Cellular mechanosignaling in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a vasculopathy characterized by sustained elevated pulmonary arterial pressures in which the pulmonary vasculature undergoes significant structural and functional remodeling. To better understand disease mechanisms, in this review article we highlight recent insights into the regulation of pulmonary arterial cells by mechanical cues associated with PAH. Specifically, the mechanobiology of pulmonary arterial endothelial cells (PAECs), smooth muscle cells (PASMCs) and adventitial fibroblasts (PAAFs) has been investigated in vivo, in vitro, and in silico. Increased pulmonary arterial pressure increases vessel wall stress and strain and endothelial fluid shear stress. These mechanical cues promote vasoconstriction and fibrosis that contribute further to hypertension and alter the mechanical milieu and regulation of pulmonary arterial cells.     

Adenosine and blood platelets

Adenosine is an important regulatory metabolite and an inhibitor of platelet activation. Adenosine released from different cells or generated through the activity of cell-surface ectoenzymes exerts its effects through the binding of four different G-protein-coupled adenosine receptors. In platelets, binding of A₂ subtypes (A_2A or A_2B) leads to consequent elevation of intracellular cyclic adenosine monophosphate, an inhibitor of platelet activation. The significance of this ligand and its receptors for platelet activation is addressed in this review, including how adenosine metabolism and its A₂ subtype receptors impact the expression and activity of adenosine diphosphate receptors. The expression of A₂ adenosine receptors is induced by conditions such as oxidative stress, a hallmark of aging. The effect of adenosine receptors on platelet activation during aging is also discussed, as well as potential therapeutic applications.     

Adenosine A2A and A2B receptor expression in neuroendocrine tumours: potential targets for therapy

The clinical management of neuroendocrine tumours is complex. Such tumours are highly vascular suggesting tumour-related angiogenesis. Adenosine, released during cellular stress, damage and hypoxia, is a major regulator of angiogenesis. Herein, we describe the expression and function of adenosine receptors (A(1), A(2A), A(2B) and A(3)) in neuroendocrine tumours. Expression of adenosine receptors was investigated in archival human neuroendocrine tumour sections and in two human tumour cell lines, BON-1 (pancreatic) and KRJ-I (intestinal). Their function, with respect to growth and chromogranin A secretion was carried out in vitro. Immunocytochemical data showed that A(2A) and A(2B) receptors were strongly expressed in 15/15 and 13/18 archival tumour sections. Staining for A(1) (4/18) and A(3) (6/18) receptors was either very weak or absent. In vitro data showed that adenosine stimulated a three- to fourfold increase in cAMP levels in BON-1 and KRJ-1 cells. The non-selective adenosine receptor agonist (adenosine-5'N-ethylcarboxamide, NECA) and the A(2A)R agonist (CGS21680) stimulated cell proliferation by up to 20-40% which was attenuated by A(2B) (PSB603 and MRS1754) and A(2A) (SCH442416) receptor selective antagonists but not by the A(1) receptor antagonist (PSB36). Adenosine and NECA stimulated a twofold increase in chromogranin A secretion in BON-1 cells. Our data suggest that neuroendocrine tumours predominantly express A(2A) and A(2B) adenosine receptors; their activation leads to increased proliferation and secretion of chromogranin A. Targeting adenosine signal pathways, specifically inhibition of A(2) receptors, may thus be a useful addition to the therapeutic management of neuroendocrine tumours.     

