Characterization of Ileal Vitamin B12 Binding Using Homogeneous Human and Hog Intrinsic Factors

Elucidation of the mechanism of intrinsic factor (IF)-mediated vitamin B(12) (B(12)) binding to ileal binding sites has been hampered by the use of crude or only partially purified preparations of IF in previous studies. We have used homogeneous human IF and hog IF isolated by affinity chromatography to study [(57)Co]B(12) binding to ileal mucosal homogenates. The following observations were made: (a) Human IF-B(12) and hog IF-B(12) were bound to human, monkey, hog, dog, rabbit, mouse, hamster, and guinea pig ileal, but not jejunal, homogenates in amounts significantly greater than free B(12) or B(12) bound to five other homogeneous B(12)-binding proteins; (b) only IF-mediated B(12) binding was localized to ileal homogenates and was inhibited by EDTA; (c) values for the association constant (K(a)) for the various ileal homogenates mentioned above and human IF-B(12) and hog IF-B(12) ranged from 0.3 x 10(9) M(-1) to 13.0 x 10(9) M(-1). Apparent differences in the K(a) for human IF-B(12) and hog IF-B(12) existed in most species; (d) the number of ileal IF-B(12) binding sites per gram (wet weight) of ileal mucosa ranged from 0.3 x 10(12) to 4.9 x 10(12). The same value was always obtained with human IF-B(12) and hog IF-B(12) for any given homogenate preparation; (c) 100-fold excesses of free B(12) or human IF and hog IF devoid of B(12) did not significantly inhibit human IF-B(12) and hog IF-B(12) binding to human and hog ileal homogenates.THESE EXPERIMENTS PERFORMED WITH HOMOGENEOUS IF INDICATE THAT: (a) gastric factors other than IF are not required for B(12) binding to ileal IF-B(12)-binding sites: (b) the mechanism of ileal IF-B(12) binding is different from that of free B(12) or of B(12) bound to non-IF-B(12)-binding proteins; (c) human IF and hog IF have different structures; (d) human IF-B(12) and hog IF-B(12) bind to the same ileal binding sites; and (c) human and hog ileal IF-B(12) binding sites bind free B(12) and human and hog IF devoid of B(12) poorly, if at all.     

In irradiation chimeras, K or D regions of the chimeric host, not of the donor lymphocytes, determine immune responsiveness of antiviral cytotoxic T cells

The H-2 haplotype of the chimeric host determines the responder phenotype of maturing T cells. Spleen cells of chimeric mice formed when (K(k) nonresponder to D(b) × K(b) responder to D(b) plus vaccinia)F(1) bone marrow cells were used to reconstitute K(b)D(b) (C57BL/6 D(b) responder) irradiated recipients generated high levels of D(b) plus vaccinia virus-specific cytotoxic T cells. The same stem cells used to reconstitute K(k)D(b) (B10.A (2R) D(b) nonresponder) irradiated recipients resulted in spleen cells that responded well to K plus vaccinia, but responsiveness to D(b) was low. A generally low response to D(k) plus vaccinia, which seems to be regulated by D(k), was confirmed in chimeras. Thus, K(d)D(d) (D(d) plus vaccinia responder) stem cells differentiating in a K(d)D(k) chimeric host failed to generate a measurable response to D(k) plus vaccinia. In contrast, stem cells from K(d)D(k) (D(k) plus vaccinia low responders) differentiating in a K(d)D(d) (K(d) and D(d) high responders to vaccinia) host do generate responsiveness to D(d) plus vaccinia. These results indicate that in chimeras, the Ir phenotype is independent of the donor T cell’s Ir genotype, and that thymic selection of a T cell’s restriction specificity for a particular H-2 allele of the chimeric host also defines that T cell’s/r phenotype.     

Alpha(4)beta(7)/alpha(4)beta(1) dual integrin antagonists block alpha(4)beta(7)-dependent adhesion under shear flow

The alpha(4) integrin, alpha(4)beta(7), plays an important role in recruiting circulating lymphocytes to the gastrointestinal tract, where its ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is preferentially expressed on high endothelial venules (HEVs). Dual antagonists of alpha(4)beta(1) and alpha(4)beta(7), N-(2,6-dichlorobenzoyl)-(L)-4-(2',6'-bis-methoxyphenyl)phenylalanine (TR14035) and N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-(R)-methylpropyl)-(D)-phenylalanine (compound 1), were tested for their ability to block the binding of alpha(4)beta(7)-expressing cells to soluble ligand in suspension and under in vitro and in vivo shear flow. Compound 1 and TR14035 blocked the binding of human alpha(4)beta(7) to an (125)I-MAdCAM-Ig fusion protein with IC(50) values of 2.93 and 0.75 nM, respectively. Both compounds inhibited binding of soluble ligands to alpha(4)beta(1) or alpha(4)beta(7) on cells of human or rodent origin with similar potency. Under shear flow in vitro, TR14035 and compound 1 blocked binding of human alpha(4)beta(7)-expressing RPMI-8866 cells or murine mesenteric lymph node lymphocytes to MAdCAM-Ig with IC(50) values of 0.1 and 1 microM, respectively. Intravital microscopy was used to quantitate alpha(4)-dependent adhesion of fluorescent murine lymphocytes in Peyer's patch HEVs. When cells were prestimulated with 2 mM Mn(2+) to activate alpha(4)beta(7) binding to ligand, anti-alpha(4) monoclonal antibody (mAb) [10 mg/kg (mpk) i.v.] blocked adhesion by 95%, and anti-beta(1) mAb did not block adhesion, demonstrating that this interaction was dependent on alpha(4)beta(7). TR14035 blocked adhesion to HEVs [ED(50) of 0.01-0.1 mpk i.v.], and compound 1 blocked adhesion by 47% at 10 mpk i.v. Thus, alpha(4)beta(7)/alpha(4)beta(1) antagonists blocked alpha(4)beta(7)-dependent adhesion of lymphocytes to HEVs under both in vitro and in vivo shear flow.     

