A1 adenosine receptors mediate hypoxia-induced ventriculomegaly

Periventricular leukomalacia is characterized by a reduction in brain matter and secondary ventriculomegaly and is a major cause of developmental delay and cerebral palsy in prematurely born infants. Currently, our understanding of the pathogenesis of this condition is limited. In animal models, features of periventricular leukomalacia can be induced by hypoxia and activation of A1 adenosine receptors (A1ARs). Using mice that are deficient in the A1AR gene (A1AR-/-), we show that A1ARs play a prominent role in the development of hypoxia-induced ventriculomegaly in neonates. Supporting a role for adenosine in the pathogenesis of developmental brain injury, ventriculomegaly was also observed in mice lacking the enzyme adenosine deaminase, which degrades adenosine. Thus, adenosine acting on A1ARs appears to mediate hypoxia-induced brain injury ventriculomegaly during early postnatal development.     

A1 adenosine receptors mediate hypoglycemia-induced neuronal injury

The cellular mechanisms that lead to neuronal death following glucose deprivation are not known, although it is recognized that hypoglycemia can lead to perturbations in intracellular calcium ([Ca2+]i) levels. Recently, activation of A1 adenosine receptors (A1AR) has been shown to alter [Ca2+]i and promote neuronal death. Thus, we examined if A1AR activation contributes to hypoglycemia-induced neuronal injury using rat cortical neurons. First, we observed that hypoglycemia was associated with large increases in neuronal adenosine release. Next, decreased neuronal viability was seen with progressive reduction in glucose concentration (25, 6, 3, 0.75 and 0 mM). Using the calcium-sensitive dye, Fluo-3, we observed both acute and long-term changes in relative [Ca2+]i during hypoglycemic conditions. Demonstrating a role for adenosine in this process, both the loss in neuronal viability and the early changes in [Ca2+]i were reversed by treatment with A1AR antagonists (8-cyclopentyl, 1,3-dipropylxanthine; 9-chloro-2-(2-furyl)(1,2,4)-triazolo(1,5-c)quinazolin-5-amine; and N-cyclopentyl-9-methyladenine). We also found that hypoglycemia induced the expression of the pro-apoptotic enzyme, caspase-3, and that A1AR antagonism reversed hypoglycemia-induced caspase-3 activity. Collectively, these data show that hypoglycemia induces A1ARs activation leading to alterations in [Ca2+]i, which plays a prominent role in leading to hypoglycemia-induced neuronal death.     

D1多巴胺受体与原发性高血压

多巴胺可通过对肾脏水钠重吸收的调节作用,进而发挥对血压调控的影响,多巴胺功能障碍在原发性高血压发生过程中发挥重要的作用。在多巴胺受体的5个亚型中,D1受体的作用尤其引人注目。在体内钠负荷过载的情况下,刺激肾脏D1受体可调节超过50%的钠排量。原发性高血压状态下D1受体功能障碍,其发生的原因与D1受体/G蛋白效应物复合体失偶联有关,而其失偶联的机制归因于G蛋白激酶4变异体存在所引起的G蛋白激酶活性增高。     

Dopamine receptors and hypertension

Dopamine plays an important role in regulating renal function and blood pressure. Dopamine synthesis and dopamine receptor subtypes have been shown in the kidney. Dopamine acts via cell surface receptors coupled to G proteins; the receptors are classified via pharmacologic and molecular cloning studies into two families, D₁-like and D₂-like. Two D₁-like receptors cloned in mammals, the D1 and D5 receptors (D1A and D1B in rodents), are linked to adenylyl cyclase stimulation. Three D₂-like receptors (D2, D3, and D4) have been cloned and are linked mainly to adenylyl cyclase inhibition. Activation of D₁-like receptors on the proximal tubules inhibits tubular sodium reabsorption by inhibiting Na/H-exchanger and Na/K-adenosine triphosphatase activity. Reports exist of defective renal dopamine production and/or dopamine receptor function in human primary hypertension and in genetic models of animal hypertension. In humans with essential hypertension, renal dopamine production in response to sodium loading is often impaired and may contribute to hypertension. A primary defect in D₁-like receptors and an altered signaling system in proximal tubules may reduce dopamine-mediated effects on renal sodium excretion. The molecular basis for dopamine receptor dysfunction in hypertension is being investigated, and may involve an abnormal posttranslational modification of the dopamine receptor.     

