Selective changes in angiotensin II AT1 and AT2 receptor subtypes in the rat superior colliculus following eye enucleation.

In the brain of young (two weeks old) rats, angiotensin II receptors (AT2 receptors) are found in brain nuclei which receive and integrate direct visual input from the retina, the suprachiasmatic nuclei (containing only AT1 receptors), the lateral geniculate nuclei (containing AT2 receptors) and the superior colliculus (which contains both receptor types with a majority of AT2). In adult rats, angiotensin II receptors are present in the suprachiasmatic nuclei and the superior colliculus but not in the lateral geniculate. Using quantitative autoradiography we found that, in adult rats, bilateral eye enucleation caused a significant decrease in AT2 receptor binding, but not in AT1 receptor binding, and only in the superior colliculus. Unilateral enucleation of 12-day-old pups led to a decrease in AT2 receptor binding from the contralateral superior colliculus, as early as day 2 post-enucleation. Conversely, there was a significant increase in binding to AT1 receptors in the ipsilateral superior colliculus after seven days. No changes were seen in the lateral geniculate or suprachiasmatic nuclei. Angiotensin II binding to subcellular fractions of tissue from the superior colliculus region of 19-day-old pups suggested that AT2 receptor sites were present on the plasma membrane of the postsynaptic cell body. Membrane binding studies also showed a significant decrease in AT2 receptor binding to the same subcellular fractions when 19-day-old pups, enucleated seven days earlier, were compared to sham-operated animals. Our results suggest that expression of AT1 and AT2 receptors in the superior colliculus may be regulated by retinal input.

4-Aminopyridine stimulates B-50 (GAP-43) phosphorylation in rat synaptosomes.

Recently, we have shown that stimulation of [3H]-noradrenaline release from hippocampal slices by 4-aminopyridine (4-AP) is accompanied by an enhancement of the phosphorylation of B-50, a major presynaptic substrate of protein kinase C (PKC). PKC has been implicated in the regulation of transmitter release. In this study, we investigated the effects of 4-AP on B-50 phosphorylation in synaptosomes from rat brain and compared the effects of 4-AP with those of depolarization with K+, in order to gain more insight into the mechanism of action of 4-AP. B-50 phosphorylation was stimulated by incubation with 4-AP for 2 minutes at concentrations ranging from 10 microM to 5 mM. 4-AP (100 microM) stimulated B-50 phosphorylation already within 15 seconds; longer incubations revealed a sustained increase in the presence of 4-AP. B-50 phosphorylation was also stimulated by depolarization with 30 mM K+ for 15 seconds. The effects of both 4-AP or K+ depolarization on B-50 phosphorylation were abolished at low extracellular Ca2+ concentrations. The increase in B-50 phosphorylation induced by 4-AP seemed to be dependent on the state of depolarization, since the effect of 4-AP was largest under nondepolarizing conditions. Comparing the effects of 4-AP and K+ depolarization on B-50 phosphorylation suggests that a different mechanism of action is involved. These results indicate that the stimulation of B-50 phosphorylation by 4-AP in hippocampal slices can be attributed to a direct action of 4-AP on presynaptic terminals. In addition, our results support the hypothesis that B-50 phosphorylation by PKC is involved in Ca2(+)-dependent transmitter release evoked by 4-AP.

Identification of human I1 receptors and their relationship to alpha 2-adrenoceptors.

I1 imidazoline receptors (I1R) were defined as receptors insensitive to catecholamines and highly sensitive to [3H]clonidine and analogs. By contrast, the I2R subtype is more sensitive to [3H]idazoxan. [3H]clonidine and [3H]idazoxan imidazoline specific binding sites (IBS) have been detected in crude human membranes. Pharmacologic characterization by binding assays clearly differentiates IBS from alpha 2-adrenoceptors, whereas differences between [3H]clonidine and [3H]idazoxan IBS are less clear in crude preparations. In fact, only moderate affinity for [3H]clonidine was detectable in such preparations. However, purification procedures allowed detection of high affinity [3H]clonidine IBS in the human brain, corresponding to the I1R. Difficulties in the characterization of the I1R in crude membranes are due to multiple factors including heterogeneity of IBS, their low Bmax value, the existence of allosteric modulation, and possibly the presence of natural binding inhibitors. Immunologic studies with specific anti-idiotypic antibodies revealed a 43-kD protein as the best candidate for I1R as binding activity coincides with immunodetection. No cross-reaction was found with anti-monoamine oxidase (MAO) A/B antibodies and the 43-kD protein, ruling out the possibility of this protein being an MAO-associated I2R. Neither anti-alpha 2A- nor anti-alpha 2B-specific antibodies were able to immunodetect the 43-kD protein in crude membrane preparations or in purified fractions. These results and further biochemical characterization (pHi, N-glycosylation) of the 43-kD protein definitely assessed that human brain I1R and alpha 2-adrenoceptors clearly differ physically. However, coexpression of I1R and alpha 2-adrenoceptors in synaptic plasma membranes of the bovine brainstem reinforce the possibility of a functional relationship between the two types of receptor.