ETB-mediated contraction differs between left descending coronary artery and its next branch

Pig left descending coronary artery (main artery) and its next branch (branch arteries) differ in many properties. Here we report on the receptor types and the Ca²⁺ pools utilized for endothelin (ET) contraction in 3 mm long de-endothelialized rings of the main (weight 7.38 ± 0.38 mg) and the branch (1.07 ± 0.03 mg) arteries. KCl (60 mM) contracted the main and the branch arteries with force of 41.8 ± 3.1 and 16.9 ± 1.0 mN (millinewton), respectively. Force of contraction for all the other agents was normalized taking the KCl value as 100%. We determined the total ET-induced responses using ET-1 and those mediated by ET_B using IRL1620. In Ca²⁺-containing solutions, ET-1 contracted the main arteries with pEC_B = 8.2 ± 0.1 and a maximum force of 98 ± 5%. The branch arteries also gave similar values of pEC₅₀ (8.4 ± 0.1) and maximum force (99 ± 14%). IRL1620 contracted the main and the branch arteries with pEC₅₀ = 7.9 ± 0.1 but the maximum force was significantly higher in the branch arteries (44 ± 3%) than in the main (15 ± 2%). In Ca²⁺-free solutions, the pEC₅₀ values for ET-1 or IRL-1620 did not change but the maximum force of contraction was diminished considerably in both main and branch arteries. Thus, the left coronary artery and its next branch differ in that the role of ET_B receptors is greater in the latter.     

Autophagy in pulmonary hypertension: Emerging roles and therapeutic implications

Autophagy is an important mechanism for cellular self-digestion and basal homeostasis. This gene- and modulator-regulated pathway is conserved in cells. Recently, several studies have shown that autophagic dysfunction is associated with pulmonary hypertension (PH). However, the relationship between autophagy and PH remains controversial. In this review, we mainly introduce the effects of autophagy-related genes and some regulatory molecules on PH and the relationship between autophagy and PH under the conditions of hypoxia, monocrotaline injection, thromboembolic stress, oxidative stress, and other drugs and toxins. The effects of other autophagy-related drugs, such as chloroquine, 3-methyladenine, rapamycin, and other potential therapeutic drugs and targets, in PH are also described.     

Pharmacology of endothelins: vascular preparations for studying ETA and ETB receptors

Three rabbit vessels, the carotid and pulmonary arteries and the jugular vein were investigated to identify vascular monoreceptor systems (either ET_A or ET_B) to be used in structure-activity studies on endothelins and their antagonists. The RbCA has been found to behave as a monoreceptor ETA preparation, since it shows much greater sensitivity to ET-1 than to ET-3 and is insensitive to IRL 1620. The contractile response of the RbCA to ET-1 is reduced in the presence of BQ-123 but is not influenced by BQ-788. The RbPA behaves as a pure ET_B system when stimulated with the ET_B selective agonist IRL 1620. The contractile effect of IRL 1620 is reduced in the presence of BQ-788 but is not influenced by BQ-123. The RbJV responds to ET_A and to IRL 1620 with contractions that are reduced by both BQ-123 and BQ-788, respectively. The RbJV appears to be a mixed ET_A and ET_B system in which the two functional sites play an equivalent role in the stimulatory contractile response.Thus, contractile ET_A and ET_B receptors have been found in arterial and venous vessels of the rabbit and some of these vessels provide sensitive and selective (either ET_A or ET_B) preparations that appear to be adequate for pharmacological studies on ET receptor agonists or antagonists.     

