Darawan Pinthong

AGMATINE RECOGNIZES ALPHA 2-ADRENOCEPTOR BINDING SITES BUT NEITHER ACTIVATES NOR INHIBITS ALPHA 2-ADRENOCEPTORS

It has been suggested that agmatine (decarboxylated arginine) is an endogenous clonidine-displacing substance (CDS) which recognizes α2-adrenoceptor and non-adrenoceptor, imidazoline binding sites. We have examined the effect of agmatine at α2-adrenoceptor binding sites and pre- and postjunctional α2-adrenoceptors. Agmatine produced a concentration-dependent inhibition of 1 nmol/l 3H-clonidine binding to both rat (pKi–5.10+-0.05) and bovine (pKi–4.77+-0.38) cerebral cortex membranes. However, agmatine (0.1–100 μM) failed to activate pre-junctional α2-adrenoceptors regulating transmitter release in the guinea-pig isolated ileum and rat isolated vas deferens, nor did it activate post-junctional α2-adrenoceptors of the porcine isolated palmar lateral vein which mediate contraction or inhibition of forskolin-stimulated cyclic AMP formation. High concentrations of agmatine (10–30-fold the pKi at α2-adrenoceptor binding sites) failed to influence α2-adrenoceptor activation by either clonidine or UK-14304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline bitartrate) in any of the peripheral preparations examined. Moreover, even in a preparation where an interaction with α2-adrenoceptor binding sites on cell membranes can be demonstrated, the rat cerebral cortex, agmatine failed to inhibit forskolin-stimulated cyclic AMP in the intact tissue or affect the inhibition produced by the selective α2-adrenoceptor agonist UK-14304. Agmatine was also devoid of agonist activity in two preparations, the rat isolated thoracic aorta and the rat isolated gastric fundus, in which CDS has been reported to produce non-adrenoceptor effects. Thus, we have confirmed that agmatine recognizes α2-adrenoceptor binding sites and, therefore, is a CDS. However, since agmatine is devoid of pharmacological activity at either peripheral or central α2-adrenoceptors it can not account for earlier reports suggesting that brain-derived CDS can activate α2-adrenoceptors.

The effects of imidazoline agents on the aggregation of human platelets

Clonidine (2‐[(2, 6‐dichlorophenyl)amino]‐2‐imidazoline), an imidazoline α2‐adrenoceptor agonist, is known to exert complex effects on human platelet aggregation distinct from those of the catecholamines, which are non‐imidazoline α‐adrenoceptor agonists. This study has investigated the aggregatory/anti‐aggregatory effects of various imidazolines on human platelets. Blood samples were taken from normal volunteers and platelet aggregation was assessed by a turbidimetric method using a Chronolog aggregometer. Noradrenaline (2 μm) and adenosine diphosphate (1 μm) were used as aggregating agents. The results showed that, with the exception of moxonidine, all of the imidazoline agents used (with or without α2‐adrenoceptor activity) were able to inhibit noradrenaline‐induced platelet aggregation. Compared with the non‐imidazoline α2‐adrenergic antagonist, yohimbine, the rank order of potency was: efaroxan (IC50 = 3.07 times 10−8 m) > idazoxan (IC50 = 1.74 times 10−7 m) > tolazoline (IC50 = 3.90 times 10−7 m) > clonidine (IC50 = 1.49 times 10−6 m) ≅ antazoline (IC50 = 1.77 times 10−6 m) > yohimbine (IC50 = 3.19 times 10−6 m) > rilmenidine (IC50 = 1.27 times 10−5 m) > moxonidine (IC50 > 10−4 m). Clonidine‐displacing substance (CDS), a putative endogenous ligand at imidazoline receptors, was found to inhibit noradrenaline‐induced platelet aggregation. Harmane, norharmane and agmatine, putative candidates for the active principle of CDS, had no effect on noradrenaline‐induced platelet aggregation. In contrast to noradrenaline‐induced aggregation, ADP‐induced platelet aggregation was neither potentiated nor inhibited by the imidazoline agents, with the exceptions of clonidine and moxonidine. In conclusion, most imidazoline agents effectively inhibit noradrenaline‐induced human platelet aggregation. The lack of effect of moxonidine and the proposed endogenous ligands suggested this effect was mediated by an ‘atypical’ non‐adrenoceptor imidazoline‐binding site. The results indicated an anti‐aggregatory role of imidazoline compounds on noradrenaline‐induced human platelet aggregation. In addition, CDS might be an endogenous modulator that prevented platelet hyper‐reactivity to catecholamine stimulation.

