Amylin compared with calcitonin: competitive binding studies in rat brain and antinociceptive activity

Binding studies for rat amylin (AMY) and salmon calcitonin (sCT) were performed on rat membranes prepared from pons and medulla oblongata of rats. The aim was to see whether specific binding sites for AMY and/or for sCT present in these areas could be relevant to some of the biological activities of the two peptides. Binding sites specific for [125I]AMY are present in the pons-medulla of rat brain as AMY, but not sCT, was able to displace radiolabeled AMY binding with an IC50 = 3.7+/-0.5x10(-10) M. In contrast, binding of [125I]sCT was displaced by both sCT and AMY, although with different potencies, the IC50 for sCT being 1+/-0.1x10(-11) M, and for AMY, 1.8+/-0.08x10(-7) M. The functional significance of the presence of these binding sites was evaluated in two different nociceptive tests, hot-plate and tail-flick. In the tail-flick test neither AMY (5-10 microg/rat, i.c.v.) nor sCT (10 microg/rat i.c.v.) showed antinociceptive activity, whereas in the hot-plate test AMY (10 microg/rat, i.c.v.) significantly increased the response latencies as did sCT (250 ng/rat, i.c.v.). These results demonstrated that a 40-fold greater dose of AMY is necessary to produce a comparable antinociceptive effect to that exerted by sCT. These findings are in accordance with the low affinity of AMY for sCT binding sites in rat pons-medulla. It is therefore suggested that the central inhibitory activity of AMY on pain perception involves interaction with sCT receptors whereas the selective AMY binding sites subserve other (as yet unknown) functions.     

Calcitonin gene-related peptide (hCGRP alpha) binding sites in the nucleus accumbens. Atypical structural requirements and marked phylogenic differences.

The distribution of [125I]hCGRP alpha binding sites was studied in tissue sections from rat brain and, at the level of the nucleus accumbens in the brains of 6 other species. In the rat, very high levels of binding were found in the nucleus accumbens, the amygdaloid complex and mammillary body while high amounts were localized to the superficial layers of the superior colliculus, temporal cortex, cerebellum (molecular layer), frontal cortex and inferior olive. Moderate densities of [125I]hCGRP alpha binding were observed in the medial geniculate nucleus, inferior colliculus and substantia nigra. Regional competition studies in rat brain showed that salmon calcitonin was almost as effective as hCGRP alpha in competing for [125I]hCGRP alpha binding sites in the nucleus accumbens but was mostly inactive in other regions such as the mesolimbic cortex and the striatum. On the basis of their atypical sensitivity to salmon calcitonin, [125I]hCGRP alpha binding sites in the rat nucleus accumbens, which appear between postnatal days 4 and 7, do not seem to correspond to either the CGRP1 or CGRP2 receptor subtypes. Marked species differences were observed in the distribution of [125I]hCGRP alpha binding sites, especially in the nucleus accumbens. In the mouse, low densities of hCGRP alpha sites were observed in striatum and fronto-parietal cortex while low to moderate levels were found in the medial and posterior aspects of the nucleus accumbens. A similar distribution was seen in the guinea pig brain albeit of generally higher density. In the rat, very high amounts of [125I]hCGRP alpha binding were seen in the nucleus accumbens while lower levels were found in the striatum and certain cortical areas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinct binding sites for calcitonin gene-related peptide and salmon calcitonin in rat central nervous system

Calcitonin gene-related peptide (CGRP) binding sites have been identified in homogenates from the rat brain and spinal cord. Autoradiography with [¹²⁵I]rat CGRP (rCGRP) revealed high grain density over the lateral hypothalamus, vestibular nuclei, colliculi, medial geniculate body, corpus mamillare and the molecular layer of the cerebellum which lacked binding sites for [¹²⁵I]salmon calcitonin (sCT). In contrast, no rCGRP labeling was seen over the anterior and dorsomedial hypothalamus which showed high sCT binding. The different regional distribution of rCGRP and sCT binding sites indicates that the structurally related peptides interact with separate receptors. The overlap between the localization of CGRP binding sites and endogenous CGRP in many regions of the central nervous system suggests that CGRP exerts unique physiological functions in the central nervous system.     