S18616, a highly potent, spiroimidazoline agonist at alpha(2)-adrenoceptors: I. Receptor profile, antinociceptive and hypothermic actions in comparison with dexmedetomidine and clonidine

S18616 ((S)-spiro[(1-oxa-2-amino-3-azacyclopent-2-ene)-4, 2'-(8'-chloro-1',2',3',4'-tetrahydronaphthalene)]) displayed high affinity at native rat alpha(2)-adrenoceptors (AR)s (pK(i), 9.8), native human (h)alpha(2A)-ARs (9.6), and cloned halpha(2A)- (9.5), halpha(2B)- (9.2), and halpha(2C)- (9.0) ARs. It showed 40-fold lower affinity for halpha(1A)-ARs (8.4) and >/=100-fold lower affinity for rat alpha(1)-ARs (7.1), halpha(1B)-ARs (7.7), halpha(1D)-ARs (7.6), imidazoline(1) (7.4), and imidazoline(2) (7.4) sites and >100-fold lower affinity for all other (>50) sites. At halpha(2A)-ARs, in guanosine-5'-O-(3-[(35)S]thio)triphosphate binding studies, S18616 was a potent (partial) agonist: log effective concentration (pEC(50)), 9.3/maximal effect, 51%. This observation was corroborated employing a halpha(2A)-Gi1alpha fusion protein/GTPase assay (9.0/40%) in which the actions of S18616 were blocked by pertussis toxin. Employing guanosine-5'-O-(3-[(35)S]thio)triphosphate binding assays, S18616 was also a partial agonist at halpha(2C)-ARs (8.2/63%) but a full agonist (8.4/124%) at halpha(2B)-ARs. At halpha(2A)-, halpha(2B)-, and halpha(2C)-ARs, the selective alpha(2)-AR antagonist, atipamezole, abolished the actions of S18616: pK(b) values of 9.1, 9. 1, and 9.4, respectively. As determined by depletion of membrane-bound [(3)H]phosphatidyl inositols, S18616 behaved as a (less potent) agonist (7.8/79%) at halpha(1A)-ARs, an action abolished by prazosin (pK(b), 8.9). Reflecting alpha(2)-AR agonist properties, S18616 potently (>/=1 microg/kg, s.c.) and dose dependently elicited hypothermia and antinociception (nine diverse models) in rodents. These actions were dose dependently inhibited by chemically diverse alpha(2)- versus alpha(1)-AR antagonists, atipamezole, idazoxan, RX821,002, and BRL44418 (a preferential alpha(2A)-AR ligand). In contrast, the actions of S18616 were unaffected by the alpha(1)-AR antagonists, ARC239 and prazosin (which preferentially block alpha(2B/2C)- versus alpha(2A)-ARs). Although the affinity of dexmedetomidine at alpha(2)-ARs was lower than S18616; it displayed a similar receptor and functional profile. Clonidine displayed lower efficacy than S18616, was substantially less potent, and had marked affinity for imidazoline(1) sites and alpha(1)-ARs. In conclusion, S18616 is a novel, selective, and highly potent agonist at alpha(2)-ARs.     