Comparison of the interaction of agmatine and crude methanolic extracts of bovine lung and brain with α2‐adrenoceptor binding sites

1 In the present study we have evaluated whether α2‐adrenoceptor binding sites on bovine cerebral cortex membranes labelled by [3H]‐clonidine, [3H]‐idazoxan and [3H]‐RX‐821002 can distinguish between known agonists and antagonists. This model has then been used to compare the binding profiles of the putative non‐catecholamine, clonidine‐displacing substance (CDS), agmatine and crude methanolic extracts of bovine lung and brain. 2 Saturation studies carried out in the presence and absence of noradrenaline, 10 μmol l−1, revealed that the maximum number of binding sites on bovine cerebral cortex membranes for [3H]‐idazoxan and [3H]‐RX‐821002 were approximately 60–80% greater than those for [3H]‐clonidine (62.6 fmol mg−1 protein). Rauwolscine, the selective α2‐adrenoceptor antagonist, was approximately 100 fold more potent against each of the ligands than the selective α1adrenoceptor diastereoisomer, corynanthine. Also, the pKi value for the selective α1‐adrenoceptor prazosin against each ligand was less than 6. 3 Adrenaline, UK‐14034, rauwolscine, corynanthine, RX‐811059 and prazosin produced concentration‐dependent inhibition of binding of all three 3H‐ligands. The agonists, adrenaline and UK‐14304, were approximately 5 and 10 fold less potent against [3H]‐idazoxan and [3H]‐RX‐821002, respectively, than against [3H]‐clonidine. In marked contrast, the antagonists, rauwolscine, corynanthine, RX‐811059 and prazosin exhibited a different profile, being approximately 2–3 fold more potent against sites labelled by [3H]‐RX‐821002 and [3H]‐idazoxan compared to sites labelled by [3H]‐clonidine. 4 Agmatine and histamine produced a concentration‐dependent displacement of [3H]‐clonidine, [3H]‐idazoxan and [3H]‐RX‐821002 binding to bovine cerebral cortex membranes. The pKi values for agmatine and histamine were independent of the 3H‐ligand employed, approximately 4.8 and 4.5, respectively. 5 Crude methanolic extracts of bovine brain and lung produced a concentration‐dependent inhibition of [3H]‐clonidine binding to bovine cerebral cortex membranes (>90%). Based on the volume of the extract that caused 50% inhibition of [3H]‐clonidine binding, bovine lung contains 3 fold more CDS than bovine brain. Both extracts were at least 5 fold more potent against α2‐adrenoceptor sites labelled by [3H]‐clonidine than those labelled by [3H]‐idazoxan and [3H]‐RX‐821002. 6 All three 3H‐ligands label the same population of α2‐adrenoceptor binding sites on bovine cerebral cortex membranes, but [3H]‐clonidine appears to label selectively the ‘agonist’ state of the sites: for which known agonists, adrenaline and UK‐14304, exhibit a higher affinity. Our results indicate that neither agmatine nor histamine can account for the CDS activity present in crude extracts of bovine brain and lung. Moreover, these extracts appear to possess a binding profile similar to that of adrenaline and UK‐14304, suggesting that they may possess agonist activity.

Blockade of the Renin-Angiotensin System with Delphinidin, Cyanin, and Quercetin

Overactivation of the renin-angiotensin system is one of the most important risk factors for the development of hypertension. The use of the crude extracts and/or active compounds, such as anthocyanins and quercetin, of herbal plants that have antihypertensive effects is beneficial for decreasing of blood pressure level. However, the molecular mechanisms by which anthocyanins (delphinidin and cyanin) and quercetin regulate the renin-angiotensin system are not completely understood. In this study, we demonstrate that delphinidin, cyanin, and quercetin interrupt the renin-angiotensin system signaling pathway by inhibiting the angiotensin-converting enzyme activity and decreasing its mRNA production. Furthermore, treatment with either delphinidin or cyanin significantly inhibited renin mRNA production. However, delphinidin, cyanin, and quercetin did not act as the angiotensin II type 1 receptor antagonist and did not play roles in the regulation of its internalization. The direct inhibition of components of the renin-angiotensin system advances our understanding of the antihypertensive effects of these compounds.

