ag-213 and chelerythrine

Human sperm capacitation involves complex signal transduction mechanisms during which double phosphorylation of the threonine-glutamine-tyrosine motif (P-Thr-Glu-Tyr-P) occurs in some sperm proteins. The objective of this study was to investigate the regulation of this process. Fetal cord serum ultrafiltrate (FCSu), follicular fluid ultrafiltrate (FFu), progesterone and a combination of N(6),2'-O-dibutyryl cAMP (dbcAMP; cell permeant analogue of cAMP) and 3-isobutyl-1-methylxanthine (IBMX; phosphodiesterase inhibitor) were used as inducers of capacitation alone or in combination with inhibitors of protein kinase A (H89), protein kinase C (chelerythrine), protein tyrosine kinase (tyrphostin A47, PP2) and of dual specificity kinase (MEK-like kinases; PD98059). The level of P-Thr-Glu-Tyr-P in sperm proteins of 80 and 105 kDa during capacitation induced by FCSu, FFu and progesterone was regulated by a similar signal transduction pathway and involved receptor type protein tyrosine kinase and dual specificity kinase (MEK or MEK-like) but not protein kinase A or C. However, the level of P-Thr-Glu-Tyr-P in these sperm proteins during capacitation induced by dbcAMP+IBMX was mainly mediated through protein kinase A and C and receptor type protein tyrosine kinase, but not by dual specificity kinase. In conclusion, human sperm capacitation induced by some biological and pharmacological agents is regulated through very different signal transduction pathways.

Topics: 1-Methyl-3-isobutylxanthine; Alkaloids; Amino Acid Motifs; Benzophenanthridines; Bucladesine; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Female; Fetal Blood; Flavonoids; Follicular Fluid; Humans; Isoquinolines; Male; MAP Kinase Kinase Kinase 1; Phenanthridines; Phosphorylation; Progesterone; Protein Kinase C; Protein Serine-Threonine Kinases; Seminal Plasma Proteins; Signal Transduction; Sperm Capacitation; Sulfonamides; Tyrosine; Tyrphostins

The protease, alpha-thrombin (alpha Th), affects myocardial cell contractility, a feature common among agents that induce hypertrophy. However, it is not known whether cardiac myocytes possess alpha Th receptors (alpha Th-R), or if long term treatment with alpha Th can enhance growth and gene expression. In the present study primary neonatal rat ventricular myocytes expressed a 3.6-kilobase mRNA species that hybridized with a rat alpha Th-R-specific probe. After 48 h, alpha Th induced hypertrophy, sarcomeric organization, and enhanced atrial natriuretic factor (ANF) expression, all of which were blocked by the alpha Th-selective protease inhibitor, D-Phe-Pro-Arg-chloromethyl ketone. The alpha Th-R agonist peptide, SFLLRNPND, was a potent activator of ANF expression, however, the non-agonist, FLLRNPND, was inactive. Transfection experiments showed the enhancement of ANF expression by alpha Th to be transcriptional. The abilities of alpha Th to induce myocyte hypertrophy and to augment ANF transcription and peptide production were inhibited by the protein kinase C inhibitor, chelerythrine, and by the tyrosine kinase inhibitor, tyrphostin. Thus, myocardial cell alpha Th-Rs are stimulated by the specific proteolytic actions of alpha Th, and pathways involving both protein kinase C and protein tyrosine kinases are required for subsequent hypertrophy and ANF expression. Further, these findings suggest a new role for extracellular proteases as regulators of myocardial cell gene expression and growth.

Topics: Alkaloids; Amino Acid Sequence; Animals; Animals, Newborn; Atrial Natriuretic Factor; Benzophenanthridines; Blotting, Northern; Cardiomegaly; Catechols; Cells, Cultured; Gene Expression; Heart Ventricles; Kinetics; Molecular Sequence Data; Myocardium; Nitriles; Oligopeptides; Phenanthridines; Platelet Aggregation Inhibitors; Polymerase Chain Reaction; Protease Inhibitors; Protein-Tyrosine Kinases; Radioimmunoassay; Rats; Receptors, Cell Surface; Receptors, Thrombin; RNA, Messenger; Thrombin; Tyrphostins