Quantitative autoradiography of angiotensin II AT2 receptors with [I]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]Sar¹Angiotensin 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 (K_d = 0.07−0.3nM, 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 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.     

Chemical lesion of the inferior olive reduces [I]Sarcosine–Angiotensin II binding to AT2 receptors in the cerebellar cortex of young rats

In young rats, AT₂ receptors and AT₂ receptor mRNA are discretely localized in neurons of the inferior olive, with highest expression in the medial nucleus. We previously detected AT₂ receptor binding, but not AT₂ receptor mRNA, in the molecular layer of the cerebellar cortex. To determine whether AT₂ receptors are expressed in climbing fiber terminals which arise to the molecular layer from the inferior olive and innervate Purkinje cells, we chemically destroyed olivary neurons of 2-week-old rats by intraperitoneal (i.p.) injection of the neurotoxin 3-acetylpyridine. Lesions of the inferior olive reduced [¹²⁵I]Sar¹–Ang II binding to AT₂ receptors and AT₂ receptor mRNA levels in this area by 50%, and produced a similar decrease in AT₂ receptor binding in the molecular layer of the cerebellar cortex. The extent of binding reduction was similar 3 days and 7 days after the lesion. 3-Acetylpyridine lesions did not change [¹²⁵I]Sar¹–Ang II binding to AT₁ receptors in the molecular layer of the cerebellar cortex or AT₁ receptor mRNA levels in Purkinje cells. AT₂ receptor binding and AT₂ receptor mRNA levels in the deep cerebellar nuclei were also not affected by 3-acetylpyridine. Our results support the hypothesis that AT₂ receptors are produced by inferior olivary neurons and transported through climbing fibers to the molecular layer of the cerebellar cortex. The high expression of AT₂ receptors in the inferior olivary–cerebellar pathway during a crucial time in postnatal development of climbing fiber-Purkinje cell connectivity suggest a role of AT₂ receptors in the development of this pathway.     

Changes in angiotensin II receptors in dopamine-rich regions of the mouse brain with age and ethanol consumption

The density of angiotensin II (Ang II) receptors was determined in three dopaminergic nerve terminal-rich brain regions (caudate putamen, nucleus accumbens, and ventral pallidum) of mice that were given either water (control) or 20% w/v ethanol (EtOH) to drink for either 2–8 weeks (young) or 46 weeks (old). The receptors were labeled with ¹²⁵I-sarcosine¹, isoleucine⁸ angiotensin II (¹²⁵I-SI Ang II) and measured by quantitative densitometric image analysis (receptor autoradiography) or by saturation binding assays on homogenates of these brain regions. The selective AT₂ receptor subtype antagonist PD 123319 (10 μM) was used to inhibit ¹²⁵I-SI Ang II binding to AT₂ receptors to determine AT₁ receptor density in brain sections. In young control mice the density of Ang II receptor binding sites in the caudate putamen was 407±26 fmol/g, in the nucleus accumbens the density was 346±27 fmol/g, and in the ventral pallidum the density was 317±27 fmol/g. Less than 5% of specific ¹²⁵I-SI Ang II binding was displaced by PD 123319, suggesting that nearly all of the Ang II receptors in these brain regions were the AT₁ subtype. The B_max in homogenates of these three regions in young control mice was 11.0±2.1 fmol/mg protein. The K_D was 0.49±0.13. Ang II receptors in old mouse brains were decreased, respectively, by 32%, 35% and 30% in the caudate putamen, nucleus accumbens and ventral pallidum (p<0.001). Ang II receptors were slightly, but not significantly increased in both young and old EtOH-consuming mice.     

Muscarinic acetylcholine receptors on cultured glia cells

Muscarinic acetylcholine receptors (76 fmol/mg protein) were detected on cultured glia cells (astroblasts) from embryonic chicken brain by specific [³H]quinuclidinylbenzilate (QNB) binding at physiological conditions. The QNB binding (K_d = 9.5 × 10⁻¹¹) to the intact cells seems to be cooperative (n_H= 1.98) as shown by graphical methods.     

