Electrophysiological analysis of the action of kavinton on the smooth muscles

Cavinton at a concentration of 10(-7)-10(-5) M was found to have a dose-dependent relaxing effect on bovine cerebral artery smooth muscles, without changing the resting potential and membrane resistance. Smooth muscles of the rabbit portal vein and guinea-pig taenia coli were insensitive to low cavinton concentrations. The results are consistent with the hypothesis that relaxing action of cavinton is due to the blocking of Ca2+ ions influx into the cells of cerebral artery through receptor-operated calcium channels. At higher concentrations (exceeding 10(-5) M) cavinton exerts nonspecific influence on the smooth muscles under study, inhibiting their excitability and decreasing membrane resistance resulting in the attenuation of tetanic contractions in the smooth muscles of the portal vein and taenia coli.     

Effect of calcium entry blockers and adenosine on the relaxation of large and small coronary arteries

The mechanism(s) by which adenosine causes dilation of the vascular smooth muscle is not properly understood. Several mechanisms including the inhibition of calcium influx and intracellular translocation have been suggested for its action. This study is an attempt to further elucidate the site of action of adenosine in relation to calcium by making use of calcium entry blockers. Large (1 +/- 0.2 mm, o.d.) and small (0.5 +/- 0.2 mm, o.d.) branches of bovine left anterior descending coronary artery (LADCA) contracted with 50 mM K+ were used as a model for these studies. Concentration-response curves for various calcium entry blockers were obtained and the order of potency was found to be: D-600 greater than nifedipine greater than verapamil greater than diltiazem greater than lidoflazine for large branches and nifedipine greater than D-600 greater than verapamil greater than lidoflazine greater than diltiazem for small branches of LADCA. The concentration-response relationship for adenosine (10(-6)-10(-4) M) in the presence and absence of these drugs (10(-9)-10(-7) M) was unchanged. 8-phenyltheophylline (2 X 10(-5) M), an adenosine receptor antagonist was without an effect on the relaxations induced by various calcium entry blockers, however, it antagonized the relaxing response to adenosine. Lidoflazine at concentrations of 7 X 10(-7) M and 2 X 10(-7) M potentiated the effect of adenosine in relaxing the large and small LADCA, respectively. In summary, the data show an increased sensitivity of small coronary vessels to nifedipine, D-600 and lidoflazine. The data further suggest a different site of action for adenosine and calcium entry blockers.     

Comparative effects of estradiol-17beta and estriol on uterine RNA polymerases I, II, and III in vivo.

The early and later effects of estradiol-17β and estriol on the RNA polymerase activities of uterine nuclei obtained from ovariectomized rats were compared. At 4 hr of hormone action both estradiol-17β and estriol stimulated the activity of polymerase I, but not the activities of polymerases II and III. At 24 hr, however, the effect of estriol had disappeared, whereas estradiol-17β stimulated all three polymerase activities. These results indicate that estrogen-induced growth of the uterus occurs in two phases, initiation and maintenance. Estriol initiates uterine growth, but does not maintain the process. Estradiol-17β, in contrast, does both. The differences in the effects of the two estrogens may reside in their different binding affinities.     

Effect of ethanol on thromboxane and prostacyclin synthesis by fetal platelets and umbilical artery

The effect of ethanol (10-500 mmol/l) on platelet thromboxane production and on vascular thromboxane and prostacyclin was studied in human fetal tissues. The release of thromboxane B2 (a metabolite of thromboxane A2) during thrombin-induced spontaneous aggregation of fetal platelets was inhibited by ethanol concentrations of 50 mmol/l or higher. Ethanol at concentration from 100 mmol/l also inhibited umbilical artery production of thromboxane B2 and that of 6-keto-prostaglandin F1 alpha (a metabolite of prostacyclin). However, it stimulated the conversion of exogenous arachidonic acid to thromboxane B2 in fetal platelets and to 6-keto-prostaglandin F1 alpha in the umbilical artery. This suggests that ethanol inhibits phospholipase A2, but stimulates the enzymes distal from phospholipase A2 in the prostaglandin-synthesizing enzyme cascade.     

Influence of 5alpha- and 5beta-reduced progestins on the contractility of isolated human myometrium at term.

