Structural Basis for Activation of G‐Protein‐Coupled Receptors

Abstract: Our understanding of how G‐protein‐coupled receptors (GPCRs) operate at the molecular level has been considerably improved over the last few years. The application of advanced biophysical techniques as well as the availability of high‐resolution structural information has allowed insight both into conformational changes accompanying GPCR activation and the underlying molecular mechanism governing transition of the receptor between its active and inactive states. Using the β₂‐adrenergic receptor as a model system we have obtained evidence for an evolutionary conserved activation mechanism where disruption of intramolecular interactions between TM3 and TM6 leads to a major conformational change of TM6 relative to the rest of the receptor. This conclusion was based on experiments in which environmentally sensitive, sulfhydryl‐reactive fluorophores were site‐selectively incorporated into wild‐type and mutant β₂‐adrenergic receptors purified from Sf‐9 insect cells. Our studies have also raised important questions regarding kinetics of receptors activation. These questions should be addressed in the future by application of techniques that will allow for simultaneous measurement of conformational changes and receptor activation. At the current stage we are exploring the possibility of reaching this goal by direct in situ labeling of the β₂‐adrenergic receptor in Xenopus Laevis oocytes with conformationally sensitive fluorescent probes and parallel detection of receptor activation by co‐expression with the cAMP sensitive Cl^? channel CFTR (cystic fibrosis transmembrane conductance regulator) and electrophysiological measurements.     

Functional Characterization of Endothelin Receptors in the Bovine Retractor Penis Muscle and Penile Artery

Abstract: The effects of endothelin-1 and sarafotoxin 6c on the bovine retractor penis muscle and the bovine penile artery were studied, and a functional characterization of endothelin receptors in these tissues was performed by using the ET_A-receptor antagonist BQ-123 and the ET_B-receptor antagonist IRL 1038. The retractor penis muscle and the penile artery were about equipotently contracted by endothelin-1 in a concentration-dependent manner the EC₅₀ values being 3.5 X 10^-9 M and 1.3 X 10^-9 M, respectively. In both tissues BQ-123 (10^-6 M) inhibited maximal contraction induced by endothelin-1 by about 50%. Sarafotoxin 6c substantially relaxed the retractor penis muscle, and to a lesser extent also the penile artery, whereas endothelin-1 did not relax either tissue. The sarafotoxin 6c-induced relaxation of the retractor penis muscle was totally inhibited by IRL 1038 (3 X 10^-6 M) and the nitric oxide synthase inhibitor L-NNA (10^-4 M). In both tissues L-NNA enhanced the contraction induced by endothelin-1 and lowered the threshold concentration for it. The results show that in both tissues the contraction induced by endothelin-1 was mediated primarily by ET_A-receptors. The retractor penis muscle is also equipped with ET_B-receptors, probably at least in part located on the inhibitory nerves, which mediate relaxation via activation of the L-arginine nitric oxide synthase pathway.     

Chemogenetic Modulation of G Protein‐Coupled Receptor Signalling in Visual Attention Research

Attention is a fundamental cognitive process involved in nearly all aspects of life. Abnormal attentional control is a symptom of many neurological disorders, most notably recognized in ADHD (attention deficit hyperactivity disorder). Although attentional performance and its malfunction has been a major area of investigation, it has proven difficult to accurately associate specific neuronal projections, cell types, neurotransmitter systems and receptors with distinct phenotypes owing to its complexity. In this MiniReview, we present a recently invented technology known as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). The DREADD technology is an emerging and transformative method that allows selective manipulation of G protein‐coupled receptor (GPCR) signalling, and its broad‐ranging usefulness in attention research is now beginning to emerge. We first describe the different DREADDs available and explain how unprecedented specificity of neuronal signalling can be achieved using DREADDs. We next discuss various studies performed in animal models of visual attention, where different brain regions and neuronal populations have been probed by DREADDs. We highlight the interplay between the dopamine (DA) and noradrenaline (NA) catecholamine systems in visual attention and explain why DREADD technology can untangle and help us better understand such complex systems in normal and malfunctioning conditions.     

