Molecular and binding characteristics of IP3 receptors in bovine spermatozoa.

We have shown the presence of inositol 1,4,5-triphosphate (IP3) receptors in bovine spermatozoa. These receptors are mainly localized and functionally associated with the acrosome region. Molecular characterization of these bovine IP3 receptors has shown that the functional size of the IP3 binding domain is a protein of 66+/-2 kDa, in agreement with the size of both bovine adrenal cortex and bovine adrenal medullar chromaffin cells IP3 receptors. In contrast, bovine cerebellum IP3 receptor displays molecular weight of 220+/-5 kDa, a value in agreement with data in the literature. Bovine IP3 receptors have a one-affinity state characterized by a low affinity (Kd 750 nM) and a relatively high density (7.5 pmol/mg protein). They are functional and release internal calcium upon the binding of the second messenger. Moreover, the finding that the specific A1 adenosine receptor agonist R-PIA elicits almost the same effect as IP3 might be of some help in understanding the physiological role of these inhibitory adenosine receptors in mammalian spermatozoa.

CD26 and adenosine deaminase interaction: its role in the fusion between horse membrane vesicles and spermatozoa.

Membrane vesicles of horse seminal plasma present at their surface a highly specific serine-type protease, dipeptidyl peptidase IV/CD26, a surface antigen known to characterize human prostasomes. Horse sperm cells expressed at their surface A(1) adenosine receptors (A(1)AR) and ecto-adenosine deaminase (ecto-ADA), both detected by immunoblot analysis, whereas CD26 was visualized at the equatorial segment by immunofluorescence microscopy. In addition to CD26, horse membrane vesicles showed ecto-ADA. The fusion process between horse sperm cells and vesicles was evidenced by confocal microscopy, which showed the localization of CD26 at the postacrosomal region and at the midpiece of the spermatozoa after incubation with vesicles. Moreover, a similar localization of CD26 and ecto-ADA on the spermatozoa was evidenced after fusion. Our results suggest that the interaction CD26/ecto-ADA might be responsible for fusion. Since A(1)ARs are said to be second receptors for ecto-ADA to form ecto-ADA/A(1)AR complexes, and since horse spermatozoa have A(1)ARs at their surface, the interaction CD26/ecto-ADA/A(1)AR during the fusion process cannot be ruled out.

Rabbit spermatozoa: a model system for studying ATP homeostasis and motility.

This paper studies the adenosine triphosphate (ATP) homeostasis and the motility parameters of rabbit spermatozoa. Rabbit sperm, collected by artificial vagina, were studied in various buffer systems to determine motility over time. Sperms were also extracted to measure enzyme activity. Analyses of motility by Computer Assisted Semen Analyzer system were run in parallel with energy metabolic studies of sperm cells maintained in different physiological solutions sometimes containing inhibitors of energy metabolism. Rabbit spermatozoa were shown to be able to form ATP either via glycolysis or via oxidative phosphorylation. Both these metabolic pathways were active in viable cells where creatine kinase and adenylate kinase systems were also present (1.1 and 7,000 nmol/min per 100 x 10(6), respectively) and involved in maintaining high ATP levels. A dynamic balance between ATP synthesis and ATP-hydrolyzing enzymes was suggested by the fact that rabbit sperms in their seminal plasma preserved their motility for hours. The decrease in sperm ATP content was mainly due to its hydrolysis by dynein ATPases coupled with movements. Therefore, motility of rabbit spermatozoa appeared to be dependent only on the ATP available to dynein ATPases. In fact, statistical analyses of motility parameters and the concentrations of intracellular ATP or ATP-metabolite did not show any significant correlation.

Activity of IMP- and AMP-preferring isoforms of 5'-nucleotidase from human seminal plasma with AMP analogues.

