Signalling pathways involved in the contractile response to 5-HT in the human pulmonary artery.

Serotonin (5-hydroxytryptamine; 5-HT) is a potent pulmonary vasoconstrictor and mitogenic agent whose plasma level is increased in pulmonary hypertensive patients. Thus, we explored the signalling pathways involved in the contractile response to 5-HT in human pulmonary arteries (HPAs). Intact and beta-escin permeabilised rings from HPAs mounted in an organ bath system were used to assess both tension and myofilament Ca(2+)-sensitisation. Microspectrofluorimetry was used for intracellular Ca(2+) recordings in cultured HPA smooth muscle cells. Voltage-operated Ca(2+) channel blockers (nitrendipine and nifedipine) partially reduced the contraction to 5-HT. Thapsigargin or cyclopiazonic acid (CPA), known to deplete sarcoplasmic reticulum Ca(2+) stores, also partially inhibited the contraction, whereas removal of extracellular Ca(2+) under these conditions further inhibited the contraction. Changing from Ca(2+)-free to Ca(2+) containing solution, in the presence of nitrendipine and CPA, a protocol known to stimulate store-operated Ca(2+) channels, induced HPA contractions that were blocked by nickel. Nickel or gadolinium also reduced the contraction to 5-HT. Finally, 5-HT increased intracellular Ca(2+) responses in cultured HPA smooth muscle cells and myofilament Ca(2+)-sensitisation in HPA rings. Collectively, these results indicate that voltage-operated and voltage-independent Ca(2+) channels, as well as Ca(2+) release and myofilament Ca(2+)-sensitisation, participate in 5-HT-induced contraction in HPAs.

A synthetic decapeptide from a conserved ZP3 protein domain induces the G protein-regulated acrosome reaction in bovine spermatozoa.

In some animal species, the zona pellucida protein 3 (ZP3) plays a central role during fertilization, functioning as a specific receptor for sperm and as an inducer of the acrosome reaction. On the other hand, the zona pellucida protein 2 (ZP2) acts as a secondary receptor, binding to acrosome-reacted sperm. The objective of these studies was to identify ZP2 and ZP3 domains that may be of importance for the induction of the acrosome reaction. For this purpose, we synthesized a number of ZP2 and ZP3 peptides that were either conserved among species or that were species-specific according to their respective primary structures. We identified a defined, conserved ZP3 decapeptide (ZP3-6 peptide) that bound to the surface of the acrosomal region and induced the acrosome reaction in a concentration-dependent manner in capacitated bovine sperm; this effect was significant in the nanomolar range. Pertussis toxin inhibited the ZP3-6 peptide-induced acrosome reaction but had no effect on the progesterone-induced exocytotic event. Our data are in accordance with previous studies showing that progesterone induces acrosomal exocytosis via a different pathway than ZP3 and strengthen the hypothesis that the effect of ZP3-6 peptide upon acrosomal exocytosis is G protein regulated. Despite the commonly accepted idea that glycosylation of ZP proteins is required for successful sperm-oocyte interaction, we found that acrosomal exocytosis can be induced by a synthetic ZP3 peptide that is not glycosylated. The results presented in this study may be useful for the investigation of the molecular mechanisms of sperm-egg interaction in bovine and other species.

In vitro tests for essential sperm functions using the phyto-oestrogen genistein as a test substance.

Sperm motility, binding of spermatozoa to the zona pellucida and induction of the acrosome reaction are prerequisites for successful oocyte fertilization. Examination of the physiological and nonphysiological effects of particular compounds on sperm functions requires high-quality in vitro test systems. In this short methodological overview, a reliable combined in vitro test system with bovine gametes is described. The purpose of the study was to evaluate whether aliquots of pooled post-thaw spermatozoa are suitable for examination of environmental substances that affect essential sperm functions. The combined test system includes a number of known methods for the assessment of sperm vitality and motion parameters, acrosomal status, inducibility of acrosome reaction and sperm zona pellucida binding. First observations indicate that genistein inhibits the induction of acrosomal exocytosis and binding of spermatozoa to the zona pellucida. Motility parameters and the viability of bovine spermatozoa were not affected by this substance. It is concluded that genistein, a phyto-oestrogen which is abundant in several plants, can be used as a test substance for the evaluation of effects upon essential bovine sperm functions in vitro.

Functional activity of epididymal Chinchilla laniger spermatozoa cryopreserved in different extenders.

Chinchilla laniger is an endangered species and improved cryopreservation of spermatozoa would constitute a significant advance in the development of assisted reproductive techniques in this species. The functional activity of epididymal spermatozoa from adult males was studied immediately after extrusion and after 24 hours incubation, and the ability of five extenders to protect these gametes during cryopreservation was determined. A decrease in sperm motility, viability, acrosome intact cells and response to hypo-osmotic swelling test was detected 24 hours later. The extenders here assayed showed well-defined differences in their cryoprotective ability; however such differences could not be attributed to any one of their individual components. The presence of TES plus Tris, the proportion of the individual constituents and/or the differences in metabolic substrate content could explain the above-mentioned finding. The results indicate that cryo-buffer II (TES-Tris-egg yolk-fructose-glycerol) is the most powerful protector of sperm functional activity in this species.

Activity of Chinchilla laniger spermatozoa collected by electroejaculation and cryopreserved.

Because reproductive studies and the application of assisted reproductive techniques are relevant issues for an endangered species such as Chinchila laniger, the availability of a source of viable spermatozoa becomes of utmost importance. In this paper, we evaluate several functional parameters (motility, viability, response to hypoosmotic swelling test and acrosomal integrity) of fresh or frozen-thawed spermatozoa. Electro-ejaculation trials (50-cyc/sec sinusoidal wave was applied for 5 of every 10 sec) were successful in all unanesthetized animals. After volume (108.3 +/- 12.0 microL, n = 15) and concentration (421.8 +/- 34.4 x 10(6) cells/mL, n = 15) measurements, the above mentioned parameters were determined. In frozen-thawed semen samples sperm motility, viability, hypoosmotic swelling test and acrosomal integrity were significantly lower than in fresh semen samples. The results clearly indicated that electro-ejaculation is a useful method for evaluating spermatozoa for genetic analysis or for used in Al in this species. In addition, the cryopreservation procedure in this study preserved adequate levels of functional sperm activity.

[The role of low-molecular-weight heparin in unstable angina, acute myocardial infarction and post-elective percutaneous transluminal coronary angioplasty]. / O papel da heparina de baixo peso molecular na angina instável, infarto agudo do miocárdio e pós-angioplastia percutânea transluminal coronária eletiva.

PURPOSE: To evaluate the clinical efficacy and safety of subcutaneous (SC) low molecular weight heparin (LMWH) compared to intravenous (IV) non fractioned heparin (NFH) in unstable angina, acute myocardial infarction and post-percutaneous transluminal coronary angioplasty. METHODS: From September/92 to April/94, 314 patients were randomized in two groups. Group I-- 154 patients treated with SC LMWH, using in the 1st phase SC LMWH with a dosage of 160 UaXa IC/kg/day (group IA--92 patients), and in the 2nd, a dosage of 320 UaXa IC/kg/day (group IB--62 patients). Group II--160 patients treated with IV NFH 100UI/kg (bolus), followed by 1000UI/h with adjusted dosage by activated partial thromboplastin time. RESULTS: There was not a statistically significant difference among the three groups in relation to cardiac events, hemorrhagic complications and deaths. CONCLUSION: The clinical efficacy and safety of SC LMWH in patients with unstable angina, acute myocardial infarction and post-percutaneous transluminal coronary angioplasty were similar to IV NFH with the dosages used in this study.