Endothelin‐1 and Endothelin‐3 Regulate Endothelin Receptor Expression in Rat Coronary Arteries

In ischaemic hearts, endothelin (ET) levels are increased, and vasoconstrictor responses to ET‐1 are greatly enhanced. We previously reported that ET_B receptors are up‐regulated in the smooth muscle layer of coronary arteries after myocardial ischaemia–reperfusion and that the MEK–ERK1/2 signalling pathway is involved in ET_B receptor up‐regulation. Whether ETs are directly involved in receptor regulation has not been determined. We suggest that ET‐1 and ET‐3 alter the expression/activity of ET receptors in coronary vascular smooth muscle cells. Vasoconstrictor responses were studied in endothelium‐denuded coronary artery segments from rats that were subjected to experimental ischaemia–reperfusion or in organ‐cultured segments. Post‐ischaemic and cultured coronary arteries exhibited similar increased sensitivity to ET‐3. ET_A receptor‐mediated vasoconstriction was dominant in fresh and non‐ischaemic arteries. Organ culture significantly up‐regulated ET_B receptors and down‐regulated ET_A receptor expression. Co‐incubation with ET‐1 (1 nM) or ET‐3 (100 nM) induced further down‐regulation of the ET_A receptor mRNA, while the function and protein level of ET_A remained unchanged. ET‐3 (100 nM) further up‐regulated ET_B receptor mRNA and proteins but abolished ET_B receptor‐mediated vasoconstriction, suggesting a desensitization of ET_B receptors that was not observed with ET‐3 (1 nM). In conclusion, ET‐1, which is the most prevalent isoform in the cardiovascular system, induces down‐regulation of ET_A receptor expression without changing ET_A or ET_B receptor function or protein levels. Intermediate concentrations of ET‐3 had an effect that was similar to that of ET‐1, such that high concentrations of ET‐3 (100 nM) up‐regulated the ET_B receptor at the gene and protein levels but switched off the function of the ET_B receptors via desensitization.     

Anabolic Effect of Insulin Therapy on the Bone: Osteoprotegerin and Osteocalcin Up‐Regulation in Streptozotocin‐Induced Diabetic Rats

Type 1 diabetes mellitus (T1DM) is associated with several skeletal alterations, particularly in conditions of poor glycaemic control. Insulin therapy is the major conservative treatment for T1DM; however, the effects of this hormone on bone markers of T1DM rats are limited, and the regulatory mechanisms remain elusive. Therefore, the evaluation of molecular and non‐molecular parameters in a chronic animal model of T1DM‐induced bone loss, treated with and without insulin, may help in elucidating the insulin mechanisms. Male Wistar rats were assigned into three groups: control, T1DM (T1DM rats induced with streptozotocin [STZ] at 40 mg/kg intravenously) and T1DM plus insulin therapy (T1DMI). After 8 weeks, we evaluated the serum biochemical, tibia histomorphometric and biomechanical parameters, as well as the gene expression of the receptor activator of nuclear factor kappa‐B ligand (RANKL), osteoprotegerin (OPG) and osteocalcin (OC) of femur mRNA. Compared with T1DM, the T1DMI group showed less bone loss, which was revealed by the increased trabecular width (TbWi, p < 0.001) and trabecular bone area (BAr, p < 0.01), reduced trabecular separation (TbSp, p < 0.01) and increased Young's modulus (p < 0.05). Moreover, molecular analyses indicated that the expression of OPG and OC was up‐regulated (p < 0.001 and p < 0.05, respectively). In summary, the up‐regulation of OPG and OC in the T1DMI group supports an anabolic effect of insulin, which was demonstrated by the maintenance of bone architecture and flexibility. These results suggest that insulin therapy may prevent T1DM‐induced bone loss via the effects on the bone formation.     

疏血通注射液对原发性肾病综合征患者血浆内皮素和可溶性白介素-2受体的影响

目的:探讨疏血通注射液治疗原发性肾病综合征(PNS)患者后对其血浆内皮素(ET)及可溶性白介素-2受体(sIL-2R)水平的影响。方法:将44例PNS患者随机分为常规治疗组20例及常规治疗加疏血通注射液治疗组(干预组)24例,观察治疗前、后2组ET及sIL-2R水平变化情况。结果:治疗前患者血浆ET、sIL-2R水平均高于正常对照组(P<0·01),2组治疗后与其治疗前水平比较均显著降低(P<0·05),治疗后2组比较具有显著性差异(P<0·05)。结论:PNS患者在常规治疗基础上加用疏血通注射液能更有效地改善患者血浆ET、sIL-2R水平,从而减轻肾组织的损害。     

