1B Binding Sites Research Articles

CREB-AP1 Protein Complexes Regulate Transcription of the Collagen XXIV Gene (Col24a1) in Osteoblasts

Collagen XXIV is a newly discovered and poorly characterized member of the fibril-forming family of collagen molecules, which displays unique structural features of invertebrate fibrillar collagens and is expressed predominantly in bone tissue. Here we report the characterization of the proximal promoter of the mouse gene (Col24a1) and its regulation in osteoblastic cells. Using well characterized murine models of osteoblast differentiation, we found that the Col24a1 gene is activated sometime before onset of the late differentiation marker osteocalcin. Additional analyses revealed that Col24a1 produces equal amounts of two alternatively spliced products with different 5'-untranslated sequences that originate from distinct transcriptional start sites. Cell transfection experiments in combination with DNA binding assays demonstrated that Col24a1 promoter activity in ROS17/2.8 osteosarcoma cells is under the control of an upstream cis-acting element, which is shared by both transcripts and is recognized by specific combinations of c-Jun, CREB1, ATF1, and ATF2 dimers. Consistent with these results, overexpression of c-Jun, ATF1, ATF2, or CREB1 in transiently transfected osteoblastic cells stimulated transcription from reporter gene constructs driven by the Col24a1 promoter to different degrees. Moreover, chromatin immunoprecipitation experiments showed that these nuclear factors bind the same upstream sequence of the endogenous Col24a1 gene. Collectively these data provide new information about transcriptional control of collagen fibrillogenesis, in addition to implicating for the first time CREB-AP1 protein complexes in the regulation of collagen gene expression in osteoblasts.

Autoradiographic localization of [125I-Tyr0]bradykinin binding sites in brains of Wistar-Kyoto and spontaneously hypertensive rats.

1. The present study was undertaken to localize and characterize bradykinin (BK) binding sites in brains from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). 2. Serial sections of brains were cut from adult WKY and SHR and specific [125I-Tyr0]bradykinin ([125I-Tyr0]BK) binding was determined using in vitro quantitative receptor autoradiographic techniques. 3. Specific binding of [125I Tyr0]BK was localized in the medulla oblongata to the regions of the nucleus of the solitary tract (NTS), area postrema (AP), dorsal motor nucleus of the vagus (X), and caudal subnucleus of the spinal trigeminal nucleus in both strains of rat. The specific binding (85-90% of total binding) was of high affinity and saturable with KD values in the range of 100 pM and a B(max) of 0.75 fmol per mg tissue equivalent in the NTS-X-AP complex of both the WKY and SHR. In competition studies, the rank order of potencies was similar in both strains with BK = Lys-BK > icatibant >>> DesArg9-BK. The B2 receptor antagonist icatibant inhibited [125I-Tyr0]BK binding with a Ki value of 0.63 +/ 0.19 nM in WKY and 0.91 +/- 0.73 nM in SHR, while Ki values for the B1 receptor agonist DesArg9-BK were 1475 +/- 1055 and 806 +/-362 nM in WKY and SHR, respectively. 4. Our finding of specific high-affinity [125I-Tyr0]BK B2 binding sites in the NTS, AP, and the X of WKY and SHR is important because these brain areas are associated with central cardiovascular regulation. However, alterations in BK B2 receptors in the medulla that could contribute to the hypertensive state in the SHR were not detected.

Sculpting the visual map: the distribution and function of serotonin-1A and serotonin-1B receptors in the optic tectum of the frog

