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Research ArticleNeuropharmacology

Tonically Active cAMP-Dependent Signaling in the Ventrolateral Medulla Regulates Sympathetic and Cardiac Vagal Outflows

Vikram J. Tallapragada, Cara M. Hildreth, Peter G.R. Burke, Darryl A. Raley, Sarah F. Hassan, Simon McMullan and Ann K. Goodchild
Journal of Pharmacology and Experimental Therapeutics February 2016, 356 (2) 424-433; DOI: https://doi.org/10.1124/jpet.115.227488

Abstract

The ventrolateral medulla contains presympathetic and vagal preganglionic neurons that control vasomotor and cardiac vagal tone, respectively. G protein-coupled receptors influence the activity of these neurons. Gαs activates adenylyl cyclases, which drive cyclic adenosine monophosphate (cAMP)–dependent targets: protein kinase A (PKA), the exchange protein activated by cAMP (EPAC), and hyperpolarization-activated cyclic nucleotide–gated (HCN) channels. The aim was to determine the cardiovascular effects of activating and inhibiting these targets at presympathetic and cardiac vagal preganglionic neurons. Urethane-anesthetized rats were instrumented to measure splanchnic sympathetic nerve activity (sSNA), arterial pressure (AP), heart rate (HR), as well as baroreceptor and somatosympathetic reflex function, or were spinally transected and instrumented to measure HR, AP, and cardiac baroreflex function. All drugs were injected bilaterally. In the rostral ventrolateral medulla (RVLM), Sp-cAMPs and 8-Br-cAMP, which activate PKA, as well as 8-pCPT, which activates EPAC, increased sSNA, AP, and HR. Sp-cAMPs also facilitated the reflexes tested. Sp-cAMPs also increased cardiac vagal drive and facilitated cardiac baroreflex sensitivity. Blockade of PKA, using Rp-cAMPs or H-89 in the RVLM, increased sSNA, AP, and HR and increased HR when cardiac vagal preganglionic neurons were targeted. Brefeldin A, which inhibits EPAC, and ZD7288, which inhibits HCN channels, each alone had no effect. Cumulative, sequential blockade of all three inhibitors resulted in sympathoinhibition. The major findings indicate that Gαs-linked receptors in the ventral medulla can be recruited to drive both sympathetic and parasympathetic outflows and that tonically active PKA-dependent signaling contributes to the maintenance of both sympathetic vasomotor and cardiac vagal tone.

Introduction

Key centers for the autonomic control of vasomotor tone and heart rate are located in the ventrolateral medulla oblongata. Presympathetic neurons of the rostral ventrolateral medulla (RVLM) regulate the activity of sympathetic preganglionic neurons of the spinal cord, predominantly those controlling vasomotor tone (Dampney, 1994; Pilowsky and Goodchild, 2002; Guyenet, 2006). Cardiac vagal preganglionic neurons are localized primarily in the nucleus ambiguus and innervate cardiac ganglia to control heart rate (Wang et al., 2001). The tonic activity of both of these neuronal populations in the ventrolateral medulla, as now accepted, is dependent on synaptic drive resulting from the sum of excitatory and inhibitory input (Wang et al., 2001; Lipski et al., 2002; Guyenet, 2006). Blockade of ionotropic glutamate receptors in both regions, however, fails to decrease sympathetic vasomotor tone or increase heart rate (HR), respectively, despite the fact that blockade of GABA-A receptors in these regions has clear directionally opposite responses (Dampney et al., 2003; Hildreth and Goodchild, 2010). Inputs arising from multiple brain sites are encoded by a plethora of not only ionotropic but also G protein-coupled receptors (GPCR) present in the region (Lovick, 1985; Dampney, 1994; Bowman et al., 2013). Those GPCRs that can be recruited to drive or tonically modulate these two neuronal populations have not been clearly identified.

The multitude of GPCRs are linked to heterotrimeric G proteins, whose α subunits signal via three major intracellular proteins: adenylyl cyclase, phospholipase C-β, and Rho (Brown and Sihra, 2008). Despite this convergence, the expression and functions of G protein-related signaling molecules in controlling cardiovascular autonomic functions mediated by the ventrolateral medulla are poorly understood. We have previously demonstrated that mRNA for all Gα proteins are expressed in the ventrolateral medulla, with Gαs most abundant (Parker et al., 2012). Gαs mRNA is present in all adrenergic C1 neurons, an important cardiovascular subpopulation within the region. Gαs proteins couple to adenylyl cyclases that catalyze the conversion of ATP to cyclic adenosine monophosphate (cAMP). cAMP in turn can activate three downstream targets: cAMP-dependent protein kinase A (PKA), exchange proteins activated by cAMP (EPAC), and hyperpolarization-activated cyclic nucleotide–gated (HCN) channels (Beavo and Brunton, 2002; Bos, 2003; Holz et al., 2006). Cardiovascular autonomic functions regulated by cAMP in ventrolateral medulla are the focus of this study.