Dopamine D2 and D3 receptors are linked to the actin cytoskeleton via interaction with filamin A

We have used a yeast two-hybrid approach to uncover protein interactions involving the D2-like subfamily of dopamine receptors. Using the third intracellular loop of the D2S and D3 dopamine receptors as bait to screen a human brain cDNA library, we identified filamin A (FLN-A) as a protein that interacts with both the D2 and D3 subtypes. The interaction with FLN-A was specific for the D2 and D3 receptors and was independently confirmed in pull-down and coimmunoprecipitation experiments. Deletion mapping localized the dopamine receptor-FLN-A interaction to the N-terminal segment of the D2 and D3 dopamine receptors and to repeat 19 of FLN-A. In cultures of dissociated rat striatum, FLN-A and D2 receptors colocalized throughout neuronal somata and processes as well as in astrocytes. Expression of D2 dopamine receptors in FLN-A-deficient M2 melanoma cells resulted in predominant intracellular localization of the D2 receptors, whereas in FLN-A-reconstituted cells, the D2 receptor was predominantly localized at the plasma membrane. These results suggest that FLN-A may be required for proper cell surface expression of the D2 dopamine receptors. Association of D2 and D3 dopamine receptors with FLN-A provides a mechanism whereby specific dopamine receptor subtypes may be functionally linked to downstream signaling components via the actin cytoskeleton.     

A2 adenosine receptors and vascular pathologies

Cardiovascular disease, a leading cause of death and morbidity, is regulated, among various factors, by inflammation. The level of the metabolite adenosine is augmented under stress, including inflammatory, hypoxic, or injurious events. Adenosine has been shown to affect various physiological and pathological processes, largely through 1 or more of its 4 types of receptors: the A1 and A3 adenylyl cyclase inhibitory receptors and the A2A and A2B adenylyl cyclase stimulatory receptors. This article focuses on reviewing common and distinct effects of the 2 A2-type adenosine receptors on vascular disease and the mechanisms involved. Understanding the pathogenesis of vascular disease mediated by these receptors is important to the development of therapeutics and to the prevention and management of disease.     

Different functional roles of T1R subunits in the heteromeric taste receptors

The T1R receptors, a family of taste-specific class C G protein-coupled receptors, mediate mammalian sweet and umami tastes. The structure-function relationships of T1R receptors remain largely unknown. In this study, we demonstrate the different functional roles of T1R extracellular and transmembrane domains in ligand recognition and G protein coupling. Similar to other family C G protein-coupled receptors, the N-terminal Venus flytrap domain of T1R2 is required for recognizing sweeteners, such as aspartame and neotame. The G protein coupling requires the transmembrane domain of T1R2. Surprisingly, the C-terminal transmembrane domain of T1R3 is required for recognizing sweetener cyclamate and sweet taste inhibitor lactisole. Because T1R3 is the common subunit in the sweet taste receptor and the umami taste receptor, we tested the interaction of lactisole and cyclamate with the umami taste receptor. Lactisole inhibits the activity of the human T1R1/T1R3 receptor, and, as predicted, blocked the umami taste of l-glutamate in human taste tests. Cyclamate does not activate the T1R1/T1R3 receptor by itself, but potentiates the receptor's response to l-glutamate. Taken together, these findings demonstrate the different functional roles of T1R3 and T1R2 and the presence of multiple ligand binding sites on the sweet taste receptor.     

Blood pressure-dependent inhibition of Renin secretion requires A1 adenosine receptors

Renal perfusion pressure (RPP) regulates renin release with a reduction of RPP stimulating and an elevation inhibiting renin secretion. The precise sensing and effector mechanisms by which changes in arterial pressure are linked to the exocytosis of renin are not well-defined. The present experiments were designed to study the potential role of adenosine as a mediator of this renal baroreceptor mechanism. In isolated perfused mouse kidneys a stepwise reduction of RPP from 90 mm Hg to 65 and 40 mm Hg stimulated renin secretion rates (RSR) 1.4-fold and 3.6-fold, whereas stepwise elevations of RPP from 90 mm Hg to 115 and 140 mm Hg suppressed RSR to 64% or 40% of baseline. Inactivation of A1 adenosine receptors by either pharmacological blockade (DPCPX 1 micromol/L) or genetic deletion (A1AR(-/-) mice) did not modify the stimulation of renin release by a low RPP, but completely prevented the suppression of renin secretion by higher perfusion pressures. In vivo, the induction of arterial hypertension by either acute (single subcutaneous injection) or chronic (osmotic minipump for 72 hours) application of phenylephrine significantly reduced plasma renin concentration (PRC) in wild-type mice to approximately 40% of control, whereas it did not significantly affect PRC in A1AR(-/-) mice. Together these data demonstrate that A1 adenosine receptors are indispensable for the inhibition of renin secretion by an increase in blood pressure, suggesting that formation and action of adenosine is responsible for baroreceptor-mediated inhibition of renin release. In contrast, the stimulation of the renin system by a low blood pressure appears to follow different pathways.     

