Quantitative autoradiography of 125I-[Sar1, Ile8]-angiotensin II binding in the brain of spontaneously hypertensive rats

The brain contains its own angiotensin II (AII) system. To better understand the role of central AII in cardiovascular regulation, we used 125I-[Sar1, Ile8]-AII (125I-SI-AII), radioactive AII antagonist, to autoradiographically localize putative AII receptor binding in many parts of the central nervous system of the spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. With 125I-SI-AII binding on brain membrane preparations. Scatchard analysis indicated that Kd values were from 0.10 +/- 0.04 nM to 0.13 +/- 0.05 nM, whereas Bmax values (femtomol/mg protein) were found to be from 6.95 +/- 1.60 to 15.52 +/- 4.99 among brain regions studied. Various SI-AII receptor binding activities among brain regions revealed in this study were therefore most likely due to differences in AII receptor density with high affinity binding of 125I-AII. Using 125I-SI-AII, specific binding for SI-AII was found in the nucleus tractus solitarius (NTS), paraventricular hypothalamic nucleus (PVN), subfornical organ (SFO), suprachiasmatic nucleus (SCN), area postrema, the dorsal motor nucleus of the vagus (DMX), and the nucleus of spinal tract of the trigeminal system (NSV). With quantitative receptor autoradiography in conjunction with radioactive standards, we have observed that the NTS possesses the highest SI-AII binding, followed by the PVN, SFO, NTS, DMX, and NSV. No significant differences were observed between the SHR and WKY rats in the SI-AII binding within the SFO, PVN and NTS. However, SHR at early hypertensive (7 weeks) and established hypertensive (16 weeks) stages contained significantly higher SI-AII bindings in the NSV, as compared to age-matched WKY rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative autoradiography of I-[Sar , Ile]-angiotensin II binding in the brain of spontaneously hypertensive rats

The brain contains its own angiotensin II (AII) system. To better understand the role of central AII in cardiovascular regulation, we used ¹²⁵I-[Sar¹, Ile⁸]-AII (¹²⁵I-SI-AII), radioactive AII antagonist, to autoradiographically localize putative AII receptor binding in many parts of the central nervous system of the spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. With ¹²⁵I-SI-AII binding on brain membrane preparations, Scatchard analysis indicated that K_d values were from 0.10±0.04 nM to 0.13±0.05 nM, whereas B_max values (femtomol/mg protein) were found to be from 6.95±1.60 to 15.52±4.99 among different brain regions studied. Various SI-AII receptor binding activities among brain regions revealed in this study were therefore most likely due to differences in AII receptor density with high affinity binding of ¹²⁵I-SI-AII. Using ¹²⁵I-SI-AII, specific binding for SI-AII was found in the nucleus tractus solitarius (NTS), paraventricular hypothalamic nucleus (PVN), subfornical organ (SFO), suprachiasmatic nucleus (SCN), area postrema, the dorsal motor nucleus of the vagus (DMX), and the nucleus of spinal tract of the trigeminal system (NSV). With quantitative receptor autoradiography in conjunction with radioactive standards, we have observed that the NTS possesses the highest SI-AII binding, followed by the PVN, SFO, NTS, DMX, and NSV. No significant differences were observed between the SHR and WKY rats in the SI-AII binding within the SFO, PVN and NTS. However, SHR at early hypertensive (7 weeks) and established hypertensive (16 weeks) stages contained significantly higher SI-AII bindings in the NSV, as compared to age-matched WKY rats. Furthermore, SHR rats also exhibited significantly higher SI-AII bindings in DMV at 16-week established hypertensive stage. It has been documented that the NSV and DMX are also abundant in opiate receptors. Therefore, AII in association with the opiate in the above two nuclei, may play important roles in the development and maintenance of hypertension in SHR rats.     