Agonists of serotonin (5-HT)-1 receptors modulate the synaptic strength of the connection between retinal ganglion cells and neurons of the frog optic tectum in brain slices (Brain Res. 1998;781:167–181). We have now used autoradiographic receptor binding techniques to determine the location of 5-HT 1A and 5-HT 1B binding sites in the laminated optic tectum. 5-HT 1A binding sites, as labeled with [ 3H]8-hydroxy-dipropylaminotetralin (8-OH-DPAT), were highest in the superficial, retinorecipient layers of the tectum, intermediate in layers 6 and 7 and low in the remaining layers. Binding densities in all of these layers were unaffected by optic nerve lesion. 5-HT 1B binding sites were visualized using [ 125I]iodocyanopindolol (ICYP). Binding densities were highest in the plexiform layers 5 and 7 and intermediate in layers 6 and 8. Binding sites were present at low levels in layer 9; however, optic nerve lesion resulted in a strong upregulation of these sites in this layer. Pharmacological manipulation of receptor activation resulted in changes in the activity-dependent visual map that is created at the tectum by retinal ganglion cell terminals. Chronic treatment of the tectum with SB-224289, a selective antagonist of 5-HT 1B receptors, disrupted the topographic map. In contrast, exposure to WAY-100635, a selective antagonist of 5-HT 1A receptors, refined it. We conclude that both 5-HT 1A and 5-HT 1B receptors are present in the adult frog tectum and that changes in their activation levels can produce changes in retinotectal transmission levels that drive visual plasticity in opposite directions.

Region-specific decrease in 5-HT 1A and 5-HT 1B binding sites after intra-hippocampal ibotenic acid injections in the rat

The cellular location of 5-HT 1A and 5-HT 1B binding sites in the hippocampus was investigated using a stereotaxic unilateral lesion of the CA1 field by ibotenic acid, followed by autoradiography on brain sections with the specific ligands [ 3H]8-OH-DPAT and S-CM-G[ 125I]TNH 2, respectively. As compared to the contralateral side of the lesion, the ipsilateral side exhibited a decrease in the density of 5-HT 1A binding sites in the strata oriens and radiatum of CA1, and of 5-HT 1B binding sites in the dorsal subiculum and stratum oriens of CA1. The results demonstrate that 5-HT 1A binding sites are located on dendrites whereas 5-HT 1B binding sites are located on axon terminals of CA1 pyramidal cells.

Cloning of rabbit α 1b-adrenoceptor and pharmacological comparison of α 1a-, α 1b- and α 1d-adrenoceptors in rabbit

We have isolated a cDNA clone of the rabbit α 1b-adrenoceptor which has an open reading frame of 1557 nucleotides encoding a protein of 518 amino acids. The sequence shows higher identity to those of hamster, human, and rat α 1b-adrenoceptors than to those of rabbit α 1a- and α 1d-adrenoceptors. The pharmacological binding properties of this clone expressed in Cos-7 cells showed a characteristic profile as α 1b-adrenoceptor; high affinity for prazosin (p K i=10.3), relatively high affinity for tamsulosin (9.5) and low affinity for (−)-( R)-1-(3-hydroxypropyl)-5-[2-[[2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethyl]amino]propyl]indoline-7-carboxamide (KMD3213) (8.5), 2-(2,6-dimethoxy-phenoxyethyl)-aminomethyl-1,4-benzodioxane hydrochloride (WB4101) (8.7), and 8-[2-[4-(2-methoxy-phenyl)- l-piperazinyl]-8-azaspiro[4,5]decane-7,9-dione dihydrochloride (BMY7378) (7.3). We have compared the levels of mRNA expression of three α 1-adrenoceptor subtypes in rabbit tissues using the competitive reverse transcription/polymerase chain reaction (RT/PCR) assay. In most rabbit tissues except heart, α 1a-adrenoceptor mRNA was expressed 10 folds more than the other two subtypes. However, binding experiments with [ 3H]prazosin and [ 3H]KMD3213 in rabbit tissues revealed a poor relationship between binding density and mRNA level. Especially, α 1b binding sites were exclusively predominant in spleen, whereas the α 1b subtype was minor at the mRNA level. These results indicate a high identity of structural and pharmacological profiles of three distinct α 1-adrenoceptor subtypes between rabbit and other species, but there are species differences in their distribution.

