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Angiotensin II (Ang II), acting at Ang II type 1 receptors (AT1Rs), increases the firing rate of neurons from Wistar-Kyoto (WKY) rat brain via protein kinase C (PKC)- and calcium-calmodulin kinase II (CaMKII)-dependent mechanisms. The objectives of this study were twofold; first, to compare the Ang-II-stimulated increase in firing of neurons from WKY and spontaneous hypertensive rats (SHR) and second, to elucidate the signaling mechanisms involved. Action potentials were measured in neurons cultured from SHR and WKY rat brains using the whole cell configuration of the patch-clamp technique in the current-clamp mode. Ang II (100 nM) caused three- and sixfold increases in neuronal firing rate in WKY rat and SHR neurons, respectively; effects that were abolished by the AT1R antagonist Losartan (1 μM). Co-administration of calphostin C (10 μM, a PKC inhibitor) and KN-93 (10 μM, a CaMKII inhibitor) completely blocked this Ang II action in WKY rat neurons, while they caused only a ∼50% attenuation in SHR neurons. The residual increase in firing rate produced by Ang II in SHR neurons was blocked by inhibitors of phosphatidylinositol 3 kinase (PI3-kinase), either LY 294002 (10 μM) or wortmannin (100 nM). These observations suggest that a PI3-kinase signaling pathway may be responsible for the enhanced chronotropic effect produced by Ang II in SHR neurons.

Embryonic or neonatal rat neurons retain plasticity and are readily grown in tissue culture, but neurons of the adult brain were thought to be terminally differentiated and therefore difficult to culture. Recent studies, however, suggest that it may be possible to culture differentiated neurons from the hippocampus of adult rats. We modified these procedures to grow differentiated neurons from adult rat hypothalamus and brain stem. At day 7 in tissue culture and beyond, the predominant cell types in hypothalamic and brain stem cultures had a stellate morphology and could be subdivided into two distinct groups, one of which stained with antibodies to the immature neuron marker α-internexin, while the other stained with the astrocyte marker GFAP. The α-internexin positive cells were mitotic and grew to form a characteristic two-dimensional cellular network. These α-internexin positive cells coimmunostained for the neuronal markers MAP2, type III β-tubulin, and tau, and also bound tetanus toxin, but were negative for the oligodendrocyte marker GalC and also for the neurofilament triplet proteins NF-L, NF-M, and NF-H, markers of more mature neurons. Patch-clamp analysis of these α-internexin positive cells revealed small Ca2+ currents with a peak current of −0.5 ± 0.1 pA/pF at a membrane potential of −20 mV ( n = 5) and half-maximal activation at −30 mV ( n = 5). Na+ currents with a peak current density of −154.5 ± 49.8 pA/pF at a membrane potential of −15 mV ( n = 5) were also present. We also show that these cells can be frozen and regrown in tissue culture and that they can be efficiently infected by viral vectors. These cells therefore have the immunological and electrophysiological properties of immature mitotic neurons and should be useful in a variety of future studies of neuronal differentiation and function.

Previously, we determined that angiotensin II (Ang II) elicits an Ang II type 2 (AT2) receptor–mediated increase of neuronal delayed rectifier K+( I KV) current in neuronal cultures from newborn rat hypothalamus and brain stem. This requires generation of lipoxygenase (LO) metabolites of arachidonic acid (AA) and activation of serine/threonine phosphatase type 2A (PP-2A). Enhancement of I KV results in a decrease in net inward current during the action potential (AP) upstroke as well as shortening of the refractory period, which may lead to alterations in neuronal firing rate. Thus, in the present study, we used whole-cell current clamp recording methods to investigate the AT2 receptor–mediated effects of Ang II on the firing rate of cultured neurons from the hypothalamus and brain stem. At room temperature, these neurons exhibited spontaneous APs with an amplitude of 77.72 ± 2.7 mV ( n = 20) and they fired at a frequency of 0.8 ± 0.1 Hz ( n = 11). Most cells had a prolonged early after-depolarization that followed an initial fully developed AP. Superfusion of Ang II (100 nM) plus losartan (LOS, 1 μM) to block Ang II type 1 receptors elicited a significant chronotropic effect that was reversed by the AT2 receptor inhibitor PD 123,319 (1 μM). LOS alone had no effect on any of the parameters measured. The chronotropic effect of Ang II was reversed by the general LO inhibitor 5,8,11,14-eicosatetraynoic acid (10 μM) or by the selective PP-2A inhibitor okadaic acid (1 nM) and was mimicked by the 12-LO metabolite of AA 12-(S)-hydroxy-(5Z, 8Z, 10E, 14Z)-eicosatetraynoic acid. These data indicate that Ang II elicits an AT2 receptor–mediated increase in neuronal firing rate, an effect that involves generation of LO metabolites of AA and activation of PP-2A.