Characterization of two angiotensin II binding sites in cultured mouse spinal cord neurones

Characteristics of angiotensin II (AII) binding have been determined in cultured mouse spinal cord neurones using [125I]AII and [3H]AII. The Scatchard plot of equilibrium binding was curvilinear and could be described by postulating the existence of two different classes of independent binding sites (Kd1 = 0.43 nM, Bmax1 = 12.5 fmol/1.5 X 10(6) cells; Kd2 = 25.6 nM, Bmax2 = 220 fmol/1.5 X 10(6) cells). These values are in close agreement with the Kd values obtained from kinetic studies. The high affinity binding sites appeared to be similar to the single class of sites described in other studies. The relative inhibition potency of AII-related peptides was studied. Sar1,-Leu8-AII was the most potent in inhibiting specific AII binding. The characteristics of the two AII binding sites suggest that they correspond to two receptors as described in a previous electrophysiological approach using this model in our laboratory. Taken together, these data confirm that this model of neurones in primary culture is a unique and very attractive model of receptor studies. The classical criteria necessary for positive identification of a ligand-receptor have been satisfied: saturability, reversibility, specificity and most importantly correlation of the binding parameters and biological effects of AII.     

Central and peripheral vagal transport of cholecystokinin binding sites occurs in afferent fibers

The effects of various vagal lesions on cholecystokinin (CCK) binding sites in the nucleus tractus solitarii (NTS) and area postrema (AP) and the peripheral transport of CCK binding sites in the cervical vagus were examined in rats by in vitro autoradiography with [125I]CCK-8. Unilateral supraganglionic, but not subdiaphragmatic vagotomy significantly reduced CCK binding in the ipsilateral NTS. Specific unilateral afferent, but not efferent, vagal rootlet transections also significantly reduced NTS CCK binding ipsilateral to the transections. None of the vagal lesions altered CCK binding in the AP. Infraganglionic but not supraganglionic vagotomy eliminated the peripheral transport of vagal CCK binding sites. Together these results demonstrate that CCK receptors in the NTS are located on vagal afferent terminals, that CCK receptors in the AP are likely postsynaptic to a vagal afferent input and that the peripheral and central transport of vagal CCK binding sites occurs in afferent fibers.     

Localization and characterization of somatostatin binding sites in the mouse retina

We studied the binding of [125I]Tyr11-somatostatin-14 and [125I]Leu8,D-Trp22,Tyr25-somatostatin-28 to frozen, unfixed sections of C57BL/6J mouse eyes with autoradiography. Specific binding of both ligands occurred in 3 maxima, a broad band extending from the retinal ganglion cell to the inner nuclear layers, a narrow and inconstant band over the outer plexiform layer, and a band over the retinal pigment epithelium and choroid. We quantified the label over the inner plexiform layer and found evidence for a single, saturable binding site after Scatchard analysis of saturation binding data. With [125I]Tyr11-somatostatin-14 the dissociation constant (Kd) was 1.48 nM and the total number of binding sites (Bmax) was 68 fmol/mg protein; in competition experiments the inhibitory binding constant (Ki) was 900 pM for somatostatin-14 and 350 pM for somatostatin-28. With [125I]Leu8,D-Trp22,Tyr25-somatostatin-28, Kd was 625 pM and Bmax was 69 fmol/mg protein; in competition experiments Ki was 4.58 nM for somatostatin-14 and 710 pM for somatostatin-28. These results demonstrate the existence of somatostatin receptors in the inner plexiform layer of the retina that appear to have greater specificity for somatostatin-28 than for somatostatin-14.     

Alpha-1-adrenergic receptors in the nucleus tractus solitarii region of rats with experimental and genetic hypertension

The binding of the alpha 1-adrenergic receptors antagonist, 125I-HEAT, to membranes of nucleus tractus solitarii (NTS) regions of the brains of neurogenic hypertensive, spontaneously hypertensive (SH), and deoxycorticosterone (DOCA)/salt hypertensive rats and their respective controls was studied to quantitate the expression of alpha 1-adrenergic receptors. Scatchard analysis of the binding studies revealed a 1.8-fold increase in the Bmax of alpha 1-adrenergic receptors in NTS region membranes of SH rats when compared to their Wistar-Kyoto (WKY) control without significant difference in the Kd for 125I-HEAT. A decrease in the Kd with no difference in Bmax of receptors for 125I-HEAT was observed in the NTS region membranes of neurogenic hypertensive rats when compared with their sham-operated controls. In contrast, comparison of the Bmax and Kd values for 125I-HEAT binding in NTS region membrane of the DOCA/salt hypertensive rats and its sham-operated control showed no significant differences. We suggest that alterations in baroreceptor afferent activity may be related to alterations in central alpha 1-adrenergic receptors binding in SH rats and rats with neurogenic hypertension.     