Endothelin contraction in pig coronary: Receptor types and Ca2+-mobilization

Endothelin is one of the most potent vasoconstrictors known. It plays an important role in the regulation of vascular tone and in the development of many cardiovascular diseases. This study focuses on the receptor types and the Ca²⁺ mobilization responsible for endothelin-1 (ET-1) contraction in de-endothelialized pig coronary artery rings. ET-1 contracted the artery rings with an EC₅₀ = 6.5 ± 1 nM and a maximum contraction which was 98.6 ± 9% of the contraction produced by 60 mM KCl. BQ123 (5 µM), an ET_A antagonist, reversed 78 ± 3% of the ET-1 contraction (50 nM). IRL1620, a selective ET_B agonist, produced 23 ± 3% of the total ET-1 contraction with an EC₅₀ = 12.7 ± 2 nM. More than 85% of the contraction due to 100 nM IRL 1620 was inhibited by 200 nMBQ788, an ET_B antagonist. Therefore, approximately 80% of the ET-1 contraction in this artery occurred via ET_A receptors, and the other 20% was mediated by ET_B receptors. To assess the Ca²⁺ pools utilized during the ET-1 response, ET-1 contraction was also examined in medium containing an L-type Ca²⁺ channel blocker nitrendipine, and in Ca²⁺ free medium containing 0.2 mM EGTA. In Ca²⁺ containing medium the contraction elicited by ET-1 was 98.6 ± 9% of the KCl contraction, however, in the presence 10 µM nitrendipine the ET-1 induced contraction was 54 ± 7% of the KCl contraction, and in Ca²⁺-free medium it was 13 ± 2%. Similarly, the IRL 1620 contractions in Ca²⁺ containing medium, in the presence of nitrendipine and in Ca²⁺-free medium were 22.4 ± 3%, 12 ± 3% and 11 ± 2% of the KCl response respectively. Thus, both ET_A and ET_B contractions utilize extracellular Ca²⁺ pools via L-type Ca²⁺ channels and other undefined route(s), as well as intracellular Ca²⁺ pools. In the pig coronary artery smooth muscle, ET-1 contractions occur predominantly via ET_A receptors, with ET_B receptors using similar Ca²⁺ mobilization pathways, but the ET_B receptors appear to use the intracellular Ca²⁺ stores to a greater extent.     

Analysis of T102C 5HT2A polymorphism in Brazilian psychiatric inpatients: relationship with suicidal behavior

1. Central serotonergic dysfunction and genetic factors are associated with suicidal behavior in psychiatric patients. The goal of this study was to examine the association between the 5-HT_2A gene polymorphism (102T/C) and suicide in a sample of Brazilian psychiatric inpatients.2. We studied 225 subjects. Genotypic frequencies were obtained after DNA extraction and the region of 5-HT_2A/T102C containing the polymorphic site amplified by the polymerase chain reaction and digested with the restriction enzyme HpaII.3. No differences were found between patients with and without suicide attempt history. Patients with a history of severe suicide attempts also did not exhibit different genotypic frequencies when compared with patients without a suicide attempt history.4. These results suggest that the 5HT_2A gene polymorphism (102T/C) may not be involved in the genetic susceptibility to suicidal behavior.     

Angiotensin II enhances endothelin-1-induced vasoconstriction through upregulating endothelin type A receptor

Endothelin-1 (ET-1) is the most potent vasoconstrictor by binding to endothelin receptors (ET_AR) in vascular smooth muscle cells (VSMCs). The complex of angiotensin II (Ang II) and Ang II type one receptor (AT₁R) acts as a transient constrictor of VSMCs. The synergistic effect of ET-1 and Ang II on blood pressure has been observed in rats; however, the underlying mechanism remains unclear. We hypothesize that Ang II leads to enhancing ET-1-mediated vasoconstriction through the activation of endothelin receptor in VSMCs. The ET-1-induced vasoconstriction, ET-1 binding, and endothelin receptor expression were explored in the isolated endothelium-denuded aortae and A-10 VSMCs. Ang II pretreatment enhanced ET-1-induced vasoconstriction and ET-1 binding to the aorta. Ang II enhanced ET_AR expression, but not ET_BR, in aorta and increased ET-1 binding, mainly to ET_AR in A-10 VSMCs. Moreover, Ang II-enhanced ET_AR expression was blunted and ET-1 binding was reduced by AT₁R antagonism or by inhibitors of PKC or ERK individually. In conclusion, Ang II enhances ET-1-induced vasoconstriction by upregulating ET_AR expression and ET-1/ET_AR binding, which may be because of the AngII/Ang II receptor pathways and the activation of PKC or ERK. These findings suggest the synergistic effect of Ang II and ET-1 on the pathogenic development of hypertension.     

