腺嘌呤核苷脱氨酶及核苷运转系统对精子运动的调节作用

 腺嘌呤核苷(ADO)和它的类似物2-Cl-ADO对牛附睾尾部精子的运动有刺激作用,为了探讨ADO及其类似物对精子运动调节作用的机理,我们从ADO受体,核苷运转系统(NTS)及腺苷脱氨酶(ADA)三个方面对牛附睾尾部的精子进行了研究。我们发现腺苷受体不存在于牛精子膜上,但ADA和NTS以膜蛋白的形式结合在精子膜上,并对精子体内的ADO浓度起调节作用。我们的结论是ADO及其类似物对牛精子运动的调节作用是首先通过精子膜上的ADA和NTS影响精子体内的ADO浓度,进而ADO又通过调节钙离子浓度刺激牛精子运动。     

Evidence for a role for cellular alkalinization in the cyclic adenosine 3',5'-monophosphate-mediated initiation of motility in bovine caput spermatozoa

Bicarbonate ion, the local anesthetics procaine and dibucaine, and the ionophores monensin and nigericin have been shown to markedly increase the ability of agents that elevate cyclic adenosine monophosphate (cAMP) levels to initiate motility in bovine caput spermatozoa. A number of other weak bases, including theophylline, D-600 and dipyridamole, elevate cAMP levels maximally in caput sperm at low levels but induce motility only at high levels. These compounds thus appear to have a dual role in the initiation of motility, i.e., they elevate both cAMP levels and internal pH. Confirmation of this view was provided by the demonstration that bicarbonate ion and procaine permit initiation of motility by theophylline, D-600 and dipyridamole at markedly reduced levels. Also, forskolin (a neutral adenylate cyclase activator) elevates cyclic AMP levels in caput sperm but initiates motility only in the presence of bicarbonate or procaine, and the membrane-permeant cAMP analogue 8-bromo-cAMP is capable of inducing motility only in the presence of bicarbonate. Thus, motility in caput sperm is induced only under conditions that elevate both intracellular cAMP and pH, whereas caudal sperm motility is stimulated by an elevation of either cAMP or pH. These data suggest that the epididymal development of motility requires a maturational increase in internal pH. This suggestion was confirmed by direct measurement of the internal pH of caput and caudal sperm; the internal pH of the former was found to be 5.84 +/- 0.1 and the latter 6.27 +/- 0.05.     

Signaling pathways for modulation of mouse sperm motility by adenosine and catecholamine agonists

Capacitation of mammalian sperm, including alterations in flagellar motility, is presumably modulated by chemical signals encountered in the female reproductive tract. This work investigates signaling pathways for adenosine and catecholamine agonists that stimulate sperm kinetic activity. We show that 2-chloro-2'-deoxyadenosine and isoproterenol robustly accelerate flagellar beat frequency with EC50s near 10 and 0.05 microM, respectively. The several-fold acceleration is maximal by 60 sec. Although extracellular Ca2+ is required for agonist action on the flagellar beat, agonist treatment does not elevate sperm cytosolic [Ca2+] but does increase cAMP content. Acceleration does not require the conventional transmembrane adenylyl cyclase ADCY3, since it persists in sperm of ADCY3 knockout mice and in wild-type sperm in the presence of the inhibitors of conventional adenylyl cyclases SQ-22536, MDL-12330A, or 2', 5'-dideoxyadenosine. In contrast, the acceleration by these agents is absent in sperm that lack the predominant atypical adenylyl cyclase, SACY. Responses to these agonists are also absent in sperm from mice lacking the sperm-specific Calpha2 catalytic subunit of protein kinase A (PRKACA). Agonist responses also are strongly suppressed in wild-type sperm by the protein kinase inhibitor H-89. These results show that adenosine and catecholamine analogs activate sperm motility by mechanisms that require extracellular Ca2+, the atypical sperm adenylyl cyclase, cAMP, and protein kinase A.     