It is very well known that progesterone induces uterine relaxation on myometrium contractile activity. However, little attention has been paid to the effect induced by its metabolites on human uterine contractility. Therefore, we set out to analyze the potential relaxing effect of some 5alpha- and 5beta-reduced progesterone derivatives on the spontaneous contractility of myometrium from pregnant women. Samples were obtained by caesarian section at 38-40 weeks of pregnancy. Spontaneous uterine contractions were recorded in vitro in the presence of progesterone, or progestins independently, at different non-cumulative microM concentrations. The progestins elicited an immediate relaxing effect that was concentration-dependent. With the exception of two 5alpha-reduced progestins (5alpha and 3beta,5alpha), the remaining progestins used in the present study were more potent than progesterone. The potency order with respect to their IC50 values was: 3alpha,5alpha (35 microM) > 5beta (81 microM) > 3beta,5beta (156 microM) > 3alpha,5beta (205 microM) > P4 (225 microM) > 5alpha (19 mM) > 3beta,5alpha (28 mM). When tissues were washed, the contractile activity was recovered. This rapid and reversible relaxing effect was not blocking by antiprogestin RU 486, suggesting that is not through receptor-mediated genomic action. The metabolites from progesterone may also determine the pattern of motility, ensuring the necessary quiescent environment to prevent abortion during gestation.     

The effect of ionophore A23187 on albumin internalization in cultured human umbilical vein endothelial cells

The effects of calcium and the calcium ionophore A23187 on endocytosis were studied in cultured human umbilical vein endothelial cells using iodinated human albumin to measure bulk phase endocytosis. In the absence of the ionophore, varying the levels of extracellular calcium did not affect endocytosis. In the presence of 10 μM A23187, the endocytic clearance of albumin decreased approx. 50% when exposed to physiological concentrations of extracellular calcium, but increased approx. 50% at lower calcium concentrations. Since the ionophore is known to alter cellular calcium levels, these results are compatible with a role for intracellular calcium in the modulation of endothelial cell endocytosis.     

The effect of prostacyclin on the human umbilical artery.

Prostacyclin was tested on human umbilical artery obtained after spontaneous delivery or by Cesarean section. Isometric and isotonic responses were measured on spiral preparations in Krebs-bicarbonate buffer at 37 degrees C equilibrated with 95% O2 and 5% CO2. Spiral artery strips, whether superfused or mounted in organ baths isometrically or isotonically, responded in a dose-dependent manner to both prostacyclin and serotonin; the PGI2 response was biphasic in that low doses (2.5 x 10(-8) M -1.0 x 10(-6) M) elicited a dose-dependent relaxation which changed with higher concentrations (1.0 x 10(-6) M -2.53 X 10(-5) M) to a contractile response. The maximum tension exerted was 50% less than that elicited by serotonin. The data indicate that the human umbilical artery is responsive to prostacyclin and may be involved in the regulation of fetal placenta blood flow.     

Prostaglandin release from the human umbilical artery in vitro

Release of prostaglandins from human umbilical artery preparations into the surrounding bathing fluid was studied by radioimmunoassay using PGF_2α antibodies. A significant release of prostaglandins was found under conditions where a spontaneous tone of the artery could be maintained. Indometacin reduced the prostaglandin release and the spontaneous tone of the artery. Intramural synthesis of prostaglandins in the human umbilical arteries is postulated.     

Influence of nitric oxide on agonist-mediated calcium mobilization in platelets

Recent studies have characterized endothelium-derived relaxing factor as nitric oxide. It appears to exert its effect by elevating intracellular levels of cyclic GMP. In this study we confirm that nitric oxide is a potent inhibitor of agonist-induced irreversible aggregation. At the concentrations tested nitric oxide effectively blocked thrombin-stimulated mobilization of cytosolic-free calcium in Fura 2-loaded platelets. In addition, nitric oxide prevented the inositol 1,4,5-trisphosphate-stimulated calcium rise in cytosolic calcium in saponin-permeabilized Fura 2-loaded platelets. Similar to the action of adenylate cyclase stimulators, nitric oxide facilitated lowering of calcium levels raised by the action of agonists. The specific mechanism by which it exerts its effect on intracellular levels of calcium is not clear.     