Endothelin-1 binding to endothelin receptors in the rat anterior pituitary gland: possible formation of an ETA-ETB receptor heterodimer

1. Interaction in the recognition of endothelin-1 (ET-1), a typical bivalent ET receptor-ligand, between ET_A and ET_B receptors was investigated in the rat anterior pituitary gland, using our quantitative receptor autoradiographic method with tissue sections preserving the cell-membrane structure and ET receptor-related compounds.2. In saturation binding studies with increasing concentrations (0.77–200 pM) of ¹²⁵I-ET-1 (nonselective bivalent radioligand), ¹²⁵I-ET-1 binding to the rat anterior pituitary gland was saturable and single with a K _D of 71 pM and a B _max of 120 fmol mg^–1. When 1.0 M BQ-123 (ET_A antagonist) was added to the incubation buffer, binding parameters were 8.3 pM of K _D and 8.0 fmol mg^–1 of B _max, whereas 10 nM sarafotoxin S6c (ET_B agonist) exerted little change in these binding parameters (K _D, 72 pM; B _max, 110 fmol mg^–1).3. Competition binding studies with a fixed amount (3.8 pM) of ¹²⁵I-ET-1 revealed that when 1.0 M BQ-123 was present in the incubation buffer, ET_B receptor-related compounds such as sarafotoxin S6c, ET-3, IRL1620 (ET_B agonist), and BQ-788 (ET_B antagonist) competitively inhibited ¹²⁵I-ET-1 binding with K _is of 140, 18, 350 pM, and 14 nM, respectively, however, these compounds were not significant competitors for ¹²⁵I-ET-1 binding in the case of absence of BQ-123.4. In cold-ligand saturation studies with a fixed amount (390 pM) of ¹²⁵I-IRL 1620 (ET_B radioligand), IRL1620 bound to a single population of the ET_B receptor, and no change was observed in binding characteristics in the presence of 1.0 M BQ-123. ¹²⁵I-IRL1620 binding was competitively inhibited by ET-1 and ET-3 in the absence of BQ-123, with K _is of 20 and 29 pM, respectively, the affinities being much the same as those of 29 nM, in the presence of 1.0 M BQ-123.5. Two nonbivalent ET_A antagonists, BQ-123 and PD151242, were highly sensitive and full competitors for ¹²⁵I-ET-1 binding (5.0 pM), in the presence of 10 nM sarafotoxin S6c.6. Taken together with the present finding that mRNAs encoding the rat ET_A and the ET_B receptors are expressed in the anterior pituitary gland, we tentatively conclude that although there are ET_A and ET_B receptors with a functional binding capability for ET receptor-ligands, the ET_B receptor does not independently recognize ET-1 without the aid of the ET_A receptor. If this thesis is tenable, then ET-1 can bridge between the two receptors to form an ET_A–ET_B receptor heterodimer.     

The role of activated adenosine receptors in degranulation of human LAD2 mast cells

Mast cell degranulation triggers hypersensitivity reactions at the body–environment interface. Adenosine modulates degranulation, but enhancement and inhibition have both been reported. Which of four adenosine receptors (ARs) mediate modulation, and how, remains uncertain. Also uncertain is whether adenosine reaches mast cell ARs by autocrine ATP release and ecto-enzymatic conversion. Uncertainties partly reflect species and cell heterogeneity, circumvented here by focusing on homogeneous human LAD2 cells. Quantitative PCR detected expression of A_2A, A_2B, and A₃, but not A₁, ARs. Nonselective activation of ARs with increasing NECA monotonically enhanced immunologically or C3a-stimulated degranulation. NECA alone stimulated degranulation slightly. Selective AR antagonists did not affect C3a-stimulated degranulation. NECA''s enhancement of C3a-triggered degranulation was partially inhibited by separate application of each selective antagonist, and abolished by simultaneous addition of antagonists to the three ARs. Only the A_2A antagonist separately inhibited NECA''s enhancement of immunologically stimulated degranulation, which was abolished by simultaneous addition of the three selective antagonists. Immunological or C3a activation did not stimulate ATP release. NECA also enhanced immunologically triggered degranulation of mouse bone marrow derived mast cells (BMMCs), which was partially reduced only by simultaneous addition of the three antagonists or by the nonselective antagonist {"type":"entrez-protein","attrs":{"text":"CGS15943","term_id":"875345334"}}CGS15943. BMMCs also expressed A_2A, A_2B, and A₃ ARs. but not A₁AR detectably. We conclude that (a) A₁AR is unnecessary for LAD2 degranulation or AR enhancement; (b) A_2A, A_2B, and A₃ ARs all contribute to pharmacologic AR enhancement of LAD2 and BMMC degranulation; and (c) LAD2 cells depend on microenvironmental adenosine to trigger AR modulation.     