Quantitative autoradiographic distribution of calcitonin gene-related peptide (hCGRP alpha) binding sites in the rat and monkey spinal cord.

Calcitonin gene-related peptide (CGRP) has been implicated in various spinal functions on the basis of its presence in the substantia gelatinosa and motoneurons and the biological effects induced by intrathecal CGRP injections. We investigated here the comparative distribution of [125I]hCGRP alpha binding sites in various segments of the rat and monkey spinal cord. The immunocytochemical localization of CGRP-like material in rat spinal cord was also evaluated for comparison. In the rat spinal cord, high densities of [125I]hCGRP alpha binding sites were observed in lamina I, in a U-shaped band that included lamina X and the medial parts of laminae III-IV and in the intermediolateral and intermediomedial nuclei. The substantia gelatinosa (lamina II) contained relatively lower, but still significant, densities of [125I]hCGRP alpha binding sites, while the ventral horn showed low amounts of specific labeling. CGRP-like immunoreactive fibers, on the other hand, were heavily concentrated in laminae I-II and in the reticulated portion of lamina V of the dorsal horn. Immunoreactivity to CGRP antiserum was also noted in fibers around the central canal and in a number of motoneurons of the ventral horn. In the monkey spinal cord, [125I]hCGRP alpha binding sites were present in lamina I in a U-shaped band that included lamina X and the medial portions of laminae V-VI. Relatively low levels of [125I]hCGRP alpha binding were detected in laminae II to IV of the dorsal horn, while the ventral horn was more enriched with specific [125I]hCGRP alpha binding sites. Thus, it appears that the autoradiographic distribution of [125I]hCGRP alpha sites is species dependent in the spinal cord. Additionally, some differences are observed between the localization of [125I]hCGRP alpha binding sites and immunoreactive material in the rat spinal cord. These differences may be relevant to the purported roles of CGRP-like peptides in spinal functions such as nociception, control of sympathetic output, and motor control.     

Comparison of binding sites for the calcitonin gene-related peptides I and II in man

Specific and saturable binding sites of the synthetic human calcitonin gene-related peptide-I (CGRP-I) and -II (CGRP-II) have been identified in membrane homogenates of the human central nervous system, heart and spleen. Half maximal inhibition of the binding of¹²⁵I-CGRP-II was achieved with 0.25 nM and 1.7 nM of non-radioactive CGRP-I or -II, and with 260 and 850 nM salmon calcitonin in the spinal cord and cerebellum, respectively, but 1 μM human calcitonin was not recognized. Autoradiographs of diencephalic sections revealed different distribution of¹²⁵I-CGRP-I and -II binding sites.     

Biologically active salmon calcitonin-like peptide is present in rat brain.

Although high densities of calcitonin (CT) receptors and potent CT-induced actions occur within the rat central nervous system, the physiological ligand for central receptors remains unidentified. This study identifies a salmon CT (sCT)-like peptide in extracts of rat brain. Aliquots of 1 M HCl brain extracts were reconstituted in 0.01 M acetic acid and assayed for sCT-like immunoreactivity using a radioimmunoassay (RIA) based on a guinea pig anti-sCT antibody (detection limit 0.1 ng). The assay was less than 0.1% cross-reactive with human CT, porcine CT, rat calcitonin gene-related peptide (CGRP)-I and human CGRP-I. Extracts of diencephalon revealed sCT-like activity (30-120 ng/g wet weight), while extracts from cortex, cerebellum and medulla oblongata did not contain detectable activity. Reverse-phase HPLC analysis demonstrated co-elution with synthetic sCT. Further, the peak material was active in 4 CT bioassay systems; (1) stimulation of cAMP production in UMR 106-06 cells and (2) T47D cells, (3) inhibition of 125I-sCT binding in UMR 106-06 cells and (4) inhibition of 125I-sCT binding in sheep brain membranes. The bioactivity of material in these assays was similar to that estimated through the RIA. The results identify, for the first time, the presence of sCT-like material in the rat brain and constitutes the first demonstration of biologically active sCT-like material from brain extracts. Salmon CT-like peptide may represent the endogenous ligand for central CT receptors.     