AMP analogues modified at various positions of the molecule were checked as substrates for the two soluble isoforms of 5'-nucleotidase from human seminal plasma. These isoforms were isolated to near homogeneity by affinity chromatographies. AMP derivatives were differently dephosphorylated by both the isoforms depending on the site of modification in the natural compound. Changes in the phosphate moiety reduced significantly hydrolysis by the IMP-preferring form, whereas the AMP-preferring form was less affected. The AMP-preferring form was characterized by a relatively broad specificity toward substrate analogues indicating that the binding domains for the phosphate moiety of these isoforms are not identical. Substitutions at the C-8 adenine base reduced the hydrolysis rate of both the enzymes and variations of the syn-anti conformational equilibrium resulted in different effects on catalysis by both forms. Therefore, the orientation of the heterocyclic base around the glycosidic bond may not be the crucial factor affecting binding and catalytic activity. Hydrogen bonding potential of base N-7 was essential for the binding and catalysis of the IMP- but not of the AMP-preferring form. This was the most striking difference between the studied isoforms. Modifications and substitutions of 6-amino function, better accepted by the IMP-preferring form than by the AMP-preferring form, indicated that no essential hydrogen bonding is required for catalytic activity. The binding was however significantly slowed in 6-SH-PuMP. Hydrogen bonding potential of N-1 was significant for the hydrolysis rate of the IMP- but not of the AMP-preferring form. We suggest that these human seminal plasma isoforms of soluble 5'-nucleotidase, characterized by unique features, may represent the tissue-specific expression of the polymorphic gene.

Regulation of agonist-receptor binding by G proteins and divalent cations in spermatozoa solubilized A1 adenosine receptors.

Solubilized A1 adenosine receptor (A1AR) was used to investigate the effect of several cations on agonist-binding characteristics and GTP hydrolysis. It was shown by Western blot with G beta-M14 that this preparation contains both G proteins and receptor. The role of the receptor molecule is to facilitate the activation of G proteins as alpha-GTP complex, and GTP hydrolysis has important consequences for the basic deactivation mechanism. Divalent cations, such as Mn2+, Ca2+, and Mg2+, potentiated the agonist-specific binding: Mn2+ had the highest apparent affinity with half-maximal effect at 50 microM. Binding assays, performed in the presence of 100 microM Mn2+, showed an increase in the apparent affinity of the binding sites, whereas, in the presence of 1 mM Mg2+, significant alteration of the apparent affinity, but not of the number of sites, was detected. Concentrations of 1 mM Mg2+ and 100 microM Mn2+ enhanced GTPase activity, whereas 5 mM Ca2+ resulted in the increase of Vmax values without significant alterations of K(m). In the presence of A1-specific agonists, Mn2+ and Mg2+ caused a decrease of Vmax values and an increase of GTP affinity. Other cations, such as Co2+, Cd2+, Cu2+, and Zn2+, inhibited the binding capacity but caused almost no changes in GTP hydrolysis kinetics.

Polycyclic aromatic hydrocarbons enhance the production of phorbol 12-myristate 13-acetate-induced superoxide ions in human monocytes.

Monocytes, separated from peripheral blood, preincubated with a mixture of polycyclic aromatic hydrocarbons (PAHs) show an enhanced production of superoxide ions (O2-.) when the cells are stimulated with phorbol 12-myristate 13-acetate (PMA, direct activator of protein kinase C). When opsonized-zymosan is used as a stimulus (receptor-dependent stimulus), no enhanced production of O2-. is observed. Superoxide production increases dose dependently up to a PAH concentration of 5 microg/ml. Although the effect was rather small (125-145% of the control value), it was significant and reproducible. Similar enhancing activity was also observed in the production of hydrogen peroxide (H2O2) excluding an inhibitory effect of PAHs on the enzyme superoxide dismutase (SOD). Since the effect is related to the concentration of PMA and in the absence of stimulus, the O2-. is undetectable in both the control and in the PAHs-treated cells, it is concluded that the over production of O2-. is due to an increased activity of the NADPH oxidase.

Occurrence of prostasome-like membrane vesicles in equine seminal plasma.

Equine seminal plasma was shown to contain membrane vesicles that are similar to the well characterized prostasomes in human seminal plasma. Determination of nucleoside and nucleotide concentrations of these particles have shown that ATP, ADP and adenosine are the main components of the nucleotidic pool. 5' nucleotidase, endopeptidase and dipeptidyl peptidase i.v. activities have been found on the surface of the particles. The interaction between these prostasome-like vesicles and spermatozoa was demonstrated by electron micrograph scans which revealed the steps of a fusion-like process leading to mixing of the membranes. In addition, endopeptidase activity, a marker enzyme of these seminal vesicles that is normally absent from equine spermatozoa, was shown to be acquired by these cells after interaction with the vesicles. The addition of these vesicles to equine spermatozoa resulted in the modification of adenylate catabolism. Therefore, a role in stabilizing the energy charge of the spermatozoa thus allowing longer viability is proposed for these organelles.

Hydrolysis of extracellular adenine nucleotides by equine epidydimal spermatozoa.

