Peptide mimics of intracellular loop 2 (ic2) of the human 5HT1a receptor have been studied with respect to their ability to inhibit agonist binding via interference with receptor-G-protein coupling. These peptides give shallow concentration-effect relationships. Additionally, these peptides have been studied with respect to their ability to trigger the signal transduction system of this Gi-coupled receptor. Two signaling parameters have been quantified: concentration of intracellular cAMP and changes in incorporation into the G protein of a stable analog of GTP. In both cases, peptide mimics near midloop of ic2 actually show agonist activity with efficacy falling off toward both loop termini near TM 3 and TM 4. Previous results have suggested that the loop region near the TM3/ic2 interface is primarily responsible for receptor-G-protein coupling, while the current result emphasizes the mid-ic2 loop region's ability to activate the G protein following initial coupling. A limited number of peptides from the receptor's TM5/ic3 loop vicinity were also studied regarding agonist inhibition and G-protein activation. These peptides provide additional evidence that the human 5HT1a receptor, TM5/ic3 loop region, is involved in both coupling and activation actions. Overall, these results provide further information about potential pharmacological intervention and drug development with respect to the human 5HT1a receptor/G-protein system. Finally, the structural evidence generated here provides testable models pending crystallization and X-ray analysis of the receptor.

Hormones, neurotransmitters, cytokines and a number of extracellular ligands participate in extracellular signaling mechanisms that modify cellular biochemistry. Intracellular ligands are involved in an array of intracellular signaling processes that increase or decrease the effects of extracellular ligands. Both extracellular and intracellular mechanisms are important in drug effects, maintaining health and treating chronic disease. The management of chronic diseases is becoming a very common process for many people. Many questions remain to be answered about chronic diseases. What causes them? Is it possible to prevent them? How are they best treated?

Even though the set of headaches known as migraine have been difficult to treat with either pharmacotherapy or other approaches, significant progress, particularly in acute episodes, has been made in recent decades. Regardless, much remains to be known about the genesis of migraine headaches, and their treatment, especially in the chronic, prophylactic sense. In this chapter, migraine is introduced in the context of various biochemical factors that have not been traditionally associated with headache. These include neural elements such as astrocytes and chemical parameters associated with metabolic, affective and sleep regulation. The primary focus is to suggest signaling pathways that might be tested for playing a role in the headaches and which could become targets for new drug development. Adipokines like leptin are noteworthy in this regard.

The human serotonin 1a receptor (H5HT1aR) is a highly studied member of the 7 transmembrane G protein-coupled receptors. This model receptor, negatively coupled to adenylyl cyclase via Gi, is linked to physiological processes such as cognition and mood regulation and to associated disorders like anxiety and depression. Gibb’s free energies, enthalpies, and entropies were calculated for the agonist [3H]8-OH-DPAT in the presence of synthetic peptides derived from sequences of intracellular loops 2 and 3 of the H5HT1aR. For comparative purposes, the thermodynamic parameters were also determined in the presence of a limited number of ligand-binding site substances (the partial agonist dipropyltryptamine [DPT], and the full agonist [3H]8-OH-DPAT alone). All of these thermodynamic measurements were based on binding data accumulated over a range of temperatures (0–35°C). Representative examples of binding constant experiments and van’t Hoff plots are shown to establish the thermodynamic variables. Although differences exist between the peptides themselves and the non-peptide agonists, in all situations the binding events are highly entropy driven. Differences between this information and published data for rat 5HT1aR are discussed, as are relationships to other receptor systems. Overall, the conclusions should be useful in further defining a comprehensive model of 5HT1aR, and for future development of binding-site and non-binding-site directed agents for the receptor.