Angiotensin II (Ang II) elicits an Ang II type 2 (AT2) receptor–mediated increase in voltage-dependent delayed rectifier K+current ( IKV) in neurons cultured from newborn rat hypothalamus and brain stem. In previous studies, we have determined that this effect of Ang II is mediated via a Gi protein, activation of phospholipase A2(PLA2), and generation of arachidonic acid (AA). AA is rapidly metabolized within cells via lipoxygenases (LO), cyclooxygenase (COX) or p450 monooxygenase enzymes, and the metabolic products are known regulators of K+currents and channels. Thus in the present study, we have investigated whether the AT2receptor–mediated effects of Ang II on neuronal IKVrequire AA metabolism and if so, which metabolic pathways are involved. The data presented here indicate that the stimulatory actions of Ang II and AA on neuronal IKVare attenuated by selective blockade of 12-LO enzymes. However, the effects of Ang II are not altered by blockade of 5-LO or p450 monooxygenase enzymes. Furthermore, the actions of Ang II are mimicked by a 12-LO metabolite of AA, but 5-LO metabolites such as leukotriene B4and C4do not alter neuronal IKV. These data indicate that the AT2receptor–mediated stimulation of neuronal IKVis partially mediated through 12-LO metabolites of AA.

Angiotensin II (Ang II) acts at specific receptors located on neurons in the hypothalamus and brain stem to elicit alterations in blood pressure, fluid intake, and hormone secretion. These actions of Ang II are mediated via Ang II type 1 (AT1) receptors and involve modulation of membrane ionic currents and neuronal activity. In previous studies we utilized neurons cultured from the hypothalamus and brain stem of newborn rats to investigate the AT1receptor–mediated effects of Ang II on neuronal K+currents. Our data indicate that Ang II decreases neuronal delayed rectifier (Kv) current, and that this effect is partially due to activation of protein kinase C (PKC), specifically PKCα. However, the data also indicated that another Ca2+-dependent mechanism was also involved in addition to PKC. Because Ca2+/calmodulin-dependent protein kinase II (CaM KII) is a known modulator of K+currents in neurons, we investigated the role of this enzyme in the AT1receptor–mediated reduction of neuronal Kv current by Ang II. The reduction of neuronal Kv current by Ang II was attenuated by selective inhibition of either calmodulin or CaM KII and was mimicked by intracellular application of activated (autothiophosphorylated) CaM KIIα. Concurrent inhibition of CaM KII and PKC completely abolished the reduction of neuronal Kv by Ang II. Consistent with these findings is the demonstration that Ang II increases CaM KII activity in neuronal cultures, as evidenced by increased levels of autophosphorylated CaM KIIα subunit. Last, single-cell reverse transcriptase (RT)-PCR analysis revealed the presence of AT1receptor-, CaM KIIα-, and PKCα subunit mRNAs in neurons that responded to Ang II with a decrease in Kv current. The present data indicate that the AT1receptor–mediated reduction of neuronal Kv current by Ang II involves a Ca2+/calmodulin/CaM KII pathway, in addition to the previously documented involvement of PKC.