Radioiodination of human intrinsic factor

Human intrinsic factor (IF) saturated with (60)Co-labeled cyanocobalamin ((60)CoB(12)) was purified and then iodinated with (125)I to yield (125)I-labeled IF-(60)CoB(12) preparations of high specific activity. Sephadex G200 and DEAE-cellulose chromatography of the iodinated IF-(60)CoB(12) complex showed coincidence of the major (125)I and the (60)Co radioactivity peaks. During starch-gel electrophoresis (60)Co radioactivity from noniodinated and iodinated complexes migrated to the same extent while (125)I radioactivity from the iodinated complex migrated slightly further anodally than did the (60)Co radioactivity. After the iodinated complex was mixed with antibody to the IF-B(12) complex (antibody II) the (125)I and (60)Co radioactivity were: (a) precipitated in similar amounts by antiglobulin serum. (b) eluted coincidentally in the 19S region on Sephadex G200, and (c) excluded to the same extent from starch gel during electrophoresis. After equilibrium exchange of IF "blocking" antibody (antibody I) for (60)Co-vitamin B(12) on (125)I-labeled IF. (125)I radioactivity from the IF-antibody I complex: (a) was precipitated by antiglobulin serum, (b) was eluated in the 19S region on Sephadex G200 gel filtration, and (c) migrated slowly towards the anode on starch-gel electrophoresis. Urinary excretion of (60)Co radioactivity in pernicious anemia patients after oral administration of (60)Co-vitamin B(12) bound to freshly prepared (125)I-labeled IF was similar to that obtained with noniodinated intrinsic factor.These results show that iodination of IF-(60)CoB(12) complex does not markedly alter the chromatographic, electrophoretic, antigenic, or absorption-promoting properties of IF.     

Muscarinic inhibition as a dominant role in cholinergic regulation of transmission in the caudate nucleus

The effects of cholinergic agonists and antagonists were investigated using slice preparations of the rat caudate nucleus (CN) to elucidate the role of the cholinergic system in the CN. Either carbachol (10(-7) to 10(-5) M) or muscarine (10(-7) to 10(-5) M) dose-dependently inhibited extracellular action potentials orthodromically elicited by local stimulation in the CN. A combination of acetylcholine (10(-6) to 10(-4) M) with physostigmine (10(-6) M) also inhibited the orthodromic response of CN neuron, but nicotine (10(-6) to 10(-3) M) had no effects on the neuronal activity. Atropine (10(-8) to 10(-6) M) antagonized the carbachol-induced inhibition of CN neuron with pA2 of 7.58. Carbachol (10(-5) M) or muscarine (10(-5) M) decreased the amplitude of excitatory postsynaptic potential (EPSP) without altering the resting membrane potential or input impedance of the CN neuron, whereas nicotine (10(-5) M) did not affect either the resting membrane potential or amplitude of EPSP. When carbachol (10(-5) M) was added to the bath, the number of action potentials elicited by applying a depolarizing current into the cell was increased, whereas action potentials transsynaptically elicited by local stimulation were inhibited conversely. The excitatory effects of carbachol on the postsynaptic site of the neuron were also blocked by atropine (3 x 10(-7) M). Carbachol (10(-5) M) did not affect the time courses of the rise and decay phases of EPSP induced by the local stimulation, but did reduce the amplitude of the EPSP.(ABSTRACT TRUNCATED AT 250 WORDS)     

alpha(2C)-Adrenergic receptors mediate spinal analgesia and adrenergic-opioid synergy.

The alpha(2A)-adrenergic receptor (AR) subtype mediates antinociception induced by the alpha(2)AR agonists clonidine, dexmedetomidine, norepinephrine, and 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK-14,304) as well as antinociceptive synergy of UK-14,304 with opioid agonists [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin and deltorphin II. Differential localization of alpha(2)-adrenergic (alpha(2A)-, alpha(2B)-(,) alpha(2C)-) and opioid (mu-, delta-, kappa-) subtypes suggests differential involvement of subtype pairs in opioid-adrenergic analgesic synergy. The present study applies a novel imidazoline(1)/alpha(2)-adrenergic receptor analgesic, moxonidine, to test for involvement of alpha(2B)- and alpha(2C)ARs in antinociception and antinociceptive synergy, because spinal antinociceptive activity of moxonidine shows minimal dependence on alpha(2A)AR. Intrathecal administration of moxonidine produced similar (2-3-fold) decreases in both mutant mice with a functional knockout of alpha(2A)AR (D79N-alpha(2A)AR) and alpha(2C)AR knockout (KO) mice. The potency of moxonidine was not altered in alpha(2B)KO mice, indicating that this subtype does not participate in moxonidine-induced spinal antinociception. Moxonidine-mediated antinociception was dose dependently inhibited by the selective alpha(2)-receptor antagonist SK&F 86466 in both D79N-alpha(2A) mice and alpha(2C)KO mice, indicating that alpha(2)AR activation is required in the absence of either alpha(2A)- or alpha(2C)AR. Spinal administration of antisense oligodeoxynucleotides directed against the alpha(2C)AR decreased both alpha(2C)AR immunoreactivity and the antinociceptive potency of moxonidine. Isobolographic analysis demonstrates that moxonidine-deltorphin antinociceptive synergy is present in the D79N-alpha(2A) mice but not in the alpha(2C)AR-KO mice. These results confirm that the alpha(2C)AR subtype contributes to spinal antinociception and synergy with opioids.     