Differential adaptation of brain 5-HT 1A and 5-HT 1B receptors and 5-HT transporter in rats treated chronically with fluoxetine

Quantification of receptor binding sites and their encoding mRNAs, and electrophysiological recordings, were used to assess central serotonin (5-HT) neurotransmission in rats 24 h after a 2–3 week treatment with the selective 5-HT reuptake inhibitor fluoxetine (8 mg/kg i.p., daily). Binding studies showed that this treatment affected neither 5-HT 1A nor 5-HT 1B binding sites in all brain areas examined. However, a significant decrease (−38%) in 5-HT 1A mRNA levels in the anterior raphe area (but not forebrain regions) and increases in 5-HT 1B mRNA levels in the striatum (+127%) and the cerebral cortex (+34%) were noted in fluoxetine-treated rats. Electrophysiological recordings in brain slices showed that chronic fluoxetine treatment reduced the potency of the 5-HT 1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin to inhibit neuronal activity in the dorsal raphe nucleus, but did not affect 5-HT 1A-evoked responses of CA1 pyramidal cells in the hippocampus. These data further demonstrate that fluoxetine-induced adaptive changes in 5-HT neurotransmission exhibit marked regional differences. The decrease in 5-HT 1A mRNA levels in the anterior raphe suggests that fluoxetine-induced desensitization of 5-HT 1A autoreceptors involves changes at the transcription level.

Rat renomedullary interstitial cells possess bradykinin B2 receptors in vivo and in vitro.

1. Renomedullary interstitial cells (RMIC), abundant throughout the medulla of the kidney, have been demonstrated to have binding sites for many vasoactive peptides, including atrial natriuretic peptide, endothelin, angiotensin II and bradykinin (BK). These observations would support the hypothesis that interactions between RMIC and vasoactive peptides are important in the regulation of renal function. 2. We aimed to localize the BK B2 receptor binding site to RMIC in vivo and to also demonstrate that these receptors are biologically active in vitro. 3. The present study demonstrates BK B2 binding sites on RMIC of the inner stripe of the outer medulla and the inner medulla of the rat kidney in vivo. 4. We further demonstrate that the BK B2 radioligand [125I]-HPP-Hoe140 specifically bound to rat RMIC in vitro. In addition, reverse transcription-polymerase chain reaction detected the mRNA for the BK B2 receptor subtype in cell extracts. 5. For RMIC in vitro, cAMP levels were increased at 1 min and cGMP levels were increased at 2 min after treatment with 10(-10) and 10(-7) mol/L BK, respectively. Inositol 1,4,5-trisphosphate was increased at 10 s treatment with both 10(-6) and 10(-7) mol/L BK. 6. For RMIC in vitro, BK induced an increase in cell proliferation ([3H]-thymidine incorporation) and an increase in extracellular matrix synthesis (ECM; trans-[35S] incorporation), both effects mediated by BK B2 receptors. 7. We conclude that BK B2 receptors are present on RMIC both in vivo and in vitro. These receptors are coupled to intracellular second messenger systems and, in vitro, their stimulation results in cellular proliferation and synthesis of ECM.

Post-lesion up-regulation of 5-HT 1B binding sites in the suprachiasmatic nucleus may be reversed after spontaneous or graft-induced serotonin reinnervation

We have previously reported that selective axotomy of serotoninergic neurons produced by an intraventricular injection of 5,7-dihydroxytryptamine is followed by an increase in 5-HT 1B binding sites in the suprachiasmatic nucleus of the hypothalamus. This post-lesion up-regulation is shown here to be spontaneously reversed after long-term survival in spite of an incomplete reinnervation of the nucleus. Recovery may be accelerated by fetal raphe transplants that produce more rapid reinnervation.

