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Cardiovascular Research 2002 53(4):888-901; doi:10.1016/S0008-6363(01)00542-9
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Role of P1 purinergic receptors in myocardial ischemia sensory transduction

G.W Thompsona, M Horackovaa and J.A Armoura,*

aDepartment of Physiology and Biophysics, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, B3H 4H7, Canada

jarmour{at}is.dal.ca

* Corresponding author. Tel.: +1-902-494-3382; fax: +1-902-494-1685

Received 2 April 2001; accepted 1 November 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives: To characterize the role that cardiac sensory P1 purinergic (adenosine A1 or A2) receptors play in transducing myocardial ischemia. Methods: Porcine nodose ganglion cardiac sensory neuron adenosine A1 or A2 receptor function was studied in situ during control states as well as in the presence of the peptides bradykinin and substance P or focal ventricular ischemia. The responses of porcine nodose ganglion cardiac and non-cardiac afferent neuronal somata to adenosine were also studied in vitro. Results: Local application of A1 or A2 adenosine receptor agonists modified the activity generated by ventricular sensory neurites associated with 70 and 74% of identified nodose ganglion cardiac afferent somata in situ, respectively, exciting most neurons. In contrast, adenosine reduced the excitability of nodose ganglion cardiac afferent neuronal somata in vitro. Bradykinin and substance P affected 56 and 63%, respectively, of tested afferent neurons. The capacity of ventricular sensory neurites to transduce signals relating to these peptides was virtually eliminated by the presence of P1 purinergic receptor antagonists. So was their capacity to transduce focal ventricular ischemia. Since most cardiac sensory neurites responded differently to adenosine in vivo than did cardiac afferent neuronal somata in vitro, it appears that the transduction properties of cardiac afferent neurons need to be characterized in situ. Conclusions: Most ventricular sensory neurites associated with nodose ganglion afferent neurons possess adenosine A1 and/or A2 receptors that play a primary role in transducing myocardial ischemic events to central neurons. These data support clinical observations implicating cardiac sensory purinoceptors in transducing myocardial ischemic events.

KEYWORDS Adenosine; Autonomic nervous system; Ischemia; Receptors


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Excitation of cardiac afferent neurons during myocardial ischemia has been accepted as the underlying cause of cardiac pain in humans [1]. The mechanisms involved in transducing cardiac ischemic events remain debatable. Acute studies employing anesthetized animals demonstrate that coronary artery occlusion activates nodose [2,3] and dorsal root [4] ganglion cardiac afferent neurons. Other studies have shown that coronary artery occlusion enhances the release of chemical mediators potentially responsible for activating cardiac afferent neurons that include adenosine [5,6], bradykinin [7] and substance P [8]. Clinical studies have shown that exogenously administered adenosine causes angina-like pain both in healthy patients and in patients with ischemic heart disease [9,10]. In accord with this, the competitive adenosine receptor antagonist theophylline attenuates adenosine-induced chest pain in humans [10]. In contrast, the peptide substance P plays a subsidiary role in the genesis of angina pectoris [9].

Ventricular sensory nerve terminals (neurites) associated with nodose ganglion cardiac afferent neurons transduce a broad range of neuroactive agents in situ that include purinergic agents and peptides such as bradykinin and substance P [2,11]. The somata of nodose ganglion cardiac afferent neurons express adenosine A1 [12] and A2 [13], as well as bradykinin [14] and substance P [15,16] receptors. The extracellular activity generated by nodose ganglion cardiac afferent neurons can be recorded for hours via tungsten microelectrodes [11]. This technique permits an assessment of the response characteristics displayed by individual cardiac sensory neurons to a variety of chemical stimuli applied directly to their sensory neurites in situ. Employing this technique, we studied afferent neuronal responses to specific adenosine receptor agonists. These data were compared to effects induced by the P1-purinergic receptor agonist adenosine on the somata of nodose ganglion afferent neurons in vitro. The effects induced by applying the peptides bradykinin and substance P to ventricular sensory neurites associated with nodose ganglion afferent neurons were also assessed before, and subsequent to, local application of adenosine receptor antagonists. Furthermore, the effects of transient coronary artery occlusion on the spontaneous activity generated by these cardiac afferent neurons were studied in the absence and presence of specific P1-purinergic receptor antagonists to determine whether they modify ischemia-induced cardiac afferent neuronal transduction. In this manner, we sought to clarify the importance of sensory neurite P1-purinergic receptors in the transduction of ischemic events by cardiac afferent neurons in situ.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Animal preparation
All experiments were performed in accordance with guidelines described in Guide for the Care and Use of Laboratory Animals (NIH publication 85-23, revised 1996) and the Guide to the Care and Use of Experimental Animals (Canadian Council on Animal Care, Ottawa, 1993). The institutional animal care and use committee of Dalhousie University approved the experiments.

2.2. In situ experiments
2.2.1. Animal preparation
Eighteen young Hamshire–Duvoc pigs (12–19 kg) of either sex were sedated with ketamine hydrochloride (18 mg/kg, i.m.) and 4% halothane before endotracheal intubation. During surgical preparation, pigs were anesthetized with sodium pentothal (loading dose: 20 mg/kg, i.v.; maintenance doses: 10 mg/kg, i.v. being provided to effect every 10 min). After the surgery was completed (see below), anesthesia was maintained using {alpha}-chloralose (loading dose: 100 mg/kg, i.v; maintenance doses: 15–20 mg/kg/h, i.v., as required). Heart rate was monitored via a lead II electrocardiogram throughout the experiments. Noxious stimuli were applied to a limb occasionally throughout the experiments while monitoring heart rate, jaw tone and arterial pressure in order to ascertain adequacy of anesthesia. Body temperature, measured with a Shiley (Irvine, CA, USA) rectal temperature probe, was maintained at 37–38 °C by means of a circulating hot water pad.

