Selective inhibition by dactinomycin of NANC sensory bronchoconstriction and [125I] NKA binding due to NK-2 receptor antagonism

Y.-P. Lou, P. Delay -Goyet & J. M. Lundber g. 1991. Selective inhibition by dactinomycin of NANC sensory bronchoconstriction and [¹²⁵I]NKA binding due to NK-2 receptor antagonism. Acta Physiol Scand 144 , 221–231. Received 6 May 1991, accepted 13 September 1991. ISSN 0001–6772. Department of Pharmacology, Karolinska Institutet, Stockholm. In the present study, dactinomycin (m) inhibited the non-adrenergic, non-cholinergic bronchoconstriction upon antidromic vagal nerve stimulation (1 Hz for 1 min) in the isolated perfused guinea-pig lung by 84%. The release of calcitonin gene-related peptide was unchanged, however, suggesting a postjunctional action. Dactinomycin (10^-5, 5 × 10^-5 m) also reduced non-adrenergic non-cholinergic bronchial contractions (maximally by 75%) induced by electrical field stimulation or capsaicin, while the cholinergic component and non-adrenergic non-cholinergic relaxation remained intact. The neurokinin-2 receptor antagonist l-659 877 (10^-6 m) had a similar effect as dactinomycin, inhibiting the non-adrenergic non-cholinergic bronchial contractions by 69%, while the neurokinin-1 receptor antagonist CP-96,345 (10^-6m) had no effect. The bronchoconstriction evoked by neurokinin A, the selective neurokinin-2 receptor agonist Nle¹⁰neurokinin A (4–10) and capsaicin was markedly inhibited by dactinomycin while the contraction induced by substance P (SP), the selective neurokinin-1 receptor agonist Sar⁹Met(0₂)¹¹SP, endothelin-1 and acetyl-choline was not affected. In autoradiographic experiments on guinea-pig lung, [¹²⁵I]neurokinin A-labelled sections showed dense binding in the bronchial smooth muscle layer. Dactinomycin inhibited the specific binding of [²⁵1]neurokinin A in a concentration-dependent manner (IC₅₀= 6.3 × 10^-6 m) and 66% of [¹²⁵I]neurokinin A total binding was inhibited by 10^-4 M dactinomycin. In the rat colon, [¹²⁵I]neurokinin A binding to neurokinin-2 sites on circular smooth muscle was inhibited by dactinomycin with an IC₅₀ value of 7.9 × 10^-6 M. Dactinomycin failed to reduce increased nerve-evoked contractions or those caused by Nle¹⁰neurokinin A (4–10) per se in the rat vas deferens, which are considered to be mediated by neurokinin-2 receptor activation. In the rat portal vein, dactinomycin did not influence the contractions caused by the neurokinin-3 selective agonist Pro⁷neurokinin B. In conclusion, dactinomycin selectively inhibited neurokinin-2 receptor activation in guinea-pig lung and rat colon, but not in rat vas deferens, which may depend on the existence of different neurokinin-2 receptor subtypes. Neurokinin A is most likely the main endogenous excitatory non-adrenergic non-cholinergic transmitter in guinea-pig bronchi.     

The ontogeny of 2-[125I]iodomelatonin binding sites in chicken brain.

The characteristics of the binding sites for 2-[125I]iodomelatonin were studied in chicken brain membranes during development. Specific binding, defined using cold melatonin (1 microM), was detected as early as 8-day-old embryos. Scatchard analysis of saturation experiments showed that 2-[125I]iodomelatonin binds to a single class of site at all ages tested (8-day-old embryos to 3-month-old chicks). Binding affinity (Kd) did not change during development (18-31 pM), but the maximal number of binding sites (Bmax) increased until embryonic day 18, and then remained relatively constant until 30 days of age. A further increase in Bmax was seen at 3 months of age. Guanosine 5'-triphosphate (GTP, 1 mM) inhibited 2-[125I]iodomelatonin binding at all ages suggesting that the melatonin binding site is coupled to a guanine nucleotide binding protein at a very early stage of development. Competition experiments with a number of melatonin analogues indicated that the binding site detected in the brain at embryonic day 8 was pharmacologically identical to that observed 15 days after hatching.     