Adenosine and its analogues, possibly acting at A2 receptors, stimulate mouse sperm fertilizing ability during early stages of capacitation

Earlier studies have provided indirect evidence that the availability of endogenous adenosine can modulate the fertilizing ability of mouse spermatozoa during capacitation. More direct evidence has been sought by evaluating the effect of exogenous adenosine present during the early stages of capacitation. A concentration-dependent stimulation of in-vitro fertilizing ability was observed, with 10 microM- and 100 microM-adenosine significantly increasing the proportion of eggs fertilized compared with drug-free controls. The adenosine-induced stimulation was observed in the presence of 0.01 microM- and 0.1 microM-dipyridamole, an inhibitor of adenosine uptake, suggesting that adenosine is acting at an external site. Comparison of adenosine with its analogues 2'-deoxyadenosine and 2-chloroadenosine indicated that the analogues at 10 microM were able to stimulate fertilization in a manner similar to adenosine. While neither adenosine nor 2'-deoxyadenosine was consistently effective at 1 microM, 2-chloroadenosine significantly stimulated fertilization at both 1 microM and 0.1 microM. In addition, 5'-N-ethylcarboxamidoadenosine (NECA) and (R)-N6-phenylisopropyladenosine (R-PIA), potent analogues in somatic cell systems, proved to be so with mouse sperm suspensions, NECA being stimulatory at greater than or equal to 0.01 microM and R-PIA at greater than or equal to 0.1 microM. Subjective evaluation of motility patterns indicated that more cells exhibited hyperactivated motility in the presence of stimulatory concentrations of adenosine or analogues. Assessment of capacitation state using chlortetracycline fluorescence patterns indicated that incubation in 2'-deoxyadenosine resulted in significantly fewer cells expressing the uncapacitated F pattern and significantly more cells with the capacitated AR (acrosome-reacted) pattern, compared with drug-free counterparts. It is concluded that adenosine promotes capacitation by interacting with externally-directed receptors, possibly on adenylate cyclase to increase the intracellular availability of cyclic adenosine monophosphate (cAMP); cAMP is known to stimulate mouse sperm fertilizing ability. The greater sensitivity to NECA, 2-chloroadenosine and R-PIA, relative to adenosine and 2'-deoxyadenosine, is consistent with interaction at stimulatory A2 adenosine receptors.     

Activation of spermatozoan adenylate cyclase by a low molecular weight factor in porcine seminal plasma

The ejaculated porcine spermatozoa were fractionated into the cytosol, membrane, midpiece plus tail (flagella) and head fractions, and their adenylate cyclase activities were measured. About 65% of the total activity was located in the flagella fraction. For all the fractions, Mn2+-dependent adenylate cyclase activity was about 20 times higher than Mg2+-dependent activity, and guanine nucleotides, fluoride, and other reagents tested did not activate adenylate cyclase. The results suggest that the GTP-dependent regulatory subunit is absent in porcine spermatozoa. The porcine seminal plasma was found to stimulate the adenylate cyclase activity in spermatozoa. The stimulating factor in porcine seminal plasma was partially purified by gel filtration and the molecular weight of the factor appeared to be between 200 and 300. The partially purified factor is heat stable and is not inactivated by treatment with Pronase, trypsin, phospholipase A2 or D but is inactivated by acid hydrolysis. It is easily soluble in water, partially soluble in methanol, and insoluble in ethanol, ethyl ether, chloroform, or acetone. The activation of sperm adenylate cyclase by the factor occurred without a time lag. The activating effect was dose-dependent, saturated at high dose, and ascribed to the increase of the maximal velocity (Vmax). The effect of the factor appears to be limited to adenylate cyclase in spermatozoa; the factor activated adenylate cyclase both in porcine and bovine spermatozoa but failed to activate those in other porcine tissues. The factor was shown to activate the enzyme not only in the ejaculated spermatozoa but also in the epididymal sperm. The factor was also found to elevate the cAMP level in the intact porcine spermatozoa. The factor enhanced the motility of corpus and cauda epididymal spermatozoa. These findings indicate the possibility that this factor initiates the spermatozoan motility upon ejaculation through directly activating adenylate cyclase.     