A bilateral thoracotomy was made in the fifth intercostal space and the ventral pericardium incised and retracted laterally to expose the epicardial surface of the heart. The origins of the left anterior descending coronary artery and the right main coronary artery were identified and looped with snares so that these arteries could be transiently occluded later during the experiments. Left atrial chamber pressure was measured using a PE-50 catheter inserted into the left atrial chamber via its appendage. Right ventricular chamber pressure was measured using a no. 6 French Berman (Arrow International Reading, PA, USA) angiographic catheter inserted via a femoral vein. Left ventricular chamber pressure was measured using a no. 6 French Cordis pigtail catheter (Cordis, Miami, FL, USA) inserted into that chamber via a femoral artery. Systemic arterial pressure was measured using a no. 7 French Cordis catheter placed in the descending aorta via the other femoral artery. These catheters were attached to Bentley (Irvine, CA, USA) Trantec model 800 pressure transducers. Single miniature solid-state pressure transducers measuring 5x1.2 mm (model P19; Konigsberg Instruments, Pasadena, CA, USA) were inserted into the midwall regions of the left and right ventricular ventral walls to record regional intramyocardial pressures. These sensing devices were employed since intraventricular systolic pressure represents a less sensitive index for detecting ventricular force changes induced by autonomic efferent neurons.

2.2.2. Extracellular recording of nodose ganglion afferent neurons
An incision was made in the neck to expose either nodose ganglion. The right nodose ganglion was used in most experiments for extracellular neuronal recording due to its greater accessibility and since right and left nodose ganglia possess similar populations of cardiac afferent neurons [17]. Once exposed, the ganglion was left bound to adjacent connective tissue to minimize motion during recording. It was covered with mineral oil to prevent desiccation.

Spontaneous activity generated by nodose ganglion afferent neurons was identified using a tungsten microelectrode (model ME no. 25-10-2, Frederick Haer, Bowdoninham, ME, USA), as described previously [2]. The tungsten microelectrode (250 µm shank diameter; an exposed tip of 1 µm; impedance of 9–12 M{Omega}) was placed in exposed ganglia at varying depths using a Marzhauser micromanipulator (model 25033-10, Fine Scientific Tools North Vancouver, Canada). Reference and ground electrodes were attached to adjacent tissues. Action potentials were amplified by a Princeton Applied Research Model 113 amplifier (Princeton, NJ, USA) with bandpass filters set at 300 Hz to 10 kHz and an amplification range of 100–500x. The output of this amplifier was further amplified (50–200x) and filtered (bandwidth 100 Hz–2 kHz) with an optically isolated amplifier (Applied Microelectronics Institute, Halifax, Canada). These signals were led to a Nicolet model 207 oscilloscope (Nicolet Instrument, Madison, WI, USA) and a Grass AM8 audio monitor (Grass instruments, Quincy, MA, USA) for monitoring neuronal activity. Individual neurons were identified by the amplitude and configuration of action potentials using the following criteria: (1) action potentials generated by a single neuron displayed the same configuration and amplitude over several hours; and (2) action potentials maintained the same configuration even when the microelectrode was moved micrometers away from the site of activity despite a change in amplitude. Action potentials with signal-to-noise ratios greater than 4:1 were recorded for extensive periods of time, a process facilitated by the lack of motion of nodose ganglia in situ.

Conduction velocity of axons associated with identified nodose ganglion cardiac afferent neurons was estimated by electrically stimulating (1–4 V, 1 ms, 0.1 Hz) epicardial sensory fields at the end of each experiment by means of a unipolar ball electrode. An indifferent electrode was attached to the thoracic wall. Time between stimulus artifact and the generated action potential was recorded. After estimating the distance between the stimulation and recording electrodes, axonal conduction velocity was estimated.

2.2.3. Identification of epicardial sensory fields
The nodose ganglion contains the somata of afferent neurons innervating pulmonary, gastrointestinal, pharyngeal and cardiovascular tissues. Thus, identification of cardiac afferent neurons associated with epicardial sensory neurites was required. Once the activity generated by one or two spontaneously active neurons was identified at a site in a nodose ganglion, approximately 10 min of baseline activity was recorded. Thereafter, various atrial or ventricular loci were gently probed using a saline-soaked Q-tip applicator. Loci in pulmonary, aortic as well as superior and inferior caval tissues were also probed. If mechanical stimuli did not modify the spontaneous activity of identified afferent neurons, pledgets soaked with the sodium channel agonist veratridine (5x10–6 g/cc) were applied to ventricular epicardial loci to activate the sensory terminals of such neurons. When spontaneous activity was modified by local mechanical and/or chemical stimuli, the neuron was considered to be associated with epicardial sensory nerve endings. As described above, estimations of conduction velocity provided further evidence that identified afferent neurons were associated with epicardial sensory neurites.

2.2.4. Mechanical stimuli
Various atrial and ventricular epicardial loci were gently probed with a saline-soaked Q-tip applicator to determine the mechanical sensitivity of individual nodose ganglion afferent neurons. The extent of each epicardial sensory field was determined by this means. Mechanical interventions were performed at least twice to confirm reproducibility of responses.

2.2.5. Chemical stimuli
Chemical agents (~2.0 ml) were applied for 60–120 s individually via 2x2 cm gauze squares placed on the epicardial surface of the outflow tract of the right ventricle and the cranial, ventral surface of the left ventricle adjacent to the left ventral descending coronary artery of each animal. In this manner, about 30% of the ventral surfaces of the two ventricles were investigated. These agents were obtained from Research Biochemicals International (Natick, MA, USA) or Sigma (St. Louis, MO, USA). The purinergic agonists studied were the specific adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) and the specific adenosine A2 receptor agonist 5'-N-cyclopropyl-carboxamidoadenosine (CPCAA). Three concentrations of each agonist (1, 10 and 100 µM) were tested initially on the epicardial sensory fields of six nodose ganglion afferent neurons. It was found that 10 µM concentrations of each of these chemicals elicited consistent responses without modifying monitored cardiovascular indices. Application of 1 µM concentrations elicited inconsistent neuronal responses while 100 µM concentrations induced minor reductions in chronotropism and arterial pressure in some animals. Thus, 10 µM concentrations were used in all subsequent experiments. The specific adenosine A1 receptor antagonist 8 cyclopentyl-1,3-dipropylxanthine (CPDPX; 100 µM) and the specific adenosine A2 receptor antagonist 3,7-dimethyl-1-propargylxanthine (DMPX; 100 µM) were also applied to identified epicardial sensory fields to test whether these receptor antagonists could attenuate afferent neuronal responses induced by each agonist.