Characterization of the calcitonin gene-related peptide receptor in mouse T lymphocytes

We have identified and characterized specific binding for calcitonin gene-related peptide (CGRP) in mouse T lymphocytes. The binding of 125I-CGRP to mouse lymphocytes was reversible and the rate of dissociation of 125I-CGRP increased with the addition of the guanine triphosphate nucleotide analog, Gpp(NH)p. Saturation experiments, and Scatchard analysis indicated two classes of binding sites for CGRP; the apparent dissociation constants (KD) were 3.5 X 10(-10)M for high affinity binding sites (Bmax, 265 sites/cell) and 4.8 X 10(-8)M for low affinity binding sites (Bmax, 13,000 sites/cell). The KD value for the high affinity binding sites is roughly comparable to the IC50 and ED50 values for inhibition of T lymphocyte proliferation and increase in the cyclic AMP concentration in these cells, respectively. 125I-CGRP bound to mouse T lymphocytes was displaced by unlabeled CGRP with an IC50 value of 3.2 X 10(-10)M; salmon or human calcitonins did not inhibit the specific binding up to 1 X 10(-6)m. Our studies suggest that CGRP may be an important immunoregulatory molecule, and implicate it in the regulation of T cell function.     

Altered Calcitonin Gene-related Peptide, Substance P and Enkephalin Immunoreactivities and Receptor Binding Sites in the Dorsal Spinal Cord of the Polyarthritic Rat

The dorsal horn of the spinal cord, which forms the locus of first synapses in pain pathways, is an important site of interaction between calcitonin gene-related peptide (CGRP), substance P and enkephalin—the neuropeptides considered to be especially involved in the regulation of pain perception. Since adjuvant-induced arthritic rats provide a suitable model for peripheral inflammation and hyperalgesia, the possible alterations of immunoreactive CGRP, substance P and enkephalin as well as the binding sites for [¹²⁵I]hCGRPα, [¹²⁵I]substance Plneurokinin-1, (NK1) and [¹²⁵I]FK-33-824/μ-opioid receptors were studied in the dorsal horn of the spinal cord receiving projections from the inflamed limbs. In arthritic rats compared to control animals, a bilateral increase in CGRP- and substance P-immunoreactive fibres and the presence of enkephalin-immunoreactive cell bodies were noted in the dorsal horn of the spinal cord. As for receptors, while a significant decrease in [¹²⁵I]hCGRPα and [¹²⁵I]substance P/NK1 binding sites was observed in selective layers, no measurable alteration in [¹²⁵I]FK-33-824/μ-opioid binding sites was noted in any regions of the arthritic rat dorsal horn compared to the unaffected control rats. Following unilateral section of the peripheral nerve prior to induction of arthritis, CGRP- and substance P-immunoreactive fibres were markedly depleted and no enkephalin-positive neurons were observed in the ipsilateral dorsal horn. Analysis of receptor binding sites in denervated arthritic rats, however, exhibited differential responses, i.e. a significant increase in [¹²⁵I]hCGRPα, a marked decrease in [¹²⁵I]FK-33-824/μ-opioid and apparently no alteration in [¹²⁵I]substance P/NK1 receptor binding sites were observed in the ipsilateral dorsal horn compared to the intact contralateral side. These results taken together provide anatomical evidence for a concerted role of these peptides in the regulation of adjuvant-induced hyperalgesia accompanying peripheral inflammation.     