Chemotactic peptide induces cAMP elevation in human neutrophils by amplification of the adenylate cyclase response to endogenously produced adenosine

The transient increase in human neutrophil cAMP levels induced by the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) is shown to be caused by amplification of adenylate cyclase response to endogenously produced adenosine. The FMLP-stimulated increase in neutrophil cAMP was potentiated markedly by a nonmethylxanthine cAMP phosphodiesterase inhibitor (Ro 20-1724). By inhibiting the degradation of newly formed cAMP, Ro 20-1724 rendered the FMLP-induced cAMP elevation persistent rather than transient. The role of endogenously produced adenosine in this phenomenon is demonstrated by the ability of either adenosine deaminase or theophylline, an adenosine receptor antagonist, to prevent FMLP-stimulated cAMP elevation. The general nature of the FMLP-potentiated cAMP response is indicated by the finding that FMLP-treated neutrophils, in the presence of exogenously supplied adenosine deaminase, exhibited augmented cAMP generation in response to three different types of receptor agonists: 2-chloroadenosine, prostaglandin E1, and L-isoproterenol. Moreover, like the neutrophil cAMP increase caused by FMLP alone, the ability of FMLP to augment cAMP response to 2-chloroadenosine in adenosine deaminase-treated cells was short-lived and declined after 1.0 min of exposure to FMLP. Preincubation of neutrophil suspensions with the adenylate cyclase inhibitor SQ 22,536 completely prevented FMLP-induced cAMP generation. Furthermore, when neutrophil suspensions were preincubated with concentrations of Ro 20-1724, which apparently maximally inhibit cAMP phosphodiesterase, a 30-s incubation with FMLP still resulted in substantially elevated cAMP levels. It therefore appears that FMLP raises cAMP by activating adenylate cyclase rather than inhibiting cAMP phosphodiesterase.     

Mouse neuroblastoma adenylate cyclase. Adenosine and adenosine analogues as potent effectors of adenylate cyclase activity.

1. Intact mouse neuroblastoma NS20 cells, in the presence of cyclic adenosine 3':5'-monophosphate (cAMP) phosphodiesterase inhibitor, responded to adenosine (200 muM) and 2-chloroadenosine (200 muM) with a 20-fold increase in intracellular cAMP levels. AMP (200 muM) additions caused only a 3.5-fold cAMP level elevation. ATP, ADP, guanosine, cytidine, uridine, and guanine, all at 200 muM, had no effect on the cAMP level of these cells. 2. Homogenate NS20 adenylate cyclase activity was increased 2.5- to 4-fold by addition of 200 muM adenosine, 2-chloroadenosine, 2-hydroxyadenosine, or 8-methylaminoadenosine. Prostaglandin E1 additions (1.4 muM) produced about an 8-fold stimulation of homogenate cyclase activity. The Km of homogenate cyclase activation by adenosine and 2-chloroadenosine was 67.6 and 6.7 muM, respectively. Addition of 7-deazaadenosine, tolazoline, yohimbine, guanosine, cytosine, guanine, 2-deoxy-AMP, and adenine 9-beta-D-xylopyranoside, all at 200 muM were found to be without effect on homogenate NS20 adenylate cyclase. Two classes of inhibitors of homogenate NS20 adenylate cyclase activity were observed. One class, which included AMP, adenine, and theophylline, blocked 2-chloroadenosine but not prostaglandin E1 stimulation of cyclase. Theophylline was shown to be a competitive inhibitor of 2-chloroadenosine, with a Ki of 35 muM. The second class of inhibitors, which included 2'- and 5'-deoxyadenosine, inhibited unstimulated, 2-chloroadenosine and prostaglandin E1-stimulated homogenate cyclase activity to about the same degree. 3. Activation of NS20 homogenate adenylate cyclase by adenosine appears to be noncooperative. 4. The inhibitory action of putative "purinergic" neurotransmitters is postulated to be due to their effects on adenylate cyclase activity.     

Both fertilization promoting peptide and adenosine stimulate capacitation but inhibit spontaneous acrosome loss in ejaculated boar spermatozoa in vitro