Estrone Sulfate: Production Rate and Metabolism in Man

Since estrone sulfate (E(1)S) is present at high concentration in plasma, we have examined the parameters of the plasma estrone, estradiol, E(1)S system. The metabolic clearance rate of E(1)S was 157 liter/day (range 70-292) in men and women. Estimated plasma production rates of E(1)S were (mugrams per day): men, 77; women, early follicular phase, 95; women, early luteal phase, 182. The conversion of plasma estrone and estradiol to E(1)S was measured and from these data and the metabolic clearance rates of the estrogens, the transfer factors were rho(E) (1) (E) (1) (S) = 0.54 and rho(E) (2) (E) (1) (S) = 0.65. Using average production rates, all plasma E(1)S could be shown to be derived from plasma estrone and estradiol.The conversion of plasma E(1)S to plasma estrone and estradiol was studied. The calculated transfer factors were: rho(E) (1) (SE) (1) = 0.21, rho(E) (1) (SE) (2) = 0.014. Essentially, similar data were obtained when E(1)S was given by mouth to two subjects.WE CONCLUDE: (a) E(1)S is a major circulating plasma estrogen and has a long plasma half-life; (b) the large contributions of estrone and estradiol to plasma E(1)S are more than sufficient to account for all the circulating plasma E(1)S.     

轻型β-珠蛋白生成障碍性贫血患者β-珠蛋白基因( AC)n(AT)x Ty多态性分析

目的 探讨轻型β-珠蛋白生成障碍性贫血患者β-珠蛋白基因(AC)n(AT)xTy多态性与其基因突变的相关性.方法 选取2009年2月至2010年7月深圳市宝安区人民医院89例已知基因突变类型的轻型β-珠蛋白生成障碍性贫血患者和110名中国汉族人群健康对照者.抽取所有个体外周静脉血,抽提基因组DNA,通过聚合酶链反应扩增β-珠蛋白基因BP1蛋白结合区序列,经DNA测序确定(AC)n(AT)xTy序列的多态性,分析(AC)n(AT)xTy多态性与其基因突变的关系.轻型β-珠蛋白生成障碍性贫血患者和健康对照者间(AC)n(AT)xTy多态性单倍型频率的比较,以及同一单倍型患者的不同突变类型发生率间的比较采用x2检验.结果 在轻型β-珠蛋白生成障碍性贫血患者的β-珠蛋白基因BP1蛋白结合区存在9种(AC)n(AT)xTy多态性序列,分别是(AC)2(AT)7T7、(AC)2 (AT)8T5、(AC)3 (AT)7T5、(AC)2 (AT)9T5、(AC)2 (AT)8T9、(AC)3 (AT)8T5、(AC)2(AT)10T3、(AC)2(AT)7T5和(AC)2(AT) 11T3,其中(AC)2 (AT)7T7和(AC)2(AT)8T5是常见单倍型.轻型β-珠蛋白生成障碍性贫血患者的(AC)2(AT)7T7、(AC)3 (AT)7T5和(AC)2(AT)8T9单倍型频率分别为38.8％( 69/178)、11.8％( 21/178)、9.0％( 16/178),显著高于健康对照组的24.1％(53/220)、5.4％ (12/220)、3.2％ (7/220),差异有统计学意义(x2=9.966、4.371、6.093,P＜0.05);(AC)2 (AT)9T5单倍型频率为10.1％(18/178),显著低于健康对照组的33.2％ (73/220),差异有统计学意义(x2=29.691,P＜0.01);而(AC)2 (AT)8T5单倍型频率在患者组和健康对照组分别为25.3％ (45/178)和29.1％ (64/220),差异无统计学意义(x2 =0.718,P＞0.05).在(AC)2 (AT)7T7单倍型患者中,codon41/42(-TTCT)和IVS-II-654(C→T)的突变率分别为59％( 10/17)和29％(5/17),差异无统计学意义(x2=2.982,P＞0.05);在(AC)2( AT)8T5单倍型患者中,codon41/42(-TTCT)和IVS-Ⅱ-654(C→T)的突变率分别为29％(4/14)和57％ (8/14),差异无统计学意义(x2=2.333,P＞0.05).结论 β-珠蛋白基因BP1蛋白结合区的(AC)2 (AT) 7T7、(AC)3 (AT)7T5和(AC)2(AT)8T9单倍型与轻型β-珠蛋白生成障碍性贫血存在连锁不平衡.在轻型β-珠蛋白生成障碍性贫血患者中,携带( AC)2( AT)7T7单倍型和(AC)2(AT) 8T5单倍型患者的主要致病突变分别为codon41/42 (-TTCT)和IVS-II-654(C→T).     

t(8;21)急性髓系白血病的免疫表型特点

目的研究用流式细胞术免疫表型分析t(8;21)(q22;q22)急性髓系白血病(AML)的免疫表型特点.方法采用常规细胞形态学/细胞化学、流式细胞术间接免疫荧光标记技术和R显带核型分析(MIC)进行分型,部分加用RT-PCR检测AML1/ETO融合基因.结果①294例初治AML患者中,64例t(8;21)AML,占21.8%,在M2中高达54.7%.64例t(8;21)AML中M2占81.3%;②与对照组比较,t(8;21)AML高表达CD19和CD34,低表达CD33(P＜0.001);③以20%阳性细胞作为阳性标准,CD19阳性率在AML为13.6%(294例中40例),t(8;21)AML为50%(64例中32例),显著高于无t(8;21)AML组的3.5%(230例中8例)(P＜0.001);④在t(8;21)AML患者中,CD19+和(或)CD34+58例(90.6%)、CD19-和(或)CD34-6例(9.4%).结论t(8;21)AML特别是M2/t(8;21)高表达CD19与CD34,CD19与t(8;21)密切相关,CD19是预测t(8;21)的指标之一.     