Four concentrations of bradykinin or substance P (1, 10, 100 and 250 µM) were applied individually to the epicardial sensory fields associated with five nodose ganglion afferent neurons. As 10 µM concentrations of these peptides elicited maximal responses and no further augmentation of neuronal activity at higher concentrations, that was the concentration tested thereafter. Lower concentrations induced inconsistent responses while higher doses induced systemic vascular changes. Each adenosine receptor agonist (2 ml, 10 µM) or peptide (2 ml, 10 µM) was administered into the descending aorta to determine whether each agent would affect monitored cardiovascular indices.

2.2.6. Myocardial ischemia
Either before or following completion of the interventions described above, the left anterior descending coronary artery or the right main coronary artery was occluded individually for 30–90 s. Longer duration ischemic periods were avoided to prevent gross changes in cardiac and vascular variables. Thereafter, the other coronary artery was occluded for 30–90 s. The sequence of coronary artery occlusion was random among experiments. A minimum of 15 min elapsed between each occlusion to allow neuronal and cardiovascular variables to return to preocclusion values. Coronary artery occlusions were repeated to test whether ischemia-induced responses were reproducible. Thereafter, coronary artery occlusions were performed during epicardial application of each specific purinoceptor antagonist. Coronary artery occlusions were performed at least 30 s after antagonist application and terminated when the antagonist was removed.

2.3. In vitro experiments
2.3.1. Anatomical tracing of vagal afferent pathways
Young pigs (13–16 kg) were sedated, anesthetized and intubated as described previously. Employing anatomical techniques similar to those utilized to identify nodose ganglion afferent neurons associated with aortic mechanosensory neurites [18], the neuronal retrograde fluorescent tracing dye fast blue (Sigma) was injected transthoracically under sterile conditions into the left ventricular ventral and lateral walls using a Hamilton syringe (Hamilton; Reno, NV, USA). To confirm placement of the injecting syringe, one lead of a two lead ECG was attached to the syringe. The second lead and ground electrodes were attached directly to the animal. Large deflections in the ECG tracing, as well as physical palpation, indicated that the syringe was in direct contact with the epicardium. Fast blue, mixed as a 4% weight per volume solution, was injected in 25-µl quantities into three separate myocardial sites, for a total of 75 µl. Antibiotics were administered to the animals as they recovered.

After 21 days recovery time, these animals were sedated with ketamine hydrochloride (18 mg/kg, i.m.) and anesthetized with sodium pentothal (50 mg/kg, i.v.). One nodose ganglion (either left or right) was excised from each animal via a midline neck incision and used for acute dissociation, culture, and electrophysiological recording (see below). Thereafter, a midline thoracotomy was performed and each animal was perfused transcardially with saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The heart, the remaining nodose ganglion and the brainstem were then removed. The heart was examined to confirm the location of injection sites. Adjacent thoracic tissues (i.e. esophagus, mediastinum, trachea and lungs) were examined postfixation to determine whether dye could be visualized in these tissues. Nodose ganglia and brainstem were postfixed in 4% paraformaldehyde overnight and transferred into 0.1 M phosphate buffer containing 30% sucrose for protection until processing. These tissues were cut into 40 µm frozen sections using a Leitz sledge microtome (Leitz, New York, USA). Tissue sections were wet mounted in buffer on chrome alum subbed slides.

2.3.2. Immunohistochemistry of nodose ganglion tissue
Immunolocalization using the general neuronal marker protein gene product 9.5 (PGP 9.5) was performed on fixed nodose ganglion tissues [19] to visualize fast blue-positive labeled afferent neurons. The 40-µm sections were incubated overnight at 4 °C with rabbit polyclonal antibody against human PGP 9.5 (Ultraclone, Wellow, Isle of Wight, UK) that had been diluted 1:400 in PBS with 2% Triton X-100. After washing, tissue sections were exposed for 2 h at room temperature to a secondary antibody (1:20 FITC-labelled sheep anti-mouse IgG). After several washes, PGP-9.5-labeled sections were mounted and viewed with a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss, Thornwood, NY, USA) employing reflected light fluorescence using filter cubes (Carsen Group, Markham, Canada) for ultraviolet (U-MWU with excitation at 345–370 nm and emission at 425–480 nm) and PGP-FITC (bandpass filter 575–640 nm). Images were captured on Ectachrome P1600 color negative film.

2.3.3. Neuronal cell cultures
The effects of adenosine were studied on nodose ganglion cardiac (labeled) and non-cardiac (unlabeled) afferent neurons in vitro. The nodose ganglion not employed for the anatomy study was removed from the four pigs, immediately rinsed in Tyrode's solution several times and then cut into small pieces (~1–2 mm3). These nodose ganglion tissue blocks were placed in Dulbecco's modified Eagle's medium (Gibco) containing 0.15% trypsin (type III, Cat. No. T8253, Sigma) and collagenase (Boehringer Mannheim, Laval, Quebec, Canada, or Worthington type II, Freehold, NJ, USA) and then incubated in a 35 °C shaker bath for 1 h followed by trituration using a Pasteur pipette. Thereafter, treated tissues were centrifuged (1000 rpm for 5 min), washed and resuspended in culture media containing 1.8 mM Ca2+. The basic culture medium used was Eagle's minimum essential medium with Earle's salts (Gibco), supplemented (including 5% fetal bovine serum) as described previously [20]. Cytosine 1-β-D-arabinofuranoside (10 µM) was added to minimize the growth of fibroblasts. Nerve growth factor (100 ng/ml; Collaborative Biochemical Products, Bedford, MA, USA) was added to cultures to promote the survival of neurons and neurite growth [20]. Laminin was used to coat the coverslips (diameter=1.2 cm). To form neuronal cultures, we distributed neurons dissociated from nodose ganglia of each animal into 4–6 wells, aiming for 10–20 neurons/culture. Plated cells were maintained in an incubator at 37 °C under a 95% air–5% CO2 atmosphere for 24–48 h before recording neuronal transmembrane potentials.

2.3.4. In vitro recordings from cultured neurons
The plates containing nodose ganglion cardiac afferent neurons were placed in a small bath perfused with Tyrode's solution at 3 ml/min at 36 °C. The membrane potentials of these neurons were recorded using conventional microelectrodes (Rel=40–60 M{Omega}) filled with 2.4 M KCl. An Ag–AgCl bridge connected the pipette to the input stage of an amplifier. Then current pulses of varying durations and amplitudes (0.1–0.5 nA) were delivered to neurons at a frequency of 0.1 Hz. Current clamp stimulation protocols were utilized to study action potentials generated by neurons recorded by means of PCLAMP software (Axon Instruments) on a PC-AT 386 microcomputer at a sampling frequency of 1 kHz. These experiments were performed in the absence and presence of adenosine (10–5 M). Immediately after recording their electrical activity, we examined the cultured neurons for ultraviolet fluorescence using fluorescent microscopy (see above). Using these methods, we were able to record the electrical activity generated by five labeled and six unlabeled nodose ganglion afferent neurons in vitro.

