Receptors & channels最新文献

With completion of the sequencing of the human and mouse genomes, the primary sequences of close to 400 non-olfactory G protein-coupled receptors (GPCRs) have been determined. There are intensive efforts within the pharmaceutical industry to discover and develop new therapeutic agents acting via GPCRs. In addition, there is a concerted effort to identify potential new drug targets from the remaining 150+orphan GPCRs through the identification of their ligands. Access to functionally expressed recombinant receptors underpins both of these key drug discovery activities. Typically, GPCR drug discovery screening activities are carried out using mammalian cell lines stably expressing the target of interest. The influx of new receptor sequences originating from genomic sequencing efforts has caused a shift toward wider applications of transient rather than stable expression systems, especially in support of assays for orphan receptor ligand screening. Recombinant baculoviruses in which the polyhedrin promoter has been replaced with a mammalian promoter, termed BacMam viruses, were originally designed as potential new gene therapy delivery vehicles. This same technology offers numerous advantages as a transient expression system in the assay of membrane-expressed drug targets, including GPCRs. Data presented show that BacMam can be used rapidly to generate robust and pharmacologically authentic GPCR assays in several formats, with the potential to transform drug discovery screening processes for this gene family.

Sex and age influence morphine analgesia in humans and animals. Mature rats show greater morphine analgesia in males than in females. Ultra-low doses of naltrexone enhance morphine analgesia. In mature rats (18-22 weeks), naltrexone (0.002-2.0 mg/kg)-morphine (2 mg/kg) cotreatment enhanced morphine analgesia in females, an effect inversely related to naltrexone dose. Conversely, in mature male rats, naltrexone tended to decrease morphine analgesia with increasing dose. In young rats (8-10 weeks), morphine analgesia was unrelated to sex and in both sexes the naltrexone-morphine interaction was negligible. These data show that dose, age, and sex alter the naltrexone-morphine interaction in rats.

Tissue levels of nineteen amino acids and total free amino acids, were assayed by gas-liquid chromatography in cytosols of rat atrial and ventricular muscle cardiomyocytes. The tissues were assayed after the rats had been administered IP with the three cardioactive drugs, exerting a significant effect on their heart action: propranolol, pentylenetetrazol and reserpine. It was demonstrated that while in the atrial and ventricular cardiac muscle cytosols of control rats, arginine, glutamine and cysteine were detected in high levels (35.1% and 17.6%; 14.8% and 51.6%; 9.9% and 0.25% of the total free amino acids, respectively), all three drugs significantly reduced the total amounts of cytosolic free amino acids in both atrial and ventricular heart muscles. All three drugs (with reserpine in particular) modified the levels of arginine, cysteine, phenylalanine, tryptophan, isoleucine and tyrosine. The role of these amino acids in the heart muscle cytosol, and their involvement in the mechanism of action of these three cardioactive drugs, is discussed.

A novel, membrane potential sensitive dye and a fluorescence imaging plate reader (FLIPR) have been used to characterize the pharmacological properties of rat Na(v)1.8 voltage-gated sodium channels (VGSC) in parallel with rat Na(v)1.2a and human Na(v)1.5 VGSC subtypes, respectively. The sensitivity of recombinant Na(v)1.2a-CHO, Na(v)1.5-293-EBNA, and Na(v)1.8-F-11 cells to VGSC activators was subtype dependent. Veratridine evoked depolarization of Na(v)1.2a-CHO and Na(v)1.5-293-EBNA cells with pEC(50) values of 4.78 +/- 0.13 and 4.84 +/- 0.12, respectively (n = 3), but had negligible effect on Na(v)1.8-F-11 cells (pEC(50) < 4.5). Type I pyrethroids were without significant effect at all subtypes. In contrast, the type II pyrethroids deltamethrin and fenvalerate evoked direct depolarization of Na(v)1.8-F-11 and Na(v)1.5-293-EBNA cells. Deltamethrin potentiated the veratridine-evoked response in Na(v)1.8-F-11 cells by > or =20-fold, in contrast to a

