Angiotensin AT₁-Receptors Depolarize Neonatal Spinal Motoneurons and Other Ventral Horn Neurons Via Two Different Conductances

Abstract

Angiotensin receptors are highly expressed in neonatal spinal cord. To identify their influence on neuronal excitability, we used patch-clamp recordings in spinal cord slices to assess responses of neonatal rat (5-12 days) ventral horn neurons to bath-applied angiotensin II (ANG II; 1 microM). In 14/34 identified motoneurons tested under current clamp, ANG II induced a slowly rising and prolonged membrane depolarization, blockable with Losartan (n = 5) and (Sar(1), Val(5), Ala(8))-ANG II (Saralasin, n = 4) but not PD123319 (1 microM each; n = 4). Under voltage clamp (V(H) -65 mV), 7/22 motoneurons displayed an ANG-II-induced tetrodotoxin-resistant inward current (-128 +/- 31 pA) with a similar time course, an associated reduction in membrane conductance and net current reversal at -98.8 +/- 3.9 mV. Losartan-sensitive ANG II responses were also evoked in 27/78 tested ventral horn "interneurons." By contrast with motoneurons, their ANG-II-induced inward current was smaller (-39.9 +/- 5.2 pA) and analysis of their I-V plots revealed three patterns. In eight cells, membrane conductance decreased with net inward current reversing at -103.8 +/- 4.1 mV. In seven cells, membrane conductance increased with net current reversing at -37.9 +/- 3.6 mV. In 12 cells, I-V lines remained parallel with no reversal within the current range tested. Intracellular dialysis with GTP-gamma-S significantly prolonged the ANG II effect in seven responsive interneurons and GDP-beta-S significantly reduced the ANG II response in four other cells. Peak inward currents were significantly reduced in all 13 responding neurons recorded in slices incubated in pertussis toxin (5 microgram/ml) for 12-18 h or in 12 neurons perfused with N-ethylmaleimide. Of 29 interneurons sensitive to pertussis toxin or N-ethylmaleimide treatment, 9 cells displayed a decrease in membrane conductance that reversed at -101.3 +/- 3.8 mV. In eight cells, membrane conductance increased and reversed at -38.7 +/- 3.4 mV. In 12 cells, the I-V lines remained parallel with no reversal within the current range tested, suggesting that both conductances are modulated by pertussis toxin-sensitive G proteins. These observations reveal a direct, G-protein-mediated depolarizing action of ANG II on neonatal rat ventral horn neurons. They also imply involvement of two distinct conductances that are differentially distributed among different cell types.

Presynaptic Angiotensin II AT1 Receptors Enhance Inhibitory and Excitatory Synaptic Neurotransmission to Motoneurons and Other Ventral Horn Neurons in Neonatal Rat Spinal Cord

“…Also noted was an associated increase in baseline thickness (see Fig. 1 in Oz and Renaud 2002). The present analysis examined the origin of this latter feature.…”

“…We recently reported that exogenous application of ANG II acts at postsynaptic AT 1 type receptors to engage two separate conductances that result in a prolonged membrane depolarization and inward current in a subpopulation of ventral horn motoand interneurons (Oz and Renaud 2002). Also noted was an associated increase in baseline thickness (see Fig.…”

“…The octapeptide angiotensin (ANG II) and other angiotensin fragments are considered to function in neurotransmitter or neuromodulatory roles mediated by AT 1, AT 2 , and AT 4 receptor subtypes (for reviews, see McKinley et al 2003;Phillips and Sumners 1998). A role for angiotensin receptors, in particular the AT 1 subtype, in regulating neuronal excitability can be inferred from electrophysiological and imaging observations of neuronal responsivity to exogenously applied angiotensin peptides in a variety of neurons (e.g., Bai and Renaud 1998;Barnes et al 2003;Ferguson and Washburn 1998;Gebke et al 1998;Li et al 2003;Oz and Renaud 2002;Shapiro et al 1994;Yang et al 1992). Binding studies and receptor-expression analyses also indicate a form of developmental plasticity within the central renin-angiotensin system with a high level of expression during the first and second postnatal week, changing during ontogeny (Milan et al 1991;Tsutsumi and Saavedra 1991).…”

Angiotensin II Attenuates Synaptic GABA Release and Excites Paraventricular-Rostral Ventrolateral Medulla Output Neurons

“…A general G protein inhibitor, 1 mM guanosine 5Ј-O-(2-thiodiphosphate) (GDP-␤-S) was added into the recording pipette solution to block the possible postsynaptic response mediated by G proteins coupled to angiotensin AT 1 receptors (Li et al, 2003). The effective concentration of GDP-␤-S to block the postsynaptic effect of Ang II has been shown in previous studies (Ohya and Sperelakis, 1991;Oz and Renaud, 2002). To study the evoked IPSCs and EPSCs (eIPSCs/ eEPSCs) in labeled PVN neurons, synaptic currents were evoked by electrical stimulation (0.1 ms, 0.3-0.8 mA, and 0.1 Hz) through a bipolar tungsten electrode connected to a stimulator (Grass Instruments, West Warwick, RI).…”

ASIC3 and ASIC1 Mediate FMRFamide-Related Peptide Enhancement of H+-Gated Currents in Cultured Dorsal Root Ganglion Neurons

“…This observation raised the question of whether FMRFamide and related peptides might act on DRG H ϩ -gated currents through a second messenger effect. To test this possibility, we included GDP-␤-S, a competitive G protein inhibitor, in the internal pipette solution at a 1-2 mM concentration (Oz and Renaud 2002); then, after obtaining the whole cell configuration and allowing 5 min for the GDP-␤-S to diffuse into the cell, we tested the effects of FRRFamide and RFRP-2 on H ϩ -gated currents. GDP-␤-S did not alter the current response to either peptide (Fig.…”