Characterization of calcitonin gene-related peptide receptors and adenylate cyclase response in the murine macrophage cell line P388 D1.

Specific receptors for calcitonin gene-related peptide (CGRP) were identified and characterized on plasma membranes from the interleukin-1 secreting murine macrophage-like cells line P388 D1. The binding of [125I]-rat CGRP I was time-dependent, reversible and the rate of dissociation of [125I]-rat CGRP I increased in the presence of GTP. Scatchard analysis was consistent with a single class of binding sites, with an apparent dissociation constant of 1.76 nM and a maximal binding capacity of 85.48 fmol/mg protein. In competitive displacement studies, rat CGRP I, human CGRP I and human CGRP II were equipotent to inhibit the binding of [125I]-rat CGRP I (IC50 = 4 nM) while rat CGRP II and the synthetic analogue [tyr(o)]-human CGRP I were ten-fold less potent. Porcine calcitonin and VIP did not inhibit tracer binding. In the presence of GTP, CGRP stimulation of adenylate cyclase was dose-dependent and strongly correlated with receptor occupation. These results indicate that the P388 D1 macrophage-like cell line expresses CGRP specific receptors functionally coupled to adenylate cyclase, which may be involved in CGRP-mediated macrophage immune response.     

Changes in [125I]hCGRP binding in rat spinal cord in an experimental model of acute, peripheral inflammation.

[125I]Human calcitonin gene-related peptide ([125I]hCGRP) binding in the dorsal horn of the spinal cord exhibited differential changes among laminae over time in response to unilateral adjuvant-induced inflammation. In laminae I/II, 4 days after induction of inflammation, the binding decreased 36% on the side of the spinal cord ipsilateral to the inflammation, while there was no change on the contralateral side. The decrease ipsilateral to inflammation was due primarily to a decrease in the Bmax of the high affinity binding site for CGRP. In lamina V, the binding increased 18% on both sides of the spinal cord at the same time point. In lamina X, the binding increased 16% on both sides of the spinal cord at 2 days after induction of inflammation and remained increased at 8 days. The increases in [125]hCGRP binding in laminae V and X were primarily due to a decrease in the Kd of the low affinity binding site for CGRP. the accompanying hyperalgesia was first measured at 2 days after induction of inflammation and persisted at 8 days. Because the changes in [125I]hCGRP binding did not parallel the hyperalgesia accompanying the unilateral adjuvant-induced inflammation, we believe that CGRP receptors are not directly involved with the hyperalgesia but may be involved with other plastic changes observed in the spinal cord during unilateral adjuvant-induced inflammation.     

In vitro autoradiographic localization of calcitonin binding sites in human medulla oblongata

In this study we examined the distribution of calcitonin (CT) binding sites in the human medulla oblongata by in vitro autoradiography. In competition studies, the rank order of potency for calcitonins competing for ¹²⁵I-salmon CT binding was salmon CT > porcine CT > human CT, which is consistent with physiologically relevant CT receptors in other systems. For the determination of CT binding in the human medulla, 20-μm cryostat sections were incubated with ¹²⁵I-salmon CT in the presence or absence of 10^?6 M unlabelled salmon CT to map specific CT binding sites. Punctate binding was observed over discrete nuclei of the medulla. High binding densities were seen over subnuclei of the dorsal motor nucleus of the vagus, the nucleus of the solitary tract, the intermediate reticular zone, the gigantocellular and dorsal paragigantocellular nuclei, and the raphe obscurus nucleus. Moderate levels of binding were observed over the lateral paragigantocellular nucleus and the rostral extent of the epiolivary nucleus. The cuneate and gracile nuclei and the fiber tracts did not contain detectable binding, while the inferior olivary nucleus had moderate levels of nonspecific binding. The localization of calcitonin binding sites in the human presents similarities but also important differences to the distribution in rat and cat. The most notable difference is the extreme binding densities in the intermediate reticular zone of the human. The location of binding sites suggests involvement of calcitonin in regulation of autonomic function.     