The objective is to determine whether Gαs-linked receptors can drive and/or tonically modulate outputs from the ventral medulla. Specifically the aims are to determine: the effects of 1) activating or 2) inhibiting cAMP-dependent effectors on splanchnic sympathetic outflow, blood pressure, heart rate, and baroreceptor and somatosympathetic reflex functions mediated by RVLM presympathetic and cardiac vagal pathways originating in the ventrolateral medulla.

Materials and Methods

All experiments were approved by the Macquarie University Animal Ethics Committee (Protocol Number 2009-019) and conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

Surgical Preparation

A total of 47 male Sprague-Dawley rats (350–450 g) were used. Rats were anesthetized with urethane (1.2–1.3 g/kg, i.p.) and depth of anesthesia was assessed every 30–40 minutes by monitoring withdrawal, respiratory, or blood pressure responses to firm pinch of the hind paw. Additional doses of urethane (20–30 mg, i.v.) were given as required. Core temperature was maintained between 36.5°C and 37.0°C with a feedback-controlled heating blanket (Harvard Apparatus, Holliston, MA).

Both femoral veins and the right femoral artery were cannulated for the administration of drugs and fluids and for the measurement of arterial blood pressure, respectively. Heart rate was derived from the R-wave of the electrocardiogram (ECG) obtained from leads attached to both forepaws and one hind limb. A tracheotomy was performed to permit artificial ventilation. Rats were secured in a stereotaxic frame.

Procedures Specific for Assessment of RVLM Vasomotor Function.

Rats were vagotomized. The left greater splanchnic nerve was dissected via a retroperitoneal approach and cut at the distal end to permit recording of efferent nerve activity. The sciatic nerve was isolated, cut at the distal end, and stimulated to drive the somatosympathetic reflex response. Rats were paralyzed with pancuronium bromide (0.4 mg given as a 0.2-ml bolus, i.v., then an infusion of 20% pancuronium in 0.5% glucose in saline at 1.5 ml/h) and artificially ventilated with oxygen-enriched room air. End-tidal CO2 was monitored and blood gases measured regularly; ventilation was adjusted to maintain PaCO2 and pH within a physiologic range (PaCO2 40 ± 3 mmHg; pH 7.35–7.45). The dorsal medullary surface was exposed by occipital craniotomy and nerves were mounted on bipolar silver wire electrodes and covered in paraffin oil.

The RVLM was mapped on both sides by pneumatic microinjection of glutamate, as previously described (Burke et al., 2008). Pressor sites were located 1.8–2.2 mm rostral and 1.6–2.0 mm lateral to the calamus scriptorius and between 3.3–3.8 mm ventral to the brainstem surface. A site was considered to be within the RVLM if a 50-nl microinjection of 100 mM glutamate caused a rise in blood pressure ≥35 mmHg.

Procedures Specific for Assessment of Cardiac Vagal Function.

Rats were spinally transected between cervical segments 7 and 8 and cardioinhibitory regions of both sides of the brain were identified by glutamate microinjection (50 nl, 100 mM), as described previously (Hildreth and Goodchild, 2010). Sites in and around the nucleus ambiguus that produced bradycardic responses greater than 50 bpm were selected for injection of drugs.

Experimental Protocols

In the RVLM, the cumulative dose response evoked by drugs was performed in initial studies to determine effective doses to be used. Bilateral injections of 50 nl per side of each drug were made with increasing doses. Only one drug and vehicle was used in each animal.

Each drug was then assessed using the same protocol. Following a control period of recording, bilateral microinjections of 50 nl per side of the test drug (or vehicle) were made into the selected sites. Supramaximal somatosympathetic (2–15 V sciatic nerve stimulation, 50 × 0.1-millisecond pulses at 1 Hz) and baroreceptor reflexes [sequential injection of sodium nitroprusside (SNP; 10 μg in 0.4 ml saline) and phenylephrine (PE; 10 μg in 0.4 ml saline) via two different femoral venous cannulae, as previously described (Burke et al., 2008)] were activated before and every 5–10 minutes after drug injection for up to 1 hour. Only one drug was tested in each animal with the exception of one study in which the three inhibitors were sequentially injected. Parameters measured were sSNA, arterial pressure (AP), HR, and sympathetic baroreflex and somatosympathetic reflex functions. Cardiac baroreflex function was not measured in these animals.

For experiments investigating cardiac vagal pathways, two 100-nl injections of the vehicle followed by the test drug were made on each side, approximately 600 μm apart, to effectively target cardiac vagal preganglionic neurons [as described previously (Hildreth and Goodchild, 2010)]. Injection of PE (10 μg/kg) permitted calculation of heart rate baroreflex sensitivity (BRS) before and after vehicle and drug injections; effects on HR were also monitored.

At the conclusion of recordings, injection sites were marked with 50–100 nl of ink/dye and the animal was euthanized (0.8 ml of 3 M KCl, i.v.). Brainstems were removed, drop-fixed in 4% formaldehyde overnight, and cryopreserved until histologic processing. Coronal sections (100 μm) were cut on a vibratome and injection sites verified.