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.

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.

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)

Expression of a novel angiotensin II receptor subtype in gerbil brain.

Angiotensin II receptors are highly localized in adult gerbil brain. Apparent receptor number is high in subfornical organ, vascular organ of the lamina terminalis, nucleus of the solitary tract, hippocampus, and in the anterior pituitary gland. In the hippocampus, binding is localized to the stratum oriens, radiatum, the lacunar molecular layers of the CA1 subfield, and the molecular layer of the gyrus dentatus, with a medial to lateral and anterior to posterior gradient in receptor expression. Binding is absent from the pyramidal layer of the CA1 subfield and from the granular cell layer of the gyrus dentatus, areas rich in angiotensin IV binding. Characterization in the hippocampus revealed the presence of a high affinity receptor, sensitive to incubation with the guanine nucleotide GTP gamma S, and displaced by angiotensin II = angiotensin III < Sar1-Ile8-angiotensin II, but not by angiotensin IV or other angiotensin fragments, the AT1 receptor antagonist losartan, or the AT2 ligands CGP 42112 or PD 123177. In other brain areas, binding was equally insensitive to displacement by AT1 or AT2 ligands, with the exception of binding in the olfactory bulb, which was totally displaced by CGP 42112 and PD 123177, but not by losartan. In the gerbil, most of the brain and pituitary angiotensin II receptors are different from the AT1, AT2 and AT4 subtypes, and should be considered 'atypical' until further characterization.

Activation of protein kinase C by 4-aminopyridine dependent on Na+ channel activity in rat hippocampal slices.

The convulsant drug 4-aminopyridine (4-AP) stimulates the phosphorylation of the neuron-specific presynaptic protein B-50 in hippocampal slices. This effect could be attenuated by the protein kinase C (PKC) inhibitor staurosporine. Moreover, the endogenous phosphorylation of B-50 was found to be restricted to the 15 kDa Staphylococcus aureus protease fragment of B-50, known to contain the PKC acceptor site. The effect of 4-AP on B-50 phosphorylation was sensitive to the Na+ channel blocker tetrodotoxin. These results indicate that 4-AP stimulates PKC activity in hippocampal slices by a mechanism dependent on Na+ channel activity.

Protein kinase C phosphorylates Ser152, Ser156 and Ser163 but not Ser160 of MARCKS in rat brain.

MARCKS is one of the major physiological substrates of PKC and was reported to be phosphorylated by PKC at 4 serine residues that are within the CaM-binding region (Graff et al., J. Biol. Chem. 264, 11912, 1989). Using MARCKS from rat brain and a synthetic peptide of 25 amino acids containing all 4 of the serine residues, we investigate the differences in phosphorylation by PKC isozymes I, II and III. Tryptic peptide analysis of PKC phosphorylated MARCKS or peptide, we found 32P was in peptides of (K)S152FK, (R)FS156FK and LS160GFS163FK. Further digestion of LSGFSFK with alpha-chymotrypsin revealed that 32P incorporation occurred only at Ser163 but not at Ser160. The initial rates and stoichiomatry of phosphorylation of Ser152 and Ser156 were twice as those of Ser163 using either one of the three PKC isozymes. These results indicate that in vitro, PKC phosphorylates MARCKS only at three sites, but not at Ser160 as that reported previously, and there was no preferential phosphorylation of MARCKS by either PKC isozyme I, II or III.

Purification and characterization of angiotensin II AT2 receptors from neonatal rat kidney.

Angiotensin II (Ang II) AT2 receptors were purified 40,000-fold to a nearly homogeneous state after solubilization from neonatal rat kidney membranes with 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane-sulfonic acid. Comparable IC50 values for the soluble extract (0.32 nM) and membranes (0.31 nM) were obtained by competition curves with 125I-labeled CGP42112, a selective AT2 ligand. Binding to AT2 receptors in the soluble extract was not sensitive to dithiothreitol. AT2 receptors were further purified by gel filtration and a CGP42112 Sepharose affinity column. Ang II AT2 receptors were selectively eluted with 5 microM CGP42112 at 4 degrees C, and a single band with an apparent molecular mass of 71 kDa was obtained after SDS/PAGE. Two-dimensional electrophoresis confirmed the purity of the protein and an isoelectric point of 5.3-5.5 was obtained. A highly selective elution of the AT2 receptors from the affinity column was performed with 5 nM 125I-labeled CGP42112 at room temperature after the column was treated with 1 microM losartan in the presence of high salt. After cross-linking, a major labeled protein with similar molecular mass and isoelectric point was obtained. Dissociation of the radiolabeled protein was insensitive to losartan but was enhanced by CGP42112, PD123177, Ang II, and [Sar1]Ang II. In summary, Ang II AT2 receptors were purified by CGP42112 affinity chromatography and selective elution and retain the pharmacological specificity of particulate receptors.