B1 Bradykinin Receptors and Carboxypeptidase M Are Both Upregulated in the Aorta of Pigs after LPS Infusion

Bradykinin receptor subtypes were characterized in aortic cryosections obtained from healthy normal pigs, animals that were given an LPS infusion, and animals that came with a pre-existing infection or inflammation to the laboratory by binding studies andin vitroautoradiography. In control aorta a single class of high affinity B2 binding sites, located within the endothelium, but with no significant binding of B1 ligand were identified. No major changes in the expression of B2 BK receptors were noted in inflammed tissues. In cryosections of inflammed vascular tissue a markedly increased endothelial carboxypeptidase M activity was verified that parallelled an upregulation of B1 receptors in the aortic smooth muscle layer. In crosstalk between endothelial cells and smooth muscle cells B1 receptor mediated functional responses may counteract some of the detrimental effects of inflammation.

Human vascular kinin receptors of the B2 type characterized by radioligand binding.

1. The human umbilical vein responds to bradykinin (BK) with contractions that are mediated by B2 receptors. In the present study, the corresponding vascular smooth muscle B2 binding sites have been investigated. 2. [3H]-BK, a full agonist labelled ligand, was used to demonstrate a single binding site giving a Kd value of 0.51+/-0.02 nM and a Bmax of 24+/-1 fmol mg(-1) protein. Scatchard plots were linear (r=0.98) in the 0.05-5 nM range of concentrations. Non-specific binding was found to be 30% of total binding. 3. Competition binding curves gave the following order of potency for various B2 receptor agonists: BK-[Hyp3]-BK > or = Lys-BK >> [Aib7]-BK >>> [desArg9]-BK, which is typical of B2 receptors. There was no binding to B1 receptors since the selective B1 receptor ligand, Lys-[desArg9]BK was inactive up to 10 microM (n=4). 4. Characterization of the binding site with antagonists, performed with three chemically distinct series of peptide and non-peptide compounds, revealed a high affinity of Hoe 140 (D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK) (Ki 0.17 nM; n=4) which was more potent that FR 173657 ([(E)-3-(6-acetamido-3-pyridyl)-N-[N-[2,4-dichloro-3-[(2-methyl-8-quinol inyl)oxymethyl]phenyl]-N-methylaminocarbonylmethyl] acrylamide]) (Ki 1.94 nM; n=4), D-Arg-[Hyp3,D-Phe7,Leu8]-BK (Ki 256 nM; n=4) and Win 64338 (phosphonium, [[4[[2[[bis(cyclohexylamino)methylene]amino]-3-(2-naphthalenyl)-1-oxopro pyl]amino]phenyl]methyl]tributyl, chloride, monohydrochloride) (Ki 1,450 nM; n=4). 5. The present study describes and characterises B2 receptor binding sites in the vascular smooth muscle of the human umbilical vein. The binding assay appears to be suitable for studying new agonists or antagonists designed to activate or block the B2 receptor class that mediate the majority of the physiopathological effects of kinins in man.

Localization of bradykinin B2 binding sites in rat kidney following chronic ACE inhibitor treatment

Bradykinin exerts important influences on renal hemodynamics and tubular function by acting on renal bradykinin B2 receptors. However, the precise sites and mechanisms of its actions on the kidney are not known. To help elucidate the mechanisms of renal actions of bradykinin in vivo, we have employed high resolution electron microscopic autoradiography to localize bradykinin B2 binding sites in the rat kidney following intravenous administration of a radiolabeled ligand, 125I-HPP-Hoe140 (3-4-Hydroxyphenyl-propionyl-DArg0-[Hyp3-Thi5-D-Tic 7-Oic8]-bradykinin), a derivative of the highly selective bradykinin B2 receptor antagonist, Hoe140. In non-treated rats, bradykinin B2 binding sites were localized to the cell bodies and the luminal brush border of the proximal convoluted tubules in the cortex. In the medulla (except for the outer stripe of the outer medulla), binding occurred in the distal tubules, thin limbs of the loop of Henle, collecting ducts, peritubular capillary endothelium and renomedullary interstitial cells. To exclude the possibility that the radioligand may bind to angiotensin converting enzyme, rats were pretreated with the angiotensin converting enzyme inhibitor, perindopril. In these rats, binding to the cell bodies and the luminal brush border of the proximal convoluted tubules in the cortex was completely abolished, while binding remained unaltered in the medulla. Further studies using high performance liquid chromatography revealed that while the radioligand was degraded following systemic administration in nontreated rats, the degradation was significantly reduced in the rats pretreated chronically with perindopril. These results indicate that binding detected in the proximal tubules in the normal rats is due primarily to the tubular uptake of the degraded radioligand, and that bradykinin B2 binding sites occur predominantly in the renal tubules, vascular endothelium, and renomedullary interstitial cells of the renal medulla.