Ectoenzymic activities capable of hydrolyzing ATP sequentially to adenosine are present on equine epidydimal spermatozoa membranes. Kinetic parameters for ATPase, ADPase and 5'-nucleotidase were obtained by analysis of progress reactions curve when ATP, ADP and AMP were supplied as initial substrates. These values are not different from those found when the substrates were supplied from the preceding reactions. Feed-forward inhibition on 5'-nucleotidase by ATP/ADP was taken into account to fit simulated data to the experimental results. None of the substrates supplied by the preceding reactions showed a preferential delivery to ADPase and/or 5'-nucleotidase. We therefore conclude that the model that fits the equine spermatozoa is that already proposed for pig aortic endothelial cells.

Human seminal plasma soluble 5'-nucleotidase: regulatory aspects of the dephosphorylation of nucleoside 5'-monophosphates.

Human seminal plasma contains two enzyme activities both capable of dephosphorylating all nucleoside 5-monophosphates with different efficiency and specificity. Broad-spectrum soluble 5'-nucleotidase is the object of this paper which deals with the definition of the response of this enzyme to effectors, some physiological and others not naturally occurring. The enzyme did not show any product regulation as all the nucleosides tested caused a moderate effect on the hydrolysis of the substrates. Theophylline and other xanthine derivatives had no effect on enzyme activity, whereas glycerate 2,3-bisphosphate, like other soluble 5'-nucleotidases, caused a stimulation of the enzyme, especially toward CMP and UMP. 5-Deoxy-5-isobutylthiadenosine resulted in no inhibition of the hydrolysis of AMP and IMP. The enzyme was affected neither by monovanadate nor by decavanadate, whereas it was strongly inhibited by Ap5 A. Variations in adenylate energy charge did not cause any alteration of the enzyme activity toward AMP and only a slight decrease of the hydrolysis of IMP. These regulatory properties, distinct from those of other soluble 5'-nucleotidases, show that this form, newly isolated from human seminal plasma, is subject to an almost unique, tissue-specific regulation.

[3H]-(R)-N6-Phenylisopropyladenosine agonist binding to the solubilized A1 adenosine receptor from bovine epididymal spermatozoa.

The high-affinity agonist radioligand [3H]-(R)-N6-phenylisopropyladenosine ([3H]-R-PIA) was used to investigate agonist A1 adenosine receptors interactions in soluble preparations from bovine spermatozoa. The digitonin-solubilized receptor shows a high-affinity state with a Kd of 5.32+/-1.17 nM and a Bmax of 460+/-33 fmol/mg protein. The binding capacity, higher than that of the membrane-bound form, indicates that the soluble preparation is likely enriched with binding sites. In the presence of guanylyl-5'-imidodiphosphate (Gpp(NH)p), [3H]-R-PIA binds to the soluble receptor with a Kd of 7.97+/-1.44 nM and a Bmax of 400.8+/-27 fmol/mg protein. The radioligand rapidly dissociates with a K(-1) of 0.125 min(-1) although specific [3H]-R-PIA is still found in solubilized A1 receptor. The A1 agonist N6-cyclopentyladenosine differentiates two affinity states, whereas Gpp(NH)p shifts the agonist curve to the right and all the receptors are in the low-affinity state. In the presence of NaCl, the agonist still recognizes two affinity states with a lower affinity than that observed in the absence of NaCl. Analyses of sedimentation profiles show the existence of a population of A1 receptors tightly coupled to Gi, the pertussin toxin-sensitive component of the G protein family.

Effect of airborne particulate extracts on monocyte oxidative metabolism.

Alveolar macrophages lie on the air side of the alveolar-capillary barrier of the lung. They originate from circulating monocytes and are an important first-line host defense against inhaled microorganisms. In monocytes and macrophages, phagocytosis is associated with an increase in O2 consumption and superoxide anion (O2-) generation, that is, "the respiratory burst". O2- is the precursor of highly reactive, oxygen-derived free radicals that are used to kill potential pathogens. Although it is well known that airborne particulate matter inhibits the phagocytic activity of alveolar macrophages, very little is known about the effect of airborne particulate extracts on the respiratory burst. In this study, monocytes isolated from the peripheral blood were incubated for 2 hr at 37 degrees C with increasing concentrations of particulate extract and then stimulated for 30 min with phorbol 12-myristate 13 acetate (PMA) or with Zymosan. The released O2- was measured by the superoxide dismutase inhibitable reduction of cytochrome C. The results cleary showed that, at a particulate concentration of 0.17 mg/mL, the production of O2- was reduced to 22% and 40% of the control values when the cells were stimulated with PMA and Zymosan, respectively. Concomitantly, there was a release of LDH in the supernatant (50% of the total), indicating that a large proportion of cells were damaged by the treatment with the environmental pollutants, and some cytosolic components were released from the cells. Giemsa staining of the treated monocytes revealed the presence of many cells with a dispersed cytosol; the nucleus, although not destroyed, had a different shape. It was suggested that the airborne particulate matter has a toxic effect that induces the disintegration of the plasma membrane. Cytosolic factors (proteins and coenzymes) necessary for O2- production leak from the cells and superoxide generation is therefore reduced. It remains to be determined whether this phenomenon also occurs in vivo.