Rat brain cells in primary culture: Characterization of angiotensin II binding sites

The binding kinetics of angiotensin II (ANG II) have been studied in primary cultures from fetal rat brain. Binding of [125I]ANG II to rat brain cells in culture is time-, pH- and cell concentration-dependent. The binding is saturable, reversible, and 90--95% specific. Binding follows first-order kinetics, with values for K1 and K-1 of 4.9 x 10(6)M-1 S-1 and 3.33 x 10(4)S-1 respectively. Scatchard analysis reveals the presence of a single class of binding sites with Ka of 1.0 x 10(9)M-1 and an average of approximately 6 x 10(3) sites per cell. [125I]ANG II recovered from incubation medium under the conditions of the binding assay or after dissociation from cells is not significantly degraded as judged by gel filtration on Sephadex G-25 and radioreceptor assay. ANG II analogs compete with [125I]ANG II for binding, with potencies in general paralleling previously established biological activities. Of 5 analogs tested, (Ile8)-ANG II was almost equipotent with ANG II while (Dval3)-ANG II was least potent in the competitive binding assay. These data fulfill criteria for the identification of specific angiotensin II receptors in cells from mammalian brain.     

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.     

Regional and subcellular distribution of [125I]endothelin binding sites in rat brain.

The binding of [125I]endothelin-1 (125I-ET-1) to membranes from whole rat brain, from individual brain regions, and derived from subcellular fractionation of whole rat brain was investigated. 125I-ET-1 binding to whole rat brain membranes was rapid, concentration-dependent, saturable, and characterized as irreversible because it was not displaced by unlabeled endothelin-1 (ET-1) and different concentrations of ligand produced, with time, a similar magnitude of binding. The maximum binding site capacity and second-order forward rate association constant of binding were 1,946 +/- 147 fm/mg protein and 5.53 +/- 1.72 x 10(6) M-1 s-1. Removal of either extramembranal calcium or membrane-bound calcium and calcium binding proteins did not affect the binding of 125I-ET-1 to whole rat brain membranes. The brain stem and cerebellum contained the highest levels of 125I-ET-1 binding sites, whereas the cerebral cortex, striatum, and hippocampus contained binding site levels three- to fourfold less. Subcellular fractionation of whole rat brain and subsequent analyses of the distribution of 125I-ET-1 binding demonstrated a twofold enrichment of binding sites in the synaptosomal fraction compared to the homogenate. The myelin fraction contained a similar density of binding sites compared to the homogenate, while the mitochondrial and microsomal fractions contained considerably less binding sites. The ribosomal fraction did not contain any 125I-ET-1 binding sites. The subcellular distribution of 125I-ET-1 binding sites did not correlate with the distribution of 5'-nucleotidase, cytochrome-C oxidase, phosphodiesterase, and alkaline phosphatase. Depletion of extracellular calcium increased 125I-ET-1 binding in the synaptosomal fraction but not in the myelin and mitochondrial fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of prolactin receptor in human brain and choroid plexus

We have studied the binding of¹²⁵I-labeled human prolactin (PRL) to membranes from various regions of the human brain (hypothalamus, cerebral cortex, cerebellum and choroid plexus) derived from autopsy specimens. Among the various regions studied, the choroid plexus of both male and female subjects showed the highest specific binding and a clearly detectable specific binding was also observed in the hypothalamus of both sexes, whereas it was very low in other brain regions. No significant sex differences in PRL binding to various brain regions were observed except for the hypothalamus where a higher binding was seen in female subjects. The binding did not vary with the age of the subjects. Moreover, the cause of death and the time elapsed from death to autopsy in this study did not affect the binding significantly. The binding of¹²⁵I-labeled human PRL to hypothalamus and choroid plexus membranes from female specimens was inhibited in a dose-dependent manner by both unlabeled human and ovine PRL and by human growth hormone (GH), but not by other polypeptide hormones. Scatchard analysis of the binding revealed the presence of saturable binding sites with low capacity and high affinity for human PRL ligand. These results provide strong preliminary evidence for the presence of PRL binding sites in the human brain.     