Increased cardiac morbidity and mortality are associated with air pollution. Mexico City (MC) residents are exposed to high concentrations of fine particulate matter and endotoxin. South MC residents are exposed to higher endotoxin v North (Rosas 2007). SMC exposed mice had the highest CD14 and IL1beta mRNA in myocardium v N (Villarreal 2009). To test the hypothesis that residency within MC is a key factor for the degree of myocardial inflammation, we measured inflammatory mediator genes in autopsy samples from R and L ventricles of 6 South and 15 North age‐matched MC residents 18.5±5.2y. South residents had significant up‐regulation of CD14, IL1beta and TNFalpha v North (p<0.001). An anti‐inflammatory biventricular response was also present: IL10 (RV p=0.001, LV p=0.02). Increases in myocardial IL1beta and TNFalpha have been implicated in the pathogenesis of myocardial dysfunction and cardiomyocyte death in ischemia‐reperfusion injury, sepsis, chronic heart failure, etc. Exposure to polluted urban air is associated with myocardial inflammation in young people, the RV responds differently from the LV and endotoxin is a key player in the inflammatory response. Occult cardiotoxicity (Golomb et al., 2009) may be a deleterious effect in young urbanites and the long‐term impact of sustained myocardial inflammation is uncertain.

A bioactive synthetic 11 amino acid peptide probe (P11) was constructed according to the published sequence of the human 5HT1a receptor. The probe was used to enhance understanding of cytoplasmic loop 2/G protein coupling and activation. Additionally, two peptides (P8, P9) from the cytoplasmic loop 3 region were synthesized and studied. These probes were tested in a model system of human 5HT1a receptor stably expressed in Chinese Hamster Ovary cells. In agonist inhibition studies, P11 was active in all three receptor preparations tested: whole cells, membrane bound, and solubilized. In analyses of the membrane bound receptor system, P11 demonstrated uncompetitive inhibition characteristics. When forskolin-stimulated cAMP levels were measured, P11 was inactive in this negatively coupled system. Utilizing a [35S]γ-S-GTP incorporation assay, P11 was unable to stimulate G protein incorporation of GTP. While P8 and P9 were also broadly active as non-competitive agonist inhibitors, their characteristics differed in the signal transduction system. P8 and P9 did not significantly change forskolin-stimulated cAMP levels. However, P8 increased [35S]γ-S-GTP incorporation, while P9 decreased incorporation. Thus, P11, a synthetic peptide from the TM3/i2 region of the receptor, provides suggestive evidence that this receptor region is involved in G protein coupling but not activation. On the other hand, P8 and P9 activities suggest that the TM5/i3 region is involved in both coupling to and regulation of G protein activity. The current evidence from these cytoplasmic loop regions is discussed in the overall context of an emerging model for human 5HT1a receptor–G protein interactions.

Cannabidiol (CBD) is a major, biologically active, but psycho-inactive component of cannabis. In this cell culture-based report, CBD is shown to displace the agonist, [3H]8-OH-DPAT from the cloned human 5-HT1a receptor in a concentration-dependent manner. In contrast, the major psychoactive component of cannabis, tetrahydrocannabinol (THC) does not displace agonist from the receptor in the same micromolar concentration range. In signal transduction studies, CBD acts as an agonist at the human 5-HT1a receptor as demonstrated in two related approaches. First, CBD increases [35S]GTPγS binding in this G protein coupled receptor system, as does the known agonist serotonin. Second, in this GPCR system, that is negatively coupled to cAMP production, both CBD and 5-HT decrease cAMP concentration at similar apparent levels of receptor occupancy, based upon displacement data. Preliminary comparative data is also presented from the cloned rat 5-HT2a receptor suggesting that CBD is active, but less so, relative to the human 5-HT1a receptor, in binding analyses. Overall, these studies demonstrate that CBD is a modest affinity agonist at the human 5-HT1a receptor. Additional work is required to compare CBD’s potential at other serotonin receptors and in other species. Finally, the results indicate that cannabidiol may have interesting and useful potential beyond the realm of cannabinoid receptors.

It is unclear whether the two enantiomeric forms (R & S) of lipoic acid (LA) share similar pharmacological activity and the exact cellular targets of LA are not well identified. We oxidatively stressed 3 cell culture systems representing different cell types. Mitochondrial metabolism was the primary endpoint. When C6 glioma was damaged by hydrogen peroxide (H2O2), all forms of LA protected. Racemic and S-LA were less effective than the R-isomer that was also protective in tertiary butyl hydroperoxide (TBHP)-damaged C6 glioma. In PC12 cells, little damage was produced by TBHP; R-LA increased mitochondrial metabolism above the level of non-damaged control. In H2O2 damaged PC12 cells, R-LA and racemic LA (but not S-LA) not only protected against damage, but increased mitochondrial metabolism above the non-damaged control level. When BAE cells were damaged with H2O2, R- and racemic LA protected while S-LA was ineffective.