2.4. Data analysis
During the in situ experiments, afferent neuronal activity, heart rate, left atrial pressure, right and left ventricular intramyocardial systolic pressures, right and left ventricular chamber systolic pressures and aortic pressure were recorded simultaneously on an Astro-Med model MT 9500 eight-channel rectilinear recorder (Astro-Med, West Warwick, RI, USA). These data were stored for later analysis on VHS tape (T-120 Fuji Photo Film USA) using a videocassette recorder (A.R. Vetter, Co. Model 820, Rebersberg, PA, USA). Heart rate, left atrial pressure, intramyocardial pressures, as well as chamber pressures were measured for ten consecutive beats before and during maximal responses for each intervention. The means±S.E.M. of the monitored cardiovascular indices were calculated. Spontaneous fluctuations of hemodynamic indices were minimal over 1-min periods. For example, heart rate changes were less than 5 beats/min (~5%) and changes in systolic pressure were less than 5 mmHg (~4–6%) during control conditions. Thus, thresholds for classifying induced changes in cardiovascular variables were chosen to be above these ranges.

Action potentials generated by nodose ganglion cardiac afferent neurons in situ were counted during 60-s periods (to determine average activity) immediately before a stimulus was applied and when the response to a stimulus had reached steady state. Fluctuations in the amplitude of action potentials generated by a unit varied by less than 10 µV over several minutes, action potentials retaining the same configurations over time. Thus, action potentials recorded in a given locus with the same configuration and amplitude (±10 µV) were considered to be generated by a single unit. Action potentials with signal-to-noise ratios greater than 3:1 were analyzed. A change in neuronal activity was defined as occurring when activity differed by more than 25% from control values. When multiple action potentials were recorded from a ganglionic site, individual units were separated based on their amplitudes. Action potential data were grouped according to whether activity increased, decreased, or remained unchanged during an intervention. When more than two neurons generated activity at an identified locus, the activity generated by individual units was analyzed by means of a window discriminator (C.J. Hartley Instrumentation Development Laboratories, Baylor College of Medicine, Houston, TX, USA). The activity generated by identified neurons in active loci was converted into average activity (impulses/min). Neuronal activity and cardiovascular responses were evaluated by comparing mean (±S.E.M.) data recorded immediately before administration of a chemical with data obtained at the point of maximum change after administration of the agent. One-way ANOVA and paired t-test with Bonferroni correction for multiple tests were used for statistical analysis. A value of P<0.01 or 0.001 were considered to represent significant differences from control values.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. In situ experiments
One to three spontaneously active neurons associated with epicardial sensory fields were identified within individual loci of a nodose ganglion in each animal studied. When the recording microelectrode tip was placed in the adjacent vagus nerve either no action potentials were identified or action potentials with signal-to-noise ratios of <2:1 were present. In total, 62 primary cardiac afferent neurons that were associated with epicardial sensory fields were identified in nodose ganglia. During control states, identified cardiac afferent neurons generated, on average, 10±6 to 97±30 impulses/min (ipm) (Table 1). The activity generated by afferent neurons identified in either sex displayed similar response characteristics. Spontaneous activity was sporadic in nature and thus did not directly correlate to the cardiac cycle in the majority of neurons studied. The estimated conduction velocity of their associated axons was, on average, 1.9±0.3 m/s. Altering the frequency of the electrical stimuli delivered to their sensory fields did not alter the latencies of action potential generation displayed by individual neurons. The heart rate was 118±11 beats/min during control states, while left atrial pressure was 4±6 mmHg; right ventricular chamber systolic pressure was 18±5 mmHg, left ventricular chamber systolic pressure was 108±7 mmHg and aortic pressure 111±6/88±4 mmHg. Right ventricular intramyocardial systolic pressure was 21±4 mmHg and left ventricular intramyocardial systolic pressure was 113±7 mmHg during control states. These indices did not change significantly during epicardial application of chemicals.


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  		Table 1 Changes in neuronal activity (average±S.E.M.) grouped according to the intervention studied

 
3.1.1. Mechanical stimuli
Focal epicardial mechanical stimuli modified the spontaneous activity generated by 32% of the identified afferent neurons (Table 1). Mechanical stimuli initiated responses immediately after they were applied, responses subsiding rapidly after removal of the stimuli. Mechanical stimuli increased the activity generated by 29% of afferent neurons tested by an average of 150%; mechanical stimuli decreasing the activity generated by 3% of studied neurons (Table 1). Most mechanosensory fields were located in the right (58%) or left (33%) ventricular epicardium; 9% were also associated with atrial sensory fields. Identified epicardial sensory fields covered approximately 1x1 cm areas.

3.1.2. Adenosine receptors agonists
Epicardial application (30–60 s) of the specific adenosine A1 receptor agonist CPA or the specific adenosine A2 receptor agonist CPCAA modified the activity generated by 70% (42/60) and 74% (40/54) of afferent neurons tested, respectively (Table 1). The mean latency of onset to neuronal responses was 28±10 s for CPA and 29±12 s for CPCAA. Excitatory responses elicited by either agonist persisted for up to 25 min following their removal. Fewer depressor than excitatory responses were elicited; depressor responses took much longer to develop and lasted for longer periods after agonist removal.