Binding sites for [3H][Sar9, Met(O2)11]substance P in rat brain and guinea pig ileum.

[3H][Sar9,Met(O2)11]substance P (SP) with high specific activity (32 Ci/mmol) was used to study neurokinin-1 (NK-1) binding sites on rat brain and smooth muscle membranes of the guinea pig ileum. The specific binding of [3H][Sar9,Met(O2)11]SP was shown to be saturable, reversible and increased in parallel with the protein concentration. Scatchard analyses of equilibrium binding experiments revealed that [3H][Sar9,Met(O2)11]SP binds to a class of non-interacting binding sites in rat brain membranes (Kd = 2 nM, Bmax = 56 fmol/mg of protein) and ileum muscle membranes (Kd = 2 nM, Bmax = 194 fmol/mg of protein). Competition of [3H][Sar9,Met(O2)11]SP, 125I-BH[Sar9,Met(O2)11]SP and 125I-BH.SP with different tachykinin-related peptides gave the following rank order of potencies: SP greater than physalaemin greater than [Sar9,Met(O2)11]SP greater than N-Ac[Arg6,Sar9,Met(O2)11]SP(6-11) greater than neurokinin A (NKA) greater than or equal to eledoisin greater than or equal to neurokinin B (NKB) greater than [MePhe7]NKB (4-10) greater than [beta-Ala8]NKA(4-10). A very similar pattern was observed on ileum muscle membranes. [3H][Sar9,Met(O2)11]SP was found to be highly selective for NK-1 binding sites in rat brain and in the intestinal tissue. Binding showed good correlation with the biological activity of tachykinins and related peptides. From these data it can be suggested that (a) the NK-1 receptor characterized in the central nervous system is identical to the one in the periphery, (b) the NK-1 binding site of the muscle membranes appears to be similar to the contractile receptor of the guinea pig ileum and (c) the functional site mediating relaxation of the dog carotid artery is similar to the contractile receptor of the guinea pig ileum.     

Alteration of endothelin-1 receptor in the choroid plexus of rats with kaolin-induced experimental hydrocephalus]

Specific binding sites for endothelin-1 (ET-1) in the choroid plexus of rats with kaolin-induced hydrocephalus were analyzed using quantitative receptor autoradiographic technique with 125I-ET-1. Unlabeled ET-1 and its natural analog ET-3 inhibited the binding of 125I-ET-1 to the choroid plexus of control rats with similar high potencies. However, possibly related substances, such as ion channel regulators (omega-conotoxin GVIA, nitrendipine, verapamil, diltiazem, alpha-bungatmtoxin, aconitine, apamin), ouabain and atrial natriuretic peptide did not affect the binding. Scatchard analysis revealed the presence of a single class and high affinity binding sites for ET-1 in the choroid plexus. The number of 125I-ET-1 binding sites in the choroid plexus of rats with kaolininduced hydrocephalus was significantly lower, when compared with those in the age-matched control rats; maximum number of binding sites (Bmax) was 16.3 +/- 0.6 and 36.2 +/- 2.5 fmol/mg, respectively (p less than 0.01, n = 5). There was no significant difference in the binding affinities; affinity constants (Ka) was 2.6 +/- 0.3 x 10(9) M in control rats and 3.5 +/- 0.5 x 10(9) in hydrocephalic rats (n = 5). These results suggest that ET receptors may play a role in the regulation of cerebrospinal fluid production.     

