Direct Receptor Activation Research Articles

Maintained tubuloglomerular feedback responses during acute inhibition of P2 purinergic receptors in mice

Tubuloglomerular feedback (TGF), the change of afferent arteriolar resistance initiated by changes of luminal NaCl concentration, is thought to be related to NaCl-dependent release of ATP by macula densa cells. In the present study, we have explored the possibility that the released ATP may directly interact with vasoconstrictor P2 purinergic receptors in the vicinity of the glomerular vascular pole. In two different strains of wild-type mice (SWR/J and FVB), TGF responses were determined in vivo by measuring the stop flow pressure (P(SF)) change caused by a saturating increase in loop of Henle flow rate before and during the administration of the P2 receptor inhibitors PPADS (12 mg/kg + 35 mg·kg(-1)·h(-1) iv) or suramin (50 mg/kg + 150 mg·kg(-1)·h(-1)). Both agents significantly reduced the blood pressure response to the P2X agonist α,β-methylene ATP. In SWR/J and FVB mice, elevating flow to 30 nl/min reduced P(SF) by 16.4 ± 2.2 and 17.1 ± 1.8%. During infusion of PPADS, P(SF) fell by 18.8 ± 2 (P = 0.4) and 16.5 ± 1.5% (P = 0.82) in the two strains of mice. During suramin infusion, P(SF) decreased by 14.7 ± 2.4 (P = 0.62) and 15 ± 1.3% (P = 0.4) in SWR/J and FVB mice, respectively. Including PPADS (10(-4) M) in the loop perfusate did not significantly alter the P(SF) response (18.9 ± 1.8%; P = 0.54). Arterial blood pressure was not systematically affected by the P2 inhibitors. As measured by free-flow micropuncture, PPADS significantly reduced proximal tubular fluid reabsorption in both fractional and absolute terms. These results indicate that the direct activation of P2 purinergic receptors by ATP is not a major cause of TGF-induced vasoconstriction in vivo.

Melatonin, a Potential Therapeutic Agent for Smooth Muscle-Related Pathological Conditions and Aging

Increases or decreases in the contractile response of smooth muscle underlie important pathological conditions such as hypertension, incontinence and altered gastrointestinal transit. These disorders are also frequently encountered in the aged population. Oxidative stress and inflammation are key features in the initiation, progression, and clinical manifestations of smooth muscle disorders. Melatonin, the major secretory product of the pineal gland, has free radical scavenging and antioxidative properties and protects against oxidative insult. Recently, widespread interest has grown regarding the apparent protective effects of melatonin on smooth muscle dysfunction. "In vitro" studies have shown that melatonin decreased vascular tone of vascular beds from control, hypertensive or aged animals, through the reduction of adrenergic contraction and the increase in acetylcholine-induced relaxation. "In vivo", melatonin also attenuates sympathetic tone by direct activation of melatonin receptors, scavenging free radicals or increasing NO availability in the central nervous system. In the gastrointestinal tract, melatonin treatment improves age-related impairments in gallbladder contractility and prevents deleterious effects of cholecystitis on smooth muscle and the enteric nervous system through suppression of oxidative stress. In addition, melatonin improves colonic transit time in constipation-predominant IBS patients. Melatonin is also able to restore impaired contractility of the detrusor muscle from old animals through normalization of Ca(2+) dependent and independent contraction, mitochondrial polarity, neuromuscular function and oxidative stress, which would explain the effects of melatonin counteracting cystometric changes in senescent animals. It also reverses bladder damage following ischemia/reperfusion. In conclusion, melatonin may be a promising candidate for future research of agents that modulate smooth muscle motility.

