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Impaired Cyclic Nucleotide Signalling in Pre‐Hypertensive Spontaneously Hypertensive Rats: A Dichotomy of Pathways?
Author(s) -
Bardsley Emma,
Paterson David J
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1006.5
Subject(s) - ibmx , forskolin , medicine , endocrinology , cyclic adenosine monophosphate , chemistry , guanosine , cyclic nucleotide , protein kinase a , adenosine , förster resonance energy transfer , phosphodiesterase , nucleotide , receptor , kinase , biochemistry , enzyme , gene , physics , quantum mechanics , fluorescence
Background Spontaneously hypertensive rats (SHRs) and young pre‐hypertensive SHRs (pre‐SHR) have enhanced cardiac sympathetic activity; a feature underpinned by abnormal calcium‐dependent exocytosis. The crosstalk between cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in peripheral sympathetic stellate neurons is poorly understood. Aim We used Förster Resonance Energy Transfer (FRET) to test whether there is a difference in cAMP and protein kinase A (PKA) activity in post‐ganglionic sympathetic neurons (PGSNs) obtained from 4‐week pre‐SHR compared with normotensive controls. Furthermore, we sought to determine whether activation of the cGMP‐PKG signalling pathway modulates cytosolic cAMP concentrations and its effector PKA, in sympathetic stellate neurons obtained from Wistar and pre‐SHR. Method Cardiac stellate neurons were isolated from male Wistar rats and pre‐SHR at 4‐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 (0.1–25μM) and saturating concentrations of 3‐isobutyl‐1‐methylxanthine (IBMX; 100μM). In alternative experiments, neurons were perfused with 8‐bromoguanosine (8‐br‐cGMP; 100μM) prior to saturation with forskolin (25μM) and IBMX (100μM). All raw percentage FRET changes were subsequently normalised to the maximal FRET change at saturation. Results There was no significant difference between cAMP or PKA activity in response to forskolin administration in 4‐week stellates obtained from Wistar and pre‐SHR; however, cAMP and PKA responses to 8‐br‐cGMP were significantly different in PGSNs from the two strains. In neurons obtained from pre‐SHRs there was no change in PKA in response to 8‐br‐cGMP. Conversely, in Wistar stellate neurons, administration of the cGMP analogue facilitated a rapid increase in PKA activity (66%). Here we show that the responses to 8‐br‐cGMP results in a dichotomy in cyclic nucleotide signalling pathways in PGSNs from young Wistar versus pre‐SHR neurons. These findings suggest that the interaction between the two cyclic nucleotide signalling pathways may differ in PGSNs from the two phenotypes, prior to the onset of hypertension; either as a result of differential phosphodiesterase (PDE) profiles, or physiological uncoupling in response to diverse signalling microdomains. Support or Funding Information This work was supported by the Wellcome Trust Ion Channels and Disease (OXION) Initiative