VIP receptor subtypes in mouse cerebral cortex: evidence for a differential localization in astrocytes, microvessels and synaptosomal membranes.

The binding characteristics of a monoiodinated form of vasoactive intestinal peptide (M-[125I]VIP) to the membranes of astrocytes, intraparenchymal microvessels and synaptosomes were analyzed in mouse cerebral cortex. Binding to astrocytes, studied in primary cultures, indicates the presence of a single class of high affinity binding sites with a Kd of 3.3 nM and a Bmax of 565 fmol/mg protein. The structurally related peptide secretin does not compete for sites labeled by M-[125I]VIP. In cultured astrocytes, VIP has been previously shown to promote glycogenolysis. Secretin, despite its lack of interaction with sites labeled by M-[125I]VIP, stimulates glycogenolysis with an EC50 of 0.5 nM, thus demonstrating the presence in astrocytes of functional secretin receptors independent from those for VIP. Trypsinization of the primary astrocyte cultures followed by replating as secondary cultures, reveals a second class of low affinity binding sites, with a Kd of 41.3 nM and a Bmax of 881 fmol/mg protein. Secretin does not compete for this class of low affinity binding sites either. Binding of M-[125I]VIP to intraparenchymal microvessels reveals the presence of two classes of binding sites with Kd of 1.4 and 30.3 nM, and Bmax of 7.1 and 73.8 pmol/mg protein, respectively. Similar to what is observed in primary or secondary astrocyte cultures, secretin does not interact with these sites. In this cell type VIP stimulates cAMP formation with an EC50 of 18 nM, while secretin is ineffective. Finally, in agreement with previous reports in rat and guinea pig cerebral cortex, two classes of binding sites are observed in synaptosomal membranes: a high affinity class with a Kd of 4.9 nM and a Bmax of 316 fmol/mg protein, and a low affinity class with a Kd of 42.8 nM and a Bmax of 1578 fmol/mg protein. In contrast to what is observed in non-neuronal membranes, in synaptosomal membranes, secretin effectively competes for sites labeled by M-[125I]VIP with an EC50 of approximately 150 nM. These results indicate that secretin may represent a useful tool to discriminate between neuronal and non-neuronal VIP binding sites, since it competes with M-[125I]VIP exclusively for the neuronal class of binding sites.     

Characterization and measurement of [125I]iodopindolol binding in individual rat pineal glands: existence of a 24-h rhythm in beta-adrenergic receptor density

A simple procedure has been developed that permits measurement of beta-receptors in membrane preparations from individual rat pineal glands using [125I]iodopindolol ([125I]PIN). [125I]PIN binding to pineal membranes was stereospecific and saturable. Scatchard analysis of saturation isotherms yielded a Kd of 147.3 +/- 54 pM and a Bmax of 11.1 +/- 1.5 fmol/pineal gland. Binding was linear suggesting that [125I]PIN binds to a single population of pineal beta-adrenergic receptors. This procedure was used to evaluate 24-h variations in density of pineal [125I]PIN binding sites in male rats maintained in a 14:10 h light:dark cycle. Binding remained uniformly low during the daytime, increased slightly prior to lights off and peaked after 6 h of darkness decreasing abruptly 2 h later, before lights on. In animals maintained in light at night, the number of binding sites also increased, but did not exhibit the darkness-related decrease. The results demonstrate that beta-adrenergic receptors defined via [125I]PIN binding can be measured in tissue samples equivalent to less than one pineal gland. Moreover, the technique can be used in studies concerning the noradrenergic regulation of pineal function.     

Binding characteristics and daily rhythms of melatonin receptors are distinct in the retina and the brain areas of the European sea bass retina (Dicentrarchus labrax)