Angiotensin II receptor subtypes and angiotensin-converting enzyme in the fetal rat brain

Angiotensin II (ANG II) receptor subtypes (AT₁, displaced by losartan, and AT₂, displaced by CGP 42112A) were characterized by quantitative autoraography after incubation with the ANG II agonist [¹²⁵I]Sar¹-ANG II, in specific brain nuclei of 19-day-old rat embryos. Binding to AT₁ receptors, located in the subfornical organ, paraventricular nucleus, nucleus of the solitary tract and choroid plexus, was sensitive to incubation with GTPγS. The sensitivity of AT₂ receptors to GTPγS was heterogeneous. In the ventral thalamic, rostral hypoglossal and medial geniculate nuclei, and in the locus coeruleus, binding to AT₂ receptors was sensitive to GTPγS and these areas belong to the AT_2A subgroup. Conversely, in the inferior olive, medial (fastigial) cerebellar nucleus and caudal part of the hypoglossal nucleus, areas belonging to the AT_2B subgroup, binding was insensitive to GTPγS. AT₂ receptors were also present in cerebral arteries. In the fetal anterior pituitary, AT₁ receptors predominated. The angiotensin-converting enzyme (ACE; EC 3.4.15.1) was studied by autoradiography with the selective inhibitor [¹²⁵I]351A. In 19-day-old embryos, ACE was highly expressed in chroid plexus, with high concentrations in subfornical organ, posterior pituitary and cerebral arteries. No ACE binding was detected in extrapyramidal structures or anterior pituitary in 19-day-old embryos.     

Pharmacological characterisation of the histamine H3 receptor in the rat hippocampus

The purpose of this report was to pharmacologically characterise the histamine H₃ in the rat hippocampus using radioligand binding studies with the H₃ receptor antagonist [¹²⁵I]iodophenpropit and the H₃ receptor mediated inhibition of [³H]noradrenaline release. A dissociation constant of 0.33 nM and a maximal number of binding sites of 125 fmol/mg protein were found for [¹²⁵I]iodophenpropit. Competition studies showed stereoselectivity for the (R) and (S) enantiomers of α-methylhistamine and 10 μM of GTP_γS shifted the curve of (R)-α-methylhistamine rightwards. Up to 1 μM, (R)-α-methylhistamine displaced only 30% whereas the tested H₃-antagonists displaced 50–60% of the total [¹²⁵I]iodophenpropit bound. This indicates the presence of an additional non-H₃ receptor binding site(s) for [¹²⁵I]iodophenpropit in the rat hippocampus. This secondary site shows low affinity for H₃ agonists, but high affinity for the tested H₃ antagonists. Electrically evoked [³H]acetylcholine release was shown in slices of rat hippocampus. No H₃ receptor modulation of [³H]acetylcholine release from hippocampal slices was detectable. However, H₃ receptor activation inhibited 42% of the electrically-evoked [³H]noradrenaline release in rat hippocampal slices. The inhibition of [³H]noradrenaline release was effectively antagonized by the H₃ antagonists thioperamide and burimamide. We describe the pharmacological identification of the histamine H₃ receptor in the rat hippocampus and its similarities and differences from the cortical H₃ receptor. These studies enable us to investigate changes in density and functionality of the hippocampal H₃ receptor under (patho)physiological conditions.     

α-Bungarotoxin binding sites in rat hippocampal and cortical cultures: initial characterisation, colocalisation with α7 subunits and up-regulation by chronic nicotine treatment

High density neuronal cultures from rat E18 hippocampus and cortex have been characterised with respect to cholinergic binding sites. No specific binding of [³H]nicotine or [³H]cyttine to live cells in situ was detected, although the limit for detection was estimated to be 30 fmol/mg protein. Muscarinic binding sites labelled with [³H]QNB were present at a density of 0.75 pmol/mg protein. [¹²⁵I]α-Bungarotoxin (αBgt) bound to hippocampal cultures with a B_max of 128 fmol/mg protein and a K_d of 0.6 nM; cortical cultures expressed five times fewer [¹²⁵I]α-Bgt binding sites. Fluorescence cytochemistry with rhodamine-α-Bgt indicated that 95% of hippocampal neurons were labelled, compared with only 36% of cortical neurons. Average densities of 4 × 10⁴ and 2 × 10⁴ binding sites/cell were calculated for hippocampal and cortical cultures, respectively. Double labelling experiments with mAb307 (which recognises the rat α7 nicotinic receptor subunit) and rhodamine-α-Bgt gave coincident labelling patterns, supporting the correlation between the α7 subunit and Bgt-sensitive neuronal nicotinic receptor. Treatment of hippocampal cultures with 10 μM nicotine for 14 days elicited a 40% increase in the numbers of [¹²⁵I]α-Bgt binding sites, mimicking the up-regulation observed in vivo studies. Primary cultures offer a useful in in vitro system for investigating the expression and regulation of brain α-Bgt-sensitive receptors.     

