Efficacy of B-Type Natriuretic Peptide Is Coupled to Phosphodiesterase 2A in Cardiac Sympathetic Neurons

Abstract

Elevated B-type natriuretic peptide (BNP) regulates cGMP-phosphodiesterase activity. Its elevation is regarded as an early compensatory response to cardiac failure where it can facilitate sympathovagal balance and cardiorenal homeostasis. However, recent reports suggest a paradoxical proadrenergic action of BNP. Because phosphodiesterase activity is altered in cardiovascular disease, we tested the hypothesis that BNP might lose its efficacy by minimizing the action of cGMP on downstream pathways coupled to neurotransmission. BNP decreased norepinephrine release from atrial preparations in response to field stimulation and also significantly reduced the heart rate responses to sympathetic nerve stimulation in vitro. Using electrophysiological recording and fluorescence imaging, BNP also reduced the depolarization evoked calcium current and intracellular calcium transient in isolated cardiac sympathetic neurons. Pharmacological manipulations suggested that the reduction in the calcium transient was regulated by a cGMP/protein kinase G pathway. Fluorescence resonance energy transfer measurements for cAMP, and an immunoassay for cGMP, showed that BNP increased cGMP, but not cAMP. In addition, overexpression of phosphodiesterase 2A after adenoviral gene transfer markedly decreased BNP stimulation of cGMP and abrogated the BNP responses to the calcium current, intracellular calcium transient, and neurotransmitter release. These effects were reversed on inhibition of phosphodiesterase 2A. Moreover, phosphodiesterase 2A activity was significantly elevated in stellate neurons from the prohypertensive rat compared with the normotensive control. Our data suggest that abnormally high levels of phosphodiesterase 2A may provide a brake against the inhibitory action of BNP on sympathetic transmission.

Keywords: calcium; natriuretic peptide, brain; sympathetic nervous system; synaptic transmission.

Figure 1

A, Representative raw data traces (i) and grouped data (ii) showing the heart rate responses to sympathetic nerve stimulation (SNS) at 1, 3, 5 and 7 Hz for 30 s with 250 nmol/L B-type natriuretic peptide (BNP) compared with control. *P<0.05, **P<0.01, paired t test. B, Grouped data showing the time control (i) and with addition of 250 nmol/L BNP (ii) on [³H]-norepinephrine release from isolated atria. The atria were stimulated at 5 Hz for 1 minute at the 16th (S1) and 40th (S2) minutes. (iii) Group mean data show BNP caused a significant decrease in 5-Hz stimulation evoked [³H]-norepinephrine release (n=6 *P<0.05, 1-way ANOVA). NE indicates norepinephrine.

Figure 2

A, Bright field and immunofluorescence staining image of a cultured cardiac sympathetic neuron derived from a stellate ganglion which was stained with the catecholamine neuronal marker antityrosine hydroxylase (TH, red) and costained with the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar represents 20 μm. B, (i) Representative whole cell calcium current density traces obtained with or without 100 nmol/L B-type natriuretic peptide (BNP, 10 minutes) and after wash out. Currents were evoked by test pulses to −10 mV from a holding potential of −90 mV. (ii) Mean current density–voltage relations in the presence and absence of 100 nmol/L BNP. Wash off data were only recorded at −10mV as the quality of the recordings deteriorates over time. *P<0.05, paired t test, n=6 neurons. I_Ca indicates calcium current; pA, picoampere; and pF, picofarad. C, (i) An example recording calcium transient in a single cardiac sympathetic neuron. Neuron was exposed to KCl for 30 s to depolarize the neuron with (S2) or without (S1) BNP. (ii) Statistical data showing that concentration–effect relationship of BNP (1–250 nmol/L) changed KCl evoked increase in [Ca²⁺]_i expressed as a ratio (%) of S2 compared with S1. **P<0.01, compared with control, unpaired t test. Numbers shown in the bars indicate the number of the neurons. (iii) BNP (100 and 250 nmol/L) did not affect basal [Ca²⁺]_i in the cardiac sympathetic neurons (n=4).

Figure 3

NP-cGMP pathway inhibitors action on the intracellular calcium transient. A, Depolarization of the neuron with 50 mmol/L KCl for 30 s at 1 minute (S1, without drug) and 17 minutes (S2, with drug and 100 nmol/L BNP). B, Data showing the effect on the [Ca²⁺]_i of the natriuretic peptide receptor A (NPR-A) antagonist Isatin (10 μmol/L), the protein kinase G (PKG) inhibitor RP-8-Br-PET-cGMP (5 μmol/L), and the protein kinase A inhibitor (PKI; 1 μmol/L) with or without BNP; (C) nonspecific phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 100 and 500 μmol/L) with or without BNP; and (D) Phosphodiesterase 3 (PDE3) inhibitor Milrinone (10 μmol/L) or the PDE2 inhibitor Bay60-7550 (1 μmol/L) with or without BNP. *P<0.05, **P<0.01, ***P<0.001, compared with control, unpaired t test. †P<0.05, ††P<0.01, 1-way ANOVA. Numbers shown in the bars indicated the number of neurons. BNP indicates B-type natriuretic peptide.

