Phorbol ester does not mimic,but antagonizes the alpha-adrenoceptor-mediated positive inotropic effect in the rabbit papillary muscle

Summary The phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) was used to examine the hypothesis that phosphoinositide turnover is involved in the regulation of myocardial contractility mediated by stimulation of alpha-adrenoceptors in the mammalian cardiac muscle. Exposure of the isolated rabbit papillary muscle electrically driven at a rate of 1 Hz at a temperature of 37°C to TPA in concentrations of 10–1000 nmol/l for 30 min did not affect the basal force of contraction. The concentration-response curve for the positive inotropic effect of (–)-phenylephrine mediated by stimulation of alpha-adrenoceptors in the presence of (±)-bupranolol (100 nmol/1) was shifted to the right and downward by TPA in concentrations of 30–1000 nmol/l, while the effect of (–)-phenylephrine mediated by stimulation of beta-adrenoceptors in the presence of prazosin (100 nmol/l) was not decreased, but slightly enhanced by exposure of the muscle to relatively low concentrations of TPA (10–100 nmol/l). Incubation of the membrane fraction isolated from the rabbit ventricular muscle with TPA in vitro under the same condition as employed in the physiological experiments decreased the specific binding of [³H]prazosin but not that of [³H]CGP-12177, while the non-tumor promoting phorbol ester, PDD, was ineffective. These results indicate that activation of protein kinase C by TPA does not mimic the positive inotropic effect of catecholamines mediated by activation of myocardial alpha-adrenoceptors. on the other hand, the specific interaction of alpha-adrenoceptor-mediated processes with TPA in the rabbit papillary muscle is in line with the view that the facilitation of phosphoinositide turnover and subsequent activation of protein kinase C may play a certain role in the coupling of alpha-adrenoceptor occupation by agonists to the process leading to the positive inotropic action. Send offprint requests to M. Endoh     

An Accurate Prediction of the pH Change Due to Degradation: Correction for a “Produced” Secondary Buffering System

Esmolol hydrochloride degrades in aqueous solutions by the hydrolysis of a labile aliphatic carboxy-ester group. The products are methanol and ASL-8123. The resulting aliphatic carboxylic acid moiety (ASL-8123) has a pK of 4.80, which is within 1 pH unit of the pH of the formulation. ASL-8123 therefore acts as a secondary buffer and minimizes the change in pH due to degradation. Equations are presented to calculate the change in the pH when the primary degradation product acts as a secondary buffer. This information can be used in the development of a parenteral product to predict, a priori, the concentration of buffer necessary for optimal pH maintenance. This knowledge can reduce the number of formulation screens required to determine the necessary buffer capacity for optimal drug stability.     

Développement d’un hydrogel autodurcissant in vivo, en perspective d’un usage biomédicalA new self-hardening gel in prospect of biomedical use

Since 1995, an injectable bone substitute is developed in our laboratory, it is based on a mix of an hydrogel (hydroxypropyl methylcellulose at 3%: cellulose ether) and biphasic calcium phosphate granules (MBCP^® : 60% hydroxyapatite, 40% β-tri calcium phosphate). This first generation of injectable bone substitute is a non-self-hardening past, nevertheless it owns a great osteoconductor potential. But it shows a tendency to the discharge by its initial composition, and involved limited clinical applications. An evolution of this product is presented in this article: the modification is generated by a new self-hardening hydrogel. This hydrogel is a cellulose ether grafted by silane and diluted in an aqueous solution at basic pH. Also it will be presented the general synthesis of this cellulose derivate, the dissolution condition and the self hardening principle in function of the pH and the temperature. As to conclude by preliminary tests, the biocompatibility in vivo and in vitro of the self-hardening hydrogel mixed with biphasic calcium phosphate granules will be studied.     

Endothelin-1 causes a prolonged protein kinase C activation and acts as a co-mitogen in vascular smooth muscle cells

Endothelin-1 (ET-1) is known to act via G-protein coupled receptors. It has therefore been suggested that any mitogenic activity it may possess, is due to activation of phospholipase C and protein kinase C (PKC). We have therefore examined both the ability of ET-1 to act as a mitogen and its ability to activate PKC. We found that ET-1 significantly increased thymidine incorporation and enhanced platelet-derived growth factor-induced DNA synthesis, as well as causing a prolonged translocation of PKC to the cell membrane. ET-1 significantly increased PKC dependent phosphorylation of two specific substrates. The phosphorylation of MBP_4–14 (from myelin basic protein) was partially dependent on extracellular Ca²⁺, implicating activation of PKC-α, whereas phosphorylation of the so called ε-peptide was Ca²⁺-independent and prolonged. This could be due either to the δ or ζ isoform of PKC, known to be present in these cells. However, ET-1 induced little proliferation or PKC activity in a transformed smooth muscle cell line, DDT₁ MF-2, which lacks expression of the PKC-αisoform, but expresses the ζ-isoform. Thus, it would appear that ET-1-induced mitogenicity in smooth muscle cells may be related to the sustained, Ca²⁺-independent activation of PKC-δ.     