Distinct effects of intravascular and extravascular angiotensin II on cerebrovascular circulation of newborn pigs

Angiotensin II (AngII) is important in regulation of vascular resistance and control of blood flow among organs and tissues. The effect of AngII on the cerebral microvasculature may be mediated or altered by endothelial-derived signals. The aim of this study was to test the hypothesis that blood AngII dilates neonatal pial arterioles via an endothelial-dependent mechanism but brain AngII can constrict pial arterioles by activating smooth muscle AT1 receptors. Studies used anesthetized newborn pigs with surgically implanted closed cranial windows. AngII was given either by infusion into the carotid artery ipsilateral to the cranial window or topically. Intracarotid infusion of AngII dilated pial arterioles. The dilation was blocked by systemic administration of the AT1-receptor antagonist, losartan, but unaffected by topical losartan. Topical AngII also caused dilation, but this dilation was converted to constriction by topical losartan. In piglets pretreated with the angiotensin-converting enzyme (ACE) inhibitor, enalapril, topical AngII constricted, rather than dilated, pial arterioles. In enalapril-treated piglets, light/dye endothelial injury blocked dilation to intracarotid AngII but did not affect constriction to topical AngII. Either indomethacin or l-nitroarginine methyl ester blocked the dilation to intraluminal AngII, but neither affected constriction to topical AngII. Chromium mesoporphyrin, that inhibits heme oxygenase, did not affect responses to either topical or intravascular AngII. These data are consistent with the hypotheses that: (a) circulating AngII dilates pial arterioles via endothelial AT(1) receptor-derived relaxing factors, notably prostanoids and nitric oxide; (b) direct AT(1) receptor activation on the brain side of the blood-brain barrier by AngII causes AT(1) receptor-mediated constriction that can mask underlying AT(1) receptor-independent dilation when ACE is inhibited. Clinical manipulation of the renin-angiotensin system will have disparate actions on cerebral circulation depending on the functional integrity of the intima and ACE.

Pomolic acid of Licania pittieri elicits endothelium-dependent relaxation in rat aortic rings

Pomolic acid has recently shown hypotensive effect in rats. The purpose of this investigation was to determine the vascular effects of this triterpenoid and to examine its mode of action. Functional experiments in rat aortic rings precontracted with norepinephrine were performed to evaluate the vasorelaxant effect of pomolic acid. This triterpenoid induced a vasorelaxation (IC₅₀ = 2.45 μM) in a concentration- and endothelium-dependent manner and showed no effect on contractions evoked by KCl (25 mM). Pre-treatment of aortic rings with L-NAME (100 μM), methylene blue (100 μM) or glibenclamide (10 μM), totally prevented the vasorelaxation induced by pomolic acid, while indomethacin (10 μM) had no effect on this response. Additionally, pomolic acid relaxation was unaffected under the muscarinic- and β-adrenergic-receptor blocked ensured for atropine and propanolol respectively (10 μM each). In contrast, the vasorelaxant effect of pomolic acid was abolished under the purinergic-receptor blocked ensured for suramin (10 μM). Finally, apyrase (0.8 U/ml) an enzyme which hydrolyses ATP and ADP did not affect pomolic acid relaxation. In summary, pomolic acid has a potent endothelium-dependent vasorelaxant effect, possibly acting through the direct activation of endothelial purinergic receptors via NO-cGMP signaling pathway, which could be part of the mechanism underlying its hypotensive effect.

Attenuation of Persistent Experimental Pancreatitis Pain by a Bradykinin B2 Receptor Antagonist

The role of bradykinin (BK) receptors in activating and sensitizing peripheral nociceptors is well known. Recently, we showed that spinal dynorphin was pronociceptive through direct or indirect BK receptor activation. Here, we explored the potential role of BK receptors in pain associated with persistent pancreatitis in rats. Experimental pancreatitis and abdominal hypersensitivity were induced by intravenous administrations of dibutyltin dichloride (DBTC). [des-Arg-Leu]BK (B1 antagonist) and HOE 140 (B2 antagonist) were given by intraperitoneal or intrathecal injection. Dynorphin antiserum was given intrathecally. Reverse transcription-polymerase chain reaction was used to detect spinal mRNA for BK receptors. Dibutyltin dichloride-induced pancreatitis upregulated B1 and B2 mRNA in the thoracic dorsal root ganglion and B2, but not B1, in the pancreas. No changes in spinal B1 or B2 mRNA were observed. Intraperitoneal or intrathecal administration of HOE 140 dose dependently abolished DBTC-induced abdominal hypersensitivity, whereas [des-Arg-Leu]BK was without effect by either route of administration. Antiserum to dynorphin (intrathecal) abolished DBTC-induced hypersensitivity. These results suggest that blockade of peripheral or spinal BK B2 receptors may be an effective approach for diminishing pain associated with pancreatitis. Moreover, it is suggested that spinal dynorphin may maintain pancreatitis pain through direct or indirect activation of BK B2 receptors in the spinal cord.