Roles of cyclic AMP in regulation of phototaxis in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii swims toward or away from light (phototaxis) in a graded way depending on various conditions. Activation of rhodopsin provides signals to control the steering of this unicellular organism relative to a light source and to up-regulate rhodopsin biosynthesis. Intracellular cAMP and cGMP concentrations were measured in positive (1117, swims toward light) and negative (806, swims away from light) phototactic strains with and without light stimulation or 3-isobutyl-1-methylxanthine (IBMX). In the dark, the levels of cAMP and cGMP were significantly higher in the strain with positive phototaxis than in the strain with negative phototaxis. To test whether either cyclic nucleotide influenced the direction, their pre-stimulus levels were pharmacologically manipulated. Higher pre-stimulus levels of cAMP biased the cells to swim toward green light and lower levels biased the cells to swim away. In addition, green-light activation of rhodopsin or addition of IBMX causes a sustained increase in cAMP in both strains. As a consequence of this increase in cAMP, carotenogenesis is induced, as shown by recovery of phototaxis in a carotenoid mutant. Thus, two functions for cAMP were identified: high pre-stimulus level biases swimming toward a light source and sustained elevation following rhodopsin activation increases rhodopsin biosynthesis.

Association of CYP3A4/5 , ABCB1 and ABCC2 polymorphisms and clinical outcomes of Thai breast cancer patients treated with tamoxifen

Background Pharmacogenetic study of cytochrome P450 (CYP) gene CYP2D6 and tamoxifen outcomes remain controversial. Apart from CYP2D6, other drug-metabolizing enzymes and transporters also play a role in tamoxifen metabolic pathways. The aim of this study is to investigate the impact of CYP3A4/5, ABCB1, and ABCC2 polymorphisms on the risk of recurrence in Thai patients who received tamoxifen adjuvant therapy. Methods Patients with early-stage breast cancer who received tamoxifen adjuvant therapy were recruited in this study. All six single-nucleotide polymorphisms (SNPs), including CYP3A4*1B (−392 A>G)/*18(878 T>C), CYP3A5*3(6986 G>A), ABCB1 3435 C>T, ABCC2*1C(−24 C>T), and ABCC2 68231 A>G, were genotyped using real-time polymerase chain reaction assays. The impacts of genetic variants on disease-free survival (DFS) were analyzed using the Kaplan–Meier method and Cox regression analysis. Results The ABCB1 3435 C>T was found to have the highest allele frequency among other variants; however, CYP3A4*1B/*18 could not be found in this study. Patients with heterozygous ABCB1 3435 CT genotype showed significantly shorter DFS than those with homozygous 3435 CC genotype (P = 0.041). In contrast, patients who carried homozygous 3435 TT genotype showed no difference in DFS from wild-type 3435 CC patients. Cox regression analysis showed that the relative risk of recurrence was increased by five times (P = 0.043; hazard ratio = 5.11; 95% confidence interval: 1.05–24.74) in those patients carrying ABCB1 3435 CT genotype compared to those with ABCB1 3435 CC. Conclusion ABCB1 3435 C>T is likely to have a clinically significant impact on recurrence risk in Thai patients with breast cancer who receive tamoxifen adjuvant therapy.

Sustained βAR Stimulation Mediates Cardiac Insulin Resistance in a PKA-Dependent Manner

Insulin resistance is a condition in which cells are defective in response to the actions of insulin in tissue glucose uptake. Overstimulation of β-adrenergic receptors (βARs) leads to the development of heart failure and is associated with the pathogenesis of insulin resistance in the heart. However, the mechanisms by which sustained βAR stimulation affects insulin resistance in the heart are incompletely understood. In this study, we demonstrate that sustained βAR stimulation resulted in the inhibition of insulin-induced glucose uptake, and a reduction of insulin induced glucose transporter (GLUT)4 expression that were mediated by the β2AR subtype in cardiomyocytes and heart tissue. Overstimulation of β2AR inhibited the insulin-induced translocation of GLUT4 to the plasma membrane of cardiomyocytes. Additionally, βAR mediated cardiac insulin resistance by reducing glucose uptake and GLUT4 expression via the cAMP-dependent and protein kinase A-dependent pathways. Treatment with β-blockers, including propranolol and metoprolol antagonized isoproterenol-mediated insulin resistance in the heart. The data in this present study confirm a critical role for protein kinase A in βAR-mediated insulin resistance.