CPA increased the activity generated by 57% (34/60) of tested neurons (Fig. 1A) by, on average, 430%; peak firing frequency reached 10 Hz (i.e. 600 ipm). CPCAA increased the activity (Fig. 2A) generated by 48% (26/54) of the afferent neurons tested by an average of 176% (Table 1); peak firing frequency reached 365 ipm. On the other hand, epicardial application of CPA decreased (–68%) the activity generated by 13% (8/60 neurons) of the afferent neurons tested while CPCAA suppressed (–73%) the activity generated by 26% (14/54) of the neurons (Table 1). Thirty-two of fifty (64%) purinergic-sensitive afferent neurons responded to both CPA and CPCAA. Sixty-six percent of these neurons (21/32) generated similar responses when exposed to either agonist; eleven neurons (34%) responded differently when exposed to either agonist. In other words, epicardial application of CPA decreased the activity generated by some neurons that were excited by epicardial application of CPCAA, or vice versa. Monitored cardiac variables did not change overall during epicardial application of CPA or CPCAA. For instance, heart rate (118±11 beats/min) as well as right atrial (6±2 mmHg), right ventricular (18±5 mmHg) and left ventricular (122±10 mmHg) chamber pressures remained unaffected by these interventions. Furthermore, systemic administration of CPA or CPCAA did not modify neuronal activity or monitored cardiovascular variables.


Figure 1
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  		Fig. 1 (A) Epicardial application of the specific adenosine A1-receptor agonist CPA (arrow below) activated nodose ganglion afferent neurons. (B) Epicardial application of the specific adenosine A1-receptor antagonist CPDPX did not affect afferent neuronal activity. (C) Epicardial application of CPA in the presence of CPDPX no longer excited afferent neurons. After waiting about 20 min after their removal, reapplication of CPA initiated a similar afferent neuronal response as before (not shown).

 

Figure 2
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  		Fig. 2 (A) Epicardial application of the specific adenosine A2-receptor agonist CPCAA (arrow below) activated afferent neurons. (B) Epicardial application of the specific adenosine A2-receptor antagonist DMPX did not alter ongoing afferent neuronal activity. (C) Epicardial application of CPCAA in the presence of DMPX failed to initiate activity changes. Once these chemicals were removed, reapplication of CPCAA after a period of time initiated a similar afferent neuronal response as found in (A) (not shown).

 
3.1.3. Adenosine receptors antagonists
Epicardial application of the specific adenosine A1 receptor antagonist CPDPX excited 13% (8/62 neurons) of the afferent neurons tested (+51%; 45±28–68±28 ipm). CPDPX decreased (–55%; 78±20–35±17 ipm) the spontaneous activity generated by 34% (n=21) of the afferent neurons tested. Most of these latter afferent neurons were excited by the adenosine A1 receptor agonist. Of the remaining neurons tested (33/62; 53%), CPDPX elicited no change in neuronal activity. Epicardial application of the specific adenosine A2 receptor antagonist DMPX excited 11 of 58 (19%) afferent neurons (+140%; 10±3–24±6 ipm), while suppressing the activity (–80%; 45±13–9±4 ipm) generated by another 17 (29%) afferent neurons. Many of the afferent neurons depressed by DMPX were excited by the adenosine A2 receptor agonist CPCAA. Overall, 52% of afferent neurons tested (30/58) were unaffected by DMPX. Cardiovascular indices were unaffected by epicardial application of CPDPX or DMPX.

Due to the fact that excitatory effects induced by an agonist predominated and were brisk in nature compared to depressor effects, the responses of antagonists to agonist-induced excitation were examined statistically. Fourteen of the sensory neurons that were activated by CPA no longer responded to that selective adenosine A1 receptor agonist when applied in the presence of the adenosine A1 receptor antagonist CPDPX (Fig. 1; Table 2). Likewise, excitatory responses induced by the specific adenosine A2 receptor agonist CPCAA in 14 other animals were no longer elicited when this agonist was applied in the presence of the adenosine A2 receptor antagonist DMPX (Fig. 2; Table 3). The selectivity of each adenosine antagonist to block its specific receptor agonist was tested in six other afferent neurons. Similar excitatory responses were elicited whether the adenosine A1 receptor agonist CPA was applied alone (14±9 to 35±19 ipm; P<0.01) or in the presence of the specific adenosine A2 receptor antagonist DMPX (18±11 to 41±16 ipm; P<0.01). Activity increases induced in these neurons by the adenosine A2 receptor agonist CPCAA were also similar whether tested in the absence (17±9 to 51±15 ipm; P<0.01) or presence (22±8 to 57±18 ipm; P<0.01) of the specific adenosine A1 receptor antagonist CPDPX. In other words, prior application of the adenosine A1 or A2 receptor antagonists did not attenuate afferent neuronal responses induced by specific adenosine A2 or A1 receptor agonists, respectively.


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  		Table 2 Changes in neuronal activity (average±S.E.M.) elicited by epicardial application of CPA, bradykinin or substance P, as well as during ventricular ischemia in the absence or presence of the specific adenosine A1 receptor antagonist CPDPX. Interventions modified the activity generated by afferent neurons in the absence of CPDPX, but not in its presence

 

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  		Table 3 Changes in neuronal activity (average±S.E.M.) elicited by epicardial application of CPCAA, bradykinin or substance P, as well as during ventricular ischemia in the absence or presence of the specific adenosine A2 receptor antagonist DMPX. Each intervention modified the activity generated by afferent neurons in the absence, but not the presence of DMPX. *=P<0.01; **=P<0.001

 
3.1.4. Peptides
Sensory field application of the neuropeptides bradykinin or substance P modified the spontaneous activity generated by the majority of identified cardiac afferent neurons (Fig. 3; Table 1). The mean latency of responses elicited by bradykinin was 6±4 s, activity reaching a peak firing frequency above 16 Hz. The mean latency of responses elicited by substance P was 11±5 s, activity reaching a peak firing frequency greater than 13 Hz. Monitored cardiovascular indices were unchanged overall by either peptide. Epicardial application of bradykinin activated or inhibited the spontaneous activity generated by similar numbers of neurons (Table 1). Substance P activated more neurons that it suppressed (Table 1). Twenty-four afferent neurons (39%) responded to one peptide, while 25 (40%) responded to both peptides. Most of these neurons were modified in a similar fashion by each peptide, nine (36%) responding in an opposite fashion. Thus, thirteen (21%) afferent neurons were unaffected by either peptide. Most peptidergic-insensitive afferent neurons were sensitive to the adenosine receptor agonists.


Figure 3
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  		Fig. 3 Bradykinin-induced afferent neuronal responses. (A) Application of bradykinin (10 µM; arrow below) to an epicardial sensory field activated its associated afferent neuron. (B) In the presence of the specific adenosine A1-receptor antagonist CPDPX, bradykinin (administered at arrow below) failed to elicit a response. Note that following application of CPDPX there was a gradual reduction in neuronal activity. (C) After removal of these chemicals and waiting 35 min, reapplication of bradykinin (arrow below) in the absence of CPDPX initiated an excitatory response once again.