Melatonin is synthesized, with a circadian rhythm, in the pineal organ of vertebrates, high levels being produced during the scotophase and low levels during the photophase. The retina also produces melatonin, although in the case of the European sea bass, its secretion pattern appears to be inverted. In the study described here, radioreceptor assay techniques were used to characterize the melatonin binding sites, their regional distribution and their daily variations. Brain and retina membrane preparations were used in all the binding assays and 2-[125I]iodomelatonin ([125I]Mel) as radioligand at 25 degrees C. The specific binding of [125I]Mel was seen to be saturable, reversible, specific and of high affinity. In all the tissues assayed, the power of the ligands to inhibit [125I]Mel binding decreased in the following order: melatonin>4-P-PDOT>luzindole> or =N-acetylserotonin, which points to the presence of Mel1-like receptors. The inhibition curves of 4-P-PDOT suggested the presence of two different binding sites in the brain areas, but only one type of site of low affinity in the neural retina. No daily variations in [125I]Mel binding capacity (Bmax) or affinity (Kd) were detected in the brain areas, while a clear rhythm in Kd melatonin receptor affinity and Bmax binding capacity was observed in the retina. Kd and Bmax retinal rhythms were out of phase with the lowest Kd and the highest Bmax occurring at scotophase. This result suggests that retinal melatonin is a paracrine factor able to control receptor desensitization during photophase when ocular melatonin is higher in this species.     

Autoradiographic localization of 5HT2 receptors in rat brain using [125I]-DOI, a selective psychotomimetic radioligand

1. Binding sites for the R and S enantiomers of the 5HT2 agonist DOI (2,5-dimethoxy-4-iodophenylisopropylamine) were identified in rat brain using quantitative in-vitro autoradiography and compared with [125I]-LSD binding. 2. In most regions of the brain, binding density of the less active isomer [125I]S-DOI was 15 to 85% of that exhibited by the active [125I]R-DOI isomer. 3. Cortical membrane preparations exhibited two binding sites, of the enantiomers with high (KdH) and low (KdL) affinity constants of 1.2 +/- 0.02 nM and 29 +/- 7 nM for the [125I]R-DOI and 2.1 +/- 0.2 nM and 18 +/- 4 nM for [125I]S-DOI respectively. The respective high (BmaxH) and low (BmaxL) binding densities were 92 +/- 10 and 536 +/- 164 fmol/mg protein for the [125I]R-DOI and 67 +/- 19 and 245 +/- 60 fmol/mg protein for [125I]S-DOI. 4. Our results correlate with regional distribution of 5HT2 receptors reported in previous studies and indicate that DOI and its congeners have potential clinical applications for the in-vivo localization of 5HT2 receptors.     

125I]Iodomethyllycaconitine binds to alpha7 nicotinic acetylcholine receptors in monkey brain

We examined the binding of the novel nicotinic acetylcholine receptor (nAChR) ligand [125I]iodomethyllycaconitine (iodoMLA) in the brains of M. cynomologous (macaque) monkeys. [125I]iodoMLA bound throughout the brain with the greatest density in the thalamus and moderate intensity in the basal ganglia and cortical regions. The Kd and Bmax in whole brain tissue were similar whether 1 mM nicotine (Kd 33.25 +/- 15.17 nM, Bmax 5.80 +/- 1.06 fmol/mg) or 2 microM of the alpha7-selective antagonist alpha-bungarotoxin (Kd 46.12 +/- 18.45 nM, Bmax 6.30 +/- 1.06 fmol/mg) was used for nonspecific binding. The subtype-selectivity of this ligand was further studied with competition binding studies using nicotine, alpha-bungarotoxin and noniodinated MLA. Each ligand completely inhibited [125I]iodoMLA binding throughout the monkey brain, with Ki values of 2.23 +/- 0.85 microM for nicotine, 2.72 +/- 1.71 nM for alpha-bungarotoxin and 1.83 +/- 0.35 nM MLA in the caudate and 2.03 +/- 1.14 microM, 2.65 +/- 0.86 nM and 3.32 +/- 0.71 nM, respectively, in the putamen. The alpha3beta2/alpha6*-selective antagonist alpha-conotoxin MII failed to inhibit [125I]iodoMLA binding in any brain region. In monkeys with cognitive deficits resulting from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration, [125I]iodoMLA binding was significantly increased in the striatum, similar to results previously observed for [125I]alpha-bungarotoxin. These results suggest that, under the present experimental conditions, [125I]iodoMLA was selective for alpha7-containing nAChRs and did not bind to alpha6-containing nAChRs. This radioligand may be a useful tool for selectively imaging alpha7-containing nAChRs in vivo.     