Transport of [125I] IgG and [125I] ovalbumin between lymphocyte-like and macrophage-like rat spleen cells: chromatin binding

Spleen cells from rats were incubated for 10 min in Eagle's medium containing [125I] IgG or [125I] ovalbumin. Lymphocyte-like and macrophage-like cells were separated by centrifugation in Ficoll-metrizoate solution (d = 1.095). Control spleen cells were incubated in Eagle's medium without [125I] IgG or [125I] ovalbumin and both subpopulations were separated analogically. Lymphocyte-like cells labelled with [125I] IgG or [125I] ovalbumin were incubated for 10 min with control macrophage-like cells and labelled macrophage-like cells with control lymphocyte-like cells. After the incubation time all subpopulations were separated again in Ficoll-metrizoate solution. Radioactivity of [125I] IgG or [125I] ovalbumin was found in cytoplasmic fraction, sap protein fraction (nucleoplasm) and chromatin of control lymphocyte-like and macrophage-like cells. It may be concluded that transport of these potential antigenic compounds occurs directly in lymphocyte-like and macrophage-like cells as well as transport between these two subpopulations.     

Autoradiographic localization of [125I]charybdotoxin binding sites in rat brain.

Charybdotoxin, a 37 amino acid peptide isolated from scorpion venom, is a potent inhibitor of potassium channel function. [125I]charybdotoxin was originally believed to be a selective ligand for the Ca(2+)-sensitive channel in many tissues, but it appears to bind only to a voltage-sensitive potassium channel in brain. We found high densities of [125I]charybdotoxin binding in lateral olfactory tract, interpeduncular nucleus and a variety of mesencephalic nuclei. Moderate levels were found in the cerebral cortex, medial thalamus, hypothalamus and selected thalamic nuclei. These results indicate that [125I]charybdotoxin identifies a potassium channel or channels with a unique distribution in the brain.     

Early developmental expression of leptin receptor gene and [125I]leptin binding in the rat forebrain

Leptin, via leptin receptors (Ob-R), regulates appetite and energy balance. Of the six isoforms of the receptor identified, so far, only the long form (Ob-Rb) can fully activate downstream signal transduction pathways. Although the expression and function of leptin receptors is well described in the adult brain, little is known about the ontogeny of leptin receptor system around the time of birth. In this study, the mRNA expression patterns of total leptin receptor, Ob-R, and the long signalling form of the receptor, Ob-Rb, were investigated in the brain of embryonic and newborn rats using in situ hybridisation and [125I]leptin binding. On embryonic day 18 (E18), Ob-R mRNA was detected in the choroid plexus and the ependymal layer of the third ventricle by in situ hybridisation. At E21, Ob-Rb mRNA was first observed in the arcuate and the ventral premammillary hypothalamic nuclei while at P3, receptor expression was also found in the dorsomedial nucleus. Other leptin target areas identified were the trigeminal ganglion, the thalamus and the hippocampus. Using quantitative receptor autoradiography specific [125I]leptin binding sites on the choroid plexus were found to increase with age in contrast to the ependymal layer of the third ventricle where levels decreased with age. Together these findings demonstrate that the leptin receptor system is differentially regulated during late gestation and early postnatal life in the rat.     

Lack of alpha-adrenoreceptor binding of [125I]BE2254 to rat basilar artery membranes

Rat basilar arteries do not contain classical alpha- or beta-adrenoreceptors as assessed by electrophysiological techniques even though these arteries are innervated by catecholamine-containing perivascular nerves. These arteries were therefore examined for their ability to selectively bind an alpha-adrenoceptor radioligand, [125I]BE2254 (2/beta/4-hydroxyphenyl)-ethylaminomethyl)-tetralone). For comparison, rat tail arteries were also studied as these are known to contain functional alpha-adrenoreceptors. It was found that basilar artery membranes had only one-third of the specific binding of tail artery membranes and this finding collaborates the electrophysiological data.     