Effects of stress on the functional properties of pre- and postsynaptic 5-HT 1B receptors in the rat brain

Numerous studies have clearly shown that the turnover and release of serotonin (5-hydroxytryptamine, 5-HT) are increased under acute stressful conditions. Inasmuch as this latter process is under the control of a feedback mechanism involving the stimulation of presynaptic 5-HT 1B autoreceptors, we have investigated the possible effects of acute restraint (40 min) on the functional properties of 5-HT 1B receptors. The efficacy of the selective 5-HT 1B receptor agonist 3-[1,2,5,6-tetrahydropyrid-4-yl]pyrrolo-[3,2-b]pyrid-5-one (CP-93,129) in inhibiting in vitro the K +-evoked release of [ 3H]5-HT, was significantly reduced in stressed rats as compared to naive animals. Similarly, the responsiveness of 5-HT 1B receptors inhibiting the release of [ 3H]acetylcholine (presynaptic 5-HT 1B heteroreceptors), was reduced by restraint. These effects were observed in the hippocampus, but using the inhibitory effect of CP-93,129 on forskolin-stimulated adenylyl cyclase activity as an index of 5-HT 1B receptor function, it could be shown that the 5-HT 1B receptors located in the substantia nigra are also desensitized by stress. The number as well as the apparent affinity constant of 5-HT 1B binding sites labelled by [ 125I]iodocyanopindolol, as measured by quantitative autoradiography and membrane binding, were similar in naive and restraint-stressed rats suggesting that the stress-induced desensitization of 5-HT 1B receptors is not due to a reduced number of 5-HT 1B binding sites. As stress is thought to be a causal factor for the etiology of anxiety and depression, these results support the potential involvement of 5-HT 1B receptor dysfunction in the development of these neurological disorders.

Impairment of serotoninergic transmission is followed by adaptive changes in 5HT 1B binding sites in the rat suprachiasmatic nucleus

Serotonin1B (5-HT 1B) receptor binding in the suprachiasmatic nucleus (SCN) following impairment of serotoninergic transmission was studied by quantitative autoradiography. Serotonin (5-HT) denervation with 5,7-dihydroxytryptamine (5,7-DHT) caused a significant increase in the density of 5-HT 1B receptors in both the ventral (62%) and dorsal (53%) parts of the SCN as early as 3 days after axotomy. The magnitude of this increase did not differ 3, 15 or 21 days post-lesion. An up-regulation of 5-HT 1B receptors with similar magnitude was obtained in the two parts of the SCN after inhibition of 5-HT synthesis by chronic parachlorophenylalanine treatment. In this case, up-regulation was shown to be reversible after restoration of 5-HT synthesis with l-5-hydroxytryptophan. These results indicate that 5-HT 1B receptor density in the SCN was inversely correlated with 5-HT levels. These plastic properties exhibited by 5-HT 1B receptors in the SSN are discussed in relation to the mode of 5-HT transmission and possible localization of the receptors onto the main chemically defined cell populations of the nucleus.