The isolation from human seminal plasma of a new form of soluble 5'-nucleotidase.

Soluble broad spectrum 5'-nucleotidase from human seminal plasma was purified to homogeneity by a combination of (NH4)2SO4 precipitation, affinity chromatography, and gel filtration. The pure enzyme had a specific activity of 4800 nmol min-1 mg-1. SDS-PAGE of purified enzyme preparation revealed a single polypeptide band of 53 kDa and a tetrameric structure of 203 kDa was proposed for the native enzyme. This form had modest preference for AMP as substrate; Mg2+ and Mn2+ were activators of the enzyme although its activity was not absolutely dependent on the presence of these exogenous bivalent cations. The enzyme, recovered in the nonsedimentable fraction of human seminal plasma, had a pH optimum of 7.5; ATP and ADP were inhibitors of mixed type, Pi was a potent inhibitor at nonphysiological concentrations, and Con A and adenosine 5-[alpha, beta-methylene]diphosphate had no effect on the enzyme activity. The enzyme described here therefore has some unique properties between truly cytoplasmic and membrane-bound derived forms.

The dephosphorylation of AMP and IMP by a soluble low Km 5'nucleotidase from human seminal plasma: some regulatory aspects.

In this study, a soluble low Km 5'nucleotidase, dephosphorylating IMP with a Vmax/Km ratio 10-times higher than that of AMP, has been purified from human seminal plasma. The effect of inorganic phosphate (Pi) and adenylate energy charge variations on the activity of this enzyme has also been investigated. In the physiological range, with IMP as substrate, the activity of the enzyme does not change whereas the hydrolysis of AMP increases with decreasing energy charge values. In the presence of both the substrates, phosphate exerts an inhibitory effect on the enzyme activity with a similar concentration dependence pattern. The results show that AMP-hydrolysing activity responds to variations of energy charge by increasing the AMP degradation thus protecting the value of energy charge at the expense of a decrease in the total adenylate pool. In contrast, the dephosphorylation of IMP is not regulated by changes in energy charge. This data suggests that the degradation of IMP and AMP, although carried out by the same enzyme, is controlled by different regulatory mechanisms.

Evidence of A1 adenosine receptor on epidydimal bovine spermatozoa.

3H-R-phenylisopropyladenosine (PIA) was used to characterize adenosine receptors on bovine epidydimal spermatozoa membranes. Dypiridamole, an adenosine uptake inhibitor, did not effect the radioligand binding, indicating an external site for the interaction of adenosine with spermatozoa. Steady-state binding was achieved after 45 min at 25 degrees C and lasted for at least 3 h. Scatchard plots were linear with a Kd of 6.98 +/- 1.02 nM and Bmax of 34 +/- 8 fmol/mg protein. N-6-cyclopentyladenosine (CPA), with a Ki of approx. 0.196 nM, was the most potent inhibitor of binding and the agonist order potency series was CPA > R-PIA = N-6-cyclohexyladenosine > N-6-phenyladenosine > 5'-N-ethylcarboxamidoadenosine > 2-chloroadenosine > 2-(p-2-carboxyethyl)phenylamine)-5'-N-ethylcarboxy-amidoa denosine. 1-3-Dipropyl-8-cyclopentylxanthine (DPCPX), an A1 receptor selective antagonist, produced the strongest inhibition with a Ki of 0.46 +/- 0.1 nM. Antagonist order potency series DPCPX > xanthine amine congener > cyclopentyltheophilline = theophylline > caffeine > 1-3-dipropylxanthine > 8-phenyltheophilline was consistent with A1 adenosine receptor (A1AR). Guanylyl-5'-imidodiphosphate did not decrease bound 3-H-R-PIA nor accelerate its dissociation, a behavior consistent with inhibitory receptors only. The incubation of isolated membranes with N-ethylmaleimid followed by a reduction of 57% of the ligand binding further supports the existence of A1AR on bovine epidydimal spermatozoa.