Figure 4

cAMP measurements in isolated cardiac sympathetic neurons. A, Dynamic changes on emission intensity in the cyan fluorescent protein (CFP) channel (cyan) and yellow fluorescent protein (YFP) channel (yellow). Black arrows indicate depolarization of the neuron with 50 mmol/L KCl for 1 minute at 10 minutes (S1) and 30 minutes (S2). B-type natriuretic peptide (BNP) was introduced at 20 minutes. B, Representative kinetics of cAMP-induced fluorescence resonance energy transfer (FRET) changes by ratiometric recording of CFP and YFP emission in response to depolarization. C, Mean absolute values of FRET change during depolarization in the presence or absence of BNP. D, BNP was slightly reduced basal cAMP concentration in cardiac sympathetic neurons (n=4). E, Phosphodiesterase 2 inhibitor Bay60-7550 (1 μmol/L) and 100 nmol/L BNP did not significantly affect basal FRET (n=4). Nonspecific inhibition of phosphodiesterases with 3-isobutyl-1-methylxanthine (IBMX, 100 μmol/L) enhanced the intracellular cAMP levels.

Figure 5

A, Phosphodiesterase 2 (PDE2) activity in stellate ganglia tissue from prehypertensive spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats (i), normal Sprague Dawley rats transduced with empty and Ad.PDE2A (ii). PDE2 activity was measured by colorimetric assay and defined as the production of 5′-GMP inhibited by 1 μmol/L Bay60-7550. B, Western blot showing PDE2A.mCherry expression (127 KDa) in transduced empty and PDE2A stellate ganglia tissue (with anti-PDE2A antibody). Band optical density was normalized to that of β-actin (42 KDa). C, Effect of 250 nmol/L B-type natriuretic peptide (BNP) on cGMP concentration in transduced empty and PDE2A stellate ganglia tissue. *P<0.05, **P<0.01, unpaired t test.

Figure 6

A, Representative calcium current traces (left) and mean current density–voltage relations (right) obtained before and after exposure to 100 nmol/L BNP (i and ii) or B-type natriuretic peptide (BNP) with 1 μmol/L Bay60-7550 (iii) in the transduced empty (i) and Ad.PDE2A (ii and iii) cardiac sympathetic neurons. *P<0.05, paired t test. I_Ca indicates calcium current; pA, picoampere; and pF, picofarad. B, Images of single stellate neurons transfected with empty/Ad.PDE2A after 12 hours for patch clamping. The yellow arrows point to the patch pipette attached to the transduced mCherry (red) neuron. Scale bar represents 20 μm. C, Ratio data traces (i) and statistical data (ii) showing 50 mmol/L KCl evoked intracellular calcium transient ([Ca²⁺]_i) in the transduced empty and Ad.PDE2A cardiac sympathetic neurons. (iii) Group data showing KCl evoked peak [Ca²⁺]_i changes in response to 100 nmol/L BNP and the phosphodiesterase 2 (PDE2) inhibitor Bay60-7550 (1 μmol/L). *P<0.05, unpaired t test; †P<0.05, ††P<0.01, unpaired t test, compared with control within same group.

Figure 7

A, Representative raw data traces (i and ii) and group data (iii) showing [³H]-norepinephrine release during 5-Hz field stimulation from percutaneous gene transfer to right atria with empty virus (i) or Ad.PDE2A (ii). Norepinephrine release was significantly enhanced in the Ad.PDE2A transduced group when compared with the empty control (S1 vs S1; iii). B-type natriuretic peptide (BNP, 250 nmol/L) significantly reduced norepinephrine release (S1 vs S2) in empty (i), but not in transduced Ad.PDE2A group (ii). *P<0.05, unpaired t test. Numbers indicate the number of neurons. B, Representative Western blot showing PDE2A.mCherry expression (127KDa) in percutaneous gene transfer to right atria with Ad.PDE2A or empty virus (with antimCherry antibody). Band optical density was normalized to that of β-actin (42KDa) as a loading control. *P<0.05, unpaired t test. Numbers indicate the number of atria. PDE2A indicates phosphodiesterase 2A; and NE, norepinephrine.

Figure 8

Diagram illustrating the potential signal transduction pathways mediated by B-type natriuretic peptide (BNP) in depolarized stellate neurons. BNP binds to the natriuretic peptide receptor A (NPR-A) and stimulates particulate guanylyl cyclase (pGC). Newly synthesized cGMP activates phosphodiesterase 2 (PDE2) to degrade cGMP, but not cAMP, and thus limits the increase in protein kinase G (PKG) activity. Moreover, PKG inhibits calcium current by phosphorylating voltage-gated calcium channels (VGCCs) and reduces the intracellular calcium transient. This in turn decreases norepinephrine release from synaptic vesicles and reduces the heart rate response to sympathetic stimulation. In addition, elevated intracellular calcium triggers the activation of adenylate cyclase (AC) via calmodulin (CaM) and produces cAMP. cAMP activates cAMP-dependent protein kinase (PKA), thus phosphorylation of calcium channel.