P5.25

Abstract

Background: Spontaneously hypertensive rats (SHRs) and young pre-hypertensive SHRs have enhanced cardiac sympathetic activity; a feature underpinned by impaired cyclic nucleotide signalling linked to abnormal calcium-dependent exocytosis. The crosstalk between cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in peripheral stellate neurons is poorly understood. We used Förster Resonance Energy Transfer (FRET) to test whether cAMP and protein kinase A (PKA) activity is greater in sympathetic neurons from SHR and pre-SHR compared with normotensive controls. Method: Cardiac stellate neurons were isolated from male wistar rats and SHR at 4-weeks (pre-hypertensive) or 16-weeks. On the day of culture stellates were infected with either cAMP or PKA reporter adenovirus particles for 24 hours, and FRET experiments were carried out 3-4 days post-isolation. Data were obtained from neurons expressing FRET biosensors using dual-emission imaging, where YFP and CFP emission intensities were measured. During imaging, cells were perfused continuously with Tyrode’s, or stimulated with forskolin (0.1-25 μM) and saturating concentrations of 3-isobutyl-1-methylxanthine (IBMX; 100 μM). Results: There was a significant and concentration-dependent increase in cAMP and PKA activity following forskolin administration in the absence and presence of IBMX, which was significantly higher in 16-week SHR vs. normotensive controls (PKA EC50: 1.2x10−7 M and 5.1x10−7 M; SHR, wistar respectively). Furthermore, cAMP and PKA hyperactivity was demonstrated in pre-hypertensive SHRs compared with young wistar rats (PKA EC50: 2.8x10−7 M and 5.6x10−7 M; SHR, wistar respectively). Summary: These results show that cAMP and PKA hyperactivity occurs early in the development of the cardiac sympathetic phenotype in this genetic based model of hypertension.