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We previously reported that sarpogrelate, a selective 5-HT2A antagonist, showed a potent inverse agonist activity to constitutively active mutant (C322K) of human 5-HT2A receptor (5-HT2AR). However, it remains to be unknown about the actual mechanism of this mutant for its constitutive activation as well as inverse agonist activity of sarpogrelate. Our model shows that mutation (C322K) of 5-HT2AR causes electronic repulsion between positively charged Arg173(3.50) and Lys322(6.34) residues resulting outward movement of the C-terminus of transmembrane helix (TMH) III. This motion of TMH III leads to a partially active structure of the receptor, which may be a key step in receptor activation. The structural model of the partially active receptor also indicates that the binding of sarpogrelate to the constitutively active receptor causes an inward swing of TMH III to an inactive receptor structure. Therefore, the present study may suggest that the electronic repulsion causing outward movement of the C-terminus of TMH III may be the key step for constitutive activation of mutant C322K of 5-HT2AR and the inward movement of TMH III causes the inverse agonist activity of sarpogrelate.

We obtained a new mutant of the β1-adrenergic receptor (β1-AR) by point mutations that can constitutively activate β1-AR. Aspartate104 of the β1-AR in the 2nd transmembrane was replaced with alanine. The β1-AR mutant expressed in human embryonic kidney (HEK)-293 cells displayed high level of constitutive activity with respect to wild-type (P

The purpose of the present study was to examine 5-hydroxytryptamine (5-HT)2A-receptor sarpogrelate interactions by site-directed mutagenesis. Based on molecular modeling studies, aspartic acid (Asp)155[3.32] and tryptophan (Trp)151[3.28] in transmembrane helix (TMH) III and Trp336[6.48] in TMH VI of the 5-HT2A receptor were found to interact with sarpogrelate. All of these residues were mutated to alanine (Ala). The Asp3.32Ala mutant did not exhibit any affinity for [3H]ketanserin, whereas the Trp3.28Ala mutant showed a markedly decrease in the binding affinity for [3H]ketanserin (Kd >10,000 nM). Therefore, it was not possible to find any sarpogrelate affinity to the mutants using [3H]ketanserin. The mutation also abolished agonist-stimulated formation of [3H]inositol phosphates (IP) in both cases. On the other hand, the Trp6.48Ala mutant showed reduced binding affinity for [3H]ketanserin (Kd 2.0 nM vs 0.8 nM for the wild-type receptor) and had reduced affinity for sarpogrelate (pKi value of 5.71 vs 9.06 for the wild-type receptor). The Trp6.48Ala mutant also showed the greatest decrease in sensitivity to sarpogrelate (pKb value 8.81 for wild-type and 5.11 for mutant) in inhibiting agonist-stimulated IP formation. These results provide direct evidence that Asp3.32, Trp3.28, and less importantly, Trp6.48 are responsible for the interaction between the 5-HT2A receptor and sarpogrelate. In addition, these results support the findings obtained from molecular modeling studies. Keywords:: 5-hydroxytryptamine (5-HT), 5-HT2A receptor, sarpogrelate, site-directed mutagenesis, molecular modeling

Mutations producing constitutively active G-protein coupled receptors have been found in the pathophysiology of several diseases, implying that inverse agonists at the constitutively active receptors may have preferred therapeutic applications. Because of the involvement of 5-HT2A receptors in mediating many cardiovascular diseases, constitutively active mutants of the 5-HT2A receptor may be responsible for the disease states. Thus, the purpose of the present study was to investigate the inverse agonist activity of sarpogrelate, a selective 5-HT2A-receptor antagonist, and its active metabolite, M-1; and we compared their activities with those of other 5-HT2A-receptor antagonists such as ritanserin, ketanserin, and cyproheptadine. Using a constitutively active mutant (C322K) of the human 5-HT2A receptor, we demonstrated that like other 5-HT2A-receptor antagonists, sarpogrelate acts as a potent inverse agonist by significantly reducing basal inositol phosphate levels. However, there were no significant differences between sarpogrelate and other 5-HT2A-receptor antagonists for their inverse agonist activity. Compared with the wild type receptor, mutant receptor displayed significantly higher affinity for 5-HT and lower affinity for sarpogrelate. These results indicate that stabilization of the inactive conformation of the 5-HT2A receptor may be a key component of the mechanism of action of sarpogrelate. Keywords:: 5-HT2A receptor, sarpogrelate, mutant, constitutive activity, inverse agonist