Both fertilization promoting peptide (FPP) and adenosine stimulate capacitation and inhibit spontaneous acrosome loss in epididymal mouse spermatozoa; these responses involve modulation of the adenylyl cyclase (AC)/cAMP signal transduction pathway. However, it was unclear whether these responses were restricted to the mouse or possibly common to many mammalian species. To address this question, the response of boar spermatozoa to FPP and/or adenosine was evaluated. FPP is found in nanomolar concentrations in seminal plasma of several mammals, but not the pig. When cultured in caffeine-containing Medium 199 for 2 hr, chlortetracycline fluorescence evaluation indicated that neither FPP nor adenosine stimulated boar sperm capacitation per se but did inhibit spontaneous acrosome loss. However, in caffeine-free medium, FPP and adenosine both stimulated capacitation and inhibited spontaneous acrosome loss, suggesting that boar spermatozoa have receptors for both FPP and adenosine. Gln-FPP, a competitive inhibitor of FPP in mouse spermatozoa, has recently been shown to inhibit mouse sperm responses to adenosine as well, suggesting that FPP receptors and adenosine receptors interact in some way. Used with boar spermatozoa, Gln-FPP also significantly inhibited responses to both FPP and adenosine. These responses suggest that mechanisms whereby FPP and adenosine can regulate sperm function, via AC/cAMP, are of considerable physiological significance. Mouse, human, and now boar spermatozoa have been shown to respond to FPP, suggesting that these mechanisms may be common to many mammalian species. We also suggest that the effects of FPP and adenosine could also be exploited to maximize monospermic fertilization in porcine in vitro fertilization.     

Sodium bicarbonate in seminal plasma stimulates the motility of mammalian spermatozoa through direct activation of adenylate cyclase

Recently, a low molecular weight factor, which specifically stimulates sperm adenylate cyclase, was found in porcine seminal plasma (Okamura, N., and Sugita, Y. (1983) J. Biol. Chem. 258, 13056-13062). The purified factor was analyzed by 1H NMR, 13C NMR, infrared spectroscopy, and elementary analysis and identified as sodium bicarbonate. The effects of sodium bicarbonate both on adenylate cyclase activity in porcine spermatozoa and on sperm motility have been studied. Sperm adenylate cyclase was found to be specifically activated by bicarbonate over the physiological concentration range. In contrast, the adenylate cyclase activity in other tissues was not affected. The same concentration range of bicarbonate which resulted in activation of adenylate cyclase also stimulated sperm motility. The motility and enzyme activity of spermatozoa in all species so far tested (human, bovine, rat, mouse, and dog) were found to be similarly sensitive to bicarbonate concentration. These results show that the bicarbonate-sensitive adenylate cyclase system regulates sperm motility and suggest that this system is common to all mammals.     

Effects of oxytocin and methacholine on cyclic nucleotide levels of rabbit myometrium

The effects of oxytocin and methacholine on cyclic nucleotide levels in estrogen-primed rabbit myometrium were studied in the presence and absence of 1-methyl-3-isobutyl xanthine (MIX), a phosphodiesterase inhibitor. In the absence of MIX, methacholine increased guanosine 3',5'-cyclic monophosphate (cGMP) levels at a time when contraction was decreasing, but had no influence on adenosine 3',5'-cyclic monophosphate (cAMP) levels. In contrast, oxytocin did not elevate cGMP, but rapidly decreased cAMP levels. MIX (1 mM) increased both cAMP and cGMP levels. Oxytocin or methacholine further increased cGMP, indicating activation of guanylate cyclase. Oxytocin- but not methacholine-induced stimulation of guanylate cyclase was abolished in Ca2+-free solution. Oxytocin increased cAMP over the levels produced by MIX alone, whereas methacholine decreased cAMP below the MIX control values; these effects were insensitive to indomethacin. Tissue levels of cGMP and cAMP did not directly correlate with isometric tension. The results also indicate that both oxytocin and methacholine stimulate guanylate cyclase but have opposing effects on adenylate cyclase of rabbit myometrium.     

Manganese and manganese-ATP interactions with bovine sperm adenylate cyclase

The adenylate cyclase of mammalian spermatozoa shares some of the properties of the isolated catalytic component from somatic cell adenylate cyclases. One of these properties is the large apparent stimulation by Mn2+. We have used the direct linear plot according to Eisenthal and Cornish-Bowden to explore whether this apparent stimulation is due to direct stimulation by Mn2+ or due to complexation of free ATP, a postulated inhibitor of cyclase activity. We have observed the activity of the particulate adenylate cyclase from bovine caudal epididymal spermatozoa as a function of calculated equilibrium values for the concentrations of Mn2+, free ATP, and the enzyme's substrate, the manganese-ATP complex. Direct linear plots for activity and substrate concentration over the apparent inhibitory concentration range of free ATP give the pattern expected for a hyperbolic substrate response. By contrast, direct linear plots in which Mn2+ concentration varies over its apparent stimulatory range show that as Mn2+ concentration increases, activities are higher than would be predicted for a hyperbolic substrate response. We conclude that for particulate bovine sperm adenylate cyclase, free ATP is not strongly inhibitory, and Mn2+ is a positive effector, reaching half-maximal stimulation at 0.2 mM. The unique nature of the sperm adenylate cyclase and its possible regulation by Mn2+ under physiological conditions is discussed.     

