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In this study, we have investigated the effects of angiotensin II (ANG II) on glucose uptake into astroglia cultured from adult rat hypothalamus and brain stem. ANG II (30 min to 4 h; 10(-9) to 10(-6) M) stimulated time- and concentration-dependent increases in the uptake of 2-deoxy-D-[3H]glucose into cultured astroglia. This effect of ANG II (10(-7) M) is via AT1 receptors and protein kinase C (PKC), since it was inhibited by losartan (10(-6) M) and staurosporine (10(-6) M), respectively. Furthermore, this ANG II action was inhibited by both cycloheximide (1 microgram/ml) and actinomycin D (10(-6) M), indicating that synthesis of new glucose transporters is involved. This was confirmed by the finding that ANG II (30 min to 4 h; 10(-9) to 10(-5) M) stimulated time- and concentration-dependent increases in the steady-state levels of glucose transporter-1 (GLUT-1) mRNA in these cultures. In addition, the increase in steady-state levels of GLUT-1 mRNA elicited by ANG II was mediated by AT1 receptors and PKC. These data suggest that ANG II stimulates glucose uptake into cultured astroglia via a pathway that involves AT1 receptors, PKC, and increased steady-state levels of GLUT-1 mRNA.

Angiotensin II (ANG II) stimulates the delayed rectifier K+ current (IK) in neurons cultured from rat hypothalamus and brain stem via AT2 receptors, and this effect involves activation of a Gi protein and protein phosphatase 2A (PP2A). However, there was no evidence that the AT2 receptor involved in this response was the same as the recently cloned AT2 receptor. In the present study, intracellular injection of a 22-amino acid peptide (PEP-22) corresponding to the putative third intracellular loop of the cloned AT2 receptor elicited an increase in IK in cultured neurons that was similar to the effect produced by ANG II. Furthermore, this effect of PEP-22 was abolished by pertussis toxin (200 ng/ml, 24 h) pretreatment and also by superfusion of the PP2A inhibitor okadaic acid (10 nM), suggesting the involvement of Gi protein and PP2A, respectively. Intracellular injection of a random peptide or normal pipette solution did not affect neuronal IK. This is direct evidence to link the cloned AT2 receptor to a defined response elicited by ANG II.

The effects of adrenergic drugs on the release of immunoreactive angiotensin II (ANG II-ir) from brain cells in culture were examined. In neuronal cultures, basal release of Ang II-ir was 43.65 +/- 7.44 pg/5-min incubation period (n = 14 experiments; 52 individual determinations), and in astrocytic glial cultures, it was 21.76 +/- 5.7 pg (n = 8 experiments; 24 individual determinations) when cells were exposed to buffer alone. Incubation of neuronal cultures with the alpha 2-adrenergic antagonist yohimbine (0.1-50 microM, 5 min) caused concentration-dependent increases in ANG II-ir release above basal levels. Analysis of the released material by high-pressure liquid chromatography revealed that authentic ANG II was present. No increase in the release of ANG II-ir was seen from glial cells. Experiments using neuronal cultures revealed that the yohimbine-induced release of ANG II-ir may be secondary to increased norepinephrine (NE) release. Incubation of neuronal cultures with NE (10 nM-50 microM) caused concentration-dependent increases in the release of ANG II-ir. This effect of NE was not inhibited by the alpha 1-adrenergic blocker prazosin. However, a weaker release of ANG II-ir from neuronal cultures was stimulated by the beta-adrenergic agonist isoproterenol at 100 microM. These data show that ANG II-ir can be released from neuronal but not glial cell cultures by adrenergic receptor-mediated mechanisms.