Airway smooth muscle prostaglandin-EP1 receptors directly modulate beta2-adrenergic receptors within a unique heterodimeric complex

Multiple and paradoxical effects of airway smooth muscle (ASM) 7-transmembrane-spanning receptors activated during asthma, or by treatment with bronchodilators such as beta(2)-adrenergic receptor (beta(2)AR) agonists, indicate extensive receptor crosstalk. We examined the signaling of the prostanoid-EP(1) receptor, since its endogenous agonist prostaglandin E(2) is abundant in the airway, but its functional implications are poorly defined. Activation of EP(1) failed to elicit ASM contraction in mouse trachea via this G(alphaq)-coupled receptor. However, EP(1) activation markedly reduced the bronchodilatory function of beta(2)AR agonist, but not forskolin, indicating an early pathway interaction. Activation of EP(1) reduced beta(2)AR-stimulated cAMP in ASM but did not promote or augment beta(2)AR phosphorylation or alter beta(2)AR trafficking. Bioluminescence resonant energy transfer showed EP(1) and beta(2)AR formed heterodimers, which were further modified by EP(1) agonist. In cell membrane [(35)S]GTPgammaS binding studies, the presence of the EP(1) component of the dimer uncoupled beta(2)AR from G(alphas), an effect accentuated by EP(1) agonist activation. Thus alone, EP(1) does not appear to have a significant direct effect on airway tone but acts as a modulator of the beta(2)AR, altering G(alphas) coupling via steric interactions imposed by the EP(1):beta(2)AR heterodimeric signaling complex and ultimately affecting beta(2)AR-mediated bronchial relaxation. This mechanism may contribute to beta-agonist resistance found in asthma.     

Adrenoceptor-mediated mechanical responses of canine tracheal smooth muscle

The effect of tone on responses of canine tracheal smooth muscle (TSM) to norepinephrine (NE) was studied to elucidate the role of sympathetic innervation and adrenoceptors in the control of the airways. Electrical field stimulation produced contraction of TSM in vitro which was augmented by eserine, depressed by phentolamine, potentiated by propranolol in the presence of K+ (14 mM) and almost eliminated by tetrodotoxin or atropine. Resting TSM did not contract in response to NE (10(-8) to 10(-4) M) in the presence or absence of propranolol (10(-5)M). The addition of NE (10(-8) to 10(-6) M) at the plateau of contraction produced by K+ (22.8 mM), histamine (10(-6) M) or acetylcholine (5 X 10(-8) M) produced a further phentolamine-sensitive contraction which was potentiated by beta adrenoceptor blockade with propranolol (10(-5) M). The addition of tyramine (10(-5) to 10(-4) M) at the plateau of contraction produced by K+ (22.8 mM) produced a further contraction which was potentiated by propranolol (10(-5) M) and reduced by phentolamine (10(-5) M). Although the response to NE in the presence of elevated tone was contractile at low concentrations of NE (10(-8) to 10(-6) M), a propranolol-sensitive relaxant response was elicited at higher NE concentrations (10(-5) and 10(-4) M). Maximum contractions to NE in the absence or presence of beta-blockade were dependent on the tone of the muscle. These findings suggest a functional adrenergic innervation of canine TSM and the presence of alpha and beta adrenoceptors which mediate contractile and relaxant responses, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

The bulky N6 substituent of cabergoline is responsible for agonism of this drug at 5-hydroxytryptamine 5-HT2A and 5-HT2B receptors and thus is a determinant of valvular heart disease