On the role of extracellular Ca2+ for prolactin release and adenosine 3′:5′-monophosphate formation induced by thyroliberin in cultured rat pituitary cells

In cultured rat pituitary tumour cells (GH₃ cells) the absence of extracellular Ca⁺⁺ or addition of NaEGTA reduced spontaneous prolactin (PRL) release and abolished the stimulatory effect of thyroliberin (TRH). Readdition of CaCl₂, but not of equimolar concentrations of MgCl₂ increased spontaneous hormone release, and restored the effect of TRH. The calcium ionophore, A-23187, induced PRL release during normal calcium conditions, but not when an excess NaEGTA was present. TRH increased cyclic AMP accumulation in the presence and the absence of extracellular calcium. The effect of TRH on PRL release and cyclic AMP formation occured concomitantly with an increased efflux of ⁴⁵Ca²⁺. Intracellular electrophysiological recordings from the same single cells before and after TRH activation showed increased frequency and duration of the Ca²⁺ dependent action potentials. We conclude that TRH elevates the Ca²⁺ influx which depends on the depolarizing action current, and this effect is probably linked to formation of cyclic AMP and PRL release.     

Regulation of acrosomal matrix dispersion in digitonin-permeabilized guinea pig spermatozoa

Digitonin-permeabilized guinea pig spermatozoa undergo acrosomal matrix dispersion in response to 2.0 mM CaCl2. In this report, the effects of pH and metal ions on matrix dispersion in permeabilized spermatozoa are examined. Calcium-induced dispersion of the acrosomal matrix was dependent on the calcium concentration; the response was not observed at concentrations of CaCl2 less than 50 microM. Magnesium could not substitute for calcium and, in fact, had a retarding effect on the calcium-induced response. Matrix dispersion was also found to be pH-dependent. The induction of matrix dispersion was inhibited at pH 5.6 and pH 9.5 relative to the responses observed at pH 6.3 and pH 7.8. Nigericin induced acrosomal matrix dispersion in the absence of added calcium, indicating a possible role of Na+/H+ exchange across the outer acrosomal membrane in initiating the matrix modification. Sodium was required for the action of nigericin; the ionophore was ineffective in medium in which choline chloride or sucrose was substituted for NaCl. In contrast, the calcium-induced dispersion of the acrosomal matrix occurred in the absence of sodium. Furthermore, low concentrations of calcium inhibited an adenosine triphosphatase activity associated with isolated acrosomal apical segments. These data are consistent with the hypothesis that calcium induces alkalinization of the acrosome, leading to matrix dispersion. However, permeabilized spermatozoa incubated at either pH 9.5 or in the presence of 50 mM NH4Cl at pH 7.5 failed to undergo spontaneous matrix dispersion, suggesting that elevated intraacrosomal pH alone was not sufficient to initiate the reaction. The proposed alternative hypothesis is that calcium initiates matrix dispersion by a mechanism in which elevated intraacrosomal pH may be a secondary response.     

Mechanisms of the effect of nitroglycerine on contraction of smooth muscles of the rat femoral artery

In experiments on isolated segments of the rat femoral artery, we demonstrated that a donor of nitric oxide, nitroglycerine (NG), suppresses KCl-and phenylephrine-induced contractions of smooth muscles (SMs) of the vascular wall in a dose-dependent manner. The relaxing effect of NG on SMs is based on several mechanisms. In a series of experiments on intact preparations, we found that potassium channels of two types, Ca-dependent big-conductance and inward rectifying channels, are involved in the relaxing effect of NG. Experiments on skinned preparations showed that interaction between the contractile apparatus of SM cells and calcium ions is disturbed under the action of NG. Neirofiziologiya/Neurophysiology, Vol. 39, No. 3, pp. 208–213, May–June, 2007.     

体外培养的人脐动脉平滑肌细胞可自发成骨细胞分化并钙化

研究脐动脉来源的平滑肌细胞在体外培养时是否会自发成骨细胞分化,以深入了解该细胞的生物学特点.采用MTT法研究了脐动脉平滑肌细胞的生长曲线:用形态学方法观察了脐动脉平滑肌细胞自发细胞结节的形成过程;用Hoechst 33258染色法及TUNEL染色法研究了细胞结节中的凋亡现象;采用免疫组化染色法研究了细胞结节中碱性磷酸酶的表达.采用茜素红S染色及透射电镜研究了脐动脉平滑肌细胞的自发钙化.研究发现,体外培养的脐动脉平滑肌细胞传代后约7天细胞即可汇合,汇合后的细胞表现为典型的"峰、谷"样生长状态,随着培养时间的延长.在"峰"形生长区域,细胞聚集生长,自发形成细胞结节,经4-5周培养,细胞结节不断增大并钙化,免疫组化发现结节中有大量碱性磷酸酶表达.同时,在结节形成过程中伴随有大量平滑肌细胞凋亡.我们的研究表明,体外培养的脐动脉来源的平滑肌细胞同主动脉来源的平滑肌细胞一样可以自发成骨细胞分化.而平滑肌细胞凋亡可能参与了该过程.     