Dopamine D3 receptors as a therapeutic target for methamphetamine dependence

Background: Methamphetamine (MA) use disorders are major public health problems nationally and worldwide and treatment remains an unmet need. Objectives: (1) To review preclinical and clinical studies identifying the dopamine D3 receptor as a therapeutic target for substance use disorders (SUDs), including MA dependence, (2) to consider buspirone (Buspar®) as a potential medication based on its dopamine D3 receptor antagonist properties, and (3) to evaluate the safety and initial efficacy of buspirone in a pilot study of MA-dependent individuals. Methods: Literature on the dopamine D3 receptor as a therapeutic target and on the potential of buspirone as a novel therapy for MA dependence was reviewed. The cardiovascular and subjective effects of intravenous MA challenge were assessed in five non-treatment seeking individuals. Participants met DSM-IV criteria for MA dependence and were treated subacutely (9 days) with buspirone (60?mg daily). Results: The literature identified the dopamine D3 receptor as a therapeutic target for MA dependence, a safe and approved medication, and a valuable opportunity to re-purpose buspirone for treating MA dependence and perhaps other SUDs. Pilot data (n?=?5) indicated that buspirone is safe in MA-using individuals and comparison against historical placebo data from this laboratory suggested that at least some aspects of the subjective properties of MA may be diminished during buspirone treatment. Conclusion: Future studies should include a small-scale, placebo-controlled Phase IIa trial of buspirone in MA dependence.     

Pharmacological evidence for A1 and A2 adenosine receptors in the skin microcirculation

To characterize adenosine-mediated vascular responses, synthetic A1 and A2 receptor agonists (N-ethyl carboxamido adenosine [NECA], 2-chloro adenosine [2CA], or cyclohexyl adenosine [CHA]), the parent compound (adenosine [ADO]), an uptake inhibitor (dipyridamole [DIPYRID]) or a nonselective, competitive antagonist (8-phenyl theophylline [8pTHEO]) were topically applied to 20-60 microns arterioles in the subcutaneous microcirculation of the hamster. Blood flow was calculated from arteriolar diameter and red blood cell velocity using intravital microscopy. At greater than 10(-8) M, the potency order for vasodilation (maximum, 170-190% of control) was NECA greater than 2CA greater than ADO; these responses were attenuated by 10(-5) M 8pTHEO. From 10(-8) to 10(-6) M, 2CA evoked vasodilation whereas ADO, which has an identical affinity at A1 and A2 receptors, evoked lesser responses. ADO-induced vasodilation was potentiated by 10(-5) M DIPYRID; this response was similar to that evoked by 2CA alone or 2CA + DIPYRID. In contrast to ADO, 2CA is a poor substrate for cellular uptake, which suggests that uptake reduces the A2 effect of exogenous ADO. From 10(-10) to 10(-8) M, CHA and ADO were equipotent antagonized by 8pTHEO. Norepinephrine was a more potent vasoconstrictor and 8pTHEO did not alter these responses. Since ADO is a metabolic substrate and a nonselective receptor agonist, while CHA is A1-selective and a poor substrate for cellular uptake, neither A2 activation nor cellular uptake altered expression of the A1 effect of exogenous ADO. Furthermore, DIPYRID had no effect on the A1 response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dopamine responsiveness is regulated by targeted sorting of D2 receptors

Aberrant dopaminergic signaling is a critical determinant in multiple psychiatric disorders, and in many disease states, dopamine receptor number is altered. Here we identify a molecular mechanism that selectively targets D2 receptors for degradation after their activation by dopamine. The degradative fate of D2 receptors is determined by an interaction with G protein coupled receptor-associated sorting protein (GASP). As a consequence of this GASP interaction, D2 responses in rat brain fail to resensitize after agonist treatment. Disruption of the D2-GASP interaction facilitates recovery of D2 responses, suggesting that modulation of the D2-GASP interaction is important for the functional down-regulation of D2 receptors.     

D1A, D1B, and D1C dopamine receptors from Xenopus laevis.