Plasma Kallikrein Promotes Epidermal Growth Factor Receptor Transactivation and Signaling in Vascular Smooth Muscle through Direct Activation of Protease-activated Receptors

The kallikrein-kinin system, along with the interlocking renin-angiotensin system, is a key regulator of vascular contractility and injury response. The principal effectors of the kallikrein-kinin system are plasma and tissue kallikreins, proteases that cleave high molecular weight kininogen to produce bradykinin. Most of the cellular actions of kallikrein (KK) are thought to be mediated by bradykinin, which acts via G protein-coupled B1 and B2 bradykinin receptors on VSMCs and endothelial cells. Here, we find that primary aortic vascular smooth muscle but not endothelial cells possess the ability to activate plasma prekallikrein. Surprisingly, exposing VSMCs to prekallikrein leads to activation of the ERK1/2 mitogen-activated protein kinase cascade via a mechanism that requires kallikrein activity but does not involve bradykinin receptors. In transfected HEK293 cells, we find that plasma kallikrein directly activates G protein-coupled protease-activated receptors (PARs) 1 and 2, which possess consensus kallikrein cleavage sites, but not PAR4. In vascular smooth muscles, KK stimulates ADAM (a disintegrin and metalloprotease) 17 activity via a PAR1/2 receptor-dependent mechanism, leading sequentially to release of the endogenous ADAM17 substrates, amphiregulin and tumor necrosis factor-α, metalloprotease-dependent transactivation of epidermal growth factor receptors, and metalloprotease and epidermal growth factor receptor-dependent ERK1/2 activation. These results suggest a novel mechanism of bradykinin-independent kallikrein action that may contribute to the regulation of vascular responses in pathophysiologic states, such as diabetes mellitus.

Targeting Astrocyte Signaling for Chronic Pain

Clinical management of chronic pain after nerve injury (neuropathic pain) and tumor invasion (cancer pain) is a real challenge due to our limited understanding of the cellular mechanisms that initiate and maintain chronic pain. It has been increasingly recognized that glial cells, such as microglia and astrocytes in the CNS play an important role in the development and maintenance of chronic pain. Notably, astrocytes make very close contacts with synapses and astrocyte reaction after nerve injury, arthritis, and tumor growth is more persistent than microglial reaction, and displays a better correlation with chronic pain behaviors. Accumulating evidence indicates that activated astrocytes can release pro-inflammatory cytokines (e.g., interleukin [IL]-1β) and chemokines (e.g., monocyte chemoattractant protein-1 [MCP-1]/also called CCL2) in the spinal cord to enhance and prolong persistent pain states. IL-1β can powerfully modulate synaptic transmission in the spinal cord by enhancing excitatory synaptic transmission and suppressing inhibitory synaptic transmission. IL-1β activation (cleavage) in the spinal cord after nerve injury requires the matrix metalloprotease-2. In particular, nerve injury and inflammation activate the c-Jun N-terminal kinase in spinal astrocytes, leading to a substantial increase in the expression and release of MCP-1. The MCP-1 increases pain sensitivity via direct activation of NMDA receptors in dorsal horn neurons. Pharmacological inhibition of the IL-1β, c-Jun N-terminal kinase, MCP-1, or matrix metalloprotease-2 signaling via spinal administration has been shown to attenuate inflammatory, neuropathic, or cancer pain. Therefore, interventions in specific signaling pathways in astrocytes may offer new approaches for the management of chronic pain.