The serotonin (5-hydroxytryptamine, 5-HT) receptors have conventionally been divided into seven subfamilies, most of which have several subtypes. Among them, 5-HT(2A) receptor is associated with the contraction of vascular smooth muscle, platelet aggregation and thrombus formation and coronary artery spasms. Accordingly, selective 5-HT(2A) antagonists may have potential in the treatment of cardiovascular diseases. Sarpogrelate, a selective 5-HT(2A) antagonist, has been introduced clinically as a therapeutic agent for the treatment of ischemic diseases associated with thrombosis. Molecular modeling studies also suggest that sarpogrelate is a 5-HT(2A) selective antagonist and is likely to have pharmacological effects beneficial in the treatment of cardiovascular diseases. This review describes the above findings as well as the signaling linkages of the 5-HT(2A) receptors and the mode of agonist binding to 5-HT(2A) receptor using data derived from molecular modeling and site-directed mutagenesis.

The aim of the present study was to investigate the binding affinities and dissociation potencies of several 5-HT(2) antagonists in M(2) muscarinic receptor of rat heart membranes using [(3)H]QNB as a radioligand. The 5-HT(2) antagonists used in this study were sarpogrelate, ketanserin and cyproheptadine. The results showed that sarpogrelate and ketanserin had very weak binding affinities to M(2) muscarinic receptor, whereas cyproheptadine had higher binding affinity to this receptor than the muscarinic receptor antagonist, atropine. All of these three 5-HT(2) antagonists as well as muscarinic receptor antagonists (atropine and pirenzepine) were readily dissociated from M(2) muscarinic receptor in rat heart membranes after washing. Therefore, the findings of the present investigation suggest that the dissociation potencies of neither 5-HT(2) antagonists nor muscarinic antagonists used correlate with their binding affinities to M(2) muscarinic receptors in rat heart.

We previously reported that sarpogrelate, a selective 5-HT2A antagonist, showed a potent inverse agonist activity to constitutively active mutant (C322K) of human 5-HT2A receptor (5-HT2AR). However, it remains to be unknown about the actual mechanism of this mutant for its constitutive activation as well as inverse agonist activity of sarpogrelate. Our model shows that mutation (C322K) of 5-HT2AR causes electronic repulsion between positively charged Arg173(3.50) and Lys322(6.34) residues resulting outward movement of the C-terminus of transmembrane helix (TMH) III. This motion of TMH III leads to a partially active structure of the receptor, which may be a key step in receptor activation. The structural model of the partially active receptor also indicates that the binding of sarpogrelate to the constitutively active receptor causes an inward swing of TMH III to an inactive receptor structure. Therefore, the present study may suggest that the electronic repulsion causing outward movement of the C-terminus of TMH III may be the key step for constitutive activation of mutant C322K of 5-HT2AR and the inward movement of TMH III causes the inverse agonist activity of sarpogrelate.

Reactive oxygen species (ROS) play an important role in normal metabolic and signaling processes. Excess ROS, however, can cause severe cardiovascular damage. Thus, the present study was designed to examine effects of H(2)O(2) and xanthine plus xanthin oxidase (X/XO) on the serotonin (5HT), histamine (His) and acetylcholine (ACh)-induced contractions of porcine coronary arteries. In addition, to explore the site of ROS formation and species of it, the inhibitory effects of edaravone and EDTA were also tested. 5HT- and His-induced contractions were suppressed by H(2)O(2) and X/XO treatment. However, these suppressions of ACh-induced contraction by H(2)O(2) treatment was relatively weak and X/XO treatment caused no suppression on ACh-induced contraction. In the presence of edaravone which is thought to be a scavenger for .OH, significant decrease of inhibition of 5HT- and His-induced contractions was observed when coronary artery strips were treated with X/XO, but not H(2)O(2). On the other hand, inhibitory effects by EDTA treatments were also observed in X/XO treatments. These results suggest that 1) ROSs produced by additions of H(2)O(2) or X/XO are considered to be responsible for several physiological functions of coronary artery contractions, 2) the site of ROS produced by X/XO system, probably .OH, was outside the cell, but the inhibitory action of H(2)O(2), was inside the cell, and 3) a low susceptibility of ACh-induced contraction to H(2)O(2) and X/XO may indicates the signal transduction pathway(s) of ACh-induced contraction is different from those of 5HT and His.