Data Acquisition and Analysis

Neurograms were amplified (gain: 10,000; CWE Incorporated, Ardmore, PA), bandpass filtered (0.1–2 kHz), sampled at 3 kHz (1401 Power mkII; CED, Cambridge, UK), and rectified and smoothed with a 2-second time constant (Spike 2; CED). For RVLM microinjections, bilateral injections of phosphate-buffered saline (PBS) preceded all drug microinjections. Peak changes in mean arterial pressure (MAP), heart rate (HR), and splanchnic sympathetic nerve activity (sSNA) were measured with respect to control data measured over 120 seconds 5 minutes prior to drug/PBS injection. For time-course analysis 120-second blocks of data were averaged every 10 minutes. sSNA activity was normalized with respect to background noise postmortem (0%) and baseline activity prior to vehicle injection (100%). sSNA response to sciatic nerve stimulation was analyzed using peristimulus waveform averaging (McMullan et al., 2008); baroreceptor-function curves were generated as previously described (Burke et al., 2008). Analyses for baroreceptor- and somatosympathetic-reflex function were conducted 5–20 minutes post–target drug injection. For CVPN microinjection, peak changes in HR and BRS were calculated as described previously (Hildreth and Goodchild, 2010).

Analysis was conducted using GraphPad Prism (v 5.0). All values are expressed as mean plus or minus standard error. One-way analysis of variance (ANOVA) or paired or unpaired Student’s t test was used to analyze drug effects on baseline and reflex parameters. P < 0.05 was considered significant.

Drugs

The following drugs were used in this study; l-glutamate disodium salt, Sp-cAMPs (Sp-diastereomer of adenosine 3′, 5′-cyclic monophosphorothioate), 8-Br-cAMP (8-bromoadenosine 3′,5′-cyclic monophosphate, Rp-cAMPs (Rp-diastereomer of adenosine 3′, 5′-cyclic monophosphorothioate), H-89 (N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide dihydrochloride), 8-pCPT (8-pCPT-2′-O-Me-cAMP), BFA (brefeldin A), ZD-7288, PE, and SNP and all were obtained from Sigma-Aldrich (St. Louis, MO). Pancuronium bromide was obtained from AstraZeneca Australia (North Ryde, NSW, Australia). Drugs were dissolved in PBS (10 mM, pH 7.4) with the exception of BFA, which was first solubilized in ethanol before dilution in PBS. Urethane, PE, and SNP were prepared in 0.9% NaCl.

Results

Activating cAMP-Dependent Pathways in the RVLM

Cell-permeable drugs that activate downstream effectors PKA, EPAC, and HCN channels were microinjected into the RVLM to determine the effects of cAMP stimulation on cardiovascular tone and reflex function.

cAMP Analogs in the RVLM: Effects on Baseline Parameters.

Bilateral cumulative microinjection of two cAMP analogs, Sp-cAMPs (0.5, 1.5, and 5 nmol, n = 4) and 8-Br-cAMP (1 and 10 nmol, n = 4) increased sSNA (Sp-cAMPs: F(3, 11) = 5.9, p = 0.011; Br-cAMP: F(2, 6) = 60.8, p = 0.0001) and MAP (Sp-cAMPs: F(3, 11) = 8.4, p = 0.0035; Br-cAMP: F(2, 6) = 20.7, p = 0.002) in a dose-dependent manner (Fig. 1). Effects were rapid and prolonged. Five-nanomolar Sp-cAMP was selected for detailed investigation.

Fig. 1.
Fig. 1.

The change in sympathetic nerve activity (SNA) and mean arterial pressure (MAP) evoked by increasing doses of analogs of cAMP microinjected bilaterally and cumulatively in the RVLM. (A) Change in sSNA elicited by vehicle (PBS), Sp-cAMPs (0.5, 1.5, 5 nmol, n = 4), and 8-Br-cAMP (1 and 10 nmol) (n = 3). (B) Change in MAP evoked by the same agents (n = 4 for both). Data are are reported as mean ± S.E.M.; *P < 0.05, **P < 0.01, ****P < 0.0001.

Microinjection of Sp-cAMPs (5 nmol, n = 6) evoked increases in sSNA, HR, and MAP that were maximal at 10–20 minutes, whereas vehicle had little effect (Fig. 2, A–D). sSNA and HR remained elevated for the remainder of the experiment (>1 hour), whereas MAP recovered within 40 minutes (Fig. 2, A–D). Sp-cAMPs (5 nmol, n = 6) evoked significant peak increases in sSNA, MAP, and HR compared with control (PBS, n = 6) (P < 0.01 for all parameters) (Fig. 2E).

Fig. 2.
Fig. 2.

