The cannabinoid CB1 receptor antagonists rimonabant (SR141716) and AM251 directly potentiate GABA(A) receptors

Rimonabant (SR141716) and the structurally related AM251 are widely used in pharmacological experiments as selective cannabinoid receptor CB(1) antagonists / inverse agonists. Concentrations of 0.5-10 µM are usually applied in in vitro experiments. We intended to show that these drugs did not act at GABA(A) receptors but found a significant positive allosteric modulation instead.

Modulation of L-α-lysophosphatidylinositol/GPR55 mitogen-activated protein kinase (MAPK) signaling by cannabinoids

GPR55 is activated by l-α-lysophosphatidylinositol (LPI) but also by certain cannabinoids. In this study, we investigated the GPR55 pharmacology of various cannabinoids, including analogues of the CB1 receptor antagonist Rimonabant®, CB2 receptor agonists, and Cannabis sativa constituents. To test ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys LPI-induced activation of GPR55, a high throughput system, was established using the AlphaScreen® SureFire® assay. Here, we show that CB1 receptor antagonists can act both as agonists alone and as inhibitors of LPI signaling under the same assay conditions. This study clarifies the controversy surrounding the GPR55-mediated actions of SR141716A; some reports indicate the compound to be an agonist and some report antagonism. In contrast, we report that the CB2 ligand GW405833 behaves as a partial agonist of GPR55 alone and enhances LPI signaling. GPR55 has been implicated in pain transmission, and thus our results suggest that this receptor may be responsible for some of the antinociceptive actions of certain CB2 receptor ligands. The phytocannabinoids Δ9-tetrahydrocannabivarin, cannabidivarin, and cannabigerovarin are also potent inhibitors of LPI. These Cannabis sativa constituents may represent novel therapeutics targeting GPR55.

Regular treatment with salmeterol and inhaled steroids for chronic asthma: serious adverse events

Epidemiological evidence has suggested a link between beta2-agonists and increased asthma mortality. There has been much debate about possible causal links for this association, and whether regular (daily) long-acting beta2-agonists are safe. This is an updated systematic review.

Evidence for dissociation of TLR mRNA expression and TLR agonist-mediated functions in bovine macrophages

Toll-like receptors are of key importance in the recognition of and response to infectious agents by cells of the innate immune system. TLR mRNA expression and TLR-mediated functions were determined in bovine macrophages (MPhi) infected with bovine viral diarrhea virus (BVDV) or stimulated with interferon-gamma (IFN-gamma) in order to see whether they are correlated under these conditions. As parameters quantitative real time RT-PCR (QRT-PCR) for TLR2, TLR3 and TLR4, NO and TNF production were measured. Triggering of bovine MPhi with bona fide TLR2 and TLR4 agonists (lipopolysaccharide, lipoteichoic acid, peptidoglycan, lipopetide) led to NO and TNF production but neither TLR3 nor TLR9 agonists (double-stranded RNA, CpG DNA) showed this effect. The mRNA expression of TLR2, TLR3 and TLR4 was neither influenced by MPhi costimulation with IFN-gamma nor by MPhi preinfection with BVDV nor by the ligands themselves. However, NO production induced by TLR2 or TLR4 agonists was strongly modulated either by IFN-gamma costimulation or BVDV preinfection. Thus costimulation of MPhi with IFN-gamma resulted in an increase of both NO synthesis and TNF expression by cells stimulated simultaneously by TLR2 or TLR4 agonists. Preinfection of bovine MPhi by BVDV resulted in upregulation of TLR2- and TLR4-mediated NO synthesis. Collectively, these data show that TLR-mediated functions may be modulated by viral infection or activation via IFN-gamma of MPhi whereas the mRNA concentrations of relevant TLR members were not significantly influenced. Thus, the amount of TLR2, TLR3 and TLR4 mRNA transcripts is stable at least under the conditions tested. More importantly, modulation of TLR-mediated responses was dissociated from mRNA expression of TLR members.

