Expression of functional Angiotensin II (Ang II) receptors types, AT1R and AT2R, in RVLM neuronal cultures from adult rat brain

Abstract

Our previous studies have demonstrated that AT1R signaling pathway regulates RVLM neuronal activity. Their increased expression and coupling to PI3K has been linked to increased Ang II‐mediated neuronal activity in the SHR‐RVLM. These findings, obtained from mixed cultures of neonatal rat hypothalamus, may not reflect those of the RVLM in adults. Thus, our objectives in this study were to establish neuronal cultures from the RVLM of adult rats, and use them to study the effects of Ang II. GFP‐AAV viral vector was injected into the RVLM of SD rats two weeks prior to isolation of neurons for anatomical confirmation. Cells were dissociated, plated onto culture dishes and allowed to establish for 7–14 days. Presence of neuron specific markers, neuN and synaptophysin in GFP expressing cells confirmed the presence of RVLM neurons. Certain fluorescent neurons expressed AT1R. Ang II effect was tested on Ca2+ currents (IBa) recorded from GFP expressing neurons. Ang II caused a 30% increase in IBa that was attenuated by 2 μM losartan. However, pretreatment with AT2R antagonist, PD123319, resulted in 2.5 fold augmentation of Ang II‐mediated increase in IBa. Together, these observations demonstrate that both AT1R and AT2R are present on the same neuron and that inhibition of the AT2R accentuates the AT1R‐mediated stimulation of IBa. They suggest a functional coupling of AT1R and AT2R in the RVLM. (Supported by NIH HL 33610 and HLT6312)