Adenosine triphosphate catabolism in bovine spermatozoa.

Adenosine triphosphate metabolism in caudal epididymis bovine spermatozoa was studied. Measurements by HPLC at appropriate time intervals of the spermatozoa content of ATP and its derivatives were carried out under different experimental conditions. In the presence of 2-D-glucose, cellular ATP was transformed almost quantitatively into ADP and AMP at a rate of 2.3 nmol/min per 10(8) cells. At the same time, ADP and AMP accumulated at a rate of 1.52 and 0.58 nmol/min per 10(8) cells, respectively. In the first 4 min, about 50% of total ATP was degraded, the AEC of the cells dropped to non-physiological values while the content of other nucleosides did not vary significantly. Inorganic P(i) content also remained unchanged. Under non-induced conditions up to 240 min, no variations of the adenylic content and of the EC value was observed. Under induced and non-induced conditions, IMP and adenosine were not detected within the spermatozoa. The lack of IMP might be ascribed either to the absence of AMP deaminase, whose activity has never been found in the spermatozoa or to the intracellular environment which down regulates the activity of the enzyme. In order to explain low levels and absence of variations of adenosine, several enzymic investigations were carried out. Adenosine kinase activity was not determined, therefore the transformation of adenosine into AMP had to be excluded. Nevertheless, enzymic activities potentially able to dephosphorylate the formed AMP are present in the spermatozoa. Our findings are indicative of the existence in the spermatozoa of acid and alkaline phosphatase and of 5'-nucleotidase membrane-derived.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of adenine nucleotides on low Km 5' nucleotidase from human seminal plasma.

1. Low Km 5' nucleotidase purified from human seminal plasma has been used in this study to investigate the response of the enzyme ot adenine nucleoside di- and triphosphates in the presence of AMP and IMP as substrates. 2. In the presence of AMP, the addition of 0.5 mM ATP to the enzyme Mg-free results into the highest Vmax/Km ratio value and other experimental combinations of effectors tested cause variation of the kinetic parameters of the enzyme, indicating a control of AMP dephosphorylation by adenine nucleotides. 3. In the presence of IMP, ATP and ADP activate the enzyme but the response to various experimental combinations of effectors shows no significant difference in the kinetic properties of the enzyme, indicating a different control of the dephosphorylation of IMP.

Purification and partial characterization of the soluble low Km 5'-nucleotidase from human seminal plasma.

Soluble low Km 5'-nucleotidase from human seminal plasma has been purified to homogeneity by one affinity and two gel-filtration chromatographic steps. The pure enzyme had a specific activity of 2000 nmol min-1 mg-1. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified low Km 5'-nucleotidase revealed a single polypeptide band of 40 +/- 7 kDa and a tetrameric structure of 160 +/- 10 kDa has been proposed for the native enzyme. The kinetic properties of low Km 5'-nucleotidase have been determined and rather unique characteristics have been found for this soluble low Km 5'-nucleotidase: the substrate efficiency was slightly higher for IMP with an optimum pH at 7.5; the enzyme showed an absolute dependence on Mg2+ ions. Ca2+ could replace Mg2+ ions for activity while other divalent cations could not substitute for Mg2+; the enzymes were equally activated by ATP and ADP up to 0.1 mM concentrations. At higher concentrations up to 1 mM, ADP was still an activator while ATP caused a gradual decrease of activation to the native activity. This effect could not be related to the Mg-ATP = complexes since the enzymic preparation Mg(2+)-free still showed the same biphasic pattern of activation.

Adenosine metabolizing enzymes in seminal plasma of bull and man: a comparative study.

1. Adenosine metabolizing enzymes in seminal plasma of man and bull have been investigated. 2. A different level of 5'-nucleotidase activity has been found in two seminal plasmas: in bull 5'-nucleotidase represents 80% of the total AMP dephosphorylating enzymes while in man 5'-nucleotidase represents only 1.3% of the total AMP dephosphorylating activities. 3. Apart from the different levels of 5'-nucleotidase activity, different kinetic parameters have been reported for 5'-nucleotidase, acid prostatic phosphatases, ADA and PNP. 4. Adenosine kinase, xanthine oxidase and AdoHcy-hydrolase have not been detected in the seminal plasma of man and bull.