Pulmonary Responses to Oil Fly Ash Particles in the Rat Differ by Virtue of Their Specific Soluble Metals

Occupational exposure to residual oil fly ash (ROFA) particulatehas been associated with adverse respiratory health effectsin humans. We hypothesized that ROFA collected at differentsites within an oil burning power plant, by virtue of its differingmetal and sulfate composition, will induce differential lunginjury. Ten ROFA samples collected at various sites within apower plant were analyzed for water- and 1.0 M HCl-leachablearsenic (As), beryllium (Be), cadmium (Cd), cobalt (Co), chromium(Cr), copper (Cu), iron (Fe), manganese (Mn), nickel (Si), lead(Pb), vanadium (V), zinc (Zn), and sulfur by inductively coupledplasma-atomic emission spectroscopy. All ROFA samples containedvariable amounts of teachable (water-extractable) and 1.0 MHCl-extractable Fe, V, and/or Ni. All other metals, except Zn(ROFA No. 1 contained 3.43 and No. 3, 6.35 µg/mg Zn),were present in negligible quantities (<1.0 µg/mg)in the water extract In vivo pulmonary injury from exposureto whole saline suspensions of these ROFA was evaluated. Male,SD rats (60 days old) were intratracheally instilled with eithersaline or saline suspension of whole ROFA (<3.0 mass medianaerodynamic diameter) at three concentrations (0.833, 3.33,or 8.33 mg/kg). After 24 h, lungs were lavaged and bronchoalveolarlavage fluid (BALF) was analyzed for cellular influx and proteincontent as well as lactate dehydro-genase (LDH) and N-acetylglucosaminidase (NAG) activity and total hemoglobin as indicatorsof lung injury. ROFA-induced increases in BALF protein and LDH,but not neutrophilic inflammation, were associated with itswater-leachable total metal, Ni, Fe, and sulfate content. However,the neutrophilic response following ROFA exposure was positivelycorrelated with its water-leachable V content Modest lung injurywas observed with the ROFA samples which contained the smallestamounts of water-leachable metals. The ability of ROFA to induceoxidative burst in alveolar macrophage (AM) was determined invitro using a chemiluminescence (CL) assay. AM CL signals invitro were greatest with ROFA containing primarily soluble Vand were less with ROFA containing Ni plus V. In summary, ROFA-inducedin vivo acute pulmonary inflammation appears to be associatedwith its water-leachable V content; however, protein leakageappears to be associated with its water-leachable Ni content.ROFA-induced in vitro activation of AM was highest with ROFAcontaining leachable V but not with Ni plus V, suggesting thatthe potency and the mechanism of pulmonary injury will differbetween emissions containing V and Ni.     

Competitive Blockade of α-Adrenergic Receptors in Rat Heart by Prazosi

Abstract: Pheiiykphriiie (PE) in presence of propranolol evokes an α-adrenergic inotroyic response in rat heart. The time course of this response is characterizcd by a transient ducrease in maximal developed tension (T_max) subcontrol levels (negative phase of the inotropic response) followed by an increase which reaches maximum after 4–5 min. (positive phase of the inotropic response). Prazosin (PRZ), a selective α₁-receptor blocker. inhibited preferentially the positive phase of the inotropic response and displaced the dose-responsecurve of PE to the right in nanomolar concentrations, indicating a competitive mechanism of inhibition. Phentolamine, a non-selrctive α-blocker. blocked both the negative and the positive phase olthe inotropicresponse toabout the same degree. PRZ appears to be a competitive α-adrenergic antagonist with high affinity in rat heart. Two populations of α-adrenergic receptors may he present: one stirnulatory (α₁) iind one inhibitory     

Involvement of 5‐HT2A Receptors in the Serotonin (5‐HT) Syndrome caused by Excessive 5‐HT Efflux in Rat Brain