NAADP-induced intracellular calcium ion is mediated by the TPCs (two-pore channels) in hypoxia-induced pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a form of obstructive vascular disease. Chronic hypoxic exposure leads to excessive proliferation of pulmonary arterial smooth muscle cells and pulmonary arterial endothelial cells. This condition can potentially be aggravated by [Ca²⁺] _i mobilization. In the present study, hypoxia exposure of rat's model was established. Two-pore segment channels (TPCs) silencing was achieved in rats' models by injecting Lsh-TPC1 or Lsh-TPC2. The effects of TPC1/2 silencing on PAH were evaluated by H&E staining detecting pulmonary artery wall thickness and ELISA assay kit detecting NAADP concentrations in lung tissues. TPC1/2 silencing was achieved in PASMCs and PAECs, and cell proliferation was detected by MTT and BrdU incorporation assays. As the results shown, NAADP-activated [Ca²⁺]_i shows to be mediated via two-pore segment channels (TPCs) in PASMCs, with TPC1 being the dominant subtype. NAADP generation and TPC1/2 mRNA and protein levels were elevated in the hypoxia-induced rat PAH model; NAADP was positively correlated with TPC1 and TPC2 expression, respectively. In vivo, Lsh-TPC1 or Lsh-TPC2 infection significantly improved the mean pulmonary artery pressure and PAH morphology. In vitro, TPC1 silencing inhibited NAADP-AM-induced PASMC proliferation and [Ca²⁺]_i in PASMCs, whereas TPC2 silencing had minor effects during this process; TPC2 silencing attenuated NAADP-AM- induced [Ca²⁺]_i and ECM in endothelial cells, whereas TPC1 silencing barely ensued any physiological changes. In conclusion, TPC1/2 might provide a unifying mechanism within pulmonary arterial hypertension, which can potentially be regarded as a therapeutic target.     

Metabolic depletion of sphingolipids enhances the mobility of the human serotonin1A receptor

Sphingolipids are essential components of eukaryotic cell membranes. We recently showed that the function of the serotonin_1A receptor is impaired upon metabolic depletion of sphingolipids using fumonisin B₁ (FB₁), a specific inhibitor of ceramide synthase. Serotonin_1A receptors belong to the family of G-protein coupled receptors and are implicated in the generation and modulation of various cognitive, behavioral and developmental functions. Since function and dynamics of membrane receptors are often coupled, we monitored the lateral dynamics of the serotonin_1A receptor utilizing fluorescence recovery after photobleaching (FRAP) under these conditions. Our results show an increase in mobile fraction of the receptor upon sphingolipid depletion, while the diffusion coefficient of the receptor did not exhibit any significant change. These novel results constitute the first report on the effect of sphingolipid depletion on the mobility of the serotonin_1A receptor. Our results assume greater relevance in the broader context of the emerging role of receptor mobility in understanding cellular signaling.     

