Investigation of the interaction of VIP binding sites with VIP and PACAP in human brain.

We have compared the binding of [125I]vasoactive intestinal polypeptide (VIP) to human brain membranes with that of [125I]PACAP27. [125I]VIP was displaced by PACAP27, VIP and two synthetic peptides, peptide-1 (N-terminal PACAP27/C-terminal VIP) and peptide-2 (N-terminal VIP/C-terminal PACAP27), but the IC50 of PACAP27 and peptide-1 were 10-20 times lower than those of VIP and peptide-2. [125I]PACAP27 was readily displaced by PACAP27 and peptide-1, with an IC50 of less than 1 nM, but poorly by VIP and peptide-2. Chemical cross-linking revealed that both labels were bound to polypeptides of Mr 66,000 and Mr 50,000. The results indicate that in human brain membranes both binding sites have a higher affinity to the N-terminal sequence of PACAP27, and VIP binding sites prefer PACAP27 to VIP itself.     

Receptive field organization and response properties of visual neurons in the pigeon nucleus semilunaris

This study has demonstrated that the short and long form of the pituitary adenylate cyclase-activating polypeptide (PACAP), i.e. PACAP(27) and PACAP(38), moderately but significantly, and in a concentration (0.5-5 microM)-dependent manner, stimulated inositol phosphates (IPs) accumulation in myo-[(3)H]inositol-prelabeled cerebral cortical and hypothalamal slices of chick and duck, and in slices of rat cerebral cortex; both peptides had no effect on IPs formation in rat hypothalamus. Vasoactive intestinal peptide (VIP; 0.5-5 microM) weakly enhanced IPs accumulation in chick hypothalamus, had no significant action in chick cerebral cortex (in fact there was a tendency to attenuate the IPs response in this tissue), and slightly, but significantly, inhibited the IPs accumulation in rat cerebral cortex. VIP showed no activity in rat hypothalamus. It is concluded that the stimulatory action of PACAP on phosphoinositide metabolism in avian cerebral cortex, similar to rat cerebral cortex, is mediated via phospholipase C-linked PAC(1) type receptors. In chick hypothalamus, however, there may be a component of VPAC type receptors stimulating IPs formation.     

Characterization of beta-adrenergic receptors in duck cerebral cortex

beta-adrenoceptor binding sites were characterized in duck cerebral cortex by an in vitro binding technique, using [3H]dihydroalprenolol ([3H]DHA) as a receptor-specific radioligand. The specific binding of [3H]DHA to duck cerebral cortical membranes was found to be rapid, stable, saturable, reversible, and of high affinity. Saturation analysis resulted in a linear Scatchard plot suggesting binding to a single class of receptor binding sites with high affinity (Kd = 1.18 nM) and high capacity (Bmax = 162 fmol/mg protein). Competition studies showed the following relative rank order of potency of various compounds to inhibit the [3H]DHA binding: antagonists--ICI 118,551 > S(-)-propranolol > betaxolol, yohimbine, WB-4101, prazosin, mianserine; agonists--isoprenaline approximately equal to fenoterol > salbutamol > clonidine, phenylephrine. The obtained data suggest that in duck cerebral cortex beta-adrenergic receptors (like those described in brains of chick and pigeon) are of the beta 2 subtype. This is in contrast to what has been reported for the mammalian brain, where--among beta-adrenoceptors--the beta 1 subtype is predominant.     

Autoradiographic mapping of [mono[125I]iodo-Tyr10, MetO17]vasoactive intestinal peptide binding sites in the rat brain