Characterization of the α1-adrenoceptor subtype mediating contraction of guinea-pig spleen

A series of α 1-adrenoceptor agonists evoked concentration-dependent contraction of isolated guinea-pig spleen strips ( (—)- adrenaline > (—)- noradrenaline ⪢ L-phenylephrine > (—)-(4 aR,10 a)-3,4,4 a,5,10 a- hexahydro-6- methoxy-4- methyl-9- methylhio-2H- naphth[2,3-b]-1,4- oxazine methoxy-4- methyl-9- methylthio2H- naphth[2,3-b]-1,4- oxazine ( SDZ NVI 085) > cirazoline) , whereas indanidine, methoxamine, oxymetazoline and UK-14.304 were ineffective. (—)-Noradrenaline-induced contractions were inhibited by chloroethylclonidine (3 × 10 −6 −6 × 10 −5 M) and partially attenuated by SZL-49 (10 −7−10 −6 M), but remained resistant to (±)-isradipine (10 −9−10 −7 M). The contractions of the splenic strips were competitively antagonized by low concentrations of the α 1B-adrenoceptor-selective antagonist, spiperone (pA 2 8.05), but by relatively high concentrations of the α 1A-adrenoceptor-selective antagonists, (+)-niguldipine (pA 2 6.32) and 5-methyl-urapidil (pA 2 6.95). The affinities of subtype-selective antagonists determined at guinea-pig spleen α-adrenoceptors significantly correlated with p K i values at rat liver α 1B binding sites ( r=0.96) and pA 2 values at putative α 1B-adrenoceptors in rat aorta ( r=0.95), but differed from p K i values at rat cortical α 1A binding sites and pA 2 values at α 1A-adrenoceptors in rat vas defens. Also no correlation was obtained between antagonist affinities at guinea-pig spleen α-adrenoceptors and α 1C binding sites in rabbit liver. Thus, from the (1) potencies of agonists, (2) affinities of subtype-selective antagonists and (3) differential sensitivity of the contractions to α 1-adrenoceptor inactivating agents and their resistance to Ca 2+ channel blockade, the α 1-adrenoceptor mediating smooth muscle contraction of guinea-pig spleen can be best characterized as being of the B subtype.

Ontogeny of [ 125I]iodocyanopindolol-labelled 5-hydroxytryptamine 1B-binding sites in the rat CNS

Little is known about the ontogeny of the 5-hydroxytryptamine 1B (5-HT 1B) receptor, a putative terminal autoreceptor in the CNS. To learn more about the regional development of 5-HT 1B sites and how early these sites may become functional, we studied [ 125I]iodocyanopindolol ([ 125I]ICYP)-labelled central 5-HT 1B sites in developing postnatal rat brain and spinal cord. Significant ontogenic changes in B max were found in neocortex, hippocampus, striatum and brainstem. Maximum binding-site density was reached by 21 days in hippocampus and cortex, but not until 28 days in striatum. The rank order of binding site density changed with rat age, but B max was consistently high in brainstem and low in cerebellum. The percent increases in B max also varied with brain region, ranging from a 2-fold increase in brainstem up to 8-fold increases in cortex and striatum. In competition studies, the K i for 5-HT and for RU 24969 was the same in 5-day-old and adult rat brain. These data indicate regional differences in the ontogeny of 5-HT 1B-binding sites and suggest that 5-HT 1B receptors are functional early in the postnatal period.

The mouse 5-hydroxytryptamine 1B receptor is localized predominantly on axon terminals