Inhibition of bovine spermatozoa by caudal epididymal fluid: II. Interaction of pH and a quiescence factor

Previous studies (Carr and Acott , 1984) indicate that bovine sperm are maintained in a quiescent state in the caudal epididymis (CE) by a pH-dependent inhibitory factor. Here, we have determined that the pH of bovine CE fluid and of CE semen is approximately 5.8, and that the motility of CE sperm in undiluted CE fluid increases as the pH is elevated. Therefore, the acidity of CE fluid may play a physiological role in the maintenance of sperm quiescence. The changes in sperm motility, in response to changes in the pH of CE fluid, are reversible and rapid. Dilution of CE fluid with buffers at either pH 5.5 or 7.6 produces a much slower initiation of motility. In buffer a significantly lower pH is required to inhibit sperm motility than is required in CE fluid. The apparent pKs for inhibition are 5.3 in buffer and 6.6 in CE fluid. However, the motility of sperm in buffers that contain lactate, shows a pH dependence similar to sperm in CE fluid. That is, lactate inactivates sperm in buffer at pH 5.5 but not at pH 7.6. Lactate, and several other permeant weak acids, have previously been shown to reduce the intracellular pH of bovine sperm and many other types of cells. We show that these permeant weak acids, but not impermeant weak acids, reversibly reduce CE sperm motility in buffer at pH 5.5 but not at pH 7.6. Weak bases, which have previously been shown to elevate intracellular pH, initiate sperm motility in CE fluid. These results suggest that intracellular pH can regulate CE sperm motility and may be the intracellular messenger for the pH-dependent quiescence factor. Although sperm cyclic AMP levels have been previously correlated with motility stimulation, cyclic AMP levels do not change when the pH of CE fluid is elevated, even though full motility is initiated.     

Adenine nucleotide changes at initiation of bull sperm motility.

Testicular and cauda epididymal sperm were obtained via catheters previously implanted in the rete testis and proximal vas deferens of bulls and were used to examine the relationships among sperm motility, cyclic adenosine 3':5'-monophosphate (cAMP) level, adenine nucleotide levels, and rates of glucose and oxygen consumption. Testicular, cauda epididymal, and ejaculated sperm contain cAMP-stimulated protein kinase, adenylate cyclase, and nucleotide phosphodiesterase. Treatment of the nonmotile testicular sperm with phosphodiesterase inhibitors resulted in a doubling of cellular cAMP concentration and a 25% increase in their glucose consumption. No change in motility, ATP level, or rate of oxygen consumption was observed. Sperm in neat cauda epididymal semen had flagellating tails but no progressive motility. Dilution of these sperm into glucose-containing buffer resulted in an increase in intracellular cAMP concentration and a decrease in ATP level with concomitant increases in ADP and AMP levels. These biochemical changes occurred within 30 s after dilution and apparently preceded the initiation of progressive motility by most cells. Since sperm in neat cauda epididymal semen became progressively motile when diluted with neat cauda epididymal plasma as well as accessory sex gland fluid or buffer, composition of the fluid surrounding the sperm is not responsible for the initiation of progressive motility upon dilution nor does cauda epididymal plasma contain an inhibitory factor. Perhaps release from contact immobilization provides the stimulation for the initial acquisition of progressive motility by cauda epididymal sperm. We conclude that during epididymal passage sperm develop from a cell physically unresponsive to changes in cAMP concentration to a form which initiates progressive motility upon changes in cAMP concentration.     

Initiation of Sperm Motility Induced by Cyclic AMP in Hamster and Boar

When the plasma membrane of hamster and boar spermatozoa was extraced by treatment with Triton X-100 and the demembranated spermatozoa were transferred to a reactivating medium containing only ATP, axonemes were initially immotile, and then gradually became motile. Under these experimental conditions, the cAMP content in the reactivating medium increased soon. This suggests that cAMP is synthesized from ATP by adenylate cyclase involved in incompletely removed or solubilized residual sperm membrane and that the autosynthesized cAMP causes the delay in motility initiation. This delayed initiation of motility did not occur when phosphodiesterase was added to the reactivating medium and the phosphodiesterase-dependent quiescent sperm became motile instantaneously at any time when excess cAMP was supplemented. Furthermore, demembranated sperm which were diluted in the reactivating medium containing ATP and cAMP, immediately became motile. cAMP levels in the cell increased during the initiation of sperm motility in both species. These results suggest that cAMP is the real factor indispensable for the initiation of sperm motility at ejaculation in mammals.     