 
The capacity of adenosine receptor antagonists to block bradykinin-induced responses was studied in 18 cardiac afferent neurons that were sensitive to bradykinin and the adenosine A1 receptor agonist CPA. Subsequent application of bradykinin in the presence of the adenosine A1 receptor antagonist CPDPX failed to elicit excitatory responses in 14 of these 18 (78%) afferent neurons (Fig. 3; Table 2). Similarly, prior application of the adenosine A2 receptor antagonist DMPX attenuated bradykinin-induced responses in 12 of 14 afferent neurons (Table 3). Prior application of either CPDPX (Table 2) or DMPX (Table 3) attenuated substance P-induced excitatory neuronal responses in 88% (21/24) and 61% (14/23) of afferent neurons tested, respectively.

3.1.5. Myocardial ischemia
Transient occlusion (30–90 s) of a coronary artery altered the activity generated by 35 of 56 (63%) tested afferent neurons (Fig. 4; Table 1). Of these 35 ischemia-sensitive cardiac afferent neurons, 28 (80%) responded to transient occlusion of the RCA while 10 (29%) neurons responded to transient occlusion of the LAD. The activity generated by three (9%) cardiac afferent neurons was sensitive to occlusion of either coronary artery. Regional myocardial ischemia increased the spontaneous activity of 25 neurons (45% of the neurons so tested) by 112%; this intervention suppressed the activity generated by another ten ischemia sensitive afferent neurons (18% of neurons) by 75% (Table 1). No changes in monitored cardiovascular indices were induced during these short-term occlusions overall. Upon reperfusion, reflex-induced alterations in monitored cardiac indices occurred in a few instances (Fig. 4).


Figure 4
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  		Fig. 4 Myocardial ischemia-induced afferent neuronal responses. (A) Transient occlusion of the right main coronary artery (between arrows below) excited an afferent neuron. Left ventricular chamber systolic pressure (LVP) increased during the early reperfusion period. (B) When the specific adenosine A1-receptor antagonist CPDPX was applied to its sensory field (arrow below), spontaneous activity was reduced. Reocclusion of the same coronary artery (RCA Occ, second arrow below) in the presence of CPDPX failed to initiate an afferent neuronal response.

 
Of the 35 ischemia-sensitive afferent neurons, 29 (83%) were modified by CPA and/or CPCAA. Thirty-one (89%) of these ischemia-sensitive afferent neurons proved to be sensitive to the peptides (bradykinin; twenty-four neurons: substance P; twenty-five neurons). Ischemia induced excitatory responses induced in eighteen afferent neurons no longer were elicited when their associated sensory neurites were previously exposed to the adenosine A1 receptor antagonist (Fig. 4; Table 2). Prior application of DMPX obtunded ischemia induced excitatory responses in seventeen afferent neurons so tested (Table 3).

3.2. In vitro experiments
3.2.1. Anatomical studies
Fast blue-labeled neurons were observed in fixed nodose ganglia tissues that were derived from all five animals studied 19–28 days following injections of tracers into the ventral and lateral walls of the left ventricle. One to ten labeled cardiac afferent neurons were identified scattered throughout each 40-µm section that contained 100–200 neurons labeled with the general neuronal marker PGP 9.5 (Fig. 5), confirming that such neurons are not viscerotopically organized or clustered within a ganglionic region [17]. The number of labeled neurons identified in left and right nodose ganglia were similar, based on observations made in tissues derived from two animals. To determine whether labeling in nodose ganglia was specific to the heart or due to dye spreading to adjacent tissues (i.e. pulmonary, thoracic and mediastinal structures), the medulla oblongata was studied in each of these animals. Labeled parasympathetic preganglionic efferent neurons were confined to the ventrolateral nucleus ambiguus (not shown), anatomically established as the major location of porcine cardiac parasympathetic efferent preganglionic neurons [17].


Figure 5
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  		Fig. 5 Twenty-four days after injecting fast blue into the ventral wall of the left ventricle, a fast blue-labeled neuron (arrow in B) was identified among PGP positive (FITC-green) neurons (A) in a nodose ganglion section. (C) Two fast blue labeled nodose ganglion neurons identified in culture that were subsequently studied in vitro. Horizontal bars: B=25 µm; C=50 µm.

 
3.2.2. Functional studies
The response characteristics of the five labeled cardiac afferent neurons and the six unlabeled afferent neurons in vitro to the application of depolarizing currents were similar. The data derived from the in vitro experiments were grouped together because all neurons displayed similar transmembrane response characteristics. Secondly, some unlabeled neurons presumably were also associated with cardiac sensory neurites as rather limited ventricular regions were injected with neuroanatomic dyes in each animal. The resting membrane potential of afferent neurons was, on average, –45.0±2.2 mV (Table 4). When 0.5 nA intracellular current pulses were delivered to labeled or unlabeled neurons, multiple (11.5±1.4) repetitive action potentials superimposed upon sustained depolarization were generated throughout the period of current injection (Fig. 6C; Table 4). In control states, occasional spontaneous action potentials were generated after these current injections were terminated (Fig. 6C).


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  		Table 4 Effects of adenosine on the excitability of isolated nodose ganglion cells in vitro

 

Figure 6
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  		Fig. 6 Intracellular recordings representative of membrane and action potential data derived from labeled nodose ganglion cardiac afferent neurons in vitro. When an intracellular current pulse (0.5 nA; upper trace) was delivered to this neuron in control states, sustained, repetitive activity was generated throughout its application. In the absence of adenosine, spontaneous action potentials sometimes occurred after terminating the depolarizing pulse. Application of adenosine (10–5 M) to the perfusate reduced the frequency of the tonic discharge activity generated by the neuron when exposed to the same intracellular current pulse (upper trace). No spontaneous action potentials were elicited during afterhypolarizations in the presence of adenosine. The resting membrane potential of this afferent neuron was –45 mV before adenosine application (C) and –47 mV during its application (A). Similar results were obtained from the other four labeled neurons. C, control; A, adenosine.