Essential role of the cAMP-cAMP response-element binding protein pathway in opiate-induced homeostatic adaptations of locus coeruleus neurons

Excessive inhibition of brain neurons in primary or slice cultures can induce homeostatic intrinsic plasticity, but the functional role and underlying molecular mechanisms of such plasticity are poorly understood. Here, we developed an ex vivo locus coeruleus (LC) slice culture system and successfully recapitulated the opiate-induced homeostatic adaptation in electrical activity of LC neurons seen in vivo. We investigated the mechanisms underlying this adaptation in LC slice cultures by use of viral-mediated gene transfer and genetic mutant mice. We found that short-term morphine treatment of slice cultures almost completely abolished the firing of LC neurons, whereas chronic morphine treatment increased LC neuronal excitability as revealed during withdrawal. This increased excitability was mediated by direct activation of opioid receptors and up-regulation of the cAMP pathway and accompanied by increased cAMP response-element binding protein (CREB) activity. Overexpression of a dominant negative CREB mutant blocked the increase in LC excitability induced by morphine- or cAMP-pathway activation. Knockdown of CREB in slice cultures from floxed CREB mice similarly decreased LC excitability. Furthermore, the ability of morphine or CREB overexpression to up-regulate LC firing was blocked by knockout of the CREB target adenylyl cyclase 8. Together, these findings provide direct evidence that prolonged exposure to morphine induces homeostatic plasticity intrinsic to LC neurons, involving up-regulation of the cAMP-CREB signaling pathway, which then enhances LC neuronal excitability.

Nicotine Exerts an Anti-inflammatory Effect in a Murine Model of Acute Lung Injury

Activation of the cholinergic anti-inflammatory pathway through direct activation of nicotinic acetylcholine receptors on immune cells can inhibit pro-inflammatory chemokine and cytokine release and thereby protect in a variety of inflammatory diseases. The aim of this study was to investigate whether nicotine treatment protected against acute lung inflammation. Mice challenged with intratracheal lipopolysaccharide (LPS, 50 μg) were treated with nicotine (0.2 or 0.4 mg/kg, sc). After 24 h, bronchoalveolar lavage fluid (BALF) was obtained to measure leukocyte infiltration, lung edema, and pro-inflammatory chemokine (MIP-1α, MIP-2, and eotaxin) and cytokine (IL-1, IL-6, and TNF-α) levels. Nicotine treatment reduced the LPS-mediated infiltration of leukocytes and edema as evidenced by decreased BALF inflammatory cells, myeloperoxidase, and protein. Nicotine also downregulated lung production of pro-inflammatory chemokines and cytokines. These data support the proposal that activation of the cholinergic anti-inflammatory pathway may represent a useful addition to the therapy of acute respiratory distress syndrome.

2‐arachidonyl glycerol activates platelets via conversion to arachidonic acid and not by direct activation of cannabinoid receptors

There are conflicting views in the literature as to whether cannabinoids have an impact on platelet activity and to what extent cannabinoid receptors are involved. This is an important issue to resolve because platelet effects of putative therapeutic cannabinoid inhibitors and stimulators will have an impact on their potential benefits and safety. The data presented in this manuscript clearly show that the endocannabinoid 2-arrachidonyl glycerol can activate platelet activity, but that the effects are mediated through an aspirin-sensitive pathway that is not affected by cannabinoid receptor antagonists or FAAH inhibition, but is abolished by MAGL inhibition. The findings question the role of cannabinoid receptors in platelet function and suggest that platelet function is unlikely to be directly affected by cannabinoid receptor antagonists, at least in the acute phase. Cannabinoid receptor-1 (CB(1)) antagonists suppress appetite and induce weight loss. Direct antagonism of CB(1) receptors on platelets might be an additional benefit for CB(1) antagonists, but the role of CB(1) receptors in platelets is controversial. We tested the hypothesis that the endocannabinoid, 2-arachidonyl glycerol (2-AG), induces platelet aggregation by a COX-mediated mechanism rather than through CB(1) receptor activation, in blood obtained from healthy volunteers and patients with coronary artery disease receiving low dose aspirin. Aggregatory responses to the cannabinoids 2-AG and Delta(9)-THC were examined in blood sampled from healthy volunteers (n= 8) and patients (n= 12) with coronary artery disease receiving aspirin using whole blood aggregometry. The effects of CB(1) (AM251) and CB(2) (AM630) antagonists, as well as fatty acid amide hydrolase (FAAH) and monoacyl glycerol lipase (MAGL) inhibitors and aspirin on 2-AG-induced aggregation were also assessed. AM251 (100 nm-30 microm) had no effect on platelet aggregation induced by either ADP (P= 0.90) or thrombin (P= 0.86). 2-AG, but not Delta(9)-THC, induced aggregation. 2-AG-induced aggregation was unaffected by AM251 and AM630 but was abolished by aspirin (P < 0.001) and by the MAGL inhibitor, URB602 (P < 0.001). Moreover, the aggregatory response to 2-AG was depressed (by >75%, P < 0.001) in blood from patients with coronary artery disease receiving aspirin compared with that from healthy volunteers. 2-AG-mediated activation of platelets is via metabolism to arachidonic acid by MAGL, and not through direct action on CB(1) or CB(2) receptors, at least in the acute phase.