The effect of diazepam on adenosine uptake and adenosine-stimulated adenylate cyclase in guinea-pig brain

We have used the adenosine-stimulated adenylate cyclase of guinea-pig brain to examine the potency of diazepam as an adenosine uptake inhibitor. Diazepam at concentrations in the range 10--500 microM stimulates the production of cAMP in incubated slices of guinea-pig cerebral cortex, with maximal fivefold stimulations over basal levels by 200 microM diazepam. The increases can be largely (but not completely) blocked by the adenosine antagonist theophylline or by addition of excess adenosine deaminase to the system. It appears that the stimulation of cAMP production is due to a blockade of adenosine uptake which results in an increase in extracellular adenosine and concomitant activation of the adenosine receptor coupled to adenylate cyclase. Since the cAMP response to standard adenosine uptake blockers (dipyridamole, dilazep) can be completely blocked by theophylline or adenosine deaminase, a small component of the diazepam response cannot be explained by an adenosine effect. The concentration of diazepam at which the first significant cAMP increase occurs is 10 microM or slightly lower. This is significantly higher than the concentration of diazepam needed to saturate the pharmacologically characterized central nervous system receptors for benzodiazepines.     

Histidine regulation of cyclic AMP metabolism in cultured renal epithelial LLC-PK1 cells.

L-Histidine and imidazole (the histidine side chain) significantly increase cAMP accumulation in intact LLC-PK1 cells. This effect is completely inhibited by isobutylmethylxanthine (IBMX). Histidine and imidazole stimulate cAMP phosphodiesterase activity in soluble and membrane fractions of LLC-PK1 cells suggesting that the IBMX-sensitive effect of these agents to stimulate cAMP formation is not due to inhibition of cAMP phosphodiesterase. Histidine and imidazole but not alanine (the histidine core structure) increase basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in LLC-PK1 membranes. Two other amino acids with charged side chains (aspartic and glutamic acids) increase AVP-stimulated but neither basal- nor forskolin-stimulated adenylate cyclase activity. This suggests that multiple amino acids with charged side chains can regulate selected aspects of adenylate cyclase activity. To better define the mechanism of histidine regulation of adenylate cyclase, membranes were detergent-solubilized which prevents histidine and imidazole potentiation of forskolin-stimulated adenylate cyclase activity and suggests that an intact plasma membrane environment is required for potentiation. Neither pertussis toxin nor indomethacin pretreatment alter imidazole potentiation of adenylate cyclase. IBMX pretreatment of LLC-PK1 membranes also prevents imidazole to potentiate adenylate cyclase activity. Since IBMX inhibits adenylate cyclase coupled adenosine receptors, LLC-PK1 cells were incubated in vitro with 5'-N-ethylcarboxyamideadenosine (NECA) which produced a homologous pattern of desensitization of NECA to stimulate adenylate cyclase activity. Despite homologous desensitization, histidine and imidazole potentiation of adenylate cyclase was unaltered. These data suggest that histidine, acting via an imidazole ring, potentiates adenylate cyclase activity and thereby increases cAMP formation in cultured LLC-PK1 epithelial cells. This potentiation requires an intact plasma membrane environment, occurs independent of a pertussis toxin-sensitive substrate and of products of cyclooxygenase, and is inhibited by IBMX. This IBMX-sensitive pathway does not involve either inhibition of cAMP phosphodiesterase activity or a stimulatory adenosine receptor coupled to adenylate cyclase.     

Effect of vasoactive intestinal polypeptide on the cyclic adenosine monophosphate generating system in rat kidney glomeruli and cortical tubules

The effect of in vitro addition of vasoactive intestinal polypeptide (VIP) on the 3'-5'-cyclic adenosine monophosphate (cAMP) generating system in rat kidney glomeruli and cortical tubules was studied. VIP did not stimulate cAMP accumulation in glomeruli; but VIP did stimulate, specifically and in a dose-dependent manner, cAMP concentrations in tubular membranes with the addition of GTP. These results are consistent with the existence of a functionally active VIP receptor coupled to adenylate cyclase in rat kidney cortical tubules.     