Three distinct genes encoding members of the D1 dopamine receptor family were isolated from Xenopus laevis. Based on the deduced amino acid sequence, two of the receptors (Xen D1A and Xen D1B) appear to be homologues of mammalian D1/D1A and D5/D1B receptors. The third receptor, termed Xen D1C, displays equal overall amino acid and nucleotide sequence identity (approximately 55%) with mammalian D1A and D1B/D5 receptors. In agreement with their structural similarities, Xen D1A and D1B receptors, when expressed in COS-7 cells, displayed pharmacological profiles that paralleled those of their mammalian counterparts, with dopamine and 2-amino-6,7-dihydroxytetralin exhibiting 10-fold higher affinity for D1B than for D1A. The Xen D1C receptor displayed an overall rank order of potency and pharmacological profile clearly indicative of a D1-like receptor, with individual affinities for most agonists higher than those for either Xen or mammalian D1/D1A and D5/D1B receptors, whereas antagonist Ki values were intermediate to those for the D1/D1A and D5/D1B receptors. All three receptors stimulated adenylate cyclase activity in response to dopamine or SKF-82526. Xen D1A, D1B, and D1C receptor mRNAs were differentially distributed, with all three receptors expressed in brain and only D1B and D1C receptors expressed in kidney. The existence of a receptor which lacks appreciable overall sequence similarity to, but displays pharmacological homology with, mammalian D1-like receptors lends strong support to the contention that additional mammalian D1-like receptor gene products may exist to allow for the expression of the full spectrum of D1-like dopamine receptor-mediated events.     

A SPIKE1 signaling complex controls actin-dependent cell morphogenesis through the heteromeric WAVE and ARP2/3 complexes

During morphogenesis, the actin cytoskeleton mediates cell-shape change in response to growth signals. In plants, actin filaments organize the cytoplasm in regions of polarized growth, and the filamentous arrays can be highly dynamic. Small GTPase signaling proteins termed Rho of plants (ROP)/RAC control actin polymerization. ROPs cycle between inactive GDP-bound and active GTP-bound forms, and it is the active form that interacts with effector proteins to mediate cytoskeletal rearrangement and cell-shape change. A class of proteins termed guanine nucleotide exchange factors (GEFs) generate GTP-ROP and positively regulate ROP signaling. However, in almost all experimental systems, it has proven difficult to unravel the complex signaling pathways from GEFs to the proteins that nucleate actin filaments. In this article, we show that the DOCK family protein SPIKE1 (SPK1) is a GEF, and that one function of SPK1 is to control actin polymerization via two heteromeric complexes termed WAVE and actin-related protein (ARP) 2/3. The genetic pathway was constructed by using a combination of highly informative spk1 alleles and detailed analyses of spk1, wave, and arp2/3 single and double mutants. Remarkably, we find that in addition to providing GEF activity, SPK1 associates with WAVE complex proteins and may spatially organize signaling. Our results describe a unique regulatory scheme for ARP2/3 regulation in cells, one that can be tested for widespread use in other multicellular organisms.     

A1 and A2 adenosine receptors regulate adenylate cyclase in cultured human lung fibroblasts

Adenosine stimulates and inhibits adenylate cyclase activity and cAMP levels in WI-38 and VA13 fibroblasts. The inhibitory effects appear to be mediated by both A1 receptors and the P-site. Results supporting these conclusions are as follows: Adenosine by itself increased cAMP accumulation in these cells. PGE1-stimulated cAMP accumulation was inhibited by adenosine in a concentration-dependent fashion. IAP treatment blocked adenosine inhibition of cAMP accumulation and adenylate cyclase activity and enhanced adenosine stimulation of cAMP accumulation in VA13 cells. Theophylline and MIX attenuated adenosine inhibition of cAMP accumulation. Adenosine analogs with substitutions in the purine ring inhibited PGE1-stimulated cAMP accumulation and adenylate cyclase activity. PGE1-stimulated cAMP accumulation was inhibited by the P-site agonist 2'5'-dideoxyadenosine, but this inhibition was not attenuated by MIX or IAP treatment. These data support the idea that adenosine may inhibit cAMP accumulation in VA13 or WI-38 cells by acting at an A1 receptor of the P-site. The decrease in cAMP accumulation mediated by the A1 receptor appeared to be due at least in part to an Ni-mediated inhibition of adenylate cyclase.     