4-Aminopyridine inhibits synaptosomal plasma membrane protein phosphorylation in vitro: effect of the selective NMDA-antagonist 2-amino-5-phosphonovalerate.

Phosphorylation of synaptosomal plasma membranes from rat hippocampus in the presence of the convulsant drug 4-aminopyridine resulted in the inhibition of the phosphorylation of the nervous tissue specific protein kinase C substrate protein B-50 (48 kDa) and the alpha-subunit of calcium/calmodulin-dependent protein kinase II (50 kDa). Preincubation of SPM with 2-amino-5-phosphonovalerate prevents the inhibition of B-50 phosphorylation by 4-aminopyridine, but had no effect on the inhibition of 50 kDa phosphorylation. 2-Amino-5-phosphonovalerate is known to be a specific N-methyl-D-aspartate antagonist and has anti-epileptic activity in vitro and in vivo. Several other anti-epileptic drugs tested did not influence the 4-aminopyridine-induced inhibition of protein phosphorylation.

Characterization of AT2 receptor expression in NIH 3T3 fibroblasts.

1. 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 GTP gamma S, 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.

Evidence for the existence of imidazoline-specific binding sites in synaptosomal plasma membranes of the bovine brainstem.

Nonadrenergic imidazoline-specific binding sites were characterized pharmacologically in crude cerebral membrane preparations, but little is known about their subcellular localization in neurons. As in the brainstem these sites are involved in cardiovascular regulation and peripherally imidazolines modulate neurotransmitter release, we tried to determine a possible (pre)synaptic localization in brainstem. We found a specific enrichment in (entire) synaptosome, purified synaptosomal plasma membrane (37 fmol/mg), and mitochondrial (83 fmol/mg) fractions as compared with other membrane fractions (3-8 fmol/mg). Synaptosomes appeared to be free of postsynaptic structures, and purified synaptosomal plasma membranes were devoid of mitochondrial material, as determined by electron microscopy and by comparison with the distribution of marker enzymes such as monoamine oxidase. These results show for the first time that these extramitochondrial imidazoline-specific sites are neuronal and are located on presynaptic terminals. We found high affinities for unlabeled p-iodoclonidine (subnanomolar), clonidine (0.2 nM), and efaroxan (11 nM), but idazoxan did not compete significantly for the p-[125I]iodoclonidine binding in these membranes. Therefore, these sites can be classified as I1 imidazoline receptors. In summary, we describe for the first time that high-affinity I1 receptors of the bovine brainstem are located on (pre)synaptic membranes.

Specific, non-angiotensin, [125I]CGP 42112 binding sites in rat spleen macrophages.

We report the novel expression of a high affinity non-angiotensin II binding site to [125I]CGP 42112, predominantly localized to the red pulp of the rat spleen and to isolated rat spleen macrophages. Binding is not inhibited by angiotensin II, related peptides or AT1 or AT2 receptor ligands, and is not affected by several growth factors and cytokines involved in macrophage function. Thus, CGP 42112 recognizes binding sites other than angiotensin II AT2 sites in rat macrophages. CGP 42112 and related peptides might influence the regulation of macrophage function.

4-Aminopyridine stimulates B-50 (GAP43) phosphorylation and [3H]noradrenaline release in rat hippocampal slices.

In situ phosphorylation of the presynaptic protein kinase C substrate B-50 was investigated in rat hippocampal slices incubated with the convulsant drug 4-aminopyridine (4-AP). Phosphorylation of B-50 was significantly enhanced 1 min after the addition of 4-AP (100 microM). This increase by 4-AP was concentration dependent (estimated EC50 30-50 microM). Concomitant with the changes in B-50 phosphorylation, 4-AP also dose-dependently stimulated [3H]noradrenaline [( 3H]NA) release from the slices. 4-AP stimulated [3H]NA release within 5 min to seven times the control level. The B-50 phosphorylation induced by 4-AP remained elevated after removal of the convulsant, this is contrast to B-50 phosphorylation induced by depolarization with K+. A similar persistent increase was observed for [3H]NA release after a 5-min incubation period with 4-AP. These results give more insight into the molecular mechanisms underlying 4-AP-induced epileptogenesis and provide further evidence for the correlation between B-50 phosphorylation and neurotransmitter release in the hippocampal slice.