Specific inhibition of endothelial cell proliferation by thrombospondin.

Angiogenesis is a multi-step event involving endothelial cell migration, attachment, and proliferation. A thrombospondin (TSP)-like protein has recently been described as a naturally-occurring inhibitor of angiogenesis. We now report that human platelet TSP inhibits the in vitro proliferation of endothelial cells from the rabbit corpus luteum, bovine adrenal cortex and pulmonary artery, and human umbilical vein. The antiproliferative effect of TSP was neutralized by monoclonal antibodies against TSP. The growth arrest seen with TSP was specific for endothelial cells since TSP actually stimulated the growth of vascular smooth muscle cells and human foreskin fibroblasts. These results imply that the angiogenesis-inhibiting effect of TSP is mediated through an inhibition of endothelial cell proliferation. Elucidation of the mechanism of action of TSP on endothelial cell proliferation may lead to potential therapeutic approaches for the control of neovascular diseases.     

Effect of prostaglandins A1, A2, B1, B2, E2 and F2 alpha on human umbilical cord vessels

Human umbilical blood vessels have the ability to close spontaneously following delivery at term. It has been suggested that prostaglandins may have a possible physiological role in its closure. This study investigates the effects of 6 naturally occurring prostaglandins (A1, A2, B1, B2, E2, F2a) on the umbilical blood vessels. Umbilical cords were collected from cases of normal spontaneous vaginal deliveries and cesarian section at term. A total of 41 strips of umbilical arteries and 26 strips of umbilical veins from 24 cords were used. A 4-point bioassay method was used to compare the potency of prostaglandins A1, A2, B1 and F2a with PGE2. The effect of Polyphloretin Phosphate (PPP) on prostaglandin-induced contractions was studied on umbilical artery strips from 12 cords. The 6 prostaglandins exerted a stimulant effect on the isolated strips of human umbilical arteries. Prostaglandin B2 was the most potent compound on the umbilical vein, followed by PGA2. PPP in the concentration range of 10 to 40 mcg/ml completely eliminated the responses of PGE2, F2a, A1, A2, and B1. Responses to PGB2 were considerably but not completely abolished. PPP (up to 40 mcg/ml) did not affect contractions induced by 5-hydroxytryptamine, suggesting the presence of discrete receptor sites in the blood vessels for different pharmacologically active compounds. This is the first report of the constrictor effect of PGA and PGB compounds. These naturally occuring prostaglandins with high potencies (compared with other prostaglandins and other vasoactive substances) may play a role in spontaneous closure of umbilical vessels. PGE1, E2, F1 and F2a are found in umbilical blood vessels obtained at term.     

Unique effect of the pregnancy hormone estriol on antigen-induced production of specific antibodies in female BALB/c mice

We investigated the modulatory effect of estriol (E(3)), an estrogen predominantly produced during human pregnancy, on the antigen-induced production of specific antibodies in female BALB/c mice, and its effect was compared with 17beta-estradiol (E(2)). Estriol (E(3)) had a very different effect than E(2) on the antigen-induced production of specific antibodies in animals immunized with two different antigens, i.e., the bovine serum albumin (BSA) and pneumococcal polysaccharide serotype-14 (PPS-14). While E(2) strongly stimulated the production of BSA-specific antibodies (mostly IgG1), E(3) had little or no effect on their production. In comparison, when the bacterial PPS-14 was the immunogen, E(3) and E(2) both strongly increased the production of PPS-14-specific antibodies (mostly IgM). E(3) and E(2) also had a similar effect on the thymus weight reduction and on the spontaneous antibody production in these animals. Our results provided an example demonstrating that the pregnancy hormone E(3) has a distinctly different profile of modulatory actions in the immune system compared to E(2), while the former strongly enhanced the body's ability to produce bacteria-specific IgM antibodies, it had no effect on the production of specific antibodies against a soluble protein. This differential effect of E(3) may be beneficial for reducing the risk of developing antibody-mediated immune attack against the maternal and fetal elements during pregnancy.     

Facile synthesis of deuterated estrogens

The acid catalyzed exchange of the aromatic protons, ortho to the phenolic hydroxyl moiety in estriol, estradiol, and estrone is described. The acidic protons adjacent to the keto function in estrone are also exchanged leading to tetradeuterioestrone (80.0 atom%). Estriol was converted to dideuterioestriol (81.2 atom%), and estradiol into dideuterioestradiol (90.5 atom%).