Endothelin synthesis and receptors in human endometrium throughout the normal menstrual cycle

This study was undertaken to investigate the presence of messengerRNA (mRNA) for prepro-endothelin-I (ET-1) and the known receptorsubtypes (ETA and ETB) in human endometrium at different stagesof the menstrual cycle obtained at hysterectomy. Northern blotanalysis revealed expression of ET-1 mRNA in human endometriumduring the normal menstrual cycle. The concentration of ET-1mRNA in endometrial tissue was greater during the menstrualand proliferative phases than during the ovulatory and secretoryphases. Immunoreactive ET-1 was secreted into the medium ofisolated endometrial stromal cells. Oestradiol and progesteronesignificantly attenuated ET-1 release in endometrial stromalcells cultured for 6 days. ETA and ETB mRNA were also presentin endometrial tissue of the normal cycle. The concentrationof ETA receptor mRNA was greater in the proliferative phasethan in the secretory phase, whereas expression of ETB mRNAincreased in menstrual phase. ET-1 significantly increased extracellularaccumulation of cyclic AMP (cAMP), intracellular generationof inositol phosphates and significantly enhanced DNA synthesisin cultured endometrial stromal cells from the proliferativephase. Our results showed that human endometrial cells synthesizedand released ET-1, and contained ETA and ETB receptors whichwere functionally coupled to phosphoinositide breakdown andto adenylate cyclase with the increase of cAMP by ET-1 stimulation.Our findings suggest that ET-1 may have a potential autocrineand/or paracrine function in human endometrial stromal cells.     

Comparative study of pituitary and bacteria-derived human growth hormone by monoclonal antibodies

We have established hybridoma lines which secrete mouse monoclonal antibodies (Mabs) to human pituitary growth hormone, hGH. Using indirect competitive ELISA and indirect passive hemagglutination inhibition twelve different Mabs were characterized with regard to cross-reactivity with the hGH-related hormones, human chorionic somatomammotropin, hCS, and human prolactin, hPRL. The reactivity of these Mabs with pituitary hGH was compared to that with either bacterially-produced methionyl-hGH or to that of reduced and S-carboxymethylated hGH, which has an altered conformation. None of the Mabs reacted with hPRL. Four did not react with hCS whereas the others showed varying degree of cross-reactivity with hCS. All Mabs reacted more weakly with reduced and S-carboxymethylated hGH than with the native form of the hormone, which was not seen with conventional rabbit antisera to hGH. Thus in the case of hGH the Mabs are superior to conventional antisera in revealing small conformational differences. However the pituitary and bacterially-derived methionyl-hGH were indistinguishable as determined by the 12 Mabs.     

Monoclonal antibodies against human granulocytes and myeloid differentiation antigens

Monoclonal antibodies (MCA) were obtained by immunizing BALB/c mice with 99% pure granulocytes from normal donors or with a whole leukocyte suspension obtained from a chronic myelogenous leukemia (CML) patient, and then fusing the mouse spleen cells with a 315–43 myeloma cell clone. Four MCA were selected and studied using ELISA, immunofluorescence, cytotoxicity assays, and FACS analysis. Antibodies 80H.1. 80H.3. and 80H.5 (from normals) and 81H.1 (from CML) detected antigens expressed on neutrophils. Antibodies 80H.1 and 80H.3 (lgG) also reacted with monocytes but not with other blood cell subsets. Antibodies 80H.5 and 81H.1 (lgM) were cytotoxic and reacted strongly with most of the cells of the neutrophil maturation sequence. i.e., myeloblasts, promyelocytes, myelocytes, and mature granulocytes. Antibodies 80H.5 and 81H.1 also inhibited BFU-GM and CFU-E. Antigens recognized by 80H.3. 80H.5, and 81H.1 were expressed both on a proportion of cells from HL.60, KG.1, ML.1, and K562 myeloid cell lines, and on a proportion of blast cells isolated from patients with acute myelogenous leukemia. They were not found on lymphoid cell lines or lymphoid leukemia cells. These MCA recognize either late differentiation antigens expressed on mature neutrophils and monocytes (80H.1 and 80H.3) or early differentiation antigens (80H.5 and 81H.1) specific to the granulocytic lineage. They may be useful for a better definition of those antigens specific to hematopoietic stem cells and their relationship with normal or neoplastic hematopoiesis.     