Proterguride, a highly potent dopamine receptor agonist promising for transdermal administration in Parkinson's disease: interactions with alpha(1)-, 5-HT(2)- and H(1)-receptors

Dopamine receptor agonists play an important role in the treatment of Parkinson's disease and hyperprolactinemic conditions. Proterguride (n-propyldihydrolisuride) was already reported to be a highly potent dopamine receptor agonist, thus its action at different non-dopaminergic monoamine receptors, alpha(1A/1B/1D), 5-HT(2A/2B)- and histamine H(1), was investigated using different functional in vitro assays. The drug behaved as an antagonist at alpha(1)-adrenoceptors without the ability to discriminate between the subtypes (pA(2) values: alpha(1A) 7.31; alpha(1B) 7.37; alpha(1D) 7.35) and showed antagonistic properties at the histamine H(1) receptor. In contrast, at serotonergic receptors (5-HT(2A), 5-HT(2B)) proterguride acted as a partial agonist. The drug stimulated 5-HT(2A) receptors of rat tail artery in lower concentrations than 5-HT itself but failed to evoke comparable efficacy (proterguride: pEC(50) 8.34, E(max) 53% related to the maximum response to 5-HT; 5-HT: pEC(50) 7.03). Agonism at 5-HT(2B) receptors is presently considered to be involved in drug-induced valvular heart disease. Activation of 5-HT(2B) receptors in porcine pulmonary arteries by proterguride (pEC(50) 7.13, E(max) 49%; E(max) (5-HT) 69%), however, occurred at concentrations much higher than plasma concentrations achieving dopaminergic efficacy in humans. The results are discussed focussing on the relevance of action at 5-HT(2B) receptors as well as their significance for a transdermal administration of proterguride. Since it is well accepted that pulsatile dopaminergic stimulation is associated with treatment-related motor complications in the dopaminergic therapy of Parkinson's disease, the transdermal route of administration is of great clinical interest due to the possibility to achieve constant plasma concentrations.     

TNF-alpha upregulates the A2B adenosine receptor gene: The role of NAD(P)H oxidase 4

Proliferation of vascular smooth muscle cells (VSMC), oxidative stress, and elevated inflammatory cytokines are some of the components that contribute to plaque formation in the vasculature. The cytokine tumor necrosis factor-alpha (TNF-α) is released during vascular injury, and contributes to lesion formation also by affecting VSMC proliferation. Recently, an A_2B adenosine receptor (A_2BAR) knockout mouse illustrated that this receptor is a tissue protector, in that it inhibits VSMC proliferation and attenuates the inflammatory response following injury, including the release of TNF-α. Here, we show a regulatory loop by which TNF-α upregulates the A_2BAR in VSMC in vitro and in vivo. The effect of this cytokine is mimicked by its known downstream target, NAD(P)H oxidase 4 (Nox4). Nox4 upregulates the A_2BAR, and Nox inhibitors dampen the effect of TNF-α. Hence, our study is the first to show that signaling associated with Nox4 is also able to upregulate the tissue protecting A_2BAR.     

Synthesis, biological activity and molecular modelling studies of tricyclic alkylimidazo-, pyrimido- and diazepinopurinediones

Syntheses and biological activities of imidazo-, pyrimido- and diazepino[2,1-f]purinediones containing N-alkyl substituents (with straight, branched or unsaturated chains) are described. Tricyclic derivatives were synthesized by the cyclization of 8-bromo-substituted 7-(2-bromoethyl)-, 7-(3-chloropropyl)- or 7-(4-bromobutyl)-theophylline with primary amines under various conditions. Compound 22 with an ethenyl substituent was synthesized by dehydrohalogenation of 9-(2-bromoethyl)-1,3-dimethyltetrahydropyrimido[2,1-f]purinedione. The obtained derivatives (5–35) were initially evaluated for their affinity at rat A₁ and A_2A adenosine receptors (AR), showing moderate affinity for both adenosine receptor subtypes. The best ligands were diazepinopurinedione 28 (K_i = 0.28 μM) with fivefold A_2A selectivity and the non-selective A₁/A_2A AR ligand pyrimidopurinedione 35 (K_i A₁ = 0.28 μM and K_i A_2A = 0.30 μM). The compounds were also evaluated for their affinity at human A₁, A_2A, A_2B and A₃ ARs. All of the obtained compounds were docked to the A_2A AR X-ray structure in complex with the xanthine-based, potent adenosine receptor antagonist—XAC. The likely interactions of imidazo-, pyrimido- and diazepino[2,1-f]purinediones with the residues forming the A_2A binding pocket were discussed. Furthermore, the new compounds were tested in vivo as anticonvulsants in maximal electroshock, subcutaneous pentylenetetrazole (ScMet) and TOX tests in mice (i.p.). Pyrimidopurinediones showed anticonvulsant activity mainly in the ScMet test. The best derivative was compound 11, showing 100 % protection at a dose of 100 mg/kg without symptoms of neurotoxicity. Compounds 6, 7, 8 and 14 with short substituents showed neurotoxicity and caused death. In rat tests (p.o.), 9 was characterized by a high protection index (>13.3). AR affinity did not apparently correlate with the antiepileptic potency of the compounds.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-013-9358-3) contains supplementary material, which is available to authorized users.     