Molecular docking based screening of triterpenoids as potential G-quadruplex stabilizing ligands with anti-cancer activity

Triterpenoids isolated from Ganoderma lucidum (GLTs) exhibit a broad spectrum of anti-cancer properties, including anti-proliferative, anti-metastatic and anti-angiogenic activities. Current research studies revealed the role by GLTs in inducing apoptosis and suppression of telomerase activity of cancer cells with much lower toxicity to healthy cells. Compounds selectively binding and stabilizing G-quadruplex structures could inhibit the telomerase or downregulate the oncogenes and may act as anti-cancer agents. Targeting human telomeric G-quadruplex DNA could be one of the mechanisms by which these GLTs exert anti-cancer activity. In this study, 208 GLTs were screened for ligands with high binding affinity and selectively to stabilize the pG4DNA by using the docking tool AutoDock4. The results showed that ganoderic acid A and ganoderic acid Df exhibit high binding affinity and selectively bind to the lateral groove of pG4DNA. Based on our findings, we suggest that the triterpenoid represents a new class of G-quadruplex groove binding ligands and thus act as potential anti-cancer agents.

Clinically relevant genetic variants of drug-metabolizing enzyme and transporter genes detected in Thai children and adolescents with autism spectrum disorder.

Single-nucleotide polymorphisms (SNPs) among drug-metabolizing enzymes and transporters (DMETs) influence the pharmacokinetic profile of drugs and exhibit intra- and interethnic variations in drug response in terms of efficacy and safety profile. The main objective of this study was to assess the frequency of allelic variants of drug absorption, distribution, metabolism, and elimination-related genes in Thai children and adolescents with autism spectrum disorder. Blood samples were drawn from 119 patients, and DNA was extracted. Genotyping was performed using the DMET Plus microarray platform. The allele frequencies of the DMET markers were generated using the DMET Console software. Thereafter, the genetic variations of significant DMET genes were assessed. The frequencies of SNPs across the genes coding for DMETs were determined. After filtering the SNPs, 489 of the 1,931 SNPs passed quality control. Many clinically relevant SNPs, including CYP2C19*2, CYP2D6*10, CYP3A5*3, and SLCO1B1*5, were found to have frequencies similar to those in the Chinese population. These data are important for further research to investigate the interpatient variability in pharmacokinetics and pharmacodynamics of drugs in clinical practice.

Pharmacogenomic Study Reveals New Variants of Drug Metabolizing Enzyme and Transporter Genes Associated with Steady-State Plasma Concentrations of Risperidone and 9-Hydroxyrisperidone in Thai Autism Spectrum Disorder Patients

The present study sought to investigate the genetic variants in drug metabolizing enzyme and transporter (DMET) genes associated with steady-state plasma concentrations of risperidone among Thai autism spectrum disorder (ASD) patients. ASD patients taking risperidone for at least 1 month were enrolled for this pharmacogenomic study. Genotyping profile was obtained using Affymetrix DMET Plus array interrogating 1931 variants in 231 genes. Steady-state plasma risperidone and 9-hydroxyrisperidone were measured using liquid chromatography/tandem mass spectrometry assay. The final analysis included 483 markers for 167 genes. Six variants, ABCB11 (c.3084A > G, c.∗420A > G, c.∗368G > A, and c.∗236G > A) and ADH7 (c.690G > A and c.-5360G > A), were found to be associated with plasma concentrations of risperidone. 9-Hydroxyrisperidone and the total active-moiety levels were associated with six gene variants, SCLO1B1 (c.-11187G > A and c.521T > C), SLCO1B3 (c.334G > T, c.699A > G, and c.1557G > A), and SLC7A5 c.∗438C > G. Polymorphisms in UGT2B4 c.∗448A > G and CYP2D6 (c.1661G > C, c.4180G > C, and c.-2178G > A) showed considerable but not significant associations with metabolic ratio. This pharmacogenomic study identifies new genetic variants of DMET genes in monitoring risperidone therapy.