Acetaminophen, proinflammatory cytokineinduced pain behaviour in mice 152 Acetylcholine 119, 210 -transferase 84 Adenosine 15, 143 -deaminase 144 -, endogenous regulator of endothelial cells 143 -receptor subtypes 143 Adenylyl cyclase 143 Adrenoceptor(s) 1, 2, 23, 157, 158 -(• 1 ) densitiy 23 -(• 1 ) function 24 -(ß) agonist 158 -(ß 1 , ß 2 , ß 3 ) antagonism 2 -(ß) antagonism, DPJ 904 1 -(ß 3 ) antagonist cynopindol 157 -(ß 3 ) human oviduct relaxation 157 Airway phlogosis 127 Akt kinase

Dipropyltryptamine (DPT) is a synthetic indolealkylamine first characterized in the 1960s. Largely forgotten since the discovery of multiple serotonin receptor subtypes, some of the properties of DPT at the cloned human 5-HT1a receptor are described here. When [H-3] 8-OH-DPAT is bound to the receptor, DPT inhibits the interaction with an IC50 of 0.1 mu mol/l. This interaction is shown to be competitive when double-reciprocal plots of the DPT/agonist interaction are analyzed. DPT's effects in the signal transduction system are complex. While DPT alone (0.1 - 1,000 mu mol/ l) activates G(i) when both cAMP and gamma-S-GTP incorporation are measured, in the presence of 5-HT ( 0.1 - 10 mu mol/l), DPT blocks the agonist effect. In combination, the findings suggest that DPT is a moderate affinity partial agonist at the human 5-HT1a receptor. These results provide evidence that DPT has potential as a versatile experimental tool at 5-HT1a receptors. Copyright (C) 2005 S. Karger AG, Basel.

Berkeley Pit Lake in Butte, Montana, is an acid mine waste reservoir rich in toxic metals. A Pithomyces sp. isolated from the Pit Lake yielded three tyrosine derivatives (1-3), one of which acts as a 5-HT(2a) receptor ligand. This type of activity has been associated with migraine preventative and antihypertensive drugs. The isolation and characterization of compounds 1-3 and three sesquiterpenes (5-7) that have been isolated previously from higher plants are reported here.

To better understand cytoplasmic loop 3/G protein coupling, variations in a bioactive synthetic peptide probe (P1) were constructed according to the published sequences of the human 5HT1a receptor. These probes were tested in a model system of human 5HT1a receptor stably expressed in Chinese hamster ovary cells. In agonist inhibition studies, peptides with amino acid substitutions of residues 6–9 from the amino terminus of loop 3 were less active than P1. Truncated peptide P4, conserving the residue 6–9 region, was also less active than P1. Truncates P5 and P6, deleting the residue 6–9 region, were inactive. When cAMP levels were measured, both substituted peptides were more active than P1 in this negatively coupled system. In contrast, the truncated peptides were without activity in the cAMP assays. Thus, P1 and its derivatives (P2–P6) constitute a small group of peptides with differential uncoupling (agonist inhibition) and signal transduction (cAMP) activities in this G-protein-linked system. It is proposed that these peptides will be useful in future studies detailing the molecular determinants at the receptor/G protein interface.

15 amino acid peptide from the transmembrane 5 - intracellular loop 3 region of the human 5HT1a receptor produced concentration-dependent decreases in agonist binding. This result is consistent with a competitive interaction between peptide, receptor, and G protein at the receptor - G protein interface. Bombesin and a 13 amino acid peptide from the carboxyl terminus region of the receptor were inactive. Additionally, the peptide decreased forskolin-mediated cAMP elevation. Overall, these results suggest that amino acid residues from this region of the receptor are involved in receptor - G protein coupling and that G protein is activated by the receptor.

Parthenolide displaces [3H]ketanserin from 5HT2A receptors from rat and rabbit brain and cloned 5HT2A receptors. Ki's are in the 100-250 microM range. These results suggest that parthenolide may be a low-affinity antagonist at 5HT receptors; it is unlikely that the entire mechanism of action can be explained by its modest 5HT2A receptor affinity.