Quantitative autoradiographic distribution of NPFF1 neuropeptide FF receptor in the rat brain and comparison with NPFF2 receptor by using [125I]YVP and [(125I]EYF as selective radioligands

The selectivity of two new radioligands, [(125)I]YVP ([(125)I]YVPNLPQRF-NH(2)) and [(125)I]EYF ([(125)I]EYWSLAAPQRF-NH(2)), for neuropeptide FF (NPFF) receptor subtypes was determined using HEK293 cells expressing hNPFF(1) and CHO cells expressing hNPFF(2) receptors. Saturation binding and displacement experiments showed that [(125)I]YVP and [(125)I]EYF bound selectively with a very high affinity, K(D)=0.18 nM and 0.06 nM, to NPFF(1) and NPFF(2) receptors respectively.By using in vitro autoradiography with these radioligands and frog pancreatic polypeptide (PP) as selective unlabelled competitor of NPFF(2) binding sites, NPFF(1) and NPFF(2) receptor distribution was analyzed throughout the rat CNS.The highest densities of [(125)I]EYF binding sites were seen in the most external layers of the dorsal horn of the spinal cord, the parafascicular thalamic nucleus, laterodorsal thalamic nucleus and presubiculum of hippocampus. All specific binding of this radioligand was inhibited by 200 nM frog PP. The density of 0.1 nM [(125)I]YVP binding was much smaller in all brain areas and frog PP-insensitive binding sites (NPFF(1) receptor subtype) were detected in septal, thalamic and hypothalamic areas but were absent in the spinal cord.The restricted distribution of NPFF(1) receptors in the CNS supports its specific role in a limited number of neuronal functions. In contrast to the rat spinal cord where the NPFF(1) system is absent, there is no strict separation between NPFF(1) and NPFF(2) system at the supraspinal level.     

Ontogeny and characterization of [125I]Bolton Hunter-eledoisin binding sites in rat spinal cord by quantitative autoradiography.

The distribution and characteristics of [125I]Bolton Hunter-eledoisin binding sites in rat lumbar spinal cord were studied during postnatal development by in vitro receptor autoradiography. At three, six and 10 days of age, specific [125I]eledoisin binding was distributed throughout the dorsal and ventral horns of the spinal cord. In contrast, from day 24 onwards, specific binding of [125I]eledoisin was confined to superficial layers of the dorsal horn, with negligible amounts of specific binding in the ventral horn. [125I]Eledoisin binding to neonatal (three day) and adult (eight to 12 weeks) spinal cord sections was characterized using tachykinin agonists. In both dorsal and ventral horns of neonatal spinal cord, the rank order of potency of agonists indicated that the majority (64%) of specific [125I]eledoisin binding was to neurokinin-3 binding sites. The identity of the non-neurokinin-3 sites labelled by [125I]eledoisin remains to be determined. In adult rat spinal cord, [125I]eledoisin appeared to bind exclusively to neurokinin-3 binding sites. These results suggest that major changes take place in the localization of neurokinin-3 receptors during postnatal ontogeny of the rat spinal cord. These changes may reflect an important role for tachykinins in neuronal plasticity of the developing spinal cord.     

Autoradiographic localization of [125I]-endothelin-1 binding sites in rat brain

Autoradiograms of [125I]-endothelin (ET) binding in the rat brain demonstrated that the receptors for endothelin are localized mainly in the brainstem, basal ganglia, and cerebellum. Among the many other nuclei in these regions, there also appeared nuclei which are considered to play important roles in the central nervous regulation of the cardiovascular system: they include the nuclei of the anteroventral third ventricle area, the supraoptic nucleus, and the subfornical organ, for example. From these findings, we suggest that ET-1 or its analogous peptide(s) may act as a neuropeptide regulating central nervous functions, including cardiovascular functions.     