Objectives were to investigate the effect of nonselective nitric oxide synthase (NOS) inhibitor, L-NAME on codeine-induced analgesia and to see the role of NO in its antinociceptive effect. Also, to see if L-NAME can potentiate the antinociceptive response of sub-effective dose of codeine and to explore if opioid receptors have some role to play in L-NAME effects. Mice were injected with selected doses of codeine or other selected agents intraperitoneally and the latency to hot plate was recorded at zero, 15, 30, and 60 min of the treatments. The antinociceptive response of codeine (10 mg/kg, i.p.) was studied in comparison to those of the NOS inhibitor, L-NAME, and of nitric oxide donor, sodium nitroprusside (SNP). Assessment of nitrates and nitrites (NOx) in the sera of treated mice were also made. Codeine (20 mg/kg dose), induced analgesia significantly and dose dependently only after 15 min. L-NAME at 20, 40, and 80 mg/kg dose levels significantly changed the nonanalgesic effect of codeine (10 mg/kg) to highly significant analgesia. The effect of L-NAME 40 mg/kg was significantly higher than the other two doses and was almost equal to that of the higher dose of codeine. Naloxone itself did not show any intrinsic effect but almost abolished the L-NAME-codeine induced analgesia. Similarly, SNP (1 mg/kg) reversed the decrease in reaction time by L-NAME-codeine to its control values, significantly. Pretreatment with L-NAME rendered the nonanalgesic dose of codeine significantly analgesic almost in an equal potency to the high dose of codeine alone and indicate that the NO modulatory effect on the opioid analgesic codeine is probably, at least in part, through opioid receptors.

In human atrial myocytes, serotonin rather than sympathetic, stimulation is more frequently associated with atrial fibrillation. So does the arrhythmogenic effect of serotonin result from the mechanism of action of the receptor or the context of its action upon cardiac myocytes? The capacity of agonists to produce cAMP followed the sequence 5-HT < Iso < Forskolin to increase ICaL with 5-HT = Iso = Forskolin. The simultaneous application of threshold concentrations of 5-HT and Iso maximally increased ICaL. We will show that the effect of 5-HT upon human atrial myocytes is an imbalance between low production of cAMP and maximal activation of ICaL.

G protein-coupled receptors induce intracellular signals via interaction of with cytosolic/peripheral membrane proteins, mainly G proteins. There has been much debate about the mode of interaction between the receptors, G proteins and effectors, their mobility and the ways of determining the specificity of interaction. Additional complexity has been added to system upon the discovery of i) coupling of single receptors to several G proteins and ii) active direction of this by different ligands (stimulus trafficking). These data suggest that the most primary unit in the signal transduction is the receptor complexed with a specific G protein, making the investigation of the mechanism of receptor-G protein selection and interaction even more important. In this review, I will summarize the general knowledge of receptor interaction with G proteins and effectors and the ways of investigating this.

Allosterism, whereby small molecule ligands produce global changes in the conformations of receptors, is a powerful mechanism for drug effect. This is illustrated by the recent data describing CCR5 antagonists as blockers of HIV infection. Allosteric effects are described in terms of a change in the tertiary conformation of the receptor. This paper outlines some unique features of allosteric antagonists as new drug entities. These include the fact that allosteric ligands have texture in antagonism (not all allosterically blocked receptors are alike), allosteric blockade is probe dependent (not all agonists and radioligands are blocked equally), and the fact that allosteric binding involves a separate site on the receptor may have relevance to duration of effect and selectivity. Dissociation between receptor function and binding also can be encountered with allosteric ligands.

The initial objective of this work was to examine the effects of an antibody (Anti-G21V) directed against the second extracellular loop of human heart 5-HT4 receptors expressed in Chinese hamster ovary (CHO) cells. The antibody anti-G21V had no effect upon either basal cAMP-or 5-HT-evoked increases in cAMP in CHO cells, whereas it had shown an agonist-like effect in COS-7 cells. Analysis of agonist fractions of h5-HT4(e) receptors in CHO and COS-7 cells revealed that equilibrium constant could underlie the different responses of the receptor toward the anti-G21V antibody. Therefore, different expression systems could give rise to functional differences in 5-HT4 receptor behavior.

When Chinese hamster ovary cells transfected with the gene for M(3)-muscarinic receptors were stimulated with carbachol continuously for 30 min, the response at the end of the stimulation period was about 20% of the early response (2-3 min after the start of the stimulation). Long-term treatment of the cells with phorbol ester abolished the response completely while desensitization was significantly reduced upon pre-treatment of the cells with GF109203X, antisense oligonucleotide against the alpha-isoform of protein kinase C and wortmannin. We conclude that in the Chinese hamster ovary expression system, desensitization of M(3)-muscarinic receptors is dependent on a fast feedback loop including the alpha-isoform of protein kinase C.