Activation of N-Methyl-d-aspartate (NMDA) Receptors in the Dorsal Vagal Complex Lowers Glucose Production

Diabetes is characterized by hyperglycemia due partly to increased hepatic glucose production. The hypothalamus regulates hepatic glucose production in rodents. However, it is currently unknown whether other regions of the brain are sufficient in glucose production regulation. The N-methyl-D-aspartate (NMDA) receptor is composed of NR1 and NR2 subunits, which are activated by co-agonist glycine and glutamate or aspartate, respectively. Here we report that direct administration of either co-agonist glycine or NMDA into the dorsal vagal complex (DVC), targeting the nucleus of the solitary tract, lowered glucose production in vivo. Direct infusion of the NMDA receptor blocker MK-801 into the DVC negated the metabolic effect of glycine. To evaluate whether NR1 subunit of the NMDA receptor mediates the effect of glycine, NR1 in the DVC was inhibited by DVC NR1 antagonist 7-chlorokynurenic acid or DVC shRNA-NR1. Pharmacological and molecular inhibition of DVC NR1 negated the metabolic effect of glycine. To evaluate whether the NMDA receptors mediate the effects of NR2 agonist NMDA, DVC NMDA receptors were inhibited by antagonist D-2-amino-5-phosphonovaleric acid (D-APV). DVC D-APV fully negated the ability of DVC NMDA to lower glucose production. Finally, hepatic vagotomy negated the DVC glycine ability to lower glucose production. These findings demonstrate that activation of NR1 and NR2 subunits of the NMDA receptors in the DVC is sufficient to trigger a brain-liver axis to lower glucose production, and suggest that DVC NMDA receptors serve as a therapeutic target for diabetes and obesity.

The role of monoamines in the changes in body temperature induced by 3,4‐methylenedioxymethamphetamine (MDMA, ecstasy) and its derivatives

Hyperthermia is probably the most widely known acute adverse event that can follow ingestion of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) by recreational users. The effect of MDMA on body temperature is complex because the drug has actions on all three major monoamine neurotransmitters [5-hydroxytryptamine (5-HT), dopamine and noradrenaline], both by amine release and by direct receptor activation. Hyperthermia and hypothermia can be induced in laboratory animals by MDMA, depending on the ambient temperature, and involve both central thermoregulation and peripheral changes in blood flow and thermogenesis. Acute 5-HT release is not directly responsible for hyperthermia, but 5-HT receptors are involved in modulating the hyperthermic response. Impairing 5-HT function with a neurotoxic dose of MDMA or p-chlorophenylalanine alters the subsequent MDMA-induced hyperthermic response. MDMA also releases dopamine, and evidence suggests that this transmitter is involved in both the hyperthermic and hypothermic effects of MDMA in rats. The noradrenergic system is also involved in the hyperthermic response to MDMA. MDMA activates central alpha(2A)-adrenoceptors and peripheral alpha(1)-adrenoceptors to produce cutaneous vasoconstriction to restrict heat loss, and beta(3)-adrenoceptors in brown adipose tissue to increase heat generation. The hyperthermia occurring in recreational users of MDMA can be fatal, but data reviewed here indicate that it is unlikely that any single pharmaceutical agent will be effective in reversing the hyperthermia, so careful body cooling remains the principal clinical approach. Crucially, educating recreational users about the potential dangers of hyperthermia and the control of ambient temperature should remain key approaches to prevent this potentially fatal problem.