Purkinje cells express Angiotensin II AT(2) receptors at different developmental stages

Angiotensin II (Ang II) binds and activates two major receptors subtypes, namely AT₁ and AT₂. In the fetus, AT₂ receptors predominate in all tissues and decline shortly after birth, being restricted to a few organs including brain. Interpretation of the function of Ang II in the cerebellum requires a thorough understanding of the localization of Ang II receptors. The aim of the present paper is to evaluate the localization of Ang II AT₂ receptors in the Purkinje cell (PC) layer during development. By binding autoradiography, a clear complementary pattern of AT₁ and AT₂ binding labeled by [¹²⁵I] Ang II was observed in young rats within the cerebellar cortex. This pattern was present at the stages P8 and P15, but not at P30 and P60, where AT₂ binding appears low and superimposed with AT₁ binding. We demonstrate that AT₂ antibodies recognized postmitotic Purkinje cells, labeling the somata of these cells at all the stages studied, from P8 to P60, suggesting that PCs express these receptors from early stages of development until adulthood. In P8 and P15 animals, we observed a clear correspondence between immunolabeling and the well-defined layer observed by binding autoradiography. Confocal analysis allowed us to discard the co-localization of AT₂ receptors with glial fibrillary acidic protein (GFAP), a glial marker. Double immunolabeling allowed us to demonstrate the co-localization of Ang II AT₂ receptors with zebrin II, a specific PC marker. Since PCs are the sole output signal from the cerebellar cortex and considering the role of cerebellum in movement control, the specific receptor localization suggests a potential role for Ang II AT₂ receptors in the cerebellar function.     

Subtypes of β-adrenergic receptors in rat cerebral microvessels

The¹²⁵I-labeled iodohydroxybenzylpindolol (IHYP) binding to β-receptors on brain microvessels is inhibited by isoproterenol, epinephrine and norepinephrine, with K_i values of 2 × 10⁻⁷M, 2.5 × 10⁻⁶M and 1.2 × 10⁻⁵M, respectively. A modified Scatchard analysis of the inhibitory effects of practolol, metroprolol and zinterol on IHYP binding has shown that the proportion of β₂-receptors in our preparation is about 80% of the total β-adrenergic receptor population. Our data indicate that the β-adrenergic receptors located on cerebral microvessels are of both β₁ and β₂ types, with a predominance of the β₂ type.     

Role of angiotensin II receptor subtypes in mediating the sympathoexcitatory effects of exogenous and endogenous angiotensin peptides in the rostral ventrolateral medulla of the rabbit

The pressor region in the rostral part of the ventrolateral medulla (VLM) in the rabbit contains a high density of the AT₁ subtype of angiotensin (Ang) II receptor. In this study in anaesthetized barodenervated rabbits, we determined the effect of microinjection into the rostral VLM of the AT₁ receptor antagonist losartan and the AT₂ receptor antagonist PD123319 on resting arterial pressure and renal sympathetic nerve activity, and on the cardiovascular responses normally evoked by exogenous Ang II or Ang III in this region. Losartan (1 nmol) abolished the pressor and sympathoexcitatory responses normally evoked by exogenous Ang II, but PD123319 (1 nmol) had little effect on these responses. Both losartan (0.1–10 nmol) and PD123319 (0.1–1 nmol) had little effect on the resting arterial pressure and renal sympathetic nerve activity, except for a transient sympathoexcitatory response at the higher doses. In confirmation of previous findings, however, microinjection of the non-selective Ang receptor antagonist [Sar¹,Thr⁸]Ang II (80 pmol) significantly decreased resting arterial pressure and sympathetic nerve activity. These results suggest that the sympathoexcitatory effects evoked by exogenous Ang II and III in the rostral VLM are mediated by AT₁ receptors, but that the tonic sympathoexcitation produced by endogenous Ang peptides in the rostral VLM of the rabbit are mediated by receptors other than AT₁ or AT₂ receptors.     