Time-course and peak responses in sSNA, MAP, and HR evoked by bilateral microinjection of the cAMP analog Sp-cAMPs (5 nmol/side) or vehicle (PBS) in the RVLM. (A) Representative example of the responses evoked by Sp-cAMPs. Stimulation of the sciatic nerve to evoke the somatosympathetic reflex (SSR) and injection of vasoactive agents phenylephrine (PE) and sodium nitroprusside (SNP) are indicated. These stimuli are applied multiple times but are indicated only once for clarity. Sp-cAMPs elicited an increase in all parameters measured. (B–D) Time course of effects on sSNA, MAP, and HR, respectively. The effects of PE and SNP on heart rate may be attributable to Starling’s law following rapid intravenous injection in a vagotomized rat, or the SNP-induced bradycardia may be caused by pressure-induced reduction in coronary perfusion pressure. HR baroreflex changes were not analyzed in this part of the study (however, see Fig 8). (E) Peak response in sSNA, MAP, and HR evoked by Sp-cAMPs compared with vehicle (PBS) (n = 6). Data are are reported as mean ± S.E.M. **P < 0.01, ****P < 0.0001.

Sp-cAMP in the RVLM: Effect on Sympathetic Reflexes.

The effect of Sp-cAMPs in the RVLM was tested on reflexes that are dependent on the GABAergic [baroreflex (Schreihofer and Guyenet, 2002) or glutamatergic (somatosympathetic reflex) (Kiely and Gordon, 1993)] synapses within the RVLM.

Microinjection of Sp-cAMPs significantly increased the upper plateau and maximum gain of the sympathetic baroreflex function curve compared with control (PBS) (Fig. 3A and Table 1). The data were acquired from experiments represented in Fig. 2. Importantly, cardiac baroreflex changes are not reported following drug injection into the RVLM (however, see Fig. 8).

Fig. 3.
Fig. 3.

Effects of Sp-cAMPs (A,B), 8-pCPT (C,D), or RP-cAMPs (E,F) injected bilaterally into the RVLM on baroreflex (A,C,E) or somatosympathetic reflex function (B,D,F). Effects are shown before (black) and 15–20 minutes after (gray) drug injection. For the somatosympathetic reflex prior to drug injection (before) two characteristic peaks in sSNA were evoked (black). The averaged responses + S.E.M. are shown. The increase in sSNA evoked by all drugs is evident.

View this table:
TABLE 1 

Baroreflex control of sSNA after drug microinjection into the RVLM

Intermittent stimulation of the sciatic nerve resulted in a characteristic two-phase excitatory response in sSNA. The total AUC (22.8 ± 4.9 versus 13.6 ± 3.1 au, Sp-cAMPs versus PBS, P = 0.0083) but not the amplitude of the two peaks [89.6 ± 21.2% (peak 1) and 87.7 ± 29.8% (peak 2) versus 120 ± 19.6% and 103.6 ± 28.8% sSNA; Sp-cAMPs versus PBS n.s.] was significantly affected by Sp-cAMPs compared with PBS (Fig. 3B). The effect of Sp-cAMPs was to increase the width of the second peak, particularly at more delayed latencies.

8-pCPT in RVLM: Effects on Baseline Parameters.

Bilateral microinjection into the RVLM of 8-pCPT (5 nmol in 50 nl, n = 6), a cAMP analog that selectively activates EPAC (Vliem et al., 2008) increased sSNA, AP, and HR (Fig. 4A). The grouped time-course data are shown in Fig. 4, B–D, and the peak responses compared with bilateral PBS injection are shown in Fig. 4E. 8-pCPT significantly increased sSNA (p < 0.01). Increases in MAP and HR were not statistically significantly different.

Fig. 4.
Fig. 4.

Time-course and peak responses in sSNA, MAP, and HR evoked by bilateral microinjection of a cAMP analog that selectively activates EPAC, 8-pCPT (5 nmol/side), in the RVLM. (A) Representative example of the responses evoked. Stimulation of the sciatic nerve to evoke the somatosympathetic reflex (SSR) and injection of vasoactive agents phenylephrine (PE) and sodium nitroprusside (SNP) are indicated. 8-pCPT elicited an increase in all parameters measured. (B–D) Time course of effects on sSNA, MAP, and HR, respectively. See legend for Fig. 1 for additional details. (E) Peak response evoked in each parameter measured compared with the effect evoked by vehicle (PBS; n = 6). Data are reported as mean ± S.E.M. **P < 0.01.

8-pCPT in RVLM: Effects on Sympathetic Reflexes.

8-pCPT significantly increased the upper plateau and the maximum gain of the sympathetic baroreflex (Fig. 3C and Table 1). In contrast, 8-pCPT evoked no significant effect on somatosympathetic reflex parameters (Fig. 3D): total AUC (8.7 ± 2.6 versus 6.3 ± 1.4 au, 8-pCPT versus PBS n.s.) and peak heights (66.9± 21.9% (peak 1) and 39.23 ± 23.9 (peak 2) versus 64.1 ± 9.7% and 44.3 ± 18.1 sSNA; 8-pCPT versus PBS, not significant).

Blocking cAMP-Dependent Pathways in the RVLM

To determine whether the downstream effectors of cAMP, PKA, EPAC, and HCN channels are tonically activated in the RVLM, their effects were individually blocked using cell-permeable, selective pharmacological agents.

Inhibition of PKA in RVLM: Effects on Baseline Parameters.