Alpha 1-adrenergic receptors in the brain: characterization in astrocytic glial cultures and comparison with neuronal cultures

Binding of [125I]HEAT to membranes prepared from primary cultures of astrocytic glial cells was time-dependent and 70-85% specific. Various adrenergic agonists and antagonists competed for [125I]HEAT binding according to the potencies of prazosin greater than, yohimbine greater than or equal to, clonidine, norepinephrine (NE), and propranolol. Scatchard analysis showed the Bmax of 209 fmol/mg protein and a Kd of 184 pM for [125I]HEAT binding by astrocytic glial membranes. Pretreatment of astrocytes with NE resulted in a dose-dependent downregulation of [125I]HEAT binding sites with a maximal response observed after 8 h at 100 microM NE. Removal of NE from cultures after pretreatment resulted in a time- and protein synthesis-dependent recovery of binding sites to control levels within 120 h. Incubation of astrocytic glial cultures with NE stimulated phosphoinositide (PI) hydrolysis in a time- and dose-dependent manner with a maximal stimulation of 2-fold observed in 60 min by 100 microM NE. Clonidine expressed differential effects on alpha 1-adrenergic receptors of the neuronal and astrocytic glial cultures. Pretreatment with 10 microM clonidine caused a 40% decrease in the Bmax of [125I]HEAT binding without influencing the Kd value in neuronal cultures. This downregulatory effect of clonidine was associated with a reduction in the ability of NE to stimulate PI hydrolysis in clonidine pretreated cells. In contrast to neuronal cultures, clonidine neither downregulated [125I]HEAT binding sites nor stimulated PI hydrolysis in glial cultures.     

Autoradiographic distribution of multiple classes of opioid receptor binding sites in human forebrain

Receptor binding parameters and autoradiographic distribution of various opioid receptor sites have been investigated in normal human brain, post-mortem. [3H]DAGO, a highly selective mu ligand, binds to a single class of high affinity (Kd = 1.1 nM), low capacity (Bmax = 160 fmol/mg protein) sites in membrane preparations of frontal cortex. These sites show a ligand selectivity profile that resembles that of the mu opioid receptor. On the other hand, [3H]bremazocine, in presence of saturating concentrations of mu and delta blockers, appears to selectively bind to a single population of kappa opioid sites (Kd = 0.13 nM; Bmax = 93.0 fmol/mg protein) in human frontal cortex. Whole hemisphere in vitro receptor autoradiography reveals that [3H]DAGO-mu, [3H]DSLET-delta and [3H]bremazocine (plus blockers)-kappa binding sites are discretely and differentially distributed in human forebrain. In the cortex, mu sites are concentrated in laminae I and IV, delta sites in laminae I and II while kappa sites are found in deeper layers (laminae V and VI). In subcortical nuclei, high densities of mu and delta sites are seen in the caudate and putamen while high amounts of kappa sites are present in the claustrum and amygdala. The nucleus basalis of Meynert is enriched in all three classes of sites while the globus pallidus only contains moderate densities of kappa sites. Thus, the possible alterations of these various classes of opioid receptors in neurological and psychiatric diseases certainly deserve further investigation.     

Novel angiotensin II binding sites in the mesopontine area of the rat brain

Angiotensin II (AII) immunoreactivity in the mesopontine area of the rat brain is distributed through several areas where co-localization of AII receptors has not been established. The current in vitro receptor autoradiography study re-examined the distribution of AII binding using 125I-Sar1,Ile8-AII ([125I]SIAII). When incubations were conducted without sulfhydryl reducing agents, [125I]SIAII binding was observed in the locus coeruleus, inferior colliculus, superior colliculus and the central gray in agreement with previous reports. Novel [125I]SIAII binding sites were detected in the parabrachial nucleus, pedunculopontine tegmental nucleus and the caudal linear raphe nucleus, corresponding with previously reported localization of AII immunoreactivity in these nuclei. [125I]SIAII binding was also found in the paragenual nucleus where the peptide has not been detected. Thus, the observation of novel AII receptors which are sensitive to sulfhydryl reducing agents, resolves several AII-AII receptor mismatches.     

Expression of mineralocorticoid type I and glucocorticoid type II receptors in astrocyte glia as a function of time in culture.