Losartan, nonpeptide angiotensin II-type 1 (AT1) receptor antagonist, attenuates pressor and sympathoexcitatory responses evoked by angiotensin II andL-glutamate in rostral ventrolateral medulla

We investigated the effect of losartan, a nonpeptide angiotensin II (Ang II)-type 1 (AT₁) receptor antagonist, on the responses evoked by Ang II andL-glutamate (L-Glu) in the rostral ventrolateral medulla (RVLM). Adult spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were anesthetized with halothane and artificially ventilated. Responses of mean arterial pressure (MAP), heart rate (HR) and splanchnic sympathetic nerve activity (SNA) to microinjection of Ang II (100 pmol) orL-Glu (2 nmol) into the RVLM were examined following microinjection of losartan (10 pmol–10 nmol). Ang II increased MAP (16 ± 1mmHg in SHR and16 ± 1mmHg in WKY) and SNA (9 ± 1%and10 ± 1%, respectively), which were significantly (P < 0.01) attenuated by pretreatment with losartan (100 pmol − 10 nmol) in both strains. In addition, the pressor and sympathoexcitatory responses evoked byL-Glu were attenuated by losartan in a dose-dependent manner. The increases of MAP evoked byL-Glu (53 ± 6mmHg in SHR and39 ± 3mmHg in WKY) were suppressed to 5 ± 3mmHg(P < 0.01) and 4 ± 2mmHg (P < 0.01), respectively, in the presence of 10 nmol of losartan. The increase of SNA was also markedly inhibited by higher doses of losartan. The cardiovascular responses evoked byL-Glu, however, were not attenuated by pretreatment with either 1 nmol of [Sar¹, Thr⁸]-Ang II or 10 nmol of potassium acetate, suggesting that the effect of losartan onL-Glu response may not be attributed to the blockade of Ang II receptor or to the high concentration of potassium. These results indicate that the AT₁ receptor is responsible, in part, for the vasomotor action of Ang II in the RVLM and losartan has an inhibitory effect on pressor and sympathoexcitatory responses evoked byL-Glu by mechanisms other than those mediated by Ang II receptors.     

Age-related modifications on the GABAA receptor binding properties from Wistar rat prefrontal cortex

In the present communication we have investigated the pharmacological properties of the GABA_A receptor from adult (3 months old) and aged (24 months old) Wistar rat prefrontal cortex. The prefrontal cortex is implicated in cognitive functions and stress and both processes seem to be altered during aging. These changes could be mediated by modifications in the GABA_A receptor properties. Our results indicated the absence of generalized age-related modifications on the pharmacological properties of the GABA_A receptor from prefrontal cortical membranes. Saturation experiments using the non-selective benzodiazepine [³H]flunitrazepam revealed that neither the K_d values or the B_max were modified during aging. Moreover, Cl 218 872 displacement of [³H]flunitrazepam showed no age-related modifications on either the K_is or the relative proportion between the Type I and Type II benzodiazepine binding sites. Therefore, the benzodiazepine binding sites are well preserved in aged prefrontal cortex. On the other hand, saturation experiments using the GABA agonist [³H]muscimol demonstrated a decrease in the B_max of the low affinity [³H]muscimol binding sites in aged rats (4.3 ± 0.8 pmol/mg protein vs. 2.3 ± 0.2 pmol/mg protein in adult and aged rats, respectively). However, no age-dependent modifications were observed in the allosteric interaction between GABA and benzodiazepine binding sites. These results demonstrate that the benzodiazepine binding sites and the GABA binding sites of the GABA_A receptor complex from rat prefrontal cortical membranes are differentially affected by the aging process.     

