RESUMO
In the female reproductive tract, the spermatozoa undergo a series of physiological and biochemical changes, prior to gaining the ability to fertilize, that result to capacitation. However, the actin polymerization and protein tyrosine phosphorylation are the two necessary steps for capacitation. In this study, we have demonstrated the actin polymerization and established the correlation between protein tyrosine phosphorylation and actin reorganization during in vitro capacitation in buffalo (Bubalus bubalis) spermatozoa. Indirect immunofluorescence and Western blot techniques were used to detect actin polymerization and tyrosine phosphorylation. The time-dependent fluorimetric studies revealed that the actin polymerization starts from the tail region and progressed towards the head region of spermatozoa during capacitation. The lysophosphatidyl choline (LPC)-induced acrosome reaction (AR) stimulated quick actin depolymerization. The inhibitor cytochalasin D (CD) blocked the in vitro capacitation by inhibiting the actin polymerization. In addition, we also performed different inhibitor (Genistein, H-89, PD9809 and GF-109) and enhancer (dbcAMP, H(2)O(2) and vanadate) studies on actin tyrosine phosphorylation and actin polymerization. The inhibitors of tyrosine phosphorylation inhibit actin tyrosine phosphorylation and polymerization, whereas enhancers of tyrosine phosphorylation stimulate F-actin formation and tyrosine phosphorylation. These observations suggest that the tyrosine phosphorylation regulates the actin polymerization, and both are coupled processes during capacitation of buffalo spermatozoa.
Assuntos
Reação Acrossômica/fisiologia , Actinas/metabolismo , Búfalos/fisiologia , Capacitação Espermática/fisiologia , Espermatozoides/fisiologia , Tirosina/metabolismo , Animais , Bucladesina/química , Peróxido de Hidrogênio/química , Isoquinolinas/química , Masculino , Fosforilação , Polimerização , Sulfonamidas/química , Teprotida/análogos & derivados , Teprotida/químicaRESUMO
Synthetic analogues of the bradykinin potentiating nonapeptide BPP9alpha indicate significantly different structural requirements for potentiation of the bradykinin (BK)-induced smooth muscle contraction (GPI) and the inhibition of isolated somatic angiotensin I-converting enzyme (ACE). The results disprove the ACE inhibition as the only single mechanism and also the direct interaction of potentiating peptides with the bradykinin receptors in transfected COS-7 cells as molecular mechanism of potentiation. Our results indicate a stimulation of inositol phosphates (IPn) formation independently from the B2 receptor. Furthermore, the results with La3+ support the role of extracellular Ca2+ and its influx through corresponding channels. The missing effect of calyculin on the GPI disproves the role of phosphatases in the potentiating action. These experimental studies should not only contribute to a better understanding of the potentiating mechanisms but also incorporate a shift in the research towards the immune system, in particular towards the immunocompetent polymorphonuclear leukocytes. The chemotaxis of these cells can be potentiated most likely by exclusive inhibition of the enzymatic degradation of bradykinin. Thus the obtained results give evidence that the potentiation of the bradykinin action can occur by different mechanisms, depending on the system and on the applied potentiating factor.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Bradicinina/farmacologia , Contração Muscular/efeitos dos fármacos , Teprotida/análogos & derivados , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/síntese química , Inibidores da Enzima Conversora de Angiotensina/química , Animais , Células COS , Cálcio/metabolismo , Chlorocebus aethiops , Sinergismo Farmacológico , Cobaias , Humanos , Fosfatos de Inositol/biossíntese , Dados de Sequência Molecular , Músculo Liso/efeitos dos fármacos , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Peptidil Dipeptidase A/efeitos dos fármacos , Fosfoproteínas Fosfatases/antagonistas & inibidores , Receptor B2 da Bradicinina/agonistas , Receptor B2 da Bradicinina/genética , Transdução de Sinais/efeitos dos fármacos , Teprotida/químicaRESUMO
The synthesis of 5 analogues of the effective inhibitor of peptidyl dipeptidase, teprotide, has been carried out. The inhibitory and bradykinin-potentiating activity of these compounds has been assayed. N-Terminal pyroglutamic acid and positive charge of arginine in position 4 were found to be essential for biological activity of the inhibitor.
Assuntos
Inibidores da Enzima Conversora de Angiotensina , Oligopeptídeos/síntese química , Teprotida/síntese química , Sequência de Aminoácidos , Animais , Bradicinina/farmacologia , Fenômenos Químicos , Química , Dicroísmo Circular , Sinergismo Farmacológico , Cobaias , Íleo/efeitos dos fármacos , Técnicas In Vitro , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Conformação Proteica , Teprotida/análogos & derivados , Teprotida/farmacologiaRESUMO
The fat of less than Glu1-3H-labelled bradykinin-potentiating peptide 9a [BPP9a; less than Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro, an inhibitor of angiotensin-converting enzyme (peptidyl dipeptidase)] was studied in the rabbit. After intravenous injection, BPP9a was rapidly removed from blood and much of the associated radioactivity was excreted in urine. Approx. 8% of the radioactivity in urine collected 2h after drug administration occurred in the form of BPP9a itself, the remainder occurring in three lower homologues: less than Glu-Trp (60%), less Glu-Trp-Pro-Arg-Pro-Gln (20%) and less than Glu-Trp-Pro-Arg-Pro-Gln-Ile (12%). Hydrolysis was not accounted for by enzymes in blood or urine. Apparently hydrolysis occurred within the kidney, as less than Gl-Trp was obtained in 60% yield in urine of isolated rat kidney perfused with [less than Glu1-3H]BPP9a.
