Endofacial competitive inhibition of the glucose transporter 1 activity by gossypol.

Gossypol is a natural disesquiterpene that blocks the activity of the mammalian facilitative hexose transporter GLUT1. In human HL-60 cells, which express GLUT1, Chinese hamster ovary cells overexpressing GLUT1, and human erythrocytes, gossypol inhibited hexose transport in a concentration-dependent fashion, indicating that blocking of GLUT1 activity is independent of cellular context. With the exception of red blood cells, the inhibition of cellular transport was instantaneous. Gossypol effect was specific for the GLUT1 transporter since it did not alter the uptake of nicotinamide by human erythrocytes. Gossypol affects the glucose-displaceable binding of cytochalasin B to GLUT1 in human erythrocyte ghost in a mixed noncompetitive way, with a K(i) value of 20 microM. Likewise, GLUT1 fluorescence was quenched approximately 80% by gossypol, while Stern-Volmer plots for quenching by iodide displayed increased slopes by gossypol addition. These effects on protein fluorescence were saturable and unaffected by the presence of D-glucose. Gossypol did not alter the affinity of D-glucose for the external substrate site on GLUT1. Kinetic analysis of transport revealed that gossypol behaves as a noncompetitive inhibitor of zero-trans (substrate outside but not inside) transport, but it acts as a competitive inhibitor of equilibrium-exchange (substrate inside and outside) transport, which is consistent with interaction at the endofacial surface, but not at the exofacial surface of the transporter. Thus, gossypol behaves as a quasi-competitive inhibitor of GLUT1 transport activity by binding to a site accessible through the internal face of the transporter, but it does not, in fact, compete with cytochalasin B binding. Our observations suggest that some effects of gossypol on cellular physiology may be related to its ability to disrupt the normal hexose flux through GLUT1, a transporter expressed in almost every kind of mammalian cell and responsible for the basal uptake of glucose.

Novel identification of peripheral dopaminergic D2 receptor in male germ cells.

Dopamine is a recognized modulator in the central nervous system (CNS) and peripheral organ functions. The presence of peripheral dopamine receptors outside the CNS has suggested an intriguing interaction between the nervous system and other functional systems, such as the reproductive system. In the present study we analyzed the expression of D2R receptors in rat testis, rat spermatogenic cells and spermatozoa, in different mammals. The RT-PCR analysis of rat testis mRNA showed specific bands corresponding to the two dopamine receptor D2R (L and S) isoforms previously described in the brain. Using Western blot analysis, we confirmed that the protein is present in rat testis, isolated spermatogenic cells and also in spermatozoa of a range of different mammals, such as rat, mouse, bull, and human. The immunohistochemistry analysis of rat adult testis showed that the receptor was expressed in all germ cells (pre- and post-meiotic phase) of the tubule with staining predominant in spermatogonia. Confocal analysis by indirect immunofluorescence revealed that in non-capacitated spermatozoa of rat, mouse, bull, and human, D2R is mainly localized in the flagellum, and is also observed in the acrosomal region of the sperm head (except in human spermatozoa). Our findings demonstrate that the two D2 receptor isoforms are expressed in rat testis and that the receptor protein is present in different mammalian spermatozoa. The presence of D2R receptors in male germ cells implies new and unsuspected roles for dopamine signaling in testicular and sperm physiology.

Differential signalling for enhanced hexose uptake by interleukin (IL)-3 and IL-5 in male germ cells.

We studied the expression and function of the IL (interleukin)-3 and IL-5 family of receptors in male germ cells. RT (reverse transcription)-PCR showed expression of mRNAs encoding the alpha and beta subunits of the IL-3 and IL-5 receptors in human testis, and the presence of IL-3 and IL-5 receptors alpha and beta proteins was confirmed by immunoblotting with anti-alpha and anti-beta antibodies. The immunolocalization studies showed expression of these receptors in the germ line in the human testis and in human and bovine ejaculated spermatozoa. Functional studies with bull spermatozoa indicated that IL-3 signalled for increased uptake of hexoses in these cells at picomolar concentrations compatible with expression of functional high-affinity IL-3 receptors in these cells. In contrast, IL-5 failed to induce increased hexose uptake in bull spermatozoa. Experiments using HL-60 eosinophils that express functional IL-3 and IL-5 receptors confirmed that IL-3, but not IL-5, signalled for increased hexose uptake. Our findings suggest that differential signalling for increased hexose uptake by heteromeric high-affinity IL-3 and IL-5 receptors in mammalian spermatozoa is a property that depends on the identity of the alpha-subunit forming part of the alphabeta-complex and is not a property specific to the germ cells.

Expression of granulocyte-macrophage colony stimulating factor (GM-CSF) in male germ cells: GM-CSF enhances sperm motility.

The granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine capable of stimulating proliferation, maturation and function of hematopoietic cells. Receptors for this cytokine are composed of two subunits, alpha and beta, and are expressed on myeloid progenitors and mature mononuclear phagocytes, monocytes, eosinophils and neutrophils, as well as in other nonhematopietic cells. We have recently demonstrated that bull spermatozoa express functional GM-CSF receptors that signal for increased glucose and Vitamin C uptake. In this study, we analyzed the expression of GM-CSF in bovine and human germ cells and its influence in bovine sperm motility. Reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization and immunoblotting analysis demonstrated that adult bovine and human testes expressed GM-CSF. In addition, immunolocalization studies confirmed the presence of GM-CSF in the germ cell line in bovine and human testes. Computer-assisted evaluation of patterns of sperm motility demonstrated that the addition of GM-CSF enhances several parameters of sperm motility in the presence of glucose or fructose substrates.