Characterisation of dopamine receptors in insect (Apis mellifera) brain

In vitro binding experiments using the vertebrate D₁ dopamine receptor ligand [³H]SCH23390 and the vertebrate D₂ dopamine receptor ligand [³H]spiperone were conducted on membrane preparations of honey bee (Apis mellifera) brain. Specific binding of [³H]SCH23390 was saturable and reversible. Analysis of saturation data gave an apparent K_d of 6.3 ± 1.0 nM and B_max of 1.9 ± 0.2 pmol/mg protein for a single class of binding sites. The specificity of high affinity [³H]SCH23390 binding was confirmed in displacement experiments using a range of dopaminergic antagonists and agonists. The rank order of potency for antagonists was: R(+)-SCH23390 > cis-(Z)-flupentixol ≥ chlorpromazine > fluphenazine> S(+)-butaclamol > spiperone. R(±)-SKF38393 and dopamine were the most effective agonists tested. [³H]SCH23390 labels a site in bee brain that is similar, but not identical to the vertebrate D₁ dopamine receptor subtype. [³H]Spiperone also bound with high affinity to bee brain homogenates. Scatchard analysis of [³H]spiperone saturation data revealed a curvilinear plot suggesting binding site heterogeneity. The high affinity site had a apparent K_d of 0.11 ± 0.02 nM and B_max of 9.2 ± 0.5 fmol/mg protein. The calculated values for the low affinity site were a K_d of 19.9 nM and B_max of 862 fmol/mg protein. Kinetic analyses also indicated that [³H]spiperone recognises a heterogeneous population of sites in bee brain. Furthermore, agonist competition studies revealed a phenolaminergic as well as a dopaminergic component to [³H]spiperone binding in bee brain. The rank order of potency of dopaminergic antagonists in competing for [³H]spiperone binding was: spiperone > fluphenazine> S(+)-butaclamol > domperidone> R(+)-SCH23390 > S(−)-sulpiride.     

ß1-Adrenergic receptors in human neuroblastoma

ß-Adrenergic receptors and adenylate cyclase activity were identified and partially characterized in 10 human neuroblastoma tumors obtained from direct biopsy, from human cell lines grown in the athymic, nude mouse or from established tumor cell lines in tissue culture. Particulate membrane fractions of eight of the tumors contained ß-adrenergic receptor sites identified by the ß-adrenergic antagonists (−)-[³H]dihyroalprenolol ((−)-[³H]DHA) and (−)-[¹²⁵I]iodohydroxybenzylphindolol ((−)-[¹²⁵I]HYP). Specific binding of high affinity sites (B_max) varied greatly among the tumors ranging from non-detectable concentrations to approximately 130 fmol/mg of crude membrane protein. Analysis of the saturation experiments with (−)-[³H]DHA resulted in curvilinear Scatchard plots in the tumors, suggesting the presence of multiple classes of sites and there was no evidence of cooperativity in dissociation experiments with (−)-[³H]DHA. The high affinity class of sites were inhibited by catecholamines, in contrast to the ineffectiveness of catecholamines in inhibiting (−)-[³H]DHA binding at the low affinity class of sites. TheK_D of the high affinity site was approximately 1–2 nM for (−)-[³H]DHA and was similar in all the tumors studied. A single class of sites was demonstrated with (−)-[¹²⁵I]HYP, in several of the tumors studied,K_D approximately 120 pM, and the number of sites determined with (—)-[¹²⁵I]HYP was identical to the number of high affinity sites determined with (−)-[³H]DHA. The properties of the receptor were characterized in several tumor lines: SK-N-MC tumor line contained both a ß-adrenergic receptor site and catecholamine sensitive adenylate cyclase activity. Agonists competed for the ß-adrenergic sites in the order of potency (−)-isoproterenol > (−)-norepinephrine = (−)-epinephrine > > (−)-dopamine characteristic of a ß-adrenergic receptor subtype. The affinity of the receptor for the selective agents, metropol (ß₁-selective antagonist) and zinterol (ß₂-selective agonist) was typical of a homogeneous ß₁-adrenergic site. Guanine nucleotides, guanine triphosphate and guanyl-5′-yl-imidophosphate, decreased catecholamine affinity for the receptor site in several tumors studied and enhanced maximal catecholamine stimulated adenylate cyclase activity in the sensitive cell line. Adenylate cyclase activity in particulate fractions of most of the neuroblastoma tumors was responsive to prostaglandins (PGE₁), guanine nucleotide and NaF. While most tumors contained ß-adrenergic receptor sites only SK-N-MC was clearly catecholamine responsive as assessed by catecholamine-sensitive adenylate cyclase activity. Human neuroblastomas, a homogenous tissue of neural crest origin, contained ß-adrenergic receptor sites which varied greatly in number (B_max) and in their ability to stimulate adenylate cyclase activity. Guanine nucleotides decreased agonist affinity for the receptor in the lines tested and enhanced prostaglandin and catecholamine sensitive activity in responsive tumor lines.     