Bilateral microinjection of the PKA inhibitor Rp-cAMPs (5 nmol in 100 nl, n = 5) into the RVLM evoked increases in sSNA, biphasic changes in AP, and small increases in HR (Fig. 5A). The grouped time-course data are shown in Fig. 5, B–D and peak changes shown in Fig. 5E. Rp-cAMPs compared with PBS evoked significant increases in sSNA (P < 0.001), MAP (P < 0.01), and HR (P < 0.05).

Fig. 5.
Fig. 5.

Time-course and peak responses in sSNA, MAP, and HR evoked by bilateral microinjection of the PKA inhibitor Rp-cAMPs (5 nmol/side) in the RVLM. (A) Representative example of the responses evoked. Stimulation of the sciatic nerve to evoke the somatosympathetic reflex (SSR) and injection of vasoactive agents phenylephrine (PE) and sodium nitroprusside (SNP) are indicated. Rp-cAMPs elicited an increase in all parameters measured. (B–D) Time course of effects on sSNA, MAP, and HR, respectively. See legend for Fig. 1 for additional details. (E) Peak response evoked in each parameter measured compared with the effect evoked by vehicle (PBS; n = 6). Data are reported as mean ± S.E.M. *P < 0.05 **P < 0.01, ***P < 0.001.

Similar effects were observed following bilateral microinjection of another inhibitor of PKA, H-89 (1 and 10 nmol in 50 nl, n = 3), although these recordings lasted only 30 minutes. There was a significant effect of H-89 on sSNA (F(2, 9) = 4.5, P = 0.04) and MAP (F(2, 9) = 16.1, P = 0.001) following one-way ANOVA. H-89 evoked peak increases in sSNA of 17 ± 5% and 35 ± 10% and MAP of 15 ± 7 mmHg and 32 ± 5 mmHg (1 and 10 nmol, respectively).

Inhibition of PKA in RVLM: Effects on Sympathetic Reflexes.

Rp-cAMPs evoked no significant effects on baroreceptor (Fig. 3E and Table 1) or somatosympathetic (Fig. 3F) reflex parameters. Total AUC (14.1 ± 3.4 versus 9.1 ± 1.2 au, Rp-cAMPs versus PBS, not significant), and peak heights [87.3 ± 15.4% (peak 1) and 95.4 ± 30.2% (peak 2) versus 100.0 ± 46.4% and 57 ± 22.2% sSNA; Rp-cAMPs versus PBS, not significant] were not altered.

Inhibition of EPAC with BFA in the RVLM Blocks the Sympathoexcitation Evoked by 8-pCPT But Alone Has No Effect.

Bilateral microinjection of BFA (100 pmol in 100 nl, n = 4), an inhibitor of EPAC, into the RVLM had no significant effect on sSNA, MAP, or HR (Fig. 6) or on somatosympathetic or the baroreceptor reflex function (data not shown). However, when injections of 8-pCPT (5 nmol in 50 nl) were preceded 5 minutes earlier by bilateral injection of BFA (n = 3), no effect was seen over 60 minutes (Fig. 6), indicating that BFA blocked the effects of 8-pCPT alone (data taken from Fig. 4).

Fig. 6.
Fig. 6.

Time-course effects on (A) sSNA, (B) MAP, and (C) HR of 8-pCPT (data taken from Fig. 4, black square), an inhibitor of EPAC, BFA (open circle, n = 4) and when 8-pCPT microinjection was preceded by BFA (open triangle, n = 3). BFA blocked the effect of 8-pCPT. Data are are reported as mean ± S.E.M.

Blocking HCN Channels in RVLM with ZD-2788 Evokes No Effects.

Microinjection of ZD-7288, a specific antagonist of HCN channels (300 pmol in 100 nl, n = 3), bilaterally into the RVLM did not alter sSNA, MAP, or HR (data not shown) as reported previously (Miyawaki et al., 2003).

Combined cAMP Effector Blockade in the RVLM Evokes Sympathoinhibition.

To determine in the RVLM the combined effect of blocking three downstream effector proteins (PKA, EPAC, and HCN channels), each was inhibited in succession (n = 5, Fig. 7). Blockade of PKA was followed by inhibition of EPAC and then blockade of HCN channels at 10-minute intervals. Figure 7A shows a representative example of sequential blockade of cAMP effectors and the grouped time-course effects are shown in Fig. 7, B–D. The summed effect of blockade evoked a fall in all parameters with a peak decrease in sSNA of –30.8 ± 7.6% (P < 0.05) but nonsignificant decreases in MAP (–15 ± 6 mmHg, P = 0.09) and HR (–20 ± 7 bpm, P = 0.08).

Fig. 7.
Fig. 7.

Time-course responses in sSNA, MAP, and HR evoked by bilateral microinjection in the RVLM of Rp-cAMPs, followed by BFA, followed by ZD7288 (300 pmol/side) at 10-minute intervals. (A) Representative example of the responses evoked. (B–D) Time course of effects on sSNA, MAP, and HR, respectively. Data are are reported as mean ± S.E.M.

Effects of Activating or Inhibiting cAMP-Dependent Pathways at Cardiac Vagal Preganglionic Neurons

To determine whether cAMP-dependent effects could be evoked in other functional pathways originating in the ventral medulla, responses from cardiac vagal preganglionic neurons were evaluated.