The nuclei of origin of brain stem enkephalin and substance P projections to the rodent nucleus raphe magnus

The sites of origin of brain stem enkephalin and substance P projections to the rodent nucleus raphe magnus were studied utilizing the combined horseradish peroxidase retrograde transport-peroxidase-antiperoxidase immunohistochemical technique. Several brain stem areas were found to contain both enkephalin- and substance P-like immunoreactive double labeled neurons following injection of horseradish peroxidase into the raphe magnus. Nuclei providing both enkephalin and substance P inputs to the raphe magnus include the nucleus reticularis paragigantocellularis, the nucleus cuneiformis, the nucleus solitarius and the trigeminal subdivision of the lateral reticular nucleus. Enkephalin projections to the raphe magnus were also found to originate from the dorsal parabrachial nucleus, the nucleus reticularis gigantocellularis pars α and from an area which corresponds to the A5 group of Dahlström &; Fuxe. Additional neurons containing substance P-like immunoreactivity and horseradish peroxidase reaction product were identified in the superior central raphe nucleus and the nucleus pontis oralis. The midbrain periaqueductal gray contributes very few enkephalin and substance P fibers to the raphe magnus.The nucleus raphe magnus is a key structure in the intrinsic analgesia system and it has also been implicated in other diverse and non-nonciceptive functions. The present study identifies several brain stem sites which provide enkephalin and substance P input to this raphe nucleus. Several of these nuclei have been implicated in central analgesic mechanisms or in non-nociceptive autonomic functions. The present investigation raises the possibility that these brain stem regions may modulate neuronal activity in the raphe magnus via enkephalin or substance P projections and thus influence the involvement of the raphe magnus in both opiate related mechanisms of pain control and non-nociceptive functions.     

Localization of class i histocompatibility molecule assembly by subfractionation of the early secretory pathway

Class I molecules of the major histocompatibility complex bind peptides derived from cytosolic proteins and display them on the cell surface. This function alerts cytotoxic T cells to the presence of intracellular pathogens. Class I molecule assembly requires the association of the heavy chain with β₂-microglobulin, accompanied by peptide loading via specific transporters. This study localizes where these assembly steps take place, using monoclonal antibodies recognizing class I molecules in different assembly states to analyze subcellular fractions of the early secretory pathway. The distribution of peptide-loaded class I molecules was more localized than the distribution of the total pool of class I molecules in the early secretory pathway. Loaded molecules colocalized with the peptide transporter, free heavy chains, and the chaperone calnexin in high density rough endoplasmic reticulum (RER) membranes. These data suggest that subunit assembly and peptide acquisition occur at the same intracellular site. Class I molecules also localized to less dense subfractions of the early secretory pathway, which contained comparatively less peptide-loaded molecules than the high density RER fractions, at steady state. Following a 15 °C temperature block, class I molecules accumulated in these less dense membrane fractions, indicating that these fractions represent the intermediate compartment where empty class I molecules are trapped in mutant cells. In the presence of cycloheximide, a pool of class I molecules recycling to the RER was detected, suggesting empty molecules recycle to acquire peptide.     

磷酸盐型低密度脂蛋白亲和吸附剂制备与体外吸附试验研究

研究目的是制备低密度脂蛋白亲和吸附剂,用于去除高脂血症患者血液中高含量的低密度脂蛋白。首先以悬浮分散法制备壳聚糖微球和纤维素微球载体,然后固定磷酸吡哆醛和磷酸盐配基,制备出三种吸附剂,并进行体外静态吸附性能测试。结果表明,壳聚糖-磷酸吡哆醛吸附剂对LDL最大吸附量为1.30mg/mL,磷酸盐型壳聚糖和纤维素吸附剂对LDL的最大吸附量分别为2.72mg/mL和3.12mg/mL,并且吸附量随配基含量的增加呈增大趋势,3种吸附剂对高密度脂蛋白均无明显吸附。对吸附剂的灭菌和储存稳定性的研究表明,磷酸盐型纤维素吸附剂具有较好的稳定性。本研究第一次报道磷酸盐型低密度脂蛋白吸附剂。