Mesangial AT1/B2 receptor heterodimers contribute to angiotensin II hyperresponsiveness in experimental hypertension

Angiotensin II plays a central role in the pathogenesis of hypertension and of related cardiovascular disorders by binding to and activating angiotensin II receptors (AT₁ receptors). Sensitization to the vasopressor response of angiotensin II is a key feature in many cardiovascular disorders. However, underlying mechanisms responsible for angiotensin II hypersensitivity are barely understood. Because angiotensin II responsiveness of AT₁ receptors can be specifically modified by AT₁/B₂ receptor dimerization, we determined the AT₁ receptor dimerization status in an experimental model of hypertension. AT₁/B₂ receptor heterodimers were abundant on renal mesangial cells isolated from spontaneously hypertensive rats compared with that on cells from normotensive controls. Heterodimerization of AT₁ with B₂ receptors was correlated with high levels of B₂ receptor protein on kidneys and on mesangial cells of hypertensive rats, as determined in immunoblot with receptor-specific antibodies. Specific inhibition of AT₁/B₂ receptor heterodimers revealed that these receptor heterodimers mediated an enhanced angiotensin II-stimulated Gα_q/11 activation and an increased endothelin-1 secretion of mesangial cells from hypertensive rats. Thus, AT₁/B₂ receptor heterodimerization contributes to angiotensin II hyperresponsiveness of mesangial cells in experimental hypertension.     

Analysis of regional variations in the affinities of muscarinic agonists in the rat brain

The brain stem of the rat has a higher affinity toward muscarinic agonists than does the forebrain. Receptor occupancy curves of both regions of the brain deviate from simple mass-action binding. The characteristics of the binding in each region are compatible with the existence of two non-interacting binding sites, and are not attributable to desensitization or to negatively cooperative binding within a small oligomer; however, the possibility of large oligomers remains to be excluded. The agonist binding data were analyzed by a linear transformation of Scatchard-like inhibition curves of the binding of the antagonist [³H]quinuclidinyl benzilate (QNB). Such analysis, based on a model of two subpopulations of receptors in each area, shows the subpopulations of the brain stem and the forebrain to be distinct. Brain stem: 44% of receptors possess high affinity with dissociation constant for carbachol,K_H = 2.8 × 10⁻⁸M, dissociation constant of low-affinity receptor,K_L = 2.3 × 10⁻⁶M; forebrain: 41% high affinity,K_H = 2.1 × 10⁻⁷M, K_L = 1.7 × 10⁻⁵M. The data suggest that whole brain contains at least three major muscarinic receptors, which can be distinguished on the basis of their affinities for agonists.     

Benzodiazepine Receptors in Fish Brain: [H]-Flunitrazepam binding and modulatory effects of GABA in rainbow trout

We have identified and partially characterised benzodiazepine binding sites in whole brain membranes of male rainbow trout. In terms of B_max and K_d values trout brain receptors are remarkably similar to those in rat and human brain. The Hill coefficient was 0.98, indicating a single binding site. GABA (10⁻⁴ M) was able to significantly elevate binding of [³H]-FNZ through a change in K_d rather than B_max. This effect was prevented by the GABA receptor antagonist bicuculline methiodide.     

I-BH[Sar,Met(O2)]-SP, a new selective ligand for the NK-1 receptor in the central nervous system

The selective agonist [Sar⁹,Met(O₂)¹¹]-SP was radioiodinated with¹²⁵I-Bolton Hunter in order to study its binding to rat brain membranes and for further comparison with¹²⁵I-BH.SP. Specific binding of¹²⁵I-BH[Sar⁹,Met(O₂¹¹]-SP was temperature-dependent, saturable and reversible. In brain homogenates,¹²⁵I-BH[Sar⁹,Met(O₂)¹¹]-SP interacted with a single class of high affinityk_d = 1.0nM) non-interacting binding sites (B_max of 15 fmol/mg protein). In the central nervous system,¹²⁵I-BH-[Sar⁹,Met(O₂)¹¹]-SP apparently labeled the same number of binding sites as¹²⁵I-BH.SP (19 fmol/mg protein). Competition studies with tachykinins, neurokinins and selective neurokinin agonists indicated that the pharmacological profile of the site labeled by¹²⁵I-BH[Sar⁹,Met(O₂¹¹]-SP is identical with that of NK-1 receptors. In dose-displacement studies made with radiolabeled SP and [Sar⁹,Met(O₂¹¹)]-SP, an excellent correlation (r = 0.96) was found for theK_i values of the different compounds tested; these findings suggest that both radioligands recognize the same receptor in rat brain. The affinity (K_i of various neurokinin-related peptides for the brain site were compared with their biological activities on various isolated organs (dog carotid artery, guinea-pig ileum, rat portal vein). NK-1 binding sites characterized in rat brain homogenates appear to be identical with those present on the dog carotid artery, a preparation known to possess exclusively the NK-1 receptor type.     