Sp-cAMPs at Cardiac Vagal Preganglionic Neurons Decreases HR and Facilitates the Cardiac Baroreflex.

Figure 8A shows the time course and effects evoked by bilateral microinjection of Sp-cAMPs (2 injections per side of 10 nmol in 100 nl, n = 5) at cardiac vagal preganglionic neurons. A decrease in HR was evoked and the hemodynamic and cardiac effects of modifying baroreceptor reflex function (PE) was seen. Sp-cAMPs decreased resting heart rate (307 ± 10 bpm before versus 273 ± 6 after, P < 0.01) (Fig. 8C) and increased BRS (0.46 ± 0.09 bpm/mmHg before versus 0.71 ± 0.12 after, P < 0.01) (Fig. 8C). Injection of vehicle at these sites evoked no significant effect on heart rate or BRS.

Fig. 8.
Fig. 8.

Effects on HR and cardiac baroreflex sensitivity of dual bilateral microinjections of Sp-cAMPs (each injection, 10 nmol) and Rp-cAMPs (each injection, 10 nmol) in the ventral medulla targeting cardiac vagal preganglionic neurons in spinally transected animals. (A) Representative example of the effects of Sp-cAMPs on HR and also the effect of the vasoactive agent phenylephrine used to determine BRS. (B) Representative example of the effects of Rp-cAMPs on HR. (C) Grouped data of the effects of SP-cAMPs on resting HR and BRS and (D) grouped data of the effects of Rp-cAMPs on resting HR and BRS. Data are reported as mean ± S.E.M. *P < 0.05 **P < 0.01.

Rp-cAMPs at Cardiac Vagal Preganglionic Neurons Decreases HR But Has No Effect of the Cardiac Baroreflex.

Figure 8B shows the time course and effects evoked by subsequent bilateral microinjection of Rp-cAMPs (2 injections per side of 10 nmol in 100 nl, n = 4) at cardiac vagal preganglionic neurons. A decrease in HR was evoked and the hemodynamic and cardiac effects of modifying cardiac baroreceptor reflex function are seen. Rp-cAMPs injection decreased resting heart rate 299 ± 8 bpm before versus 240 ± 5 after, P < 0.05) (Fig. 8D) but did not significantly alter BRS (0.65 ± 0.12 bpm/mmHg before versus 0.66 ± 0.13 after, P = 0.98) (Fig. 8D).

Discussion

This is the first study to comprehensively investigate the role that cAMP-dependent signaling pathways play in regulating neural activities in the ventrolateral medulla. The major findings are that: 1) Activation of PKA in the RVLM is sympathoexcitatory and enhances the sympathetic baroreflex and somatosympathetic reflex and at cardiac vagal preganglionic neurons evokes bradycardia and augments the cardiac baroreflexes; 2) activation of EPAC in the RVLM using 8-pCPT also evokes sympathoexcitation, which is blocked by BFA; 3) blockade of PKA within the RVLM and at cardiac vagal preganglionic neurons is also sympathoexcitatory and cardioinhibitory, respectively, but did not alter sympathetic reflex function; 4) blockade of EPAC or HCN channels in the RVLM has no effect; however, 5) in the RVLM the summed effect of sequential and cumulative blockade of PKA, EPAC, and HCN channels is sympathoinhibitory.

Our results indicate that cAMP-dependent pathways, which are probably naturally stimulated by GPCRs linked via Gαs proteins, can be recruited to activate both sympathetic and parasympathetic outflows in the ventrolateral medulla and contribute to basal levels of sympathetic vasomotor and cardiac vagal tone. Blocking PKA alone has a net excitatory effect both in the RVLM and at cardiac vagal preganglionic neurons. One simple explanation may be that inhibitory inputs to both groups of neurons are tonically driven by cAMP-dependent signaling, and blockade of PKA causes disinhibition. As sympathetic baroreflex function is unaffected by PKA blockade, such active inhibitory inputs to the RVLM are unlikely to be of baroreceptor origin. However, as sympathoinhibition follows blockade of all cAMP-dependent signaling, this suggests that excitatory as well as inhibitory substrates within the RVLM may be tonically influenced by cAMP-dependent signaling.

Methodological Considerations.

All drugs used in this study to alter cAMP-dependent signaling were cell-permeable and largely resistant to phosphodiesterases (Schaap et al., 1993; Dostmann, 1995). Both Sp-cAMPs and 8-Br-cAMP are analogs of cAMP and effectively activate all downstream effectors. Both are potent activators of PKA and EPAC (Christensen et al., 2003). Both activators evoked significant dose-related sympathoexcitation and pressor- or vagally mediated bradycardic effects in the ventral medulla. The doses of Sp-cAMPs used were similar to those used in other brain regions (Paine et al., 2009). 8-pCPT selectively activates EPAC without effect on PKA (Christensen et al., 2003; Brown et al., 2014), although it may have some nonspecific/EPAC-independent effects, at least as identified in platelets (Herfindal et al., 2013). Nevertheless, the effects of 8-pCPT were similar to those evoked by Sp-cAMPs and were blocked by prior treatment with BFA, which alone had no effect as described elsewhere (Zhong and Zucker, 2005). ZD7288 is a commonly used selective blocker of HCN channels (Harris and Constanti, 1995), although some effect on sodium channels has been suggested (Wu et al., 2012).