The 5-hydroxytryptamine 1B receptor is a serotonin receptor subtype which is expressed predominantly in the basal ganglia. It has been suggested to play a role in movement and appetite control as well as in certain pathological states such as migraine. The recent cloning of the 5-hydroxytryptamine 1B gene as well as the discovery of a radioligand that labels in rodents 5-hydroxytryptamine 1B and possibly 5-hydroxytryptamine 1Dα receptors (S-CM-G[ 125I]TNH 2) allowed us to compare the distribution of the messenger RNA and of the protein in mouse brain sections. A high 5-hydroxytryptamine 1B messenger RNA level is found in the caudate-putamen in medium spiny neurons that project to the globus pallidus and the substantia nigra. In contrast, no messenger RNA is expressed in the globus pallidus and substantia nigra although these structures reveal the highest level of 5-hydroxytryptamine 1B binding sites. In the hippocampus, 5-hydroxytryptamine 1B messenger RNA is localized in the cell bodies of pyramidal cells of the CA1 field while the protein is found predominantly in the dorsal subiculum, a projection zone for the CA1 pyramidal neurons. In the cerebellum, 5-hydroxytryptamine 1B messenger RNA is expressed in the Purkinje cells, which display no receptor binding sites. Conversely, moderate binding is found in the deep nuclei of the cerebellum, the main projection zone of the Purkinje cells. 5-Hydroxytryptamine 1B sites are also detected in the superficial gray layer of the superior colliculus and the lateral geniculate nucleus, brain regions containing the terminals of retinal ganglion cells. The soma of these ganglion cells express high levels of 5-hydroxytryptamine 1B messenger RNA while no 5-hydroxytryptamine 1B binding sites were found in the retina. This study demonstrates that the main brain regions, expressing 5-hydroxytrypamine 1B messenger RNA contain low densities of 5-hydroxytryptamine 1B binding sites. Conversely, the major projection areas of these anatomical structures do not express detectable levels of 5-hydroxytryptamine 1B messenger RNA, but present a high density of binding sites. In addition, our data suggest that the distribution of the 5-hydroxytryptamine 1Dα binding sites is different from that of the 5-hydroxytryptamine 1Dα messenger RNA. These results together with previous lesion studies, indicate that the 5-hydroxytryptamine 1B and possibly the 5-hydroxytryptamine 1Dα receptors are localized predominantly on axon terminals, while their expression is low or absent at the somatodendritic level. The 5-hydroxytryptamine 1B and 5-hydroxytryptamine 1Dα proteins might therefore contain an addressing signal allowing their transport toward nerve endings. This localization is in good agreement with the fact that the 5-hydroxytryptamine 1B receptors have been shown to inhibit neurotransmitter release from nerve terminals.

Increase of central 5-HT 1B binding sites following 5,7-dihydroxytryptamine axotomy in the adult rat

The effects of selective axotomy of serotoninergic neurons produced by an intracerebroventricular injection of 5,7-dihydroxytryptamine (200 μg free base) on 5-HT 1B binding sites labeled with S-CM-G-[ 125I]TNH 2 were investigated by quantitative autoradiography in the rat brain. Results show, 21 days after surgery, an upregulation of 5-HT 1B receptors in the entorhinal cortex and the dorsomedial and suprachiasmatic nuclei of the hypothalamus. The cellular localization of those 5-HT 1B receptors exhibiting post-lesion plastic properties is discussed.

Evidence for existence of two distinct bradykinin receptors on rat mesangial cells.

We investigated the possible presence of bradykinin (BK) B1 receptor on rat mesangial cells (MC) by binding studies and by the effect of the B1 agonist des-Arg9-BK on intracellular calcium concentration ([Ca2+]i) and DNA synthesis in comparison with the effects of BK. Binding studies demonstrated specific, saturable binding for des-Arg9-[3H]BK inhibited by B1 but not by B2 antagonists. Scatchard analysis revealed a single class of B1 binding site with a maximum density of 15 fmol/mg protein and an affinity of 8.7 +/- 2.4 nM. Saturation and competition studies of 125I-[Tyr0]BK demonstrated the presence of two classes of B2 binding sites [dissociation constant (Kd) = 0.1 and 4 nM, respectively]. On fura-2-loaded adherent MC, both des-Arg9-BK and BK induced a biphasic increase (a transient enhancement followed by a sustained phase) in [Ca2+]i, both in primary culture and in cloned MC. Both the transient and sustained phases of [Ca2+]i induced by des-Arg9-BK were dose dependent, whereas BK induced a transient dose-dependent rise in [Ca2+]i, but the sustained phase remained constant. The increases in [Ca2+]i induced by des-Arg9-BK and BK were specifically abolished by B1 and B2 receptor antagonists, respectively, and showed homologous but not heterologous desensitization. Des-Arg9-BK and BK induced a significant proliferation (tested by cell counting and [3H]thymidine incorporation) of quiescent MC. Furthermore, the effects of des-Arg9-BK and BK were additive on Ca2+ mobilization but not on mitogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