Autoradiographic localization and characterization of [125I]lysergic acid diethylamide binding to serotonin receptors in Aplysia

The sensitive serotonergic radioligand 2-[125I]lysergic acid diethylamide was used to study the distribution and pharmacological binding properties of serotonin receptors in Aplysia californica. The high specific activity of this radioligand allowed us to develop a methodology for the investigation of receptor binding properties and receptor distribution in a single ganglion. [125I]Lysergic acid diethylamide labels a population of high-affinity serotonergic sites (Kd = 0.41 nM) in Aplysia ganglia whose regional distribution matches that expected from previous electrophysiological and immunohistochemical studies. The properties of [125I]lysergic acid diethylamide binding sites in Aplysia are in general agreement with previous studies on [3H]lysergic acid diethylamide binding in this system but these sites differ from the serotonergic receptor subtypes described in the mammalian brain. Guanine nucleotides were shown to modulate agonist but not antagonist affinity for the [125I]lysergic acid diethylamide binding site in Aplysia, suggesting that this site is coupled to a G-protein. Images of serotonin receptor distribution in the Aplysia nervous system were obtained from autoradiograms of [125I]lysergic acid diethylamide binding. Serotonin receptors in ganglia tissue sections are located primarily within the neuropil. In addition, a subset of neuronal soma are specifically labeled by [125I]lysergic acid diethylamide. These studies indicate that [125I]lysergic acid diethylamide binds to sites in the Aplysia nervous system which display a regional distribution, pharmacological binding properties and evidence of coupling to a G-protein consistent with labeling of a subset of functional serotonin receptors. In addition, the techniques used in this investigation provide a general approach for rapidly characterizing the pharmacological properties and anatomical distribution of receptor binding sites in single invertebrate ganglia. Individual neurons containing these receptor subtypes can be identified by these methods and correlated with physiological responses in the same cell.     

Specific [125I]Bolton-Hunter substance P binding sites in human and rat skin

[125I]Bolton-Hunter substance P [( 125I]BH-SP) binding sites in rat and human skin were investigated, using quantitative receptor autoradiographic and emulsion autoradiographic methods. [125I]BH-SP binding sites were discretely localized in skin areas anatomically corresponding to dermal papillae, sweat glands, and hair follicles. The highest density of the binding sites was in the dermal papilla of the finger, followed by the sweat gland. [125I]BH-SP binding to the dermal papillae of the human finger pad skin and rat paw pad skin was displaced by unlabeled SP, with a high affinity, and Kd values were calculated to be 744 pM and 297 pM, respectively. The existence of [125I]BH-SP binding sites supports the idea of the neurotransmitter role of substance P in skin dermal papilla.     

Distribution and levels of [I]IGF-I, [I]IGF-II and [I]insulin receptor binding sites in the hippocampus of aged memory-unimpaired and -impaired rats

The insulin-like growth factors (IGF-I and IGF-II) and insulin are localized within distinct brain regions and their respective functions are mediated by specific membrane receptors. High densities of binding sites for these growth factors are discretely and differentially distributed throughout the brain, with prominent levels localized to the hippocampal formation. IGFs and insulin, in addition to their growth promoting actions, are considered to play important roles in the development and maintenance of normal cell functions throughout life. We compared the anatomical distribution and levels of IGF and insulin receptors in young (five month) and aged (25 month) memory-impaired and memory-unimpaired male Long–Evans rats as determined in the Morris water maze task in order to determine if alterations in IGF and insulin activity may be related to the emergence of cognitive deficits in the aged memory-impaired rat. In the hippocampus, [¹²⁵I]IGF-I receptors are concentrated primarily in the dentate gyrus (DG) and the CA3 sub-field while high amounts of [¹²⁵I]IGF-II binding sites are localized to the pyramidal cell layer, and the granular cell layer of the DG. [¹²⁵I]insulin binding sites are mostly found in the molecular layer of the DG and the CA1 sub-field. No significant differences were found in [¹²⁵I]IGF-I, [¹²⁵I]IGF-II or [¹²⁵I]insulin binding levels in any regions or laminae of the hippocampus of young vs aged rats, and deficits in cognitive performance did not relate to altered levels of these receptors in aged memory-impaired vs aged memory-unimpaired rats. Other regions, including various cortical areas, were also examined and failed to reveal any significant differences between the three groups studied.

It thus appears that IGF-I, IGF-II and insulin receptor sites are not markedly altered during the normal ageing process in the Long–Evans rat, in spite of significant learning deficits in a sub-group (memory-impaired) of aged animals. Hence, recently reported changes in IGF-I receptor messenger RNA levels in aged memory-impaired rats[42] are apparently not reflected at the level of the translated protein.     