In the present study we have examined the expression of mineralocorticoid Type I and glucocorticoid Type II receptors in astrocyte glia maintained in culture for different periods of time. Cytosolic mineralocorticoid Type I receptors were labeled with [3H]aldosterone (ALDO) in the presence of a 500-fold molar excess of the potent Type II receptor ligand RU 28362. [3H]Dexamethasone (DEX) was used to label cytosolic Type II receptors. Both Type I and Type II receptor binding was saturable in astrocyte glia that had been maintained in culture for 20 and 30 days following final plating (i.e. 20- and 30-day-old cultures). Scatchard analysis of [3H]ALDO binding revealed a single class of Type I receptors, with dissociation constants (Kd) of 0.45 +/- 0.13 nM and 0.53 +/- 0.07 nM, respectively, in 20- and 30-day-old cultures. The number of Type I receptors in 30-day-old cultures was nearly half that found in 20-day-old cultures (22.06 vs 42.64 fmol/mg protein). Linear Scatchard plots were also obtained for [3H]DEX binding to cytosol prepared from 20- and 30-day-old cultures. There were no significant differences in the Kd or Bmax values for [3H]DEX binding in 20- or 30-day-old cultures, i.e. 2.06 +/- 0.15 nM and 247.36 +/- 18.16 fmol/mg protein for 20-day-old cells and 2.3 +/- 0.74 nM and 261.02 +/- 3.08 fmol/mg protein for 30-day-old cells. These Bmax values are more than double the Bmax value for [3H]DEX binding observed in our previous studies in 10-day-old astrocyte glial cultures. Switching cultured astrocyte glial from serum-supplemented to serum-free medium had no significant effects on the Kd values of Type I or Type II receptors in all the cultures tested. However, treatment with serum-free medium increased the number of Type I receptors in 30-day-old cultures to a level similar to that found in 20-day-old cultures. Taken together, these binding data suggest that Type I and Type II receptors are expressed differently in astrocyte glia as a function of time in culture.     

Autoradiographic localization and biochemical characterization of peripheral type CCK receptors in rat CNS using highly selective nonpeptide CCK antagonists

Two potent and highly selective nonpeptide antagonists, L-365,031 [1-methyl-3-(4-bromobenzoyl)amino-5-phenyl-3H-1,4 benzodiazepin-2-one] and 3H-L-364,718 [1-methyl-3-(2-indoloyl)amino-5-phenyl-3H-1,4 benzodiazepin-2-one] were used to localize "peripheral" CCK receptors in rat brain. In autoradiographic experiments, L-365,031 displaced 125I-Bolton Hunter CCK-8 binding from the interpeduncular nucleus (IPN) (IC50 = 7 X 10(-8) M), the area postrema (AP), and the nucleus tractus solitarius (NTS) without influencing specific binding to other areas, such as the cerebral cortex or the spinal tract of the trigeminal nerve. Desulfated CCK preferentially inhibited 125I-CCK binding to cerebral cortex (IC50 = 7 X 10(-8) M) rather than IPN (IC50 greater than 1 X 10(-6) M) or AP-NTS. In the medulla the localization of 3H-L-364,718 binding was similar to L-365,031-sensitive 125I-CCK-8 binding and was found in the AP and medial, but not lateral, aspects of the NTS. In membranes prepared from IPN, NTS, and AP, 3H-364,718 binding was of high affinity (Kd = 0.14 nM), saturable (Bmax = 20 fmol/mg protein), and inhibited by compounds previously shown to act at pancreatic CCK receptors. The receptors labeled by 3H-364,718 were modulated by guanyl nucleotide, which reduced agonist affinity 10-fold without affecting antagonist binding. The localization and high density of CCK receptors in AP and NTS suggest that these receptors may play an important role in processing sensory afferent information.     

Twenty-four hour variation of alpha 1-adrenergic receptors in the pineal gland of the male Syrian hamster

Specific binding of [125I]iodo-[beta-(4-hydroxyphenyl)-ethylaminomethyl]tetralone ([ 125I]HEAT) was used to assess alpha 1-adrenergic receptors on pineal gland membranes of male Syrian hamsters (Mesocricetus auratus) housed under a 14:10 h light-dark cycle (lights on at 06.00 h). Saturation experiments with pooled pineal membrane preparations showed the presence of alpha 1-adrenergic receptor sites (dissociation constant Kd approx. 0.1 nM). Analysis of 4 time points indicated no significant change in Kd, but significant (P less than 0.01) changes of receptor density (Bmax) with a minimum recorded at night. Binding of a constant amount of [125I]HEAT (200 pM) to pineal membranes at 8 time points exhibited a circadian variation (P less than 0.001) of receptor density with lowest values around midnight and highest levels during daytime.     