The adrenoceptor agonist, SDZ NVI 085, discriminates between α1A- and α1B-adrenoceptor subtypes in vas deferens, kidney and aorta of the rat

The potency of the α 1-adrenoceptor agonist (−)-(4aR, 10aR)-3,4,4a,5,10,10a-hexahydro-6-methoxy-4-methyl-9-methylthio-2H-naphth [2,3-b]-1,4-oxazine (SDZ NVI 085) was investigated both in isolated vas deferens and perfused kidney of the rat, two tissues with α 1 A -adrenoceptor subtype characteristics, and in the rat thoracic aorta, in which the contribution of different α 1-adrenoceptor subtypes mediating contraction is controversial. In vas deferens and kidney, SDZ NVI 085 evoked smooth muscle contraction and vascular constriction and was of similar potency to L-phenylephrine. Contractions of vas deferens in response to (−)-noradrenaline and SDZ NVI 085 were resistant to chloroethylclonidine treatment (3 × 10 −5 M), sensitive to (±)-isradipine (10 −8 M) and competitively antagonized by 5-methyl-urapidil (pA 2 = 9.04 and 8.82, respectively). The potencies of a number of α 1A-/ α 1B-adrenoceptor-discriminating antagonists to reverse renal vasoconstriction due to either (−)-nora or SDZ NVI 085, and their affinities in vas deferens correlated significantly with their pK i values at α 1A binding sites in rat cortex. In rat aorta, SDZ NVI 085 up to 5 × 10 −4 M failed to evoke contraction. The affinities of subtype-selective antagonists determined in aorta correlated significantly with the pK i values at α 1B binding sites but differed from pK i values at α 1A sites in rat cortex. Thus, the contractile α 1-adrenoceptor in rat aorta can be best characterized as B subtype. SDZ NVI 085 might be a selective α 1A-adrenoceptor agonist and thus be used as a new tool either to detect (rat vas deferens and kidney) or exclude (rat aorta) a contribution of α 1A-adrenoceptors functionally involved in smooth muscle contraction.

The effects of electroconvulsive shock or imipramine on subtypes of α 1-adrenoceptors in the frontal cortex of the rat

The effects of repeated treatment (14 days) with electroconvulsive shock (ECS) or imipramine on binding sites on α 1-adrenoceptors in the rat were studied. The binding of [ 3H]prazosin studied with WB4101 and phentolamine, as binding inhibitors, showed the existence of two subtypes of α 1-adrenoceptor (α 1A and α 1B). Proportions of the α 1A and α 1B binding sites were about 3:7 in the frontal cortex and 9:1 in the hippocampus. Pretreatment of the membranes with chlorethylclonidine (CEC) almost abolished the α 1B binding sites. Inhibition of the binding of [ 3H]prazosin studied with antidepressants (imipramine, desipramine, maprotiline and mianserin) showed that these drugs bound to α 1-adrenoceptors with low affinity, in an apparent monophasic manner. The characteristics of the α 1A and α 1B binding sites were studied by the binding assay with [ 3H]prazosin, in the presence of a small concentration (2 nM) of WB4101 to mask the α 1A binding sites, as well as the assay without WB4101, for the total α 1-adrenoceptor (α 1A and α 1B) binding. Repeated treatment with electroconvulsive shock increased but that with imipramine decreased, the density of the α 1B binding sites in the frontal cortex, without change of the affinity. Neither treatment affected the α 1A binding sites in the frontal cortex. The α 1adrenoceptors (α 1A and α 1B) in the hippocampus were not affected at all by these repeated treatments. The electroconvulsive shock-induced increase in the α 1B binding sites in the frontal cortex of the rat could contribute to differences in clinical effects between electroconvulsive shock and antidepressant drugs.