Abstract: Previous studies have demonstrated that serotonin (5‐HT) syndromes, particularly for the malignant cases, can be alleviated by ice water mists, cooling blankets and many other external cooling measures. In this study, we tested the hypothesis that external cooling measures reduce the responsivity of 5‐HT_2A receptors to excessive 5‐HT efflux, which may be a possible mechanism underlying the treatment of serotonin syndrome. To test this, rat experiments were carried out in the standard and cool ambient temperature (T_amb) by administration of the 5‐HT precursor 5‐hydroxy‐l ‐tryptophan combined with the monoamine oxidase inhibitor clorgyline. The first set of experiments was to assess severity of the syndromes by measuring body temperature responses. Consistent with the hypothesis, we found that the syndrome was malignant at the standard T_amb of 22°C but alleviated at 12 or 6°C, these results being similar to those in rats pre‐treated with the 5‐HT_2A receptor antagonist ketanserin. The second set of experiments was to utilize microdialysis to determine the relationship between the syndrome severity and 5‐HT levels at the above‐mentioned T_amb. We found that excessive 5‐HT efflux consisted of primary and secondary components through two distinct mechanisms. Furthermore, the secondary component efflux, which can be ascribed to 5‐HT_2A receptor activation, was proportionally reduced at the cool T_amb of 12 and 6°C. In conclusion, results of this study support the hypothesis that cooling T_amb reduces the functional activity of 5‐HT_2A receptors, thus alleviating the malignant syndrome.     

Impairment of the Ca2+‐Permeable AMPA/Kainate Receptors by Lead Exposure in Organotypic Rat Hippocampal Slice Cultures

Previous studies have demonstrated that chronic lead exposure may impair neuronal process underlying synaptic plasticity via a direct interaction with N‐methyl‐D‐aspartate (NMDA) receptors. The present study was carried out to investigate the effects of lead exposure on non‐NMDA (α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid, AMPA/kainate) receptors of rat hippocampus. Ca²⁺‐permeable AMPA/kainate receptors in organotypic slice cultures were evaluated by using cobalt uptake, a histochemical method that identifies cells expressing Ca²⁺‐permeable non‐NMDA receptors. Ten mM L‐glutamate‐induced cobalt accumulation was enriched in area CA1, area CA3 and in dentate gyrus, which was totally blocked by 100 μM DL‐2‐amino‐5‐phosphonovaleric acid (AP5) and 100 μM 6‐cyano‐7‐nitroquinoxaline‐2, 3‐dione (CNQX). Three hundred μM NMDA‐induced cobalt accumulation was in area CA1, area dentate gyrus and was blocked by AP5 or CNQX. One hundred μM AMPA had effects in area CA1, area CA3 and in dentate gyrus, which were blocked by CNQX, not by AP5. Furthermore, cobalt accumulations induced by NMDA and AMPA in the lead‐exposed rats decreased significantly than those in the controls. The results indicate that AMPA receptors enriched in area CA1, area CA3, area dentate gyrus and kainate receptors enriched in area CA1, area dentate gyrus are impaired by lead exposure.     

Requirement of Pretone by Thromboxane A(2) for Hypoxic Pulmonary Vasoconstriction in Precision-cut Lung Slices of Rat

Hypoxic pulmonary vasoconstriction (HPV) is physiologically important response for preventing mismatching between ventilation and perfusion in lungs. The HPV of isolated pulmonary arteries (HPV-PA) usually require a partial pretone by thromboxane agonist (U46619). Because the HPV of ventilated/perfused lungs (HPV-lung) can be triggered without pretone conditioning, we suspected that a putative tissue factor might be responsible for the pretone of HPV. Here we investigated whether HPV can be also observed in precision-cut lung slices (PCLS) from rats. The HPV in PCLS also required partial contraction by U46619. In addition, K(+) channel blockers (4AP and TEA) required U46619-pretone to induce significant contraction of PA in PCLS. In contrast, the airways in PCLS showed reversible contraction in response to the K(+) channel blockers without pretone conditioning. Also, the airways showed no hypoxic constriction but a relaxation under the partial pretone by U46619. The airways in PCLS showed reliable, concentration-dependent contraction by metacholine (EC(50), ~210 nM). In summary, the HPV in PCLS is more similar to isolated PA than V/P lungs. The metacholine-induced constriction of bronchioles suggested that the PLCS might be also useful for studying airway physiology in situ.     