Calcitonin receptors in the rat mesencephalon mediate its analgesic actions: autoradiographic and behavioral analyses

Autoradiographic analyses of salmon calcitonin (sCT) binding in the rat mesencephalon revealed an exceptionally high concentration of receptors in the ventral and ventrolateral segments of the periaqueductal gray matter (PAG) extending along the entire rostral-caudal axis. Relatively heavy labeling was also seen along a band extending ventrolaterally through the mesencephalic reticular formation. Other receptor-rich areas include the nucleus linearis, pars compacta and lateralis of the substantia nigra, locus coeruleus, parabrachial nuclei and nucleus raphe pontis of the pontine reticular formation. Injections of sCT into the PAG induced a dose-dependent increase in hot-plate latencies. All rostral-caudal levels of these brain regions appeared to be equally responsive. Injections into the midline pontine reticular formation were also effective in increasing response latencies. Unilateral injections into the hypothalamus, medial thalamus, ventral thalamus and mesencephalic reticular formation proved to be ineffective. Human calcitonin (hCT) was considerably less potent. These biological effects are consistent with the potencies of both peptides in displacing 125I-sCT from slide-mounted sections of rat PAG. Naloxone failed to antagonize sCT-induced analgesia, suggesting an opiate independent mechanism for this peptide in eliciting analgesia.     

Autoradiographic visualization and characteristics of [125I]bradykinin binding sites in guinea pig brain.

The present study was undertaken to localize and characterize bradykinin (BK) binding sites in 10 microns serial sections of guinea pig brain by in vitro quantitative receptor autoradiography. Specific binding of [125I-Tyr8]bradykinin ([125I]BK) was localized in the medulla oblongata to the regions of the nucleus of the solitary tract (nTS), the area postrema (AP), the dorsal motor nucleus of the vagus (X) and the caudal subnucleus of the spinal trigeminal nucleus. No significant specific [125I]BK binding was seen in other brain regions. The specific binding (85-90% of total binding) was of high affinity and saturable with a KD of 73.5 +/- 9.9 pM and a Bmax of 27.8 +/- 1.9 amol per mm2 of tissue. In competition studies, the rank order of potencies was: BK greater than Met-Lys-BK greater than Lys-BK much greater than Des-Arg9-BK. The B2 receptor antagonist D-Arg0-Hyp3-Thi5,8-D-Phe7-BK inhibited [125I]BK binding with a Ki value of 3.5 +/- 1.5 nM while Des-Arg9-[Leu8]-BK, a B1 receptor antagonist did not significantly inhibit [125I]BK binding in concentrations up to 10 microM. Our finding of specific high affinity [125I]BK binding sites in the nTS, AP and the X is important because these brain areas are known to be involved in central cardiovascular regulation. Moreover, our results suggest that the specific [125I]BK binding sites in the guinea pig medulla are of the bradykinin B2 receptor type.     

Characterization of [125I]Tyr11-somatostatin binding sites in the rabbit retina.

We have identified specific receptors for somatostatin (SS) in the rabbit retina using the radioligand [125I]Tyr11-Somatostatin. [125I]Tyr11-SS bound with high affinity to retinal membranes as was ascertained by both kinetic and saturation experiments. Scatchard analysis of the saturation data for [125I]Tyr11-SS binding to retinal membranes suggest a single population of sites with an apparent affinity constant (KD) of 0.90 +/- 0.20 nM and a maximum number of binding sites (Bmax) of 104 +/- 52 fmol/mg protein. The specific binding of [125I]Tyr11-SS was displaced in a dose-dependent manner by SS, Tyr11-SS, SMS 201-995, SS-28 and D-Trp8-SS. The inactive SS analog SS28(1-14) as well as the peptides CRF and bombesin had no effect. In addition, the specific binding of [125I]Tyr11-SS was attenuated by GTPgS. These findings demonstrate the presence of a selective receptor for SS in the rabbit retina that is coupled to guanine nucleotide binding protein(s).