The distribution of vasoactive intestinal peptide binding sites in the rat brain was examined by in vitro autoradiography on slide-mounted sections. A fully characterized monoiodinated form of vasoactive intestinal peptide (M-[125I]VIP) previously shown to maintain in the central nervous system the full biological activity of native vasoactive intestinal peptide was used for this study. In initial kinetic and pharmacological experiments the binding of M-[125I]vasoactive intestinal peptide to slide-mounted sections was shown to be time-dependent, saturable and reversible. Association of M-[125I]VIP specific binding was maximal within 90-120 minutes. Specific binding, corresponding to approximately 50% of total binding was saturable, of high affinity (Kd of 76.6 pM) and low capacity (fmol/mg prot range). Dissociation of M-[125I]VIP was maximal at 10 minutes. Unlabeled vasoactive intestinal peptide and the two structurally related peptides "peptide-histidine-isoleucine" (PHI) and secretin competed in a concentration-dependent manner for sites labeled by M-[125I]vasoactive intestinal peptide with the following rank order of potencies: vasoactive intestinal peptide greater than PHI greater than secretin. Vasoactive intestinal peptide receptors, as revealed by quantitative autoradiography, are present at various levels of the neuraxis. High densities were observed in olfactory bulb, cerebral cortex (highest in layers I, II, IV and VI), dentate gyrus, subiculum, various thalamic and hypothalamic nuclei, superior colliculus, locus coeruleus, area postrema, subependymal layer and pineal gland. Intermediate densities were found in the amygdala, nucleus accumbens, caudate-putamen, septum, bed nucleus of the stria terminalis, CA1 to CA4 fields of the hippocampus and central gray. No specific binding of M-[125I]vasoactive intestinal peptide was observed in white matter tracts such as corpus callosum, anterior commissure, medial forebrain bundle and fornix. The mapping of M-[125I]vasoactive intestinal peptide binding sites as revealed by autoradiography on slide-mounted sections indicates an association, although not exclusive, of vasoactive intestinal peptide receptors with brain regions involved in the processing of specific sensory inputs.     

125I]-omega conotoxin binding to human frontal cortex from normal, Alzheimer's and non-Alzheimer's dementia patients

We studied the binding of the calcium antagonist neurotoxin [125I]-omega conotoxin (GVIA) in age-matched human brains from normal, Alzheimer's disease and non-Alzheimer's dementia patients. Crude preparations of plasmalemmal membranes from frontal cortex were utilized. Saturation isotherms were subjected to Scatchard analysis to determine maximal binding capacity (Bmax) and binding affinity (Kd). In all brain samples tested, [125I]-GVIA binding was homogenous to a single class of high affinity binding sites. Scatchard analysis of saturation isotherms gave the following estimates for normal brains (mean +/- S.D., n = 7): Bmax = .630 +/- .200 pmol/mg and Kd = .177 +/- .054 nM. No significant change was observed in the Kd or Bmax estimates for [125I]-omega conotoxin binding in Alzheimer's disease or non-Alzheimer's dementia brains when compared to normal brains. Although these findings do not rule out the existence of localized changes in calcium channel receptor binding in the frontal cortex of Alzheimer's disease patients, the results do suggest that the neuronal voltage sensitive calcium channel may be unaltered in Alzheimer's disease.     

Adrenergic receptors in bovine retinal microvessels: presence of alpha 2- and beta- but not alpha 1-receptors

In bovine retinal microvessels, alpha 1, alpha 2- and beta-adrenergic receptors were characterized by binding assay, using [3H]prazosin, [3H]para-aminoclonidine and [125I]iodocyanopindolol as radioligands, respectively. The microvessels were purified from bovine eyes by differential centrifugation through a high concentration of bovine serum albumin followed by use of a glass bead filtration technique. In the preparation, specific binding sites for [3H]para-aminoclonidine and [125I]iodocyanopindolol were observed, whereas [3H]prazosin binding was not detected. The [3H]para-aminoclonidine binding sites localized to the microvessels were characterized by high affinity and saturability (KD: 173 +/- 9 pM; Bmax: 394 +/- 11 fmol/mg protein) as well as the [125I]iodocyanopindolol binding sites (KD: 20 +/- 3 pM; Bmax: 43 +/- 4 fmol/mg protein). Furthermore, the specificity of both binding sites was pharmacologically evaluated by measuring the inhibitory effects of various adrenergic reagents on binding. The existence of alpha 2- and beta-adrenergic receptors which were characterized by high affinity, saturability and stereospecificity, leads to the hypothesis that the retinal microcirculation is under neuronal control.     