An Integrated Phosphoproteomics Work Flow Reveals Extensive Network Regulation in Early Lysophosphatidic Acid Signaling

Lysophosphatidic acid (LPA) induces a variety of cellular signaling pathways through the activation of its cognate G protein-coupled receptors. To investigate early LPA responses and assess the contribution of epidermal growth factor (EGF) receptor transactivation in LPA signaling, we performed phosphoproteomics analyses of both total cell lysate and protein kinase-enriched fractions as complementary strategies to monitor phosphorylation changes in A498 kidney carcinoma cells. Our integrated work flow enabled the identification and quantification of more than 5,300 phosphorylation sites of which 224 were consistently regulated by LPA. In addition to induced phosphorylation events, we also obtained evidence for early dephosphorylation reactions due to rapid phosphatase regulation upon LPA treatment. Phosphorylation changes induced by direct heparin-binding EGF-like growth factor-mediated EGF receptor activation were typically weaker and only detected on a subset of LPA-regulated sites, indicating signal integration among EGF receptor transactivation and other LPA-triggered pathways. Our results reveal rapid phosphoregulation of many proteins not yet implicated in G protein-coupled receptor signaling and point to various additional mechanisms by which LPA might regulate cell survival and migration as well as gene transcription on the molecular level. Moreover, our phosphoproteomics analysis of both total lysate and kinase-enriched fractions provided highly complementary parts of the LPA-regulated signaling network and thus represents a useful and generic strategy toward comprehensive signaling studies on a system-wide level.

The dopamine receptor D2 agonist bromocriptine inhibits glucose-stimulated insulin secretion by direct activation of the α2-adrenergic receptors in beta cells

Treatment with the dopamine receptor D2 (DRD2) agonist bromocriptine improves metabolic features in obese patients with type 2 diabetes by a still unknown mechanism. In the present study, we investigated the acute effect of bromocriptine and its underlying mechanism(s) on insulin secretion both in vivo and in vitro. For this purpose, C57Bl6/J mice were subjected to an intraperitoneal glucose tolerance test (ipGTT) and a hyperglycemic (HG) clamp 60 min after a single injection of bromocriptine or placebo. The effects of bromocriptine on glucose-stimulated insulin secretion (GSIS), cell membrane potential and intracellular cAMP levels were also determined in INS-1E beta cells. We report here that bromocriptine increased glucose levels during ipGTT in vivo, an effect associated with a dose-dependent decrease in GSIS. During the HG clamp, bromocriptine reduced both first-phase and second-phase insulin response. This inhibitory effect was also observed in INS-1E beta cells, in which therapeutic concentrations of bromocriptine (0.5–50 nM) decreased GSIS. Mechanistically, neither cellular energy state nor cell membrane depolarization was affected by bromocriptine whereas intracellular cAMP levels were significantly reduced, suggesting involvement of G-protein-coupled receptors. Surprisingly, the DRD2 antagonist domperidone did not counteract the effect of bromocriptine on GSIS, whereas yohimbine, an antagonist of the α2-adrenergic receptors, completely abolished bromocriptine-induced inhibition of GSIS. In conclusion, acute administration of bromocriptine inhibits GSIS by a DRD2-independent mechanism involving direct activation of the pancreatic α2-adrenergic receptors. We suggest that treatment with bromocriptine promotes beta cells rest, thereby preventing long-lasting hypersecretion of insulin and subsequent beta cell failure.

A Synthetic 18-Norsteroid Distinguishes between Two Neuroactive Steroid Binding Sites on GABAA Receptors