Wang, Desuo, Craig H. Gelband, Colin Sumners, and Philip Posner. Mechanisms underlying the chronotropic effect of angiotensin II on cultured neurons from rat hypothalamus and brain stem. J. Neurophysiol. 78: 1013–1020, 1997. The chronotropic effect of angiotensin II (Ang II) was studied in cultured neurons from rat hypothalamus and brain stem with the use of the patch-clamp technique. Ang II (100 nM) increased the neuronal spontaneous 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 II (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 1 (AT1) receptor antagonist losartan (1 μM). In the presence of tetrodotoxin, Ang II (100 nM) significantly increased the frequency and the amplitude of the Cd2+-sensitive subthreshold activity of the cultured neurons. Ang II also stimulated the subthreshold early afterdepolarizations (EADs) to become fully developed action potentials. Similar to the action of Ang II, the protein kinase C (PKC) activator phorbol 12-myristate 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 with PKC inhibitory peptide (PKCIP, 5 μM), PMA alone, Ang II alone, or PMA plus Ang II no longer increased the action potential firing initiated from the resting membrane potential level. However, superfusion of PMA plus Ang II or Ang II alone increased the number of EADs that reached threshold and produced action potentials even in the presence of PKCIP (5 μM, n = 4). The actions of Ang II could also be mimicked by depolarizing pulse and K+ channel blockers (tetraethylammonium chloride or 4-aminopyridine). These results indicate that Ang II by activation of AT1 receptors increases neuronal excitability and firing frequency, and that this may involve both PKC dependent and-independent mechanisms.

1. Inward rectification of a calcium-activated K+ channel in neurons cultured from the hypothalamus and brain stem of 1-day-old rats was studied by using patch-clamp techniques. A big conductance calcium-activated K+ channel with a slow gating rate was observed in inside-out patches. With symmetrical K+ across patches, inward conductance of this calcium-activated K+ channel was 216 +/- 14 (SE) pS (n = 4 patches), which changed little as different [Ca2+] was included in the bath solution. Outward conductance of this calcium-activated K+ channel was regulated by [Ca2+] in the bath solution and was 74 +/- 15 pS with 500 microM Ca2+. The higher level of Ca2+ on the intracellular side of the membrane caused the larger degree of rectification. Mg2+ only had a minor effect on rectification of this calcium-activated K+ channel.

Wang, Desuo, Colin Sumners, Philip Posner, and Craig H. Gelband. A-type K+ current in neurons cultured from neonatal rat hypothalamus and brain stem: modulation by angiotensin II. J. Neurophysiol. 78: 1021–1029, 1997. The regulation of A-type K+ current ( I A) and the single channel underlying I A in neonatal rat hypothalamus/brain stem cultured neurons were studied with the use of the patch-clamp technique. I A had a threshold of activation between −30 and −25 mV ( n = 14). Steady-state inactivation of I A occurred between −80 and −70 mV and had a membrane voltage at which I A was half-maximum of −52.2 mV ( n = 14). The mean values for the activation and inactivation (decay) time constants during a voltage step to +20 mV were 2.1 ± 0.3 (SE) ms ( n = 8) and 13.6 ± 1.9 ms ( n = 8), respectively. Single-channel recordings from outside-out patches revealed A-type K+ channels with voltage-dependent activation, 4-aminopyridine (4-AP) sensitivity, and inactivation kinetics similar to those of I A. The single-channel conductance obtained from cell-attached patches was15.8 ± 1.3 pS ( n = 4) in a physiological K+ gradient and 41.2 ± 3.7 pS ( n = 5) in symmetrical 140 mM K+. Angiotensin II (Ang II, 100 nM) reduced peak I A by ∼20% during a voltage step to +20 mV ( n = 8). Similarly, Ang II (100 nM) markedly reduced single A-type K+ channel activity by decreasing open probability ( n = 4). The actions of Ang II on I A and single A-type K+ channels were reversible either by addition of the selective angiotensin type 1 (AT1) receptor antagonist losartan (1 μM) or on washout of the peptide. Thus the activation of AT1 receptors inhibits a tetraethylammonium-chloride-resistant, 4-AP-sensitive I A and single A-type K+ channels, and this may underlie some of the actions of Ang II on electrical activity of the brain.