Structure and function of the receptor for human atrial natriuretic peptide in cultured human skin fibroblasts

Cultured human skin fibroblasts possessed the high-affinity and low-capacity binding sites for [125I]alpha-human atrial natriuretic peptide (hANP), in which the dissociation constant and maximal binding capacity were computed to 68.7 +/- 11.3 pM and 7.3 +/- 1.2 fmols/mg protein, respectively, from Scatchard plot analysis. The specific [125I] alpha-hANP binding sites of cultured human fibroblast were displaced by unlabeled atriopeptin I, a truncated analogue, to the same extent as the case of alpha-hANP. In human adrenal membrane fractions, [125I] alpha-hANP binding sites were suppressed only by unlabeled alpha-hANP, while the high concentrations of atriopeptin I could slightly inhibit the binding sites for alpha-hANP. As it was reported that atriopeptin I had more significant affinity to the low-molecular weight ANP receptor (60-70 KD) than that to the high-molecular weight form (130-140 KD), the specific bindings may be attributed by the low-molecular weight ANP receptor in cultured human fibroblasts. Furthermore, alpha-hANP up to 10(-8)M failed to induce the significant cGMP formation in cultured human skin fibroblasts. The molecular weight of [125I]alpha-hANP binding sites of human fibroblasts was identified only at the region of 67 KD and no radioactive band was visualized around the region of large molecular weight ANP receptor in the SDS gel electrophoresis of a crosslinked [125I]alpha-hANP-receptor complex. In contrast, the affinity labeling of [125I]alpha-hANP to the human adrenal membrane fractions showed that 135 KD binding sites were responsible to the human adrenal ANP receptor. In conclusion, cultured human skin fibroblasts have a high-affinity low-capacity receptor for ANP. The molecular weight of ANP receptor is approximately 67 KD, and ANP-specific guanylate cyclase may not be linked to the receptor, suggestive that so-called C receptor may be localized in cultured human fibroblasts.     

Quantitative autoradiographic analysis of the distribution of binding sites for [125I]Bolton Hunter derivatives of eledoisin and substance P in the rat brain

[125I]Bolton and Hunter eledoisin binds to a single class of non-interacting sites in rat cerebral cortex tissue sections with an apparent Kd of 9.9 nM and a Bmax of 244 fmol/mg protein. When concentrations of up to 23 nM [125I]Bolton and Hunter eledoisin were used, [125I]Bolton and Hunter eledoisin binding was specific, saturable and reversible. Kassinin, eledoisin and neurokinin B were more potent than substance P and neurokinin A in inhibiting the specific binding of [125I]Bolton and Hunter eledoisin to cerebral cortex tissue sections. These kinetic and pharmacological characteristics are consistent with results obtained from binding studies on cortical synaptosomes. When the localization of [125I]Bolton and Hunter substance P and [125I]Bolton and Hunter eledoisin binding sites were compared, differences in many areas of the brain were noted. Large differences were seen in the paraventricular and supraoptic hypothalamic nuclei, and in layers IV and V of the cerebral cortex, which were densely labeled by [125I]Bolton and Hunter eledoisin, but not by [125I]Bolton and Hunter substance P. In contrast, nuclei of the septum (diagonal band of Broca, septohippocampal nucleus, dorsal part of the lateral septal nucleus), the rostrodorsal part of the hippocampus and other discrete nuclei [endopyriform nucleus, anterior cortical amygdaloid nucleus, the vermis columns (9-10), the dorsal tegmental nucleus, the hypoglossal and ambiguus nucleus] had high levels of [125I]Bolton and Hunter substance P binding but were only labeled weakly by [125I]Bolton and Hunter eledoisin. Thus, the two ligands seem to label different sites, since these binding sites have different biochemical and pharmacological properties, and are localized in different anatomical structures.     

The D1 dopamine receptor in the rat brain: quantitative autoradiographic localization using an iodinated ligand

The distribution of dopamine D1 receptors in the rat, labeled with [125I]SCH 23982, was studied using a quantitative in-vitro light-microscopic autoradiographic method. The binding of [125I]SCH 23982 to slide-mounted tissue sections and membrane preparations of prefrontal cortex was saturable, specific and of high affinity. Scatchard analysis revealed a Kd of 1.15 +/- 0.47 nM and Bmax of 8.76 +/- 0.34 fmol/mg tissue in prefrontal cortex membranes and a Kd of 1.27 +/- 0.14 nM and Bmax of 67.6 +/- 3.75 fmol/mg tissue in slide-mounted tissue sections at the level of the striatum. [125I]SCH 23982 was found to predominantly label D1 receptors, but a small fraction of the binding was to serotonin receptors. D1 receptors were found throughout the forebrain and were concentrated in the substantia nigra pars reticulata, accumbens nucleus, caudate putamen, entopeduncular nucleus, olfactory tubercle and the major island of Calleja. [125I]SCH 23982 binding to serotonin receptors was concentrated in the cortices, dorsal raphe, central gray, anterior hypothalamic area and the molecular cell layer of the cerebellum. Knowledge of the distribution of D1 receptors may increase our understanding of the role of D1 receptors in central nervous system dopaminergic function. Furthermore, data on the potential sites of interaction of [125I]SCH 23982 with serotonin receptors may help to understand the complex physiology and pharmacology of the primarily D1 selective compound.     

