The Kinetics of Relaxin Oxidation by Hydrogen Peroxide

In this study, hydrogen peroxide was used to study the oxidation of rhRlx under various conditions. Oxidation of rhRlx occurred at both of the two methionines on the B chain, Met B(4) and Met B(25), as expected from the three-dimensional structure of the molecule, which shows that these two residues are located on the surface of the molecule and exposed to solvent. The reaction produced three different oxidized forms of rhRlx containing either Met B(4) sulfoxide, Met B(25) sulfoxide, or both residues oxidized. The corresponding sulfone was not formed under these conditions. The oxidation at the two methionines proceeded independently from each other but Met B(25) was oxidized at a significantly faster rate than Met B(4). The fact that the rate of oxidation at Met B(25) was identical to the rate of oxidation of free methionine and that of two model peptides mimicking the residues around Met B(4) and Met B(25) suggests that the lower reactivity at Met B(4) was due to steric hindrance, and at least in this case, neighboring groups do not influence the oxidation kinetics of methionine residues. The reaction was independent of pH, ionic strength, and buffer concentration in the range studied. The enthalpy of activation for the reaction was approximately 10–14 kcal mol^–1, with an entropy of activation of the order of –30 cal K^–1 mol^–1. These data are consistent with previously published mechanisms for organic sulfide oxidation by alkyl hydroperoxides.     

The Structural and Functional Role of the B‐chain C‐terminal Arginine in the Relaxin‐3 Peptide Antagonist,R3(BΔ23‐27)R/I5

Relaxin‐3, a member of the insulin superfamily, is involved in regulating stress and feeding behavior. It is highly expressed in the brain and is the endogenous ligand for the receptor RXFP3. As relaxin‐3 also interacts with the relaxin receptor RXFP1, selective agonists and antagonists are crucial for studying the physiological function(s) of the relaxin‐3/RXFP3 pair. The analog R3(BΔ23‐27)R/I5, in which a C‐terminally truncated human relaxin‐3 (H3) B‐chain is combined with the INSL5 A‐chain, is a potent selective RXFP3 antagonist and has an Arg residue remaining on the B‐chain C‐terminus as a consequence of the recombinant protein production process. To investigate the role of this residue in the RXFP3 receptor binding and activation, the analogs R3(BΔ23‐27)R/I5 and R3(BΔ23‐27)R containing the B‐chain C‐terminal Arg as well as R3(BΔ23‐27)/I5 and R3(BΔ23‐27), both lacking the Arg, were chemically assembled and their secondary structure and receptor activity assessed. The peptides generally had a similar conformation but those with the extra Arg residue displayed a significantly increased affinity for the RXFP3. Interestingly, in contrast to R3(BΔ23‐27)R and R3(BΔ23‐27)R/I5, the peptide R3(BΔ23‐27) is a weak agonist. This suggests that the C‐terminal Arg, although increasing the affinity, alters the manner in which the peptide binds to the receptor and thereby prevents activation, giving R3(BΔ23‐27)R/I5 its potent antagonistic activity.     

Interspecies Scaling of Clearance and Volume of Distribution Data for Five Therapeutic Proteins

The clearance and volume of distribution of five human proteins (recombinant CD4, CD4 immuno-globulin G, growth hormone, tissue-plasminogen activator, and relaxin) in humans and laboratory animals were analyzed as a function of body weight using allometric scaling techniques. These proteins cover a 16-fold range of molecular weight (6 to 98 kD), are produced by recombinant or synthetic methods, and may be cleared by different mechanisms. The analyses revealed that the clearance and volume data for each protein were satisfactorily described by an allometric equation (Y = a Wb). The allometric exponent (b) for clearance (ml/min) ranged from 0.65 to 0.84, the allometric exponent for the initial volume of distribution (ml) ranged from 0.83 to 1.05, and the allometric exponent for the volume of distribution at steady state (ml) ranged from 0.84 to 1.02. Exponent values from 0.6 to 0.8 for clearance and 0.8 to 1.0 for volumes are frequently cited for small molecules and are expected based on empirical interspecies relationships. When the preclinical data were analyzed separately, the pre-clinical allometric relationships were usually predictive of the human results. These findings indicate that the clearance and volume of distribution of select biomacromolecules follow well-defined, size-related physiologic relationships, and preclinical pharmacokinetic studies provide reasonable estimates of human disposition. Employing this methodology during the early phases of drug development may provide a more rational basis for dose selection in the clinical environment.     

The Pharmacokinetics and Metabolism of Human Relaxins in Rhesus Monkeys

Two forms of chemically synthesized human relaxin (hRlx and hRlx-2) were administered as 88 µg/kg intravenous bolus doses to pregnant and nonpregnant rhesus monkeys. No significant differences in pharmacokinetics were observed between pregnant and nonpregnant animals for either form of relaxin; however, clearance of hRlx (3.1–3.4 ml/min/kg) was significantly slower than clearance of hRlx-2 (6.2–6.5 ml/min/kg) in both pregnant and nonpregnant animals. Although the terminal half-lives for hRlx and hRlx-2 were similar (148–157 min), the initial and steady-state volumes of distribution were somewhat larger for hRlx-2 (71–85 and 398–418 ml/kg, respectively) than for hRlx (61–65 and 294–319 ml/kg, respectively). The metabolism of hRlx-2 was also investigated in pregnant and non-pregnant rhesus monkeys after iv bolus (0.44 mg/kg) or 60-min infusion (1.1 mg/kg) administration. Fast atom bombardment mass spectral analysis of the relaxin immunoreactivity isolated from the plasma indicated that hRlx-2 was partially degraded by removal of amino acids from the C terminus of the B chain. The percentage of intact material declined over a 60-min time course. At 60 min post-dose, intact hRlx-2 was 46–64% of the detected material. Degraded forms representing loss of one and four amino acids (hRlx) from the C terminus of the B chain were 11–13 and 19–34% of the detectable material, respectively.     