 
When 10–5 M adenosine was added to the perfusate, the resting membrane potentials of all neurons became hyperpolarized (Table 4). In the presence of adenosine, intracellular current pulses resulted in the genesis of fewer (3.0±0.3) action potentials than in control states; these occurred early on during current application (Fig. 6A). The attenuation of the repetitive activity so initiated occurred whether cells were labeled (Fig. 6) or unlabeled. These depolarizing pulses were followed by afterhyperpolarizations that were significantly greater in the presence of adenosine than in its absence (Table 4). In the presence of adenosine, these afterhyperpolarizations were not attended by spontaneous discharges.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
These experiments elucidate several important electrophysiological and functional properties concerning sensory transduction by cardiac afferent neurons. (1) Epicardial sensory neurites associated with most nodose ganglion cardiac afferent neurons possess similar populations of adenosine A1 and A2 receptors that enhance or attenuate the activity that they generate. (2) That local application of adenosine A1 and A2 receptor antagonists attenuated or eliminated the activity generated by some afferent neurons during physiological states demonstrates the dependency of some cardiac afferent neurons on purinergic mechanisms in the absence of pathology. That somata of nodose ganglion cardiac afferent neurons possess adenosine A1 receptors [12] is in accord with such data. The fact that adenosine A1 and A2 receptor antagonists also attenuated the responsiveness of these cardiac afferent neurons to the peptides bradykinin and substance P indicates a degree of overlap in their receptor structure–ligand interactions. These receptors do not necessarily utilize the same signaling pathway, although some cross talk between them may occur [21]. (4) The capacity of cardiac afferent neurites associated with nodose ganglion afferent neurons to transduce myocardial ischemic events to central neurons depends to a large extent on their purinergic receptors since their sensitivity to transient myocardial ischemia was negligible in the presence of either adenosine receptor antagonist. (5) Adenosine reversibly reduces the excitability of nodose ganglion cardiac afferent neuronal somata studied in vitro. Thus, the somata and sensory terminals of individual nodose ganglion cardiac afferent neurons can display different response characteristics to this chemical. Further studies on specific receptor density and function are needed to elucidate this issue.

The role of adenosine in cardiac sensory transduction remains controversial, particularly during myocardial ischemia. The concentration of adenosine in myocardial interstitial fluid ranges from 10 to 15 µM in physiologic states, increasing many fold in the presence of myocardial ischemia [5,22,23]. Clinical evidence indicates that exogenous administration of adenosine causes angina-like pain in both normal individuals and patients with ischemic heart disease [11]. It has been reported that adenosine does [2,4,11] or does not [24] modify primary cardiac afferent neurons in situ when applied to their epicardial sensory neurites. This could be due to complex interactions among adenosine A1 (CPA) or A2 (CPCAA) receptor subtypes within the sensory neurites. In order to minimize such interactions, the specific response characteristics of adenosine A1 (CPA) or A2 (CPCAA) receptor agonists were examined individually in situ (Figs. 1 and 2Go).

Excitatory or inhibitory responses were elicited by individual afferent neurons when their sensory neurites were exposed to either agonist, depending on the neuron studied. As has been found in the past [11], ventricular sensory neurites associated with about 20% of identified nodose ganglion cardiac afferent neurons decrease their activity when exposed to purinergic agents or, for that matter, regional ischemia (Table 1). Presumably, the different response characteristics elicited by individual afferent neurons to one agent reflects the heterogeneity of sensory transduction displayed by their sensory neurites [2,11]. Depressor responses elicited by purinergic agonists or regional ventricular ischemia took time to develop and lasted for a relatively long time after termination of the intervention. Although the role of such depressor responses remain unknown, it can be speculated that ischemia induced depressor versus excitatory responses enhances the information content provided to central neurons as occurs by, for instance, via dorsal root ganglion ventricular mechanosensory neurons [4]. Due to the fact that excitatory responses were brisk in onset and elicited more frequently and thus more reliably studied, the effects of adenosine receptor antagonists on neurons that generated excitatory responses were analyzed statistically (Tables 2 and 3Go). In general, the final type of response (excitatory or inhibitory) was probably determined by the summation of inputs from the multiple sensory neurites associated with individual afferent neurons in a given locus at a given time [11].

Excitatory responses elicited by the adenosine A1 receptor agonist CPA were completely blocked by prior application of the specific adenosine A1 receptor antagonist CPDPX, but not by the specific adenosine A2 receptor antagonist DMPX. Likewise, excitatory responses elicited by the adenosine A2 receptor agonist CPCAA were eliminated by the presence of DMPX, but not CPDPX. These data indicate the presence of specific adenosine receptors associated with sensory terminals of nodose ganglion cardiac afferent neurons. That adenosine released from cardiac tissues in physiological states tonically influences populations of cardiac afferent neurons was indicated by the fact that in many instances ongoing activity was suppressed by local application of either adenosine receptor antagonist (Fig. 3).

Bradykinin and substance P are also liberated in greater than normal amounts during myocardial ischemia [7,8]. These peptides are known to activate epicardial sensory neurites associated with nodose ganglion afferent neurons [2,11]. Membrane peptide receptors for bradykinin and substance P activate phospholipase C (PLC) via a guanine nucleotide binding protein (Gq), resulting in the phosphorylation of ion channels and membrane receptors [21,25]. Adenosine, at physiological concentrations within the interstitium, interacts with neuronal A1 and A2 adenosine receptors. While the A1 receptor antiadrenergic response so elicited indicates coupling with the Gi protein signaling pathway, mechanisms involving A2 receptor signaling remain unresolved [27]. Current information indicates that A2 receptor agonists act on the heart via c-AMP dependent and independent mechanisms [27]. Afferent neuronal effects could have been mediated by Gs- and Gq-coupled signaling pathways. In light of possible fine-tuning of signals arising from multiple receptor signaling pathways via G protein-coupled receptor crosstalk [21], interactions of peptides such as bradykinin and substance P with A1 and A2 adenosine receptors at the level of the G-coupled pathway might be expected. For instance, increases in PLC might enhance intracellular levels of inositol trisphosphate (IP3) and diacylglycerol (DAG), perhaps thereby activating protein kinase C (PKC) and Ca2+ signaling [13]. It is hypothesized that inhibition of this pathway via adenosine A1 or A2 receptor blockade might be involved in attenuating sensory neurite peptidergic receptor activation via, for instance, IP3 or PKC formation. Although at the present time we do not understand the complexity of transduction displayed by cardiac sensory neurons, these data indicate that some degree of receptor structure–ligand overlap might occur at the level of the cardiac sensory neurite. That either adenosine receptor antagonist eliminated most of the ischemia-induced excitation of investigated afferent neurons (Tables 2 and 3Go) indicates that either receptor subtype plays an important role in transducing the ischemic signal.