Fibrotic valvular heart disease (VHD) has been observed in patients with Parkinson's disease treated with dopamine receptor agonists such as pergolide and cabergoline. 5-Hydroxytryptamine(2B) receptor (5-HT(2B)R) agonism is the most likely cause, but other 5-HT receptors may also play a role in VHD. We aimed at characterizing the molecular fragment of cabergoline responsible for agonism at 5-HT(2B)R and 5-HT(2A)R. Cabergoline with an allyl substituent at N(6) behaved as a potent 5-HT(2B)R full agonist in relaxation of porcine pulmonary arteries and as a weaker 5-HT(2A)R partial agonist in contraction of coronary arteries. The same was true for cabergoline derivatives with cyclopropylmethyl, propyl, or ethyl at N(6). However, agonism was converted into antagonism, when the N(6) substituent was methyl. 6-Methylcabergoline retained agonism compared with cabergoline at human dopamine D(2LONG) and human dopamine D(2SHORT) receptors as determined by guanosine 5'-O-(3-[(35)S]thio)triphosphate binding. In porcine aortic valve cusps, 5-HT-induced contractions were inhibited by ketanserin (5-HT(2A/2C)R antagonist) but not by N-(1-methyl-1H-5-indolyl)-N'-(3-methyl-5-isothiazolyl)urea (SB204741) (5-HT(2B)R antagonist). In porcine valvular interstitial cells, cabergoline-induced activation of extracellular signal-regulated kinase (ERK) 1/2, an initiator of cellular proliferation and activity, was blocked by (R)-(+)-4-(1-hydroxy-1-(2,3-dimethoxyphenyl)methy1)-N-2-(4-fluorophenylethyl)piperidine (MDL100907) (5-HT(2A)R antagonist) and N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)-1,1'-biphenyl-4-carboxamide (GR127935) (5-HT(1B)R antagonist), whereas the stimulatory effect on [(3)H]proline and [(3)H]glucosamine incorporations (indices of extracellular matrix collagen and glycosaminoglycan) was blocked by MDL100907. We conclude that the bulky N(6) substituent of cabergoline is responsible for 5-HT(2A)R and 5-HT(2B)R agonism. The increased ERK1/2 phosphorylation and production of extracellular matrix by cabergoline are mediated by 5-HT(2A)Rs. However, the moderate potency of cabergoline at native 5-HT(2A)Rs suggests that these are not the preferential target in VHD in vivo.     

A novel phenylaminotetralin radioligand reveals a subpopulation of histamine H(1) receptors

Previously, (-)-trans-1-phenyl-3-N,N-dimethylamino-1,2,3,4-tetrahydronaphthalene ([-]-trans-H(2)-PAT) was shown to activate stereospecifically histamine H(1) receptors coupled to modulation of tyrosine hydroxylase activity in guinea pig and rat forebrain in vitro and in vivo. Furthermore, the novel radioligand [(3)H](-)-trans-H(2)-PAT was shown to label selectively H(1) receptors in guinea pig and rat brain with high affinity (K(D), ~0.1 and 0.5 nM, respectively) and a B(max) about 50 and 15%, respectively, of that observed for the H(1) antagonist radioligand [(3)H]mepyramine. In the current study, [(3)H](-)-trans-H(2)-PAT-labeled cloned guinea pig and human H(1) receptors in Chinese hamster ovary (CHO) cell membranes with high affinity (K(D), ~0.08 and 0.23 nM, respectively) and a B(max) about 15% of that observed for [(3)H]mepyramine. The binding of H(2)-PAT to H(1) receptors in both CHO-H(1) cell lines was stereoselective with the (-)-trans-isomer having affinity (K(i), ~1.5 nM) about 4-, 20-, and 50-times higher than the (-)-cis-, (+)-trans-, and (+)-cis-isomers, respectively; the affinity of (-)-trans-H(2)-PAT was unaffected by excess GTP. In functional assays, (-)-trans-H(2)-PAT was a full antagonist of histamine H(1)-mediated stimulation of phospholipase C (PLC) and [(3)H]inositol phosphates (IP) formation in CHO-H(1) cells, a full inverse agonist of constitutively active H(1) receptors in COS-7-H(1) cells, and a full competitive antagonist (pA(2) = 9.2) of histamine H(1)-mediated contraction of guinea pig ileum. It is concluded that (-)-trans-H(2)-PAT is an antagonist at H(1) receptors coupled to PLC/IP formation and smooth muscle contraction. Meanwhile, the observation that [(3)H](-)-trans-H(2)-PAT labels only a subpopulation of H(1) receptors and that (-)-trans-H(2)-PAT activates H(1) receptors coupled to modulation of tyrosine hydroxylase suggests that there may be post-translational H(1) receptor heterogeneity.     

Histamine receptors in isolated guinea pig duodenal muscle: H3 receptors inhibit cholinergic neurotransmission.

A series of histamine H3 receptor agonists and the H3 receptor antagonist thioperamide were tested in the isolated guinea pig duodenum, to investigate the role of this new receptor subtype in the intestinal contractility. At the same time the selectivity of the different compounds for the various histamine receptor subtypes was investigated. In the presence of famotidine (10(-6) M) and thioperamide (10(-5) M), histamine, N alpha-methylhistamine (NMH) and (R)-alpha-methylhistamine (alpha-MH) exerted a concentration-dependent contractile effect through activation of H1 receptors; the ratio of potency was histamine = NMH greater than alpha-MH (this last compound was approximately 500 times less potent). In the presence of pyrilamine (10(-6) M) and thioperamide (10(-5) M), histamine, dimaprit and impromidine caused a slight contractile effect, showing a high degree of tachyphylaxis; this effect was abolished by tetrodotoxin (10(-6) M) and by famotidine (10(-6) M). alpha-MH was ineffective up to 10(-4) M. The H2 receptor agonists dimaprit (10(-4) to 10(-3) M) and impromidine (10(-6) to 10(-5) M) caused a relaxant effect on the contraction elicited by acetylcholine (ACh), BaCl2 and electrical stimulation. This effect, which was unaffected by famotidine, was not mimicked by alpha-MH and not reversed by thioperamide (10(-5) M). In the presence of pyrilamine (109-6) M) and famotidine (10(-6) M), histamine, NMH and alpha-MH inhibited the twitch responses to electrical stimulation, with EC50 values of 1.17 x 10(-7), 6.76 x 10(-8) and 2.45 x 10(-8) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of xylamine on vascular neuroeffector transmission.