Rp-cAMPs, which inhibits PKA, has little effect on EPAC (Christensen et al., 2003; Brown et al., 2014) or on H-89, which also inhibits PKA-evoked similar dose-dependent effects, although H-89 actions could also occur via other kinases (Lochner and Moolman, 2006). The pressor effect evoked by H-89 in the RVLM confirm what has been previously noted (Xu and Krukoff, 2006). ZD 7288, which blocks HCN channels but is ineffective alone in the RVLM, as described previously (Miyawaki et al., 2003), contributed to inhibitory effects when preceded by other drugs. Nevertheless, as in most pharmacological studies of this type, it is possible that the effects evoked by the drugs used may not be attributable to the substrates targeted.

Heart rate, sympathetic, and blood pressure responses evoked by drug injection in vagotomized spinal-cord intact animals are interpreted as sympathetically mediated, albeit modified by competing baroreflex pathways. The splanchnic nerve innervates functionally diverse targets, including gut vasculature, gastrointestinal muscles, and adrenal gland, and cannot therefore be interpreted as a purely vasomotor output. Conversely, data from spinally transected animals are interpreted as consequences of direct drug effects on cardiac vagal motor circuits, as described previously (Hildreth and Goodchild, 2010), as all sympathetic outputs were disrupted also, thus providing conditions of maximal baroreflex unloading.

Drug interaction with medullary interneurons presynaptic to sympathetic/parasympathetic outputs are probable. We have previously shown select effects on respiratory function within subregions of the ventrolateral medulla (Burke et al., 2013), and it is possible that changes in respiratory-sympathetic coupling contribute to the effects seen here.

Sites of cAMP Activation in the Ventral Medulla.

Activation of cAMP-dependent pathways in the RVLM evoked sympathoexcitation and a pressor effect and bradycardia at cardiac vagal preganglionic neurons. This is in keeping with our finding that the Gαs subunit mRNA is abundant in the RVLM (Parker et al., 2012) and consistent with a postsynaptic site of action, as suggested previously in neonatal RVLM brain slice preparations, in which 8-Br-cAMP and the adenylyl cyclase activator forskolin increased the firing rate of RVLM “pacemaker” neurons in the presence of tetrodotoxin (Sun and Guyenet, 1990). Activation of Gαs-linked receptors in the RVLM using pituitary adenylate cyclase–activating peptide evokes sympathoexcitation and pressor responses, although reflex functions were unaffected (Farnham et al., 2012). On the other hand, cardiac vagal nerve activity is increased by systemic adenosine (da Silva et al., 2012) or by activation of β-adrenergic receptors, specifically β1, which reduces GABAergic and glycinergic (as well as glutamatergic) conductances at cardiac vagal preganglionic neurons (Bateman et al., 2012). Recently, β1 and β2 receptors have been identified on putative presympathetic RVLM neurons, and their selective activation evoked depolarization and hyperpolarization, respectively (Oshima et al., 2014). These data suggest that cAMP-dependent signaling can be elicited by catecholamine release in the ventrolateral medulla.

Injections of PKA and EPAC activators enhanced both sympathetic and cardiac baroreflex functions. This could be explained by the activation of cAMP in presympathetic neurons and/or in inhibitory inputs and in cardiac vagal preganglionic neurons and/or in excitatory inputs, respectively. The effect, at least of PKA, on the somatosympathetic reflex [mediated by glutamatergic synapses in the RVLM (Kiely and Gordon, 1993)] could indicate modulation of glutamatergic inputs or postsynaptic effects, particularly as facilitation appeared more prominent at slowly conducting possibly catecholaminergic cells in the region.

Tonically Active PKA-Dependent Signaling in the Ventral Medulla.

