated currents in sensory neurons. Nature. 1995;377:432–435.[Medline] [Order article via Infotrieve]

39. Chen CC, Akopian AN, Sivilotti L, Colquhoun D, Burnstock G, Wood JN. A P2X purinoceptor expressed by a subset of sensory neurons. Nature. 1995;377:428–431.[Medline] [Order article via Infotrieve]

40. Kovalchuk YN, Krishtal OA, Nowycky MC. The proton-activated inward current of rat sensory neurons includes a calcium component. Neurosci Lett. 1990;115:237–242.[Medline] [Order article via Infotrieve]

41. Waldmann R, Champigny G, Bassilana F, Heurteaux C, Lazdunski M. A proton-gated cation channel involved in acid-sensing. Nature. 1997;386:173–177.[Medline] [Order article via Infotrieve]

42. Tang CM, Presser F, Morad M. Amiloride selectively blocks the low threshold (T) calcium channel. Science. 1988;240:213–215.[Abstract/Free Full Text]

43. Waldmann R, Bassilana F, de Weille J, Champigny G, Heurteaux C, Lazdunski M. Molecular cloning of a non-inactivating proton-gated Na⁺ channel specific for sensory neurons. J Biol Chem. 1997;272:20975–20978.[Abstract/Free Full Text]

44. Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger J, Rossier BC. Amiloride-sensitive epithelial Na⁺ channel is made of three homologous subunits. Nature. 1994;367:463–467.[Medline] [Order article via Infotrieve]

45. Alles A, Dom RM. Peripheral sensory nerve fibers that dichotomize to supply the brachium and the pericardium in the rat: a possible morphological explanation for referred cardiac pain? Brain Res. 1985;342:382–385.[Medline] [Order article via Infotrieve]

46. Bevan S, Winter J. Nerve growth factor (NGF) differentially regulates the chemosensitivity of adult rat cultured sensory neurons. J Neurosci. 1995;15:4918–4926.[Abstract]

47. Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, Raumann BE, Basbaum AI, Julius D. The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron. 1998;21:531–543.[Medline] [Order article via Infotrieve]

48. Deanfield JE, Shea MJ, Selwyn AP. Clinical evaluation of transient myocardial ischemia during daily life. Am J Med. 1985;79(suppl 3A):18–24.

49. Malliani A. Significance of experimental models in assessing the link between myocardial ischemia and pain. Adv Cardiol. 1990;37:126–141.[Medline] [Order article via Infotrieve]

50. Rosen SD, Paulesu E, Nihoyannopoulos P, Tousoulis D, Prackowiak SJ, Frith CD, Jones T, Camici PG. Silent ischemia as a central problem: regional brain activation compared in silent and painful myocardial ischemia. Ann Intern Med. 1996;124:939–949.[Abstract/Free Full Text]

51. Thamer V, Deussen A, Schipke JD, Tolle T, Heusch G. Pain and myocardial ischemia: the role of sympathetic activation. Basic Res Cardiol. 1990;85:253–266.

52. Francis GS, Goldsmith SR, Levine TB, Olivari MT, Cohn JN. The neurohormonal axis in congestive heart failure. Ann Intern Med. 1984;101:370–377.

53. Mitrani RD, Zipes DP. Clinical neurocardiology: arrhythmias. In: Armour JA, Ardell JL, ed. Neurocardiology. New York, NY: Oxford University Press; 1994:365–395.

54. Victor RG, Bertocci LA, Pryor SL, Nunnally RL. Sympathetic nerve discharge is coupled to muscle cell pH during exercise in humans. J Clin Invest. 1988;82:1301–1305.

55. Rendig SV, Chalal PS, Longhurst JC. Cardiovascular reflex responses to ischemia during occlusion of celiac and/or superior mesenteric arteries. Am J Physiol. 1997;272:H791–H796.[Abstract/Free Full Text]

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