Reovirus type 3 and [125I]-iodocyanopindolol bind to distinct domains on the beta-adrenergic like receptor

Antireceptor antibodies have been developed as a probe to study the cellular receptor for reovirus type 3. Using this probe, a glycoprotein with a molecular weight of 65-67 kilodaltons and a pI of 5.8-6.0 was isolated and identified as the reovirus receptor. This protein was also structurally similar to the affinity-purified beta-adrenergic receptor from calf lung. In this report, we employ [125I]-iodocyanopindolol, a high affinity beta-adrenergic antagonist, to further characterize this protein. We show that R1.1, a murine thymoma cell line, possesses about 2,000 receptors per cell with high affinity for ICYP (kD = 3.3 X 10(-11) M). Competitive inhibition studies suggest that the receptor is of the beta-2 subtype. Solubilized receptor proteins from R1.1 cells bound to the antireceptor antibody were further purified by SDS-PAGE and electroelution from the gel. Five percent of the proteins thus obtained could bind ICYP with high affinity (kD = 1.6 X 10(-10) M). This suggests that the purification procedure produced a collection of forms of this 65- to 67-kilodalton protein, some of which retained the conformation for binding the beta ligands. We also demonstrate that the isolated receptor protein was able to bind ICYP even when the virus binding site was occupied by the anti-idiotype, suggesting that reovirus type 3 and the beta ligands bind to distinct domains on the receptor protein.     

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.     

Hydrophobic labelling of membrane-embedded proteins with lipophilic reagents. Incorporation of [125I]INA and [125I]TID into B lymphocyte membrane immunoglobulins

Hydrophobic labelling is frequently used in the study of membrane-inserted domains of intrinsic proteins. However, the published procedures, fail to incorporate sufficient radioactivity into membrane immunoglobulins of B lymphocytes to permit investigation of their subunit structures and associations with other proteins. In order to increase the specific radioactivity of [125I]iodonaphthylazide ([125I]INA), an improved method for the synthesis of the reagent was developed. In addition, the optimal conditions for labelling B lymphocytes with [125I]INA and the commercially available reagent 3-(trifluoromethyl)-3-(3'-[125I]iodophenyl)diazirine ([125I]TID) were elaborated. Under these optimized conditions, Ig molecules labelled with [125I]INA and [125I]TID were isolated and analysed in detail by SDS-PAGE. The usefulness of the two reagents for the investigation of lipid-embedded domains of membrane proteins is discussed.     

Development and characterization of a whole-cell radioligand binding assay for [125I]gp120 of HIV-1.

The binding of HIV-1 envelope glycoprotein, gp120, to the CD4 receptor is an important step in productive infection. The development of agents which interrupt this binding phenomenon should be of therapeutic interest. The present study characterizes a whole cell gp120/CD4 radioligand binding assay (radioligand binding assay) modified for use in a high volume screening format. Modifications include the use of human CD4 receptor stably expressed in a Chinese hamster ovary cell line and the gentle fixation (paraformaldehyde) of the CD4 receptor just prior to assay. Binding of [125I]gp120 to fixed CD4 was of high affinity (KD = 6 nM), saturable, reversible, and specific. The kinetics of binding were identical to those of viable (non-fixed) CD4 receptor. [125I]gp120 binding was inhibited by unlabeled recombinant gp120, soluble CD4, and the anti-CD4 monoclonals OKT4A and LEU3A. A number of compounds reported to inhibit gp120 binding and/or gp120 induced syncytium formation were also active in this assay. This modified radioligand binding assay was developed to initiate a rational and extensive screening program to assist in the identification of potential chemotherapeutic agents based on their ability to inhibit gp120 binding to host cells.     

Autoradiographic visualization of the binding sites for [125I]endothelin in rat and human brain

Putative receptors for endothelin were localised autoradiographically in frozen sections of rat and human brain using [125I]endothelin as a ligand. In the rat brain the highest densities were in the granular layer of the cerebellum, choroid plexus, hippocampus, and habenular nucleus. Similar brain high densities were found in the human cerebellum and hippocampus. The non-vascular pattern of distribution suggests that endothelin may have a function as a modulator of neuronal function in addition to its possible involvement in the regulation of vascular tone.