Site-directed mutagenesis was used to investigate the molecular interactions involved in sarpogrelate binding to the human 5-Hydroxytryptamine(5-HT)2C receptor. Based on molecular modeling studies, Aspartic acid (Asp)155[3.32] in transmembrane region III and Serine(Ser)361[7.46] in transmembrane region VII of the 5-HT2C receptor were found to interact with sarpogrelate. Asp3.32 and Ser7.46 were mutated to alanine (Ala) and expressed in COS-7 cells. The radioligand [3H]mesulergine did not show any binding to Asp3.32Ala mutant of 5-HT2C receptor. Therefore, it was not possible to find any sarpogrelate affinity to the mutant using [3H]mesulergine. The mutation also abolished agonist-stimulated IP formation of [3H]myo-inositol. Introduction of dual mutation at position Ser7.46 (Asp3.32Ala-Ser7.46Ala) could not restore the function disrupted by the first mutation (Asp3.32Ala). On the other hand, the Ser7.46Ala mutant showed reduced binding affinity for [3H]mesulergine (Kd 3557 pM versus 573 pM for the wild-type receptor) and had reduced affinity for sarpogrelate. Moreover, the Ser7.46Ala mutant receptor also showed a great loss of potency for sarpogrelate in inhibiting 5-HT-stimulated IP formation of [3H]myo-inositol. The results provide direct evidence that Asp3.32 and less importantly, Ser7.46 are responsible for the interaction between 5-HT2C receptor and [3H]mesulergine as well as sarpogrelate. More interestingly, Ser7.46Ala increases the receptor expression (20-fold vs. wild-type) of the mutant receptors and basal [3H]myo-inositol formation (2.5-fold vs. wild-type), which indicates that the 5-HT2C receptor could be rendered constitutively active by mutating the amino acid serine at position 7.46 to alanine.

We hypothesize that the normal immunologic responses by the maternal immune system during pregnancy are not as well-regulated in gestational diabetes (GD) patients as in healthy pregnant women.Using two-color flow cytometry we evaluated frequencies of peripheral blood lymphocytes in 20 GD patients being treated with insulin; 43 GD patients treated with dietary therapy but no insulin; 44 women experiencing normal pregnancies; and 48 non-pregnant women.When compared with healthy pregnant women, both GD cohorts showed higher percentages CD4(+)CD25(+) (P0.05), CD4(+)CD45RO(+) (P0.05) and CD4(+)CD29(+) (P0.01) but lower percentages of CD4(+)CD45RA(+) (P0.05). Higher percentages of the activated phenotypes CD8(+)CD25(+) and CD8(+)HLA-DR(+) cells in the diet-treated cohort and CD4(+)HLA-DR(+) cells in insulin-treated GB cohort, were observed compared with healthy pregnant subjects (P0.05).Expanded populations of activated peripheral blood T cells are associated with GD, suggesting that normal maternal immunosuppression is less effective in GD-afflicted women.

Toxic anticholinesterases (AC) are known contributors to negative pregnancy outcome. Impairment of detoxification mechanisms may correlate with occurrence of pregnancy disorders in Kuwait.Butyrylcholinesterase (BuChE), an enzyme which detoxifies AC was evaluated in 18 Kuwaiti women with pregnancy-induced hypertension (PIH), compared with 15 healthy pregnant and eight healthy non-pregnant women. T-lymphocyte subpopulations were measured by flow cytometry, and BuChE activity was measured by spectrophotometry.Unlike the PIH group, the normal pregnancy group exhibited a significant increase in BuChE activity compared with non-pregnant control subjects (P = 0.04). Within the PIH cohort, inverse correlations were observed between BuChE activity and percentage of CD4+ CD25+ cells (P = 0.001), and CD8+ CD25+ cells (P = 0.007).Elevated BuChE activity in normal pregnancy may correlate with better ability to clear pregnancy-threatening toxins, while lesser ability to do this in PIH women may be a contributor to disease. The fact that PIH subjects with large subpopulations of activated T cells also exhibited low BuChE activity further suggests a correlation between susceptibility to pregnancy loss and decreased activity of the enzyme.