Frank M.J. Heemskerk

Quantitative autoradiography of angiotensin II AT2 receptors with [125I]CGP 42112

Most radiolabeled ligands for angiotensin II (Ang II) receptors do not discriminate between the AT1 and AT2 receptor subtypes, which must be distinguished by displacement with selective AT1 or AT2 ligands. We compared [125I]CGP 42112 with the non-selective agonist [125I]Sar1 Angiotensin II. We studied the inferior olive, medial geniculate nucleus and the adrenal medulla, areas rich in AT2 receptors, using both ligands with quantitative autoradiography and membrane binding techniques. [125I]CGP 42112 bound with high affinity (Kd = 0.07-0.3 nM, depending on the area studied). [125I]CGP 42112 binding was selective for AT2 receptors, as determined by lack of competition with the AT1 ligand losartan, and competition by the AT2 ligands PD 123177 and unlabeled CGP 42112 and the non-selective peptides Ang II and angiotensin III (Ang III). Using [125I]CGP 42112 binding, we found the same order of potency: CGP 42112 > Ang II = Ang III > PD 123177 using both quantitative autoradiography or membrane binding methods. Our results demonstrate that [125I]CGP 42112 is the most selective, highest affinity ligand available for AT2 receptors. Because of these characteristics, and low non-specific binding, quantitative autoradiography with [125I]CGP 42112 is the method of choice to selectively characterize AT2 receptors, especially in tissues like the brain, with a highly heterogeneous distribution of receptor subtypes.

4-aminopyridine differentially affects the spontaneous release of radiolabelled transmitters from rat brain slices in vitro

4-Aminopyridine increased the release of [3H]noradrenaline from dorsal hippocampus slices in vitro in a concentration-dependent manner. When the slices were exposed to 4-aminopyridine for 5 min, the overflow of radioactivity returned to pre-exposure values within 20-25 min. When the exposure of the slices was continued, a sustained enhancement of the release of [3H]noradrenaline was observed for the duration of the exposure. 4-Aminopyridine, 10(-4) M, had an effect of similar magnitude, or an even more pronounced effect, on the release of [3H]catecholamine from cortex, septum, periaqueductal gray and striatum slices. The effects of the compound on the release of [3H]5-hydroxytryptamine and [14C]acetylcholine were less pronounced. At this concentration 4-aminopyridine had no effect on the release of [3H]D-aspartate from hippocampus or septum slices, whereas the effect on the release of this transmitter in striatal slices was marginal. The effect of 4-aminopyridine on the release of [3H]noradrenaline in hippocampus slices was largely dependent on the presence of Ca2+ in the superfusion medium. This was also the case for the effect on the release of [3H]noradrenaline from preloaded dorsal hippocampus synaptosomes. In the presence of nitrendipine the effect of 4-aminopyridine was dose-dependently reduced, but the maximal reduction, at a nitrendipine concentration of 10(-4) M, was only 40%. Cd2+ completely abolished the effect of 4-aminopyridine on the release of [3H]noradrenaline. These results confirm that the enhancing effect of 4-aminopyridine on the release of [3H]noradrenaline depends on the entry of extracellular Ca2+ into the nerve terminals.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of endothelinA receptors in cerebral and peripheral arteries of the rat

We have characterized and quantified endothelin receptors in rat brain (anterior cerebral) and peripheral (aorta, carotid, and caudal) arteries, with the use of [125I]endothelin and quantitative autoradiography. Endothelin binding was saturable, of high affinity, and totally displaced by the selective endothelin ETA antagonist BQ 123. A single class of ETA receptors is located in the medial layer of peripheral and cerebral arteries, and its quantification by autoradiography allows study of their regulation and function.

Characterization of brain angiotensin II AT2 receptor subtype using [125I] CGP 42112A

Recently two subtypes of angiotensin receptors have been described, AT1 and AT2. Currently used radiolabeled agonists and antagonists are not able to discriminate between these receptors subtypes. Here we characterize the use of [125I] CGP 42112A, a novel, specific ligand for AT2 receptors, in a membrane binding assay and in autoradiography of brain sections of 2 week old rats. [125I] CGP 42112A bound with high affinity and autoradiography revealed binding selectively localized to areas known to express the AT2 receptor subtype only. CGP 42112A, angiotensin II, angiotensin III and PD 123177 competed for [125I] CGP 42112A binding, with potencies consistent with high affinity and specific binding to AT2 receptors. Thus [125I] CGP 42112A will be a useful new tool to study AT2 receptors.

The role of protein kinase C substrate B-50 (GAP 43) in neurotransmitter release and long-term potentiation

Long-term potentiation (LTP) is a form of synaptic plasticity, which may be one of the events underlying learning and memory. LTP is triggered by brief, high-frequency stimulation of afferents, resulting in a long-lasting increase in the effectiveness of synaptic transmission (Bliss and Lomo, 1973; Bliss and Lynch, 1988; Brown et al., 1988; Matthies, 1989). Traditionally, LTP has been divided into two phases, the initiation phase and the maintenance phase. At present, three phases can be distinguished: (i) an initiation phase, including several seconds after tetanic stimulation when the events that trigger LTP begin; (ii) a transient phase, lasting about 30 min, during which there is a slow decay of potentiation, which can be evoked by local transmitter application without stimulating the presynaptic terminal; and (iii) a maintenance phase, which can last for hours (Kauer et al., 1988; Malinow et al., 1988a).

Characterization of AT2 Receptor Expression in NIH 3T3 Fibroblasts

Abstract1. A high expression of angiotensin II receptors and of angiotensin-converting enzyme (ACE) activity was detected in confluent NIH 3T3 fibroblasts.2. Characterization with selective ligands, dithiothreitol, and GTPγS, indicated that only the AT2 subtype was expressed.3. AT2 receptors and ACE expression were strictly dependent on the cell density and growth phase of the cells, with AT2 receptors being expressed earlier than ACE. In contrast, high expression of AT2 receptors irrespective of their growth state was observed in NIH 3T3 cells lacking contact inhibition upon neoplastic transformation with ras.4. Our results imply a possible relation of AT2 receptors to cell growth and cell–cell contact.

Second Messengers for Brain Angiotensin Receptor Subtypes

Angiotensin II (ANG II) is an octapeptide produced in many tissues from its precursor molecules by subsequent cleavage by the enzymes renin and angiotensin converting enzyme.1–3 In the brain all components of this renin-angiotensin system are present.1,2,3 ANG II is involved in the central regulation of cardiovascular function, drinking and fluid metabolism, salt appetite, pituitary hormone release, and stress.1,2,4,–6