子痫前期患者血浆CF6、6-Keto-PGF1α和RLX测定的意义

目的：探讨血浆CF6、6-Keto-PGF1α、RLX浓度的改变在子痫前期发生、发展中的作用及测定的临床意义。方法：血浆CF6、6-Keto-PGF1α、RLX三项血管活性物质均采用放射免疫分析。结果：血浆CF6水平轻度子痫前期组显著高于正常孕妇组（P〈0.05）;重度子痫前期组较正常孕妇组CF6水平则升高更为显著（P〈0.01）。6-Keto-PGF1α水平轻度子痫前期组较正常孕妇组（对照组）略微降低（P〉0.05）,重度子痫前期组含量则降低非常显著（P〈0.01）。RLX水平则呈现轻度子痫前期组和重度子痫前期组均显著地低于对照组（P均〈0.01）。重度子痫前期组血浆CF6与6-Keto-PGF1α水平相关性分析示,两者呈显著负相关（r=-0.058,P〈0.05）;与RLX水平亦呈显著负相关（r=-0.611,P〈0.01）。结论：结果证实,CF6、6-Keto-PGF1α、RLX三项血管活性物质水平的变化有助于了解子痫前期的发生机制及临床的预后评估。     

增生后期及分泌期子宫内膜基质的超微结构

应用 TEM 及 SEM 观察人、猫及大鼠有关周期子宫内膜基质细胞的生长、发育和分化,并从细胞超微结构的动态变化进行分析。发现人子宫内膜在分泌早期,即出现少量前蜕膜细胞和颗粒细胞。至分泌后期,两种细胞数目剧增。颗粒细胞按其形态和结构特点,可分三型。Ⅰ型为圆形颗粒细胞,在人、猫及大鼠子宫内膜均可见到。Ⅱ型为大多边形颗粒细胞,见于人及猫的子宫内膜。Ⅰ型为梭形或细长形细胞,只见于大鼠。此外,并发现全部颗粒细胞和前蜕膜细胞均与胶原蛋白的合成密切相关。并讨论了颗粒细胞的来源、机能以及与前蜕膜细胞之间的相互关系。     

ACTIONS OF SOME AUTACOIDS AND PEPTIDES, INCLUDING RELAXIN, ON COSTO-UTERINE MUSCLE FROM RATS

1. The actions of angiotensin II, bradykinin, oxytocin, arginine vasopressin, relaxin, serotonin and the prostaglandins E2 and F2 alpha were examined on preparations of costo-uterine muscle from stilboestrol-treated rats. 2. All the agonists, except relaxin, when used in concentrations which contract the rat uterus, also produced contractions of costo-uterine muscles. Concentration-response curves were steep and maximal responses to the agonists were comparable. The negative log molar EC50 values were: serotonin, 6.5; angiotensin II, 8.8; bradykinin, 8.4; PGE2, 8.3; PGF2 alpha, 7.1. The EC50 values (units/L) for oxytocin and vasopressin were 4.4 and 2.7 respectively. 3. Indomethacin (2.8 or 5 mumol/L) did not decrease the contractile effects of the peptides or serotonin. The effects of serotonin were reduced, but not reversed, by methysergide (0.94 mumol/L). 4. Porcine relaxin inhibited field stimulation-induced contractions of costo-uterine muscle and uterine horns from immature rats pretreated with oestradiol cypionate and from stilboestrol-treated mature rats. It was much less potent, and its effects were less clearly concentration-related, on costo-uterine muscle. 5. The inhibitory effects of relaxin on the uterus were unaffected by propranolol (1 mumol/L), confirming that on this tissue relaxin acts independently of the release of catecholamines. Progesterone (30 mumol/L) was also without effect on the action of relaxin on the uterus. 6. These results taken together indicate that the costo-uterine muscle of the rat: (i) contracts in response to serotonin and the peptides angiotensin II, arginine vasopressin, bradykinin and oxytocin independently of the release of the contractile prostaglandins F2 alpha and E2; and (ii) in contrast to the uterus, may lack a significant population of receptors for relaxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Effects of Centrally Administered Porcine Relaxin on Drinking Behaviour in Male and Female Rats

Of the reproductive hormones it has been suggested that relaxin may play an important role in the increased sodium appetite of pregnancy. ICV injection of porcine relaxin caused water-replete male and female Wistar rats with access to water and 0.9% or 2.7% NaCl to drink on average about 3 to 8 ml of water within 1 h of injection. By 24 h the cumulative intake of water was no different from the control intake. The amounts of water drunk were similar after doses of 50, 100, 250 and 500 ng of relaxin. A dose of 5 ng was ineffective. Male rats generally drank more water than female rats after ICV injection of angiotensin or relaxin. Male SH rats which drink more water than male WKY rats in response to ICV angiotensin also drank more after ICV relaxin. Intakes of 0.9% or 2.7% NaCl were unaffected for up to 24 h after injection of relaxin, whereas angiotensin-injected rats showed a significant increase in 0.9% NaCl 1 h after injection though this difference was no longer evident in the 24 h cumulative intake. Relaxin did not cause any increase in NaCl intake in SH rats. Insulin, which is similar in structure and molecular weight to relaxin, was without effect on drinking when doses comparable to dipsogenically effective doses of relaxin were injected ICV. In male Wistar rats treated with DOCA for 5–15 days, relaxin retained its weak stimulatory action on water intake but did not affect NaCl intake despite the increased baseline NaCl intake during DOCA. These results indicate that relaxin is a dipsogen in the rat but that it seems to have little short-term effect on sodium appetite.