The effect of xylamine on sympathetic neuroeffector transmission in rabbit isolated blood vessels was examined. Xylamine (10(-8) to 10(-6) M) caused a slowly progressing inhibition of the contractions of pulmonary artery evoked by electrical field stimulation. The inhibition was irreversible. Cocaine (3 x 10(-5) M) prevented the inhibitory action of xylamine (10(-7) M), but it did not reverse the inhibition caused by xylamine (10(-6) M). Xylamine (10(-7) to 3 x 10(-5) M) reduced the accumulation of [3H]norepinephrine (NE) (10(-8) M). In the presence of desipramine (10(-6) M), xylamine (10(-7) to 3 x 10(-5) M) and corticosterone (10(-6) to 10(-4)) reduced the 3H-accumulation. Xylamine (10(-7) to 10(-5) M) reduced the tissue NE content by up to 50%. Xylamine (10(-7) to 10(-6) M) antagonized in a noncompetitive manner the contractions of aorta evoked by NE (10(-9) to 3 x 10(-5) M), histamine (10(-6) to 3 x 10(-4) M) and 5-hydroxytryptamine (10(-8) to 3 x 10(-4) M). In the case of NE, the antagonism was more marked in the presence of either cocaine (3 x 10(-5) M) plus corticosterone (4 x 10(-5) M) or corticosterone (4 x 10(-5) M). In the presence of xylamine (10(-7) to 10(-6) M), cumulative addition of near maximally effective concentrations of NE, 5-hydroxytryptamine and histamine caused a progressive and complete relaxation of aorta. Xylamine (10(-7) to 10(-5) M) did not alter the contractions of aorta evoked by potassium (17-55 mM). The results support the contention that xylamine is an inhibitor of neuronal and extraneuronal uptake, an adrenergic neurone blocking agent and a noncompetitive antagonist of alpha-1 adrenoceptors, histamine and 5-hydroxytryptamine2 receptors. Xylamine is not a direct acting vasodilator on smooth muscle.     

Heightened alpha1A-adrenergic receptor activity suppresses ischaemia/reperfusion-induced Ins(1,4,5)P3 generation in the mouse heart: a comparison with ischaemic preconditioning

Reperfusion of ischaemic rat or mouse hearts causes NE [noradrenaline ('norepinephrine')] release, stimulation of alpha(1)-ARs (alpha(1)-adrenergic receptors), PLC (phospholipase C) activation, Ins(1,4,5)P(3) generation and the development of arrhythmias. In the present study, we examined the effect of increased alpha(1A)-AR drive on these responses. In hearts from non-transgenic mice (alpha(1A)-WT), Ins(1,4,5)P(3) generation was observed after 2 min of reperfusion following 30 min of zero-flow ischaemia. No Ins(1,4,5)P(3) response was observed in hearts from transgenic mice with 66-fold overexpression of alpha(1A)-AR (alpha(1A)-TG). This was despite the fact that alpha(1A)-TG hearts had 8-10-fold higher PLC responses to NE than alpha(1A)-WT under normoxic conditions. The immediate phospholipid precursor of Ins(1,4,5)P(3), PtdIns(4,5)P(2), responded to ischaemia and reperfusion similarly in alpha(1A)-WT and alpha(1A)-TG mice. Thus the lack of Ins(1,4,5)P(3) generation in alpha(1A)-TG mice is not caused by limited availability of PtdIns(4,5)P(2). Overall, alpha(1)-AR-mediated PLC activity was markedly enhanced in alpha(1A)-WT mice under reperfusion conditions, but responses in alpha(1A)-TG mice were not significantly different in normoxia and post-ischaemic reperfusion. Ischaemic preconditioning prevented Ins(1,4,5)P(3) generation after 30 min of ischaemic insult in alpha(1A)-WT mice. However, the precursor lipid PtdIns(4,5)P(2) was also reduced by preconditioning, whereas heightened alpha(1A)-AR activity did not influence PtdIns(4,5)P(2) responses in reperfusion. Thus preconditioning and alpha(1A)-AR overexpression have different effects on early signalling responses, even though both prevented Ins(1,4,5)P(3) generation. These studies demonstrate a selective inhibitory action of heightened alpha(1A)-AR activity on immediate post-receptor signalling responses in early post-ischaemic reperfusion.     