Blockade of PKA, using both Rp-cAMPs and H-89, evoked sympathoexcitation and vagally mediated bradycardia, indicating tonically active PKA-dependent signaling in the ventrolateral medulla. Although a pressor effect initially accompanied the sympathoexcitation elicited by both agents, at later time points a depressor response, which was not accompanied by splanchnic sympathoinhibition, was evoked by Rp-cAMPs. This biphasic effect may indicate that splanchnic sympathetic drive is counteracted by other effectors, such as inhibition of excitatory input supplying other sympathetic vasomotor outflows. Nevertheless the effects on MAP suggest that vasomotor pathways are affected as well as both sympathetic and parasympathetic pathways controlling heart rate. As the tonic activity of RVLM neurons supplying vasomotor tone is dependent on the balance of tonic excitatory and inhibitory input, we speculate that the early net excitatory action of Rp-cAMPs could be mediated by effects at inhibitory inputs; however, as the sympathetic baroreflex (mediated by inhibitory presynaptic input) was unaffected, actions at other functional inhibitory inputs would be indicated. GABA-A receptor blockade at both the RVLM and cardiac vagal preganglionic neurons indicate significant levels of tonic inhibitory input to neurons controlling vasomotor (Schreihofer and Guyenet, 2002) and cardiac (Hildreth and Goodchild, 2010) functions. Furthermore, there is some precedent for PKA-dependent disinhibition, as modulation of glycinergic release occurs in spinal cord (Katsurabayashi et al., 2004). It is possible that blocking PKA may redistribute the active pool of cAMP to other effectors; however, at least in the RVLM, blocking either EPAC or HCN channels alone had little effect. There is little evidence supporting the idea of tonically active peptides in the RVLM (Burke et al., 2008; Pilowsky et al., 2008; Farnham et al., 2012). Nevertheless the findings here suggest that a neurotransmitter acting via Gαs-linked receptor/s is active in the ventrolateral medulla. One possibility is a catecholamine acting at β receptors where, at least in the neonatal RVLM, β2-receptor blockade depolarized neurons (Oshima et al., 2014). However, an alternative explanation could be that such a receptor is constitutively active (Milligan, 2003; Costa and Cotecchia, 2005) and candidates that are Gαs-linked in the RVLM include the H2 and melanocortin 3/4 receptors (Granata and Reis, 1987; Kawabe et al., 2006). When activated in the RVLM, only the histamine 2 receptor causes sympathoinhibition, probably via excitation of an inhibitory input (Granata and Reis, 1987).

Although blockade of PKA in RVLM evoked sympathoexcitation, blockade of other cAMP effectors each had no effect. Nevertheless combined blockade resulted in sympathoinhibition, suggesting actions at both inhibitory and excitatory synaptic sites. It should be noted that the cAMP effectors are restricted to spatially separated microdomains within cell bodies and terminals in the ventral medulla (Karpen and Rich, 2004; Calebiro and Maiellaro, 2014), so sequential blockade may have disturbed the balance within intracellular compartments.

Conclusions

Our data show that cAMP-dependent pathways, signaling via PKA and EPAC, can be recruited in the ventrolateral medulla to evoke excitation in sympathetic circuitry controlling the heart, vasculature, and baroreflex, as well as excitation of the cardiac vagus and circuitry controlling the cardiac baroreflex. Importantly, the results indicate that PKA-dependent pathways are tonically active in a region controlling the basal level of sympathetic and cardiac vagal tones. These effects are in contrast to the effects of blocking excitatory ionotropic receptors in the RVLM or at cardiac vagal preganglionic neurons, which do not alter the level of sympathetic activity or HR, respectively (Dampney et al., 2003; Hildreth and Goodchild, 2010). Thus GPCRs utilizing Gαs proteins in the ventrolateral medulla contribute to setting the level of sympathetic tone including sympathetic vasomotor as well as cardiac vagal tone.

Authorship Contributions

Participated in research design: Goodchild, Hildreth, Tallapragada.

Conducted experiments: Tallapragada, Hildreth, Raley, Burke.

Performed data analysis: Tallapragada, Hildreth, Burke, Hassan.

Wrote or contributed to the writing of the manuscript: Goodchild, Tallapragada, Hildreth, Hassan, Burke, McMullan.

Footnotes

    • Received July 20, 2015.
    • Accepted November 13, 2015.

  • This work was supported by the National Health and Medical Research Council [APP1028183, APP1030301], the Australian Research Council [DP120100920], and the Hillcrest Foundation [FR2013/1308, FR2014/0781]. Dr. Darryl Raley died before the completion of this study.

  • dx.doi.org/10.1124/jpet.115.227488.

Abbreviations

8-Br-cAMP
8-bromoadenosine 3′,5′-cyclic monophosphate
8-pCPT
8-pCPT-2′-O-Me-cAMP
AP
arterial pressure
BFA
brefeldin A
BRS
baroreflex sensitivity
cAMP
cyclic adenosine monophosphate
EPAC
exchange protein activated by cAMP
GPCR
G protein-coupled receptors
H-89
N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide dihydrochloride
HCN
hyperpolarization-activated cyclic nucleotide–gated
HR
heart rate
MAP
mean arterial pressure
PBS
phosphate-buffered saline
PE
phenylephrine
PKA
protein kinase A
Rp-cAMPs
Rp-diastereomer of adenosine 3′, 5′-cyclic monophosphorothioate
RVLM
rostral ventrolateral medulla
SNP
sodium nitroprusside
Sp-cAMP
Sp-diastereomer of adenosine 3′, 5′-cyclic monophosphorothioate
sSNA
splanchnic sympathetic nerve activity
ZD-7288
4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride

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Research ArticleNeuropharmacology

cAMP Signaling in the Ventrolateral Medulla

Vikram J. Tallapragada, Cara M. Hildreth, Peter G.R. Burke, Darryl A. Raley, Sarah F. Hassan, Simon McMullan and Ann K. Goodchild
Journal of Pharmacology and Experimental Therapeutics February 1, 2016, 356 (2) 424-433; DOI: https://doi.org/10.1124/jpet.115.227488
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