Rp-cAMPS has ho effect on adenosine A1 receptors in guinea-pig cardiomyocytes

Rp-cAMPS, a protein kinase A inhibitor, is used in the investigation of the cAMP-dependent systems. A report by Musgrave et al. (11) has suggested that Rp-cAMPS may also act on adenosine receptors. To determine whether this occurs in guinea-pig ventricular myocytes, Rp-cAMPS was applied in the presence and absence of DCPCX, an adenosine A₁ receptor antagonist. The isoprenaline-induced response was significantly decreased by Rp-cAMPS and the effect was not altered by the presence of DCPCX. Therefore Rp-cAMPS has no effect on cell contraction via adenosine A₁ receptors and can reliably be used to investigate cyclic AMP-dependent systems in isolated cardiac myocytes. Received: 23 April 1999, Returned for 1. revision: 26 May 1999, 1. Revision received: 23 August 1999, Returned for 2. revision: 28 September 1999, 2. Revision received: 15 November 1999, Accepted: 17 November 1999     

Decrease in A1 adenosine receptors in adipocytes from spontaneously hypertensive rats

We have investigated whether the insulin resistance reported to occur in hypertension is due to decreased insulin receptors or to adenosine receptors in adipocyte membranes. Membranes were isolated from adipocytes from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKy) rats and assayed for insulin receptors and A1 adenosine receptors. The two groups of membranes bound 125I-insulin equally, but in contrast the SHR membranes bound approximately 25% less 125I-HPIA ([(-)-N6-p-hydroxyphenylisopropyl adenosine], an A1 adenosine receptor agonist) than the WKy (P less than .005). Scatchard analysis demonstrated that this was due to a lower number of receptors in the SHR. The affinity of the receptor for HPIA was approximately 0.7 nmol/L in both groups. 5'-Nucleotidase activity was approximately 40% higher in membranes from SHR than WKy (P less than .001), indicating that adipocytes from SHR have a higher capacity for adenosine production. This may cause increased adenosine concentrations in the SHR adipose tissue, leading to adenosine receptor down-regulation. Since we have previously demonstrated that adenosine receptor down-regulation can lead to insulin resistance, these findings may partly account for the insulin resistance of hypertension.     

Dopamine D1 receptors involved in locomotor activity and accumbens neural responses to prediction of reward associated with place

Predicting reward is essential in learning approach behaviors. Dopaminergic activity has been implicated in reward, movement, and cognitive processes, all essential elements in learning. The nucleus accumbens (NAc) receives converging inputs from corticolimbic information-processing areas and from mesolimbic dopamine neurons originating in the ventral tegmental area. Previously, we reported that in mice, a dopamine D2 receptor knockout (D2R-KO) eliminated the prereward inhibitory response, increased place-field size of NAc neurons, and reduced locomotor activity without marked change in intracranial self-stimulation (ICSS) behavior. The present study investigated the specific contribution of dopamine D1 receptor (D1R) in mediating reward, locomotor activity, and spatial associative processes and in regulating NAc neural responses. In contrast to D2R-KO animals, here we find D1R-KO in mice selectively eliminated the prereward excitatory response and decreased place-field size of NAc neurons. Furthermore, D1R-KO impaired ICSS behavior, seriously reduced locomotor activity, and retarded acquisition of a place learning task. Thus, the present results suggest that D1R may be an important determinant in brain stimulation reward (ICSS) and participates in coding for a type of reward prediction of NAc neurons and in spatial learning.     

戊四氮点燃腺苷A1受体敲除小鼠脑内P-糖蛋白的动态表达变化

目的:观察戊四氮(PTZ)点燃腺苷A1受体敲除小鼠脑内P-糖蛋白(PGP)的动态表达变化,评价腺苷A1受体在难治性癫痫治疗中的作用。方法:采用腹腔注射PTZ制备慢性点燃癫痫模型,将动物分为野生型组(40只)和敲除鼠组(40只),再根据有无接受点燃及点燃后不同时间点再分为点燃前和点燃后24 h、7 d、30 d亚组,采用RTPCR法、免疫荧光组织化学法观察各组小鼠大脑皮层和海马PGP的表达情况。结果:PTZ点燃后24 h,野生型组小鼠大脑皮层和海马PGP的表达与点燃前比较无显著统计学差异(P0.05),敲除鼠组PGP的表达显著高于点燃前(P0.05),2组小鼠点燃后7 d和30 d时PGP的表达均显著高于对照组(均P0.05),且点燃后30 d PGP显著高于7 d(P0.05);点燃后7 d时显著高于24 h时(P0.05),说明PTZ点燃后PGP的表达随时间延长而增高;敲除鼠组PTZ点燃后同一时间点PGP的表达均显著高于野生型组(均P0.05)。结论:腺苷A1受体激活可下调PGP的表达,调控腺苷系统功能紊乱可能成为治疗耐药性癫痫的新方法。