In the absence of GABA, neuroactive steroids that enhance GABA-mediated currents modulate binding of [35S]t-butylbicyclophosphorothionate in a biphasic manner, with enhancement of binding at low concentrations (site NS1) and inhibition at higher concentrations (site NS2). In the current study, compound (3alpha,5beta,17beta)-3-hydroxy-18-norandrostane-17-carbonitrile (3alpha5beta-18-norACN), an 18-norsteroid, is shown to be a full agonist at site NS1 and a weak partial agonist at site NS2 in both rat brain membranes and heterologously expressed GABAA receptors. 3alpha5beta-18-norACN also inhibits the action of a full neurosteroid agonist, (3alpha,5alpha,17beta)-3-hydroxy-17-carbonitrile (3alpha5alphaACN), at site NS2. Structure-activity studies demonstrate that absence of the C18 methyl group and the 5beta-reduced configuration both contribute to the weak agonist effect at the NS2 site. Electrophysiological studies using heterologously expressed GABAA receptors show that 3alpha5beta-18-norACN potently and efficaciously potentiates the GABA currents elicited by low concentrations of GABA but that it has low efficacy as a direct activator of GABAA receptors. 3alpha5beta-18-norACN also inhibits direct activation of GABAA receptors by 3alpha5alphaACN. 3alpha5beta-18-norACN also produces loss of righting reflex in tadpoles and mice, indicating that action at NS1 is sufficient to mediate the sedative effects of neurosteroids. These data provide insight into the pharmacophore required for neurosteroid efficacy at the NS2 site and may prove useful in the development of selective agonists and antagonists for neurosteroid sites on the GABAA receptor.

Modulation of immune responses through direct activation of Toll-like receptors to T cells

Toll-like receptors (TLRs), which are a family of pattern recognition receptors (PRRs), are involved critically in the generation and regulation of innate immunity as well as initiation of subsequent adaptive immune responses. However, recent research results showed that different subsets of T cells express certain types of TLRs during development and activation stages. Importantly, TLRs participate in the direct regulation of adaptive immune response, possibly as co-stimulatory molecules. In this review we summarize recent studies about the novel regulation of TLRs on the homeostasis and immunity of different T cell subtypes including CD4+CD25+T regulatory cells (Treg) and interleukin (IL)-17-producing CD4+T cells (T helper type 17). The direct involvement of TLRs in T cell-mediated immunity prompted us to reconsider the role of TLRs in the occurrence of autoimmune diseases, infectious diseases and graft rejection. The important effects of TLRs in T cell-intrinsic components also prompt us to explore novel vaccine adjuvants for modifying desired immune responses in an efficient way.

Increased Protein Stability of FGF1 Can Compensate for Its Reduced Affinity for Heparin

Human FGF1 (fibroblast growth factor 1) is a powerful signaling molecule with a short half-life in vivo and a denaturation temperature close to physiological. Binding to heparin increases the stability of FGF1 and is believed to be important in the formation of FGF1·fibroblast growth factor receptor (FGFR) active complex. In order to reveal the function of heparin in FGF1·FGFR complex formation and signaling, we constructed several FGF1 variants with reduced affinity for heparin and with diverse stability. We determined their biophysical properties and biological activities as well as their ability to translocate across cellular membranes. Our study showed that increased thermodynamic stability of FGF1 nicely compensates for decreased binding of heparin in FGFR activation, induction of DNA synthesis, and cell proliferation. By stepwise introduction of stabilizing mutations into the K118E (K132E) FGF1 variant that shows reduced affinity for heparin and is inactive in stimulation of DNA synthesis, we were able to restore the full mitogenic activity of this mutant. Our results indicate that the main role of heparin in FGF-induced signaling is to protect this naturally unstable protein against heat and/or proteolytic degradation and that heparin is not essential for a direct FGF1-FGFR interaction and receptor activation.

A metabonomic study of inhibition of GABA uptake in the cerebral cortex

GABAergic activity is regulated by rapid, high affinity uptake of GABA from the synapse. Perturbation of GABA reuptake has been implicated in neurological disease and inhibitors of GABA transporters (GAT) have been used therapeutically but little detail is known about the ramifications of GAT inhibition on brain neurochemistry. Here, we incubated Guinea pig cortical tissue slices with [3-13C]pyruvate and major, currently available GABA uptake inhibitors. Metabolic fingerprints were generated from these experiments using 13C/1H NMR spectroscopy. These fingerprints were analyzed using multivariate statistical approaches and compared with an existing library of fingerprints of activity at GABA receptors. This approach identified five distinct clusters of metabolic activity induced by blocking GABA uptake. Inhibition of GABA uptake via GAT1 produced patterns similar to activity at mainstream GABAergic synapses in particular those containing α1-subunits but still statistically separable. This indicated that inhibition of GABA uptake, an indirect method of activating GABA receptors, produces different effects to direct receptor activation or to exogenous GABA. The mechanism of inhibitor function also produced different outcomes, with the channel blocker SKF 89976A yielding a unique metabolic response. Blocking GAT1 and GAT3 simultaneously induces a large metabolic response consistent with induction of tonic inhibition via high affinity GABA receptors. Blocking BGT produces patterns similar to activity at less common receptors such as those containing α5 subunits. This approach is useful for determining where in the spectrum of GABAergic responses a particular GABA transport inhibitor is effective.