Lovastatin Induces Relaxation and Inhibits L‐Type Ca2+ Current in the Rat Basilar Artery

Abstract: Statins inhibit cholesterol biosynthesis and protect against ischaemic stroke. It has become increasingly apparent that the beneficial effects of statin therapy may extend beyond lowering of serum cholesterol. The present study was done to explore possible pleiotropic statin effects at the level of the cerebral vascular smooth muscle. Lovastatin, lovastatin acid, simvastatin and pravastatin, were added to segments of the rat basilar artery and effects on contraction and Ca²⁺ handling were examined. Pravastatin had no effect on contraction. Simvastatin, lovastatin, and, to a lesser degree, lovastatin acid, caused relaxation (IC₅₀=0.8, 1.9 and 22 μmol/l) of both intact and denuded arteries precontracted with 5‐HT or high‐K⁺. This effect was not reversed by mevalonate, suggesting that it was not related to cholesterol or isoprenoid metabolism. Relaxation was associated with a reduction of the intracellular Ca²⁺ concentration measured with Fura 2 and with a reduced Mn²⁺ quench rate, suggesting a direct effect on ion channels in the smooth muscle cell membrane. Current measurements in isolated and voltage clamped basilar artery muscle cells demonstrated that both lovastatin and lovastatin acid inhibit L‐type Ca²⁺ current. We propose that lipophilicity is an important factor behind the effects of statins on vascular tone and that Ca²⁺ current inhibition is the likely mechanism of action.     

Searching for Dual Inhibitors of the MDM2‐p53 and MDMX‐p53 Protein–Protein Interaction by a Scaffold‐Hopping Approach

Two libraries of substituted benzimidazoles were designed using a ‘scaffold‐hopping’ approach based on reported MDM2‐p53 inhibitors. Substituents were chosen following library enumeration and docking into an MDM2 X‐ray structure. Benzimidazole libraries were prepared using an efficient solution‐phase approach and screened for inhibition of the MDM2‐p53 and MDMX‐p53 protein–protein interactions. Key examples showed inhibitory activity against both targets.     

A Pharmacological Comparison of the Receptors Mediating Contractile Responses to 5-Hydroxytryptamine in the Rat Isolated Caudal Artery and Fundic Strip

Abstract— Contractile responses to 5-hydroxytryptamine (5-HT) and to a number of 5-HT-receptor agonists have been compared on the rat isolated caudal artery and stomach fundic strip. On the caudal artery, 5-HT was the most potent agonist tested. The 5-HT₁-like agonist, 5-carboxamidotryptamine (5-CT), was less potent than 5-HT and produced a lower maximum response. 8-Hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) and RU24969 (5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)1H indole) were inactive as agonists and 8-OH-DPAT was not an antagonist. Ketanserin, ICI 169,369 (2-(2-dimethylaminoethyl-thio)-3-phenylquinoline hydrochloride) and ICI 170,809 (2-(2-dimethylamino-2-methylpropylthio)-3-phenylquinoline hydrochloride) were competitive antagonists of 5-HT on this preparation, indicating that 5-HT is acting via 5-HT₂ receptors. In contrast, all the agonists produced contractions of the fundic strip (rank order of potency, 5-HT = 5-CT > RU24969 > 8-OH-DPAT). The maximum response to RU24969 was significantly lower than the maximum responses to the other agonists. Ketanserin was only a weak antagonist of 5-HT in the fundic strip, demonstrating that 5-HT₂ receptors were not involved, but ICI 169,369 and ICI 170,809 were non-surmountable antagonists of 5-HT responses, as were methysergide and methiothepin. Since ICI 169,369 and ICI 170,809 are devoid of activity at 5-HT₃ and 5-HT₄ receptors, then these two subtypes would not appear to be implicated, a view that was confirmed in the case of 5-HT₃ receptors by experiments using ondansetron. Since radioligand binding studies have shown that ICI 169,369 and ICI 170,809 have high affinity for 5-HT_1C sites, α-methyl-5-HT, an agonist claimed to be selective for 5-HT_1C and 5-HT₂ receptors, was also tested on the fundic strip in the presence of ketanserin. This compound acted as an agonist and was antagonized in a competitive manner by ICI 170,809. Therefore, these results suggest that the receptors mediating contractile responses to 5-HT in the rat fundic strip are more similar to the 5-HT_1C subtype than any of the other subtypes that have been described previously.