Increased phosphorylation of a distinct subcellular pool of protein phosphatase, PP1gamma2, during epididymal sperm maturation

The enzyme PP1gamma2 is a testis- and sperm-specific isoform of type 1 protein phosphatase (PP1), and it is the only isoform of PP1 in spermatozoa. The enzyme PP1gamma2 is essential for spermatogenesis and is also a key enzyme in the development and regulation of sperm motility. The carboxy terminus of the enzyme contains a consensus amino acid sequence for phosphorylation by cyclin-dependent kinases. Using antibodies specific to this phosphorylated amino acid sequence domain, we found that phosphorylated PP1gamma2 is present in bovine epididymal spermatozoa. The level of phosphorylated PP1gamma2 is significantly higher in motile caudal compared to immotile caput epididymal spermatozoa. A number of treatments, such as 2-chloro adenosine, cAMP analogues, cAMP phosphodiesterase inhibitors, and calcium, which stimulate sperm motility, did not alter the level of phosphorylated PP1gamma2. However, calyculin A, which is an inhibitor of protein phosphatase subtypes PP1 and PP2A, significantly increases the level of phosphorylated PP1gamma2 in both caput and caudal epididymal spermatozoa. Partial purification by column chromatography showed that phosphorylated PP1gamma2 is catalytically active. Phosphorylated PP1gamma2 is the only spontaneously catalytically active form of the enzyme in caudal sperm extracts. Western blot analysis shows that the enzyme cyclin-dependent kinase 2, one of the enzymes that phosphorylates the consensus domain at the carboxy terminus in PP1 isoforms, is present in spermatozoa. Western blot analysis of proteins extracted from purified head and tail fragments of spermatozoa showed that phosphorylated PP1gamma2 is present predominantly in the sperm head. Fluorescence immunocytochemistry also showed that phosphorylated PP1gamma2 is present predominantly in the posterior region of the sperm head. The distinct subcellular localization and changes in its level during sperm maturation suggest a possible role for sperm phosphorylated PP1gamma2 in signaling events during fertilization.     

Efflux of cyclic adenosine monophosphate from cells: mechanisms and physiological implications

Although cyclic nucleotides are hydrophilic compounds, extracellular cAMP (cAMPo) rapidly accumulates during the activation of adenylate cyclase. This review considers the kinetic characteristics of cAMP transport through the plasma membrane and its physiological implications. The influx and efflux of cAMP occur via different carriers. At physiological concentrations of cAMPo, the influx of cAMP does not significantly contribute to regulation of the intracellular content of the cyclic nucleotide, but it is responsible for the accumulation of cAMPi in experiments at [cAMP]o approximately 1 mM. In contrast, the high rate of cAMP efflux is mainly responsible for normalization of [cAMP]i during long-term activation of adenylate cyclase. The possible involvement of ATP-binding cassette proteins (ABC proteins) in the efflux of cAMP from the cell is considered. In procaryotes cAMPo is a signal molecule during the generation of cell colonies, acting on special receptors that interact with GTP-binding proteins. Such receptors have not been found in vertebrates, and in most cases the signal functions of cAMPo are mediated by its degradation by extracellular enzymes with subsequent activation of adenosine receptors.     

Inhibition of the catalytic subunit of ram sperm adenylate cyclase by adenosine

Adenosine inhibits ram sperm adenylate cyclase activity which is membrane-bound and comprises only the catalytic subunit. The inhibition parameters of adenylate cyclase by adenosine were not modified when the enzyme was purified 3 to 5,000 fold. Optimal inhibition by adenosine was found to require a high concentration of manganese, and exhibited a noncompetitive pattern up to a concentration of 1 mM adenosine. Adenosine was the most potent inhibitor among various analogs tested with the following rank order of potencies: adenosine greater than 2'O-methyladenosine greater than 2'deoxyadenosine much greater than 2 chloroadenosine. Studies with agonists and antagonists of the "R"-type adenosine receptor led us to conclude that adenosine inhibits ram sperm adenylate cyclase via a "P"-site carried by the catalytic subunit itself.