Agonist specific L-type Ca(2+)-current stimulation in ventricular myocytes by a novel steroid-like compound

In cardiac muscle the amplitude of Ca(2+) transients can be increased by enhancing Ca(2+) influx. Among the processes leading to increased Ca(2+) influx, agonists of the L-type Ca(2+)-channel can play an important role. Known pharmacological Ca(2+)-channel agonists act on different binding sites on the channel protein, which may lead not only to enhanced peak currents, but also to distinct changes in other biophysical characteristics of the current. In this study, membrane currents were recorded with the patch-clamp technique in the whole-cell configuration in guinea pig isolated ventricular myocytes in combination with confocal fluorescence Ca(2+) imaging techniques and a variety of pharmacological tools. Testing a new positive inotropic steroid-like compound, we found that it increased the L-type Ca(2+)-current by 2.5-fold by shifting the voltage-dependence of activation by 20.2 mV towards negative potentials. The dose-response relationship revealed two vastly different affinities (EC(50(high-affinity))=4.5+/-1.7 nM, EC(50(low-affinity))=8.0+/-1.1 microM) exhibiting differential pharmacological interactions with three classes of Ca(2+)-current antagonists, suggesting more than one binding site on the channel protein. Therefore, we identified and characterized a novel positive inotropic compound (F90927) as a member of a new class of Ca(2+)-channel agonists exhibiting unique features, which set it apart from other presently known L-type Ca(2+)-channel agonists.

Targets of emerging therapies for viral hepatitis B and C

Viral hepatitis B and C, structurally two completely different viruses, commonly infect human hepatocytes and cause similar clinical manifestations. Since their discovery, IFN has been a pillar in the treatment. However, because of the different natures of the viruses, therapeutic approaches diverge and new treatment targets are tailored specifically for each virus. Herein, the authors analyse therapeutic approaches for hepatitis B virus (HBV) and hepatitis C virus (HCV) and focus on emerging concepts that are under clinical evaluation. In particular, promising viral inhibitors for HBV and HCV are reviewed and the current status of research for gene therapy for HCV is described. Immune therapy is a fast-moving field with fascinating results which include therapeutic vaccines and toll-like receptor agonists that could improve tomorrow's treatment approaches.

Human monocytoid cells as a model to study Toll-like receptor-mediated activation

THP-1 2A9, a subclone of the monocytoid cell line THP-1 and known to be exquisitely sensitive to LPS, was tested for TNF production following triggering by excess doses of TLR ligands. TLR2, TLR4 and TLR5 agonists, but neither TLR3 nor TLR9 agonists, induced TNF production. When used at lower concentrations, priming by calcitriol strongly influenced the sensitivity of cells to LPS and different TLR2 triggers (lipoteichoic acid (LTA), trispalmitoyl-cysteyl-seryl-lysyl-lysyl-lysyl-lysine (Pam3Cys) and peptidoglycan (PGN)). Priming by calcitriol failed to modulate TLR2 and TLR4 mRNA and cell surface expression of these receptors. TNF signals elicited by TLR2 agonists were blocked by the TLR-specific antibody 2392. CD14-specific antibodies showed variable effects. CD14-specific antibodies inhibited TNF induction by LTA. High concentrations partially inhibited TNF induction by Pam3Cys. The same antibodies failed to inhibit TNF induction by PGN. Thus, THP-1 2A9 cells respond by TNF production to some, but not all TLR agonists, and the wide variety of putative TLR2 agonists interact to variable degrees also with other cell-surface-expressed binding sites such as CD14. THP-1 2A9 cells might provide a model by which to investigate in more detail the interaction of pathogen-associated molecular patterns and monocytoid cell-surface-expressed pattern recognition receptors.

[Simultaneous defect of visual fields and loss of libido--a coincidence?]

We present the case of a 60 year old male patient with incidentally detected visual abnormalities. Detailed personal history revealed a hypogonadism that had been present for several years. Further investigations established the diagnosis of an infiltrative macroadenoma. Medical treatment with cabergoline led to a rapid regression of ophthalmologic symptoms and, subsequently, of tumor size. In male subjects symptoms of hypogonadism are often reported only late in the course of the disease, thereby leading to a generally larger tumor size at the point of diagnosis. In contrast to other pituitary tumors that are mainly treated by surgery, medical treatment with dopamine agonists is the principal therapeutic option in prolactinomas.