125I]iodomelatonin binding sites in spleens of birds and mammals.

The specific binding of [125I]iodomelatonin to duck spleen membrane preparations was studied in detail. These binding sites were stable, saturable, reversible and of high affinity. Scatchard analysis of the binding revealed a equilibrium binding constant (Kd) of 73.1 +/- 5.4 pM and a total number of binding sites (Bmax) of 3.64 +/- 1.38 fmol/mg protein. Studies on the relative binding capacities of [125I]iodomelatonin to the spleen in different species showed the following order: duck greater than chicken greater than guinea pig greater than pigeon greater than mouse. No binding site was detected in the rat spleen. The presence of [125I]iodomelatonin binding sites in the spleen of birds and mammals suggested a direct action of pineal melatonin on the immune system.     

Differential distribution of somatostatin receptor subtypes in rat brain revealed by newly developed somatostatin analogs.

Somatostatin receptor subtypes were labeled with the somatostatin analogs [125I]CGP 23996 and [125I]MK 678 and the distribution of these receptors in rat brain was investigated using quantitative autoradiographic techniques. [125I]CGP 23996 and [125I]MK 678 specifically label different populations of somatostatin receptors in rat brain. In a number of brain regions striking differences in the distribution of the somatostatin receptor subtypes labeled by each peptide were observed. High levels of binding sites for both [125I]CGP 23996 and [125I]MK 678 were present in the cerebral cortex, CA1 region and subiculum of the hippocampus. In contrast, high levels of [125I]MK 678 binding were found in the dentate gyrus of the hippocampus while few [125I]CGP 23996 binding sites were observed in this brain region. [125I]CGP 23996 binding was detected in the central region of the interpeduncular nucleus whereas the dorsal and lateral subnuclei of this brain area expressed mainly somatostatin receptors with high affinity for MK 678. The locus coeruleus and regions of the superior colliculus and hypothalamus selectively express [125I]MK 678-sensitive somatostatin receptors. Furthermore, limbic structures such as the lateral septum, the nucleus accumbens and ventromedial striatum had much higher levels of [125I]MK 678 binding sites than [125I]CGP 23996 binding sites. Differences in the expression of the somatostatin receptor subtypes were also detected in the substantia nigra. [125I]CGP 23996 binding was present in the pars reticulata but not the pars compacta whereas the reverse distribution for [125I]MK 678 binding sites was observed. The differential distribution of [125I]CGP 23996 and [125I]MK 678 binding sites in rat brain supports the hypothesis that these peptides selectively label different somatostatin receptor subtypes in the central nervous system.     

Localization of binding sites for calcitonin gene-related peptide in rat brain by in vitro autoradiography

The distribution of binding sites for calcitonin gene-related peptide (CGRP) in rat brain were studied using in vitro autoradiography. In a radioreceptor assay using [125I]human calcitonin gene-related peptide as the radioligand, with cerebellar cortical membranes, rat calcitonin gene-related peptide had a binding affinity constant of 1.16 +/- 0.23 X 10(10) M-1 and a site concentration of 43.4 +/- 3.4 fmol/mg protein. In this system, human calcitonin gene-related peptide had a binding affinity constant of 3.9 +/- 0.7 X 10(9) M-1 whereas salmon calcitonin was very weak with a binding affinity constant of only 6.8 +/- 4.0 X 10(5) M-1. CGRP binding localized by in vitro autoradiography, using [125I]rat calcitonin gene-related peptide, had a characteristic distinct distribution in the rat brain. There were high concentrations of binding found over the accumbens nucleus, the organum vasculosum of the lamina terminalis, ventral caudate putamen, median eminence, the arcuate nucleus, lateral amygdaloid nucleus and lateral mammillary nucleus, the superior and inferior colliculi, pontine nuclei, molecular and Purkinje cell layers of the cerebellar cortex, the nucleus of the solitary tract, the inferior olivary nuclei, hypoglossal complex and the vestibular and cochlear nuclei. The distribution of these binding sites suggests multiple roles for CGRP in the central nervous system including auditory, visual, gustatory and somatosensory processing, and in neuroendocrine control.     