Increased Expression of Type 1 Angiotensin II Receptors in the Hypothalamic Paraventricular Nucleus following Stress and Glucocorticoid Administration

Double staining in situ hybridization studies have shown that angiotensin II (All) type 1 receptors (AT1) in the hypothalamic paraventricular nucleus (PVN) are located primarily in corticotropin releasing hormone (CRH) neurons of the parvicellular subdivision. The purpose of these studies was to investigate the role of All regulating the hypothalamic-pituitary adrenal (HPA) axis, by correlating AT₁ receptor expression levels in the PVN with the known changes in activity of the HPA axis under different stress paradigms, and manipulation of circulating glucocorticoids. AT₁ receptor mRNA was measured by in situ hybridization using ³⁵S-labelled cRNA probes and All binding by autoradiography using ¹²⁵I[Sar¹,lle⁸]All in slide mounted hypothalamic sections. AT₁ receptor mRNA levels and All binding in the PVN were reduced by about 20% 18 h after adrenalectomy remaining at these levels up to 6 days after. This effect was prevented by corticosterone administration in the drinking water, or dexamethasone injection (100 mg, s.c., daily). Conversely, dexamethasone injection in intact rats caused a 20% increase in AT₁ receptor mRNA in the PVN. AT₁ receptor mRNA and binding in the PVN increased 4 h after exposure to stress paradigms associated with activation of the HPA axis (immobilization for 1 h, or i.p. injection of 1.5 M NaCl), and remained elevated after repeated daily stress for 14 days. Unexpectedly, two osmotic stress models associated with inhibition of the HPA axis (60 h water deprivation or 12 days of 2% saline intake) also resulted in increased AT₁ receptor mRNA levels and All binding in the parvicellular PVN. In intact rats, the stimulatory effect of acute stress on AT₁ receptor mRNA in the PVN was significantly enhanced by dexamethasone administration (100 μg, s.c., 14 h and 1 h prior to stress), while in adrenalectomized rats, with or without glucocorticoid replacement, stress reduced rather than increased, AT₁ receptor mRNA. Dexamethasone, 100 μg, injected sc within 1 min the beginning of immobilization in adrenalectomized rats, increased AT₁ receptor mRNA in the PVN to levels significantly higher than those after dexamethasone alone, indicating that the stress induced glucocorticoid surge is required for the stimulatory effect of stress on AT₁ receptor mRNA. The data suggest that AT₁ receptor expression in the PVN is under dual control during stress: stress-activated inhibitory pathways and the stimulatory effect of glucocorticoids. The lack of specificity of the changes in AT₁ receptor expression in the PVN following stressors with opposite effects on ACTH secretion (osmotic and physical-psychological stress) does not support a role for All as a major determinant of the response of the HPA axis during stress.     

Specific Binding of [H] phencyclidine in rat central nervous tissue: further characterization and technical considerations

The interaction of phencyclidine (PCP) with its specific receptor sites in the central nervous system has been further characterized. Kinetic association and dissociation rate constants of 2.9 × 10⁶ M⁻¹ and 4.8 × 10⁻¹ were determined, yielding a kinetic K_D of 1.6 × 10⁻⁷ M, in agreement with the K_D previously determined at equilibrium. Permissible separation time of 13 s was calculated from the kinetic data, well above the actual separation time of less than 10 s in the rapid filtration assay. Presoaking of filters in 0.01% poly-l-lysine eliminated displacable [³H]PCP adsorption to filter material. Binding data obtained via centrifigation assays was identical to that obtained with the rapid filtration method. Stereospecificity of the PCP receptor was demonstrated by the finding that (+)-ketamine is four-fold more potent than (−)-ketamine in displacing specifically bound [³H]PCP. Several proteolytic enzymes including trypsin, papain and thermolysin potently inactivated PCP receptors. Detailed regional distribution studies showed highest density of PCP receptors in subicular cortex and hippocampus, intermediate levels in hypothalamus, striatum, frontal cortex and cerebellum, lower levels in brainstem and spinal cord, and negligible levels in corpus callosum, a white- matter control area. Benzomorphan opiates with PCP-like behavioral effects interact with the PCP receptor. These data support the pharmacological relevance of the PCP receptor site as demonstrated by the rapid filtration method.