Analgesic activity of a non-peptide imidazolidinedione somatostatin agonist: in vitro and in vivo studies in rat

Several lines of evidence support an important role for somatostatin receptors (SSTRs) in pain modulation. The therapeutic use of established SSTR peptide agonists for this indication is limited by their broad range of effects, need for intrathecal delivery, and short half-life. Therefore, the goal of the present study was to investigate the analgesic effect of SCR007, a new, highly selective SSTR2 non-peptide agonist. Behavioral studies demonstrated that paw withdrawal latencies to heat were significantly increased following intraplantar SCR007. Furthermore, both intraperitoneal and intraplantar injection of SCR007 significantly reduced formalin- and capsaicin-induced flinching and lifting/licking nociceptive behaviors. Recordings from nociceptors using an in vitro glabrous skin-nerve preparation showed that SCR007 reduced heat responses in a dose-dependent fashion, bradykinin-induced excitation, heat sensitization and capsaicin-induced excitation. In both the behavioral and single fiber studies, the SCR007 effects were reversed by the SSTR antagonist cyclo-somatostatin, demonstrating receptor specificity. In the single fiber studies, the opioid antagonist naloxone did not reverse SCR007-induced anti-nociception suggesting that SCR007 did not exert its effects through activation of opioid receptors. Analysis of cAMP/protein kinase A (PKA) involvement demonstrated that SCR007 prevented forskolin- and Sp-8-Br-cAMPS (a PKA activator)-induced heat sensitization, supporting the hypothesis that SCR007-induced inhibition could involve a down-regulation of the cAMP/PKA pathway. These data provide several lines of evidence that the non-peptide imidazolidinedione SSTR2 agonist SCR007 is a promising anti-nociceptive and analgesic agent for the treatment of pain of peripheral and/or central origin.

[Platelet inhibition and activation. Mechanisms and clinical implications.]

The sensitivity to activation of platelets is part of the delicate equilibrium differentiating hemostasis from thrombosis. Under physiological conditions it is maintained by downregulating platelet activity and removing agonists. Under pathologic conditions the clinician tries to restore this equilibrium with pharmaceutical drugs. The results obtained by such treatments are steadily improving but there is still need for better knowledge of the mechanisms involved and for alternative inhibitors.

Platelet activation induces metalloproteinase-dependent GP VI cleavage to down-regulate platelet reactivity to collagen

Glycoprotein (GP) VI, the primary collagen receptor on platelets, has been shown to have variable expression, possibly as a consequence of immune modulation. The present study was designed to determine the mechanism by which GP VI clearance occurs. We found that direct activation of GP VI both by a GP VI-specific antibody and by GP VI ligands (collagen and convulxin) reduced binding of biotinylated convulxin to the stimulated platelets. Analysis of immunoblots of platelets and supernatants showed that the stimulated platelets contained less GP VI, while the soluble fraction contained a 57-kDa cleavage product. Stimulation of platelets with PAR-1 agonists (TRAP peptide and thrombin) also caused GP VI cleavage, although the amount of GP VI loss was less than that observed with direct GP VI ligands. The metalloproteinase (MMP) inhibitors GM6001 and TAPI prevented both the clearance of GP VI from the platelet surface and the appearance of the soluble cleavage product. Induction of GP VI cleavage caused specific down-regulation of collagen-induced platelet aggregation, providing a mechanism for the modulation of platelet responsiveness to this important platelet agonist.

The GPIb thrombin-binding site is essential for thrombin-induced platelet procoagulant activity

The role of the platelet glycoprotein (GP) Ib-V-IX receptor in thrombin activation of platelets has remained controversial although good evidence suggests that blocking this receptor affects platelet responses to this agonist. The mechanism of expression of procoagulant activity in response to platelet agonists is also still obscure. Here, the binding site for thrombin on GPIb is shown to have a key role in the exposure of negatively charged phospholipids on the platelet surface and thrombin generation, in response to thrombin, which also requires protease-activated receptor-1, GPIIb-IIIa, and platelet-platelet contact. Von Willebrand factor binding to GPIb is not essential to initiate development of platelet procoagulant activity. Inhibition of fibrinogen binding to GPIIb-IIIa also failed to block platelet procoagulant activity. Both heparin and low molecular weight heparin block thrombin-induced platelet procoagulant activity, which may account for part of their clinical efficacy. This study demonstrates a new, critical role for platelet GPIb in hemostasis, showing that platelet activation and coagulation are tightly interwoven, which may have implications for alternative therapies for thrombotic diseases.