Calcitonin gene-related peptide (CGRP) receptors are linked to cyclic adenosine monophosphate production in SK-N-MC human neuroblastoma cells

Calcitonin gene-related peptide (CGRP) stimulated cyclic adenosine monophosphate (cAMP) levels in SK-N-MC human neuroblastoma cells in a time- and concentration-dependent manner. The efficacy order for CGRPs was human alpha-CGRP = human beta-CGRP = chick CGRP greater than rat CGRP greater than human [Tyr0]CGRP. Calcitonin (CT) failed to influence cAMP production in SK-N-MC cells. [Tyr0]CGRP27-37 which by itself did not affect cAMP levels antagonized CGRP action. Saturation analysis using [125I]CGRP showed a homogeneous population of binding sites. CGRP but not CT, vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) inhibited radioligand binding. Our results provide evidence that human neuroblastoma SK-N-MC cells contain highly specific CGRP receptors which are positively coupled to cAMP generation.     

Alpha- and beta-adrenergic receptors of the rat cerebral cortex and cerebral microvessels in aging, and their response to denervation.

We studied the effects of aging and norepinephrine depletion 2 weeks after unilateral locus ceruleus lesion on alpha 1-, alpha 2- and beta-adrenergic receptors by ligand binding methods in the ipsilateral and contralateral cerebral cortex of Fischer-344 rats. We also studied the effects of aging and noradrenergic denervation on beta-adrenergic receptors in isolated cerebral microvessels. We found that specific [125I]HEAT binding to alpha 1-adrenergic receptors was not affected by aging or by norepinephrine depletion. Although aging also had no effect on the density or affinity of [3H]UK-14,304 and [125I]pindolol binding to alpha 2- and beta-adrenergic receptors, the density of receptors increased significantly in all age groups after noradrenergic denervation. beta-Adrenergic receptors of cerebral microvessels also were unaffected by aging, but increased their density after noradrenergic denervation at all ages. In all instances, there were no significant effects on the affinity of ligand binding. We conclude that aging does not affect the density or the affinity of adrenergic receptors in the cerebral cortex of Fischer-344 rats, nor does it affect the response of these receptors to norepinephrine depletion.     

Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on cyclic AMP formation in the duck and goose brain

Two molecular forms of pituitary adenylate cyclase-activating polypeptide (PACAP), i.e., PACAP27 and PACAP38 (0.0001-1 microM), as well as vasoactive intestinal polypeptide (VIP; 0.1-3 microM), have been studied for their effects on cyclic AMP formation in the hypothalamus and cerebral cortex of duck and goose. All three peptides concentration-dependently stimulated cyclic AMP production in the tested brain regions of 2-3-weeks-old (young) ducks, with VIP showing at least one order of magnitude weaker activity than PACAP. This characteristics suggests the existence in the duck's brain of adenylyl cyclase-linked PAC1 receptors. Both forms of PACAP also stimulated the nucleotide formation in the cerebral cortex and hypothalamus of 5-6-months-old (adult) ducks or geese grown under natural environment. The peptides-evoked effects in adult and young ducks were comparable, and clearly greater than those found in adult geese. The present data extend our recent observations made on chicks, and suggest PACAP to be a potent stimulator of the cyclic AMP generation in the avian central nervous system.     

Thymopoietin, a polypeptide ligand for the alpha-bungarotoxin binding site in brain: an autoradiographic study.