Nociceptin/Orphanin FQ Receptor Activation Attenuates Antinociception Induced by Mixed Nociceptin/Orphanin FQ/μ-Opioid Receptor Agonists

Activation of brain nociceptin/orphanin FQ (NOP) receptors leads to attenuation of mu-opioid receptor (MOP receptor)-mediated antinociception. Buprenorphine, a high-affinity partial MOP receptor agonist also binds to NOP receptors with 80 nM affinity. The buprenorphine-induced inverted U-shaped dose-response curve for antinociception may be due to NOP receptor activation, given that, in the presence of the NOP receptor antagonist, 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J113397), or in NOP receptor knockout mice, buprenorphine has a steeper dose-response curve and acts as a full agonist. To further explore the involvement of the direct activation of NOP receptors by buprenorphine and other compounds that activate both NOP and MOP receptors, the antinociceptive effects of 1-(1-(2,3,3alpha,4,5,6-hexahydro-1H-phenalen-1-yl)piperidin-4-yl)-indolin-2-one. (SR16435), 3-ethyl-1-(1-(4-isopropylcyclohexyl)piperidin-4-yl)-indolin-2-one (SR16507), buprenorphine, pentazocine, and morphine, compounds with varying levels of MOP and NOP receptor affinity and efficacy, were assessed in mice using the tail-flick assay. The ability of the selective NOP receptor antagonist (-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111) to potentiate antinociception induced by the above compounds was examined to investigate whether activation of NOP receptors leads to attenuation of MOP receptor-mediated antinociception. SB-612111 potentiated antinociception induced by buprenorphine and the other mixed NOP/MOP receptor agonists SR16435 and SR16507. However, SB-612111 had no effect on pentazocine or morphine antinociception, two compounds with no NOP receptor-binding affinity. These results further support the hypothesis that activation of NOP receptors can lead to attenuation of MOP receptor-mediated antinociception elicited by mixed NOP/MOP receptor compounds such as buprenorphine, SR16435, and SR16507 and that, although buprenorphine has low efficacy in vitro, it has significant NOP receptor agonist activity in vivo.

Interaction of androsterone and progesterone with inhibitory ligand-gated ion channels: a patch clamp study

Gamma-aminobutyric acid receptor type A (GABA(A)) receptor channels mediate fast inhibitory neurotransmission throughout the central nervous system while the expression of ionotropic glycine receptors is mainly restricted to the spinal cord and brain stem. Neuroactive steroids are well known as positive allosteric modulators of GABA(A) receptor function. Furthermore, there have been hints for an interaction of neuroactive steroids with ionotropic glycine receptors. The aim of the study was to characterize the effect of androsterone and progesterone on alpha(1) and alpha(1)beta glycine receptor and alpha(1)beta(2)gamma(2) GABA(A) receptor channels and to examine the molecular interactions between ligands and receptors. Electrophysiological recordings were performed on HEK 293 cells using the patch clamp technique in combination with an ultrafast perfusion system. A direct activation of inhibitory ionotropic receptors was observed for androsterone at GABA(A) receptor channels. A coactivation of currents elicited by nonsaturating agonist concentrations was observed with androsterone and progesterone at glycine and GABA(A) receptor channels. We could show that association of beta subunits with alpha subunits affects the sensitivity of glycine receptors to androsterone. In contrast to previous reports in which recombinant glycine receptors were inhibited by progesterone, a potentiating effect was revealed by our experiments. At concentrations of 0.1 mM and higher, there were also hints to a channel block-like mechanism. In conclusion, different molecular mechanisms of interaction between neuroactive steroids and GABA as well as glycine receptors could be identified and quantitatively described. Our data clarify the role of steroid compounds in the modulation of inhibitory receptor channel function.