Up-Regulation of 5-HT2B Receptor Density and Receptor-Mediated Glycogenolysis in Mouse Astrocytes by Long-Term Fluoxetine Administration

The effects were studied of short-term (1 week) versus long-term (2-3 weeks) fluoxetine treatment of primary cultures of mouse astrocytes, differentiated by treatment with dibutyryl cyclic AMP. From previous experiments it is known that acute treatment with fluoxetine stimulates glycogenolysis and increases free cytosolic Ca²⁺ concentration ([Ca²⁺]_i]) in these cultures, whereas short-term (one week) treatment with 10 M down-regulates the effects on glycogen and [Ca²⁺]_i, when fluoxetine administration is renewed (or when serotonin is administered). Moreover, antagonist studies have shown that these responses are evoked by activation of a 5-HT₂ receptor that is different from the 5-HT_2A receptor and therefore at that time tentatively were interpreted as being exerted on 5-HT_2C receptors. In the present study the cultures were found by RT-PCR to express mRNA for 5-HT_2A and 5-HT_2B receptors, but not for the 5-HT_2C receptor, identifying the 5-HT₂ receptor activated by fluoxetine as the 5-HT_2B receptor, the most recently cloned 5-HT₂ receptor and a 5-HT receptor known to be more abundant in human, than in rodent, brain. Both short-term and long-term treatment with fluoxetine increased the specific binding of [³H]mesulergine, a ligand for all three 5-HT₂ receptors. Long-term treatment with fluoxetine caused an agonist-induced up-regulation of the glycogenolytic response to renewed administration of fluoxetine, whereas short-term treatment abolished the fluoxetine-induced hydrolysis of glycogen. Thus, during a treatment period similar to that required for fluoxetine's clinical response to occur, 5-HT_2B-mediated effects are initially down-regulated and subsequently up-regulated.     

Endothelin-1 Binding to Endothelin Receptors in the Rat Anterior Pituitary Gland: Interaction in the Recognition of Endothelin-1 Between ETA and ETB Receptors

1. ¹²⁵I-Endothelin (ET)-1 binding to the rat anterior pituitary gland was saturable and single, with a K _d of 71 pM and a B _max of 120 fmol/mg.2. When 1.0 M BQ-123 (ET_A antagonist) was added to the incubation buffer, the binding parameters were 8.3 pM and 8.0 fmol/mg, whereas 10 nM sarafotoxin S6c (ET_Bagonist) exerted little change in these binding parameters (K _d,72pM;B _max, 110 fmol/mg).3. ET_B receptor-related compounds such as sarafotoxin S6c, ET-3, IRL1620, and BQ-788 competitively inhibited ¹²⁵I-ET-1 binding, only when 1.0 M BQ-123 was present in the incubation buffer.4. Thus, the ET_B receptor is capable of binding ET-1 when the ET_A receptor is being occupied by BQ-123. A collaboration mechanism between the ET_A and the ET_B receptor may function in the recognition of ET-1, a typical bivalent ligand.