Thymopoietin, a 48-49-amino acid polypeptide present in the thymus gland, was investigated as a potential ligand for the neuronal nicotinic alpha-bungarotoxin binding site in rat brain. Binding of [125I]alpha-bungarotoxin to whole rat brain sections was inhibited by thymopoietin in a concentration-dependent manner with an IC50 of 30.0 +/- 8.2 nM as compared to 1.1 +/- 0.3 nM for alpha-bungarotoxin. However, at concentrations of thymopoietin of up to 1 microM, [3H]nicotine binding to high affinity sites was not inhibited. Thysplenin, a polypeptide with considerable homology to thymopoietin did not affect [125I]alpha-bungarotoxin binding. These results suggest that thymopoietin selectively interacts with the nicotinic alpha-bungarotoxin binding site labelled by [125I]alpha-bungarotoxin rather than the neuronal nicotinic receptor(s) labelled by [3H]nicotine. Autoradiographic studies revealed that 1 microM thymopoietin almost completely inhibited [125I]alpha-bungarotoxin binding in all brain regions. Computer-assisted image analysis of displacement curves was performed on various brain areas rich in alpha-bungarotoxin binding, such as the dorsal endopiriform nucleus, fields 1 and 2 of Ammon's horn, the polymorph cell layer of the dentate gyrus and cortical layers 4 and 5. Thymopoietin inhibited [125I]alpha-bungarotoxin binding with similar potency in all these regions, suggesting that it interacted at the same site in the different brain areas. The IC50 values averaged over the six regions were 24.6 +/- 2.8 nM for thymopoietin and 1.2 +/- 0.2 nM for alpha-bungarotoxin. These results show that thymopoietin specifically interacted with the alpha-bungarotoxin site with a similar potency in different brain regions. It is suggested that thymopoietin represents a selective ligand for alpha-bungarotoxin binding sites in brain.     

Prostaglandin E2 binding sites in human renal tissue: characterization and localization by radioligand binding and autoradiography

The prostaglandin E2 (PGE2) binding site in human kidney was characterized in membrane preparations from cortex, outer medulla and inner medulla using radioligand binding techniques. The localization of the binding sites for [3H]PGE2 was visualized autoradiographically. In the membrane suspensions, the highest level of specific [3H]PGE2 binding was detected in the outer medulla (Bmax = 335 +/- 28 fmol mg-1 protein) followed by the inner medulla (Bmax = 258 +/- 21 fmol mg-1 protein) and the cortex (Bmax = 143 +/- 22 fmol mg-1 protein). The binding was of high affinity with KD values between 3.7 and 6.2 nM in the various regions. Unlabelled prostaglandins competed for the [3H]PGE2 binding sites in the following rank order of potency: PGE2 approximately PGE1 greater than PGF2 alpha approximately PGA2 greater than PGB2 greater than PGI2 approximately PGD2. Autoradiographs revealed that a high density of [3H]PGE2 (2 nM) binding sites were located on the distal tubule, particularly on the thick ascending limbs of Henle. Lower densities of [3H]PGE2 binding sites were found on the medullary collecting ducts and possibly on the thin loops of Henle. In contrast, no specific [3H]PGE2 binding could be found on the proximal tubule, glomeruli or on blood vessels. This distribution is in accordance with the assumed site of action for the salt and water regulatory function of PGE2.     

Reconstitution of the purified gamma-aminobutyric acid-benzodiazepine receptor complex from bovine cerebral cortex into phospholipid vesicles

The purified gamma-aminobutyric acid-benzodiazepine receptor complex from bovine cerebral cortex has been reconstituted into phospholipid vesicles by a cholate dialysis procedure. The reconstituted receptor bound [3H]flunitrazepam at a single class of sites with dissociation constant Kd = 2.9 +/- 0.3 nM, an increase in affinity to the membrane level from the 4-fold weakening found in detergent solution. It also bound [3H]muscimol with a Kd for the high-affinity sites of approximately 50 nM. [35S]tert.-Butyl-bicyclophosphorothionate, for which there is evidence in membranes for binding to a channel gating site on this receptor, showed similar binding to the reconstituted receptor.     

Specific atrial natriuretic peptide binding sites in rat cerebral capillaries

We examined the specific rat 125I-alpha-rat atrial natriuretic peptide(1-28)[ANF-(99-126)] (125I-rANP) binding sites in the cerebral capillaries from the cerebral cortex of male adult Wistar rats. The binding of 125I-rANP at 37 degrees C was saturable and of high affinity with a Kd of approximately 100 pM and Bmax of 152 fmol/mg protein. Divalent cations, Mn2+ (2.2 mM) and Ca2+ (1.8 mM) potently inhibited the binding. The rank order for inhibition of the binding was rANP, alpha-human ANP and ANF-(101-126) greater than ANF-(103-126) and ANF-(103-125)"ANF-(103-123). These data on specific binding sites of ANP in cerebral capillaries suggest a possible role for ANP in the blood-brain permeability of water and electrolytes.