MECHANISMS UNDERLYING THE CHRONOTROPIC EFFECT OF ANGIOTENSIN-II ON CULTURED NEURONS FROM RAT HYPOTHALAMUS AND BRAIN-STEM

The chronotropic effect of angiotensin II (Ang II) was studied in cult ured neurons from rat hypothalamus and brain stem with the use of the patchclamp technique. Ang II (100 nM) increased the neuronal spontaneo us firing rate from 0.8 +/- 0.3 (SE) Hz in control to 1.3 +/- 0.4 Hz ( n = 7, P < 0.05). The amplitude of threshold stimulation was decreased by Ang IT (100 nM) from 82 +/- 4 pA to 62 +/- 5 pA (n = 4, P < 0.05). These actions of Ang II were reversed by the angiotensin type I (AT(1 )) receptor antagonist losartan (1 mu M). In the presence of tetrodoto xin, Ang II (100 nM) significantly increased the frequency and the amp litude of the Cd2+-sensitive subthreshold activity of the cultured neu rons. Ang II also stimulated the subthreshold early afterdepolarizatio ns (EADs) to become fully developed action potentials. Similar to the action of Ang II, the protein kinase C (PKC) activator phorbol 12-myri state 13-acetate (PMA, 100 nM) increased the firing rate from 0.76 +/- 0.3 Hz to 2.3 +/- 0.5 Hz (n = 6, P < 0.05) and increased the neuronal subthreshold activity. After neurons were intracellularly dialyzed wi th PKC inhibitory peptide (PKCIP, 5 mu M), PMA alone, Ang II alone, or PMA plus Ang II no longer increased the action potential firing initi ated from the resting membrane potential level. However, superfusion o f PMA plus Ang II or Ang II alone increased the number of EADs that re ached threshold and produced action potentials even in the presence of PKCIP (5 mu M, n = 4). The actions of Ang IT could also be mimicked b y depolarizing pulse and K+ channel blockers (tetraethylammonium chlor ide or 4-aminopyridine). These results indicate that Ang II by activat ion of AT, receptors increases neuronal excitability and firing freque ncy, and that this may involve both PKC dependent and -independent mec hanisms.