Characterization of angiotensin II binding sites on neuroblastoma × glioma hybrid cells

Mouse neuroblastoma X rat glioma hybrid cells, NG108-15, have recently been shown to contain immunoreactive angiotensin II (AII). In the present study, we have examined this hybrid cell line for the presence of specific AII binding sites using [125I] AII. Specific AII binding was saturable and reversible. Scatchard analysis revealed a linear plot with an affinity constant (Kd) of 0.323 nM and a binding capacity (Bmax) of 7.13 fmol/mg protein. Kinetic studies demonstrated an association rate constant (K+1) of 3.55 X 10(6) M(-1) sec-1 and a dissociation rate constant (K-1) of 4.18 X 10(-4) sec-1. Displacement curves, using concentrations of 10(-11) M to 10(-4) M of unlabeled AII, revealed high and low affinity components of the AII binding site with IC50's of 0.46 nM and 1.75 microM respectively. The AII antagonist, saralasin, had approximately equal potency with unlabeled AII at the high affinity site. Furthermore, structurally related and unrelated peptides had no significant inhibitory effect on AII binding. This study demonstrates that specific AII binding sites are present on NG108-15 cells, and that these binding sites are similar in kinetic character to the AII receptor that has been previously identified in membranes from mammalian brain. It is concluded that the NG108-15 hybrid cells may provide a useful continuous cell line model for investigating both the biochemical and molecular properties of the AII binding site.     

Evidence for heterogeneity of muscarinic receptors in the mollusc Pleurobranchaea

The properties of the specific binding of the muscarinic antagonist [125I]3-quinuclidinyl-4-iodobenzilate ([125I]4IQNB] to nervous tissue of Pleurobranchaea california were characterized. The specific binding of [125I]4IQNB to Pleurobranchaea nervous tissue was characterized by its high affinity (Kd = 0.61 +/- 0.11 nM) and saturability (Bmax = 602 +/- 46 fmol/mg protein). A comparison of the numbers of binding sites recognized by [125I]4IQNB and l-[3H]QNB in nervous tissue of three invertebrate species indicated that in Aplysia and Cancer magister (crab) ganglia membranes the two radioligands labeled comparable numbers of binding sites; however, in Pleurobranchaea membranes l-[3H]QNB recognized only a subpopulation (8-10%) of the total number of [125I]4IQNB binding sites. The disparity in the numbers of binding sites labeled by these radioligands was consistent with our finding of a heterogeneity of muscarinic antagonist binding sites in l-QNB competition experiments in Pleurobranchaea. Computer-assisted analysis of l-QNB competition of [125I]4IQNB specific binding demonstrated that these data were best described by a two-site model with high- and low-affinity sites for l-QNB. The high-affinity site recognized by l-QNB possessed an IC50 value of 0.2 nM and comprised 18% of the total specific binding, while the lower affinity site had an IC50 value of 55.6 nM and comprised the remaining 82% of the total population of [125I]4IQNB recognition sites. The IC50 value for l-QNB at the high-affinity site in Pleurobranchaea membranes is in excellent agreement with Kd values for l-[3H]QNB labeling of classical muscarinic receptors in a variety of invertebrate and vertebrate species.     

Development of the rat pineal alpha 1-adrenoceptor

Pineal alpha 1-adrenoceptors in rats from 19 days of gestation until 11 months of age were studied using [125I]iodo-2-[beta-(4-hydroxyphenyl)ethylaminomethyl]tetralone ( [125I]HEAT). The number of specific [125I]HEAT binding sites increased markedly between 18 days of gestation (101.7 +/- 13.1 fmol/mg protein) and 10 days of age (336.2 +/- 34.3 fmol/mg protein). A significant decline occurred after 1 month of age. A saturation study showed similar changes in receptor density with age (Bmax; 20 days of gestation, 130.5 fmol/mg protein; 35 days old, 288.1 fmol/mg protein) but no difference in Kd (58.4 pM at both -1 and +35 days). The developmental appearance of the pineal alpha 1-adrenoceptor and the decline in its density with age are remarkably similar to changes reported for pineal beta-adrenoceptors.