Cathepsin B-sensitive polymers for compartment-specific degradation and nucleic acid release

Degradable cationic polymers are desirable for in vivo nucleic acid delivery because they offer significantly decreased toxicity over non-degradable counterparts. Peptide linkers provide chemical stability and high specificity for particular endopeptidases but have not been extensively studied for nucleic acid delivery applications. In this work, enzymatically degradable peptide-HPMA copolymers were synthesized by RAFT polymerization of HPMA with methacrylamido-terminated peptide macromonomers, resulting in polymers with low polydispersity and near quantitative incorporation of peptides. Three peptide-HPMA copolymers were evaluated: (i) pHCathK(10), containing peptides composed of the linker phe-lys-phe-leu (FKFL), a substrate of the endosomal/lysosomal endopeptidase cathepsin B, connected to oligo-(L)-lysine for nucleic acid binding, (ii) pHCath(D)K(10), containing the FKFL linker with oligo-(D)-lysine, and (iii) pH(D)Cath(D)K(10), containing all (D) amino acids. Cathepsin B degraded copolymers pHCathK(10) and pHCath(D)K(10) within 1 h while no degradation of pH(D)Cath(D)K(10) was observed. Polyplexes formed with pHCathK(10) copolymers show DNA release by 4 h of treatment with cathepsin B; comparatively, polyplexes formed with pHCath(D)K(10) and pH(D)Cath(D)K(10) show no DNA release within 8 h. Transfection efficiency in HeLa and NIH/3T3 cells were comparable between the copolymers but pHCathK(10) was less toxic. This work demonstrates the successful application of peptide linkers for degradable cationic polymers and DNA release.     

Inhibition of Dihydropteroate Synthetase from Escherichia coli by Sulfones and Sulfonamides

The inhibitory action of various diphenylsulfones and sulfonamides on dihydropteroate synthetase partially purified from Escherichia coli was examined. 4,4'-Diaminodiphenylsulfone (DDS; I(50) = 2 x 10(-5) M) and the monosubstituted derivatives 4-amino-4'-formamidodiphenylsulfone (I(50) = 5.8 x 10(-5) M) and 4-amino-4'-acetamidodiphenylsulfone (I(50) = 5.2 x 10(-5) M) were effective inhibitors of dihydropteroate synthetase activity. Disubstitution of the arylamine groups of DDS (4,4'-diformamidodiphenylsulfone and 4,4'-diacetamidodiphenylsulfone) resulted in complete loss of inhibitory activity. Both DDS (K(I) = 5.9 x 10(-6) M) and sulfadiazine (K(I) = 2.5 x 10(-6) M) were found to be competitive inhibitors of dihydropteroate synthetase. These findings are discussed in regard to the Bell and Roblin theory of structure-activity relationships for p-aminobenzoic acid antagonists.     

Differential actions of antiparkinson agents at multiple classes of monoaminergic receptor. II. Agonist and antagonist properties at subtypes of dopamine D(2)-like receptor and alpha(1)/alpha(2)-adrenoceptor

The accompanying multivariate analysis of the binding profiles of antiparkinson agents revealed contrasting patterns of affinities at diverse classes of monoaminergic receptor. Herein, we characterized efficacies at human (h)D(2SHORT(S)), hD(2LONG(L)), hD(3), and hD(4.4) receptors and at halpha(2A)-, halpha(2B)-, halpha(2C)-, and halpha(1A)-adrenoceptors (ARs). As determined by guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding, no ligand displayed "full" efficacy relative to dopamine (100%) at all "D(2)-like" sites. However, at hD(2S) receptors quinpirole, pramipexole, ropinirole, quinerolane, pergolide, and cabergoline were as efficacious as dopamine (E(max)100%); TL99, talipexole, and apomorphine were highly efficacious (79-92%); piribedil, lisuride, bromocriptine, and terguride showed intermediate efficacy (40-55%); and roxindole displayed low efficacy (11%). For all drugs, efficacies were lower at hD(2L) receptors, with terguride and roxindole acting as antagonists. At hD(3) receptors, efficacies ranged from 33% (roxindole) to 94% (TL99), whereas, for hD(4) receptors, highest efficacies (approximately 70%) were seen for quinerolane, quinpirole, and TL99, whereas piribedil and terguride behaved as antagonists and bromocriptine was inactive. Although efficacies at hD(2S) versus hD(2L) sites were highly correlated (r = 0.79), they correlated only modestly to hD(3)/hD(4) sites (r = 0.44-0.59). In [(35)S]GTPgammaS studies of halpha(2A)-ARs, TL99 (108%), pramipexole (52%), talipexole (51%), pergolide (31%), apomorphine (16%), and quinerolane (11%) were agonists and ropinirole and roxindole were inactive, whereas piribedil and other agents were antagonists. Similar findings were obtained at halpha(2B)- and halpha(2C)-ARs. Using [(3)H]phosphatidylinositol depletion, roxindole, bromocriptine, lisuride, and terguride displayed potent antagonist properties at halpha(1A)-ARs. In conclusion, antiparkinson agents display diverse agonist and antagonist properties at multiple subtypes of D(2)-like receptor and alpha(1)/alpha(2)-AR, actions, which likely contribute to their contrasting functional profiles.