Most cardiac sensory neurites associated with canine nodose ganglion afferent neurons are known to be sensitive to myocardial ischemia [2]. It is proposed that both adenosine A1 and A2 receptors on cardiac sensory neurites associated with nodose ganglion afferent neurons play a major role in the transduction of myocardial ischemia to central neurons. This is based on the evidence that either selective P1 purinergic receptor antagonist alone attenuated excitatory afferent neuronal responses elicited by transient coronary artery ligation (Fig. 4; Tables 2 and 3Go). These data support the hypothesis that both adenosine receptors are central in the capacity of sensory neurites to transduce signals arising from purines locally released during ischemic states. Chemicals that are liberated in increasing quantities during focal ventricular ischemia arise from many cell types, including cardiomyocytes [6,26]. Whatever their source, such chemicals appear to exert differential effects on sensory neurite transduction in situ.

Although transient occlusion of a major coronary artery induces ischemic changes primarily in the mid myocardium and endocardium it does affect the transduction properties of sensory neurites located in the epicardium [2,4], presumably reflecting to some degree epicardial ischemia or alterations in the local chemical milieu. It is known that substances like adenosine liberated within the myocardium quickly reach the epicardial surface [28]. As a matter of fact, the quantity of adenosine applied to epicardial sensory fields was similar to that in fluid recovered from the canine ventricular epicardium [28]. Epicardial sensory neurites were tested in order to ensure that locally applied chemicals reached them since greater doses of these agents are required it sensory neurites located deeper within the myocardium are to be consistently affected [2]. Presumably the superficial nature of investigated sensory neurites is the reason why application of adenosine receptor blocking agents adjacent to them modified their transduction capabilities, even in the presence of regional myocardial ischemia.

To compare the effects of adenosine applied to the cell bodies of nodose ganglion cardiac afferent neurons with those elicited when adenosine agonists were applied to their sensory terminals, we used anatomical tracing techniques whereby the fluorescent neuronal tracer fast blue was injected into the myocardium to retrogradely label cardiac afferent neurons (Fig. 5). The somata of anatomically identified neurons associated with cardiac sensory terminals were dispersed throughout nodose ganglia bilaterally, as reported previously [17]. That a topographical organization of cardiac afferent neurons was not found in nodose ganglia was confirmed by our physiological studies. That labeled neurons were cardiac in function was established anatomically by the presence of retrogradely labeled preganglionic parasympathetic efferent neurons confined to the external formation of the nucleus ambiguus. The presence of labeled neurons outside this medullary region would indicate that labeling was not specific to cardiac tissues [17].

As has been shown in rat nodose ganglion cardiac afferent neurons in vitro [12], adenosine exerted negligible effects on single action potentials generated by cultured cardiac afferent neurons following short-duration (1 ms) depolarizing pulses (not illustrated). On the other hand, adenosine modified nodose ganglion neurons when the latter were activated by long-duration depolarizing pulses in vitro (Fig. 6). In the absence of adenosine, cardiac afferent neurons produced bursts of action potentials superimposed upon sustained depolarization during long-duration depolarizing pulses (Table 4). These repetitive action potentials were followed by afterhyperpolarizations once the stimulus ceased. The repetitive discharges induced by labeled or unlabeled afferent neurons during prolonged depolarization were attenuated significantly by the presence of adenosine (Table 4). Presumably that was due to a decreased excitability secondary to the significant hyperpolarization of neurons induced by adenosine. Further support for such attenuation of excitability by adenosine was provided by the fact that no poststimulation spontaneous activity occurred in its presence (Fig. 6A).

That adenosine exerted some of its inhibitory effects via A1 adenosine receptors is in agreement with the inhibitory effects that adenosine exerts on rat brain neurons via their adenosine A1 receptors [23]. That adenosine so affects cardiac afferent neuronal somata in vitro is in agreement with the fact that adenosine inhibits neuronal firing within the peripheral and central nervous systems [for a review, see 14]. These in situ and in vitro data demonstrate that purinergic agents may elicit differential effects on cardiac sensory neurons, depending on whether applied to their somata or sensory neurites. The discrepancy of these in vitro and in vivo data could have been due to a number of factors. They may have been due to the relative densities of the A1 and A2 receptors on cardiac sensory neurites associated with nodose ganglion afferent neurons versus their somata, currently an unresolved issue. Furthermore, it is likely that this discrepancy was due, at least in part, to ongoing inputs from other signaling pathways that occur in vivo, but would not occur in vitro.

The limitations of this study include the specificity of the chemicals employed, as well as the necessity to apply multiple chemicals to the sensory neurites on individual cardiac afferent neurons. There is a finite number of interventions that each cardiac sensory neurons can be expected to respond to functionally. Thus, one limitation of this study is the relatively limited number of chemicals that could be applied to sensory neurites of individual afferent neurons. As the state of individual sensory neurons (relatively low or high spontaneous activity levels) presumably affects their transduction capabilities [11], this represents another uncontrolled variable.

Clinical and laboratory evidence indicates that adenosine and peptides, both released in increasing quantities by the ischemic myocardium, function in an autocrine and paracrine fashion to modulate cardiac afferent neuronal activity. The results of the present study demonstrate that purinergic agents exert differential effects on neuronal excitability, depending on whether they are applied to cardiac afferent somata in vitro or their associated sensory terminals in vivo. These results also suggest that various endogenous neuroactive agents released from myocardial tissues act synergistically to contribute to sensory transduction during normal and pathological states. The interactions among various chemical stimuli at the level of the sensory neurites associated with cardiac afferent neurons must be understood before targeted pharmacological strategies can be developed to treat myocardial ischemia from a neurocardiological perspective.

Time for primary review 25 days.


   Acknowledgements
 
The authors gratefully acknowledge the technical assistance of Richard Livingston. A Doctoral Research Award from the Medical Research Council of Canada (GWT), as well as grants from the Nova Scotia and New Brunswick Heart and Stroke Foundations and the Medical Research Council of Canada (JAA and MH) supported this work.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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