Surface expression and functional characterization of alpha-granule factor V in human platelets: effects of ionophore A23187, thrombin, collagen, and convulxin

Factor V (FV) present in platelet alpha-granules has a significant but incompletely understood role in hemostasis. This report demonstrates that a fraction of platelets express very high levels of surface-bound, alpha-granule FV on simultaneous activation with 2 agonists, thrombin and convulxin, an activator of the collagen receptor glycoprotein VI. This subpopulation of activated platelets represents 30.7% +/- 4.7% of the total population and is referred to as convulxin and thrombin-induced-FV (COAT-FV) platelets. COAT-FV platelets are also observed on activation with thrombin plus collagen types I, V, or VI, but not with type III. No single agonist examined was able to produce COAT-FV platelets, although ionophore A23187 in conjunction with either thrombin or convulxin did generate this population. COAT-FV platelets bound annexin-V, indicating exposure of aminophospholipids and were enriched in young platelets as identified by the binding of thiazole orange. The functional significance of COAT-FV platelets was investigated by demonstrating that factor Xa preferentially bound to COAT-FV platelets, that COAT-FV platelets had more FV activity than either thrombin or A23187-activated platelets, and that COAT-FV platelets were capable of generating more prothrombinase activity than any other physiologic agonist examined. Microparticle production by dual stimulation with thrombin and convulxin was less than that observed with A23187, indicating that microparticles were not responsible for all the activities observed. These data demonstrate a new procoagulant component produced from dual stimulation of platelets with thrombin and collagen. COAT-FV platelets may explain the unique role of alpha-granule FV and the hemostatic effectiveness of young platelets. (Blood. 2000;95:1694-1702)

Glycoprotein VI is a major collagen receptor for platelet activation: it recognizes the platelet-activating quaternary structure of collagen, whereas CD36, glycoprotein IIb/IIIa, and von Willebrand factor do not

Simple collagen-related peptides (CRPs) containing a repeat Gly-Pro-Hyp sequence are highly potent platelet agonists. Like collagen, they must exhibit tertiary (triple-helical) and quaternary (polymeric) structure to activate platelets. Platelet signaling events induced by the peptides are the same as most of those induced by collagen. The peptides do not recognize the alpha 2 beta 1 integrin. To identify the signaling receptor involved, we have evaluated the response to the CRP, Gly-Lys-Hyp(Gly-Pro-Hyp)10-Gly-Lys-Hyp-Gly of platelets with defined functional deficiencies. These studies exclude a primary recognition role for CD36, von Willebrand factor (vWF), or glycoprotein (GP) IIb/IIIa. Thus, both CD36 and vWF-deficient platelets exhibited normal aggregation, normal fibrinogen binding, and normal expression of CD62 and CD63, measured by flow cytometry, in response to the peptide, and there was normal expression of CD62 and CD63 on thrombasthenic platelets. In contrast, GPVI-deficient platelets were totally unresponsive to the peptide, indicating that this receptor recognizes the Gly-Pro-Hyp sequence in collagen. GPVI-deficient platelets showed some fibrinogen binding in response to collagen but failed to aggregate and to express CD62 and CD63. Collagen, but not CRP-XL, contains binding sites for alpha 2 beta 1. Therefore, it is possible that collagen still induces some signaling via alpha 2 beta 1, leading to activation of GPIIb/IIIa. Our findings are consistent with a two-site, two-step model of collagen interaction with platelets involving recognition of specific sequences in collagen by an adhesive receptor such as alpha 2 beta 1 to arrest platelets under flow and subsequent recognition of another specific collagen sequence by an activatory receptor, namely GPVI.

Human group II 14 kDa phospholipase A2 activates human platelets

Recombinant human group II phospholipase A2 (sPLA2) added to human platelets in the low microg/ml range induced platelet activation, as demonstrated by measurement of platelet aggregation, thromboxane A2 generation and influx of intracellular free Ca2+ concentration and by detection of time-dependent tyrosine phosphorylation of platelet proteins. The presence of Ca2+ at low millimolar concentrations is a prerequisite for the activation of platelets by sPLA2. Mg2+ cannot replace Ca2+. Mg2+, given in addition to the necessary Ca2+, inhibits sPLA2-induced platelet activation. Pre-exposure to sPLA2 completely blocked the aggregating effect of a second dose of sPLA2. Albumin or indomethacin inhibited sPLA2-induced aggregation, similarly to the inhibition of arachidonic acid-induced aggregation. Platelets pre-treated with heparitinase or phosphatidylinositol-specific phospholipase C lost their ability to aggregate in response to sPLA2, although they still responded to other agonists. This suggests that a glycophosphatidylinositol-anchored platelet-membrane heparan sulphate proteoglycan is the binding site for sPLA2 on platelets. Previous reports have stated that sPLA2 is unable to activate platelets. The inhibitory effect of albumin and Mg2+, frequently used in aggregation studies, and the fact that isolated platelets lose their responsiveness to sPLA2 relatively quickly, may explain why the platelet-activating effects of sPLA2 have not been reported earlier.