P1 and P2 purinergic receptor signal transduction in rat type II pneumocytes

Extracellular ATP is a potent agonist of surfactant phosphatidylcholine (PC) exocytosis from type II pneumocytes in culture. We studied P1 and P2 receptor signal transduction in type II pneumocytes. The EC50 for ATP on PC exocytosis was 10(-6) M, whereas the EC50 for ADP, AMP, adenosine, and the nonmetabolizable ATP analogue alpha,beta-methylene ATP was 10(-4) M. The rank order of agonists for PC exocytosis was ATP greater than ADP greater than AMP greater than adenosine greater than alpha,beta-methylene ATP. The rank order of agonists for phosphatidylinositol (PI) hydrolysis was ATP greater than ADP, whereas AMP, adenosine, and alpha,beta-methylene ATP did not stimulate PI hydrolysis. ATP (10(-4) M) caused a 15-fold increase in adenosine 3',5'-cyclic monophosphate (cAMP) production, and the nonmetabolizable adenosine analogue 5'-N-ethylcarboxyamidoadenosine (10(-6) M) increased cAMP production threefold. The effects of both these agonists on cAMP production were completely inhibited by the adenosine antagonist 8-phenyltheophylline (10(-5) M). The effects of ATP (10(-4) M) on PC exocytosis were inhibited 38% by 10(-5) M 8-phenyltheophylline. Thus, ATP regulates PC exocytosis by activating P2 receptors, which stimulate PI hydrolysis to inositol phosphate, as well as by activating P1 receptors, which stimulate cAMP production. Interactions between the P1 and P2 pathways may explain the high potency of extracellular ATP as an agonist of PC exocytosis.     

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.     

肝素处理山羊精子体外获能的研究

系统研究了作用浓度、时间和温度以及输卵管上皮细胞和卵丘细胞对肝素处理山羊精子体外获能后的精子活力、质膜完整性、顶体完整率、获能比例及受精和卵裂的影响,为改善山羊精子体外获能效果和研究获能机理提供了必要的数据。主要实验结果如下：1、在获能液中添加5、10、25、50和100μg/mL肝素处理45min时,添加50和100μg/mL肝素精子获能比率最高（分别为55%和56%）,但添加100μg/mL肝素处理后顶体完整率明显（P<0.05）低于对照组。说明山羊精子获能的最佳肝素浓度为50μg/mL。2、肝素作用时间（0, 10, 20, 30, 45, 60 和120 min）的延长,获能精子比例逐渐提高。其中,肝素处理45～120 min各组的获能精子比例差异不显著（P>0.05）,处理120 min组的精子活力和质膜完整率显著低于其它各组。说明50μg/mL肝素处理精子获能的最佳时间是45～60 min。3、在42℃和38.5℃下处理时,获能精子比例显著高于15℃和37℃,但42℃处理后精子活力和顶体完整率显著低于其它温度。因此,385℃为山羊精子获能的最佳温度。4、与输卵管上皮细胞共培养获能精子比例显著高于对照组和卵丘细胞组,但精子活力、质膜完整率和顶体完整率差异不显著。输卵管上皮组的受精率（91.3％）和卵裂率（72.2％）显著高于对照组（81.2％,65.0％）。说明与输卵管上皮细胞共培养能显著提高肝素处理山羊精子体外获能的效果。     

Purine metabolites suppress proliferation of human NK cells through a lineage-specific purine receptor.

NK cell proliferation is suppressed in some patients with cancer by unknown mechanisms. Because purine metabolites released into the extracellular space during cell lysis may affect cell function, we hypothesized that these metabolites could serve as feedback regulators of NK cell proliferation. Sorted NK (CD56+/CD3-) cells were incubated with IL-2 (1000 U/ml) in a 4-day thymidine uptake assay with or without 10-10,000 microM of nucleotides. Adenine nucleotides inhibited NK cell proliferation, with ATP = ADP > 5'-adenylylimidodiphosphate > AMP = adenosine; ADP-ribose and nicotinamide adenine dinucleotide, but not nicotinamide or UTP, caused a dose-dependent suppression of thymidine uptake. A total of 100 microM ATP, a concentration that induced a maximal (80%) inhibition of thymidine uptake, did not inhibit cytotoxic activity against K562 targets. Because NK cells retained the ability to lyse K562 targets 4 days after exposure to 500 microM ATP or 1000 microM adenosine, inhibition of thymidine uptake was not due to cell death. Incubation of NK cells with dibutyryl cAMP and forskolin also suppressed thymidine uptake. Cholera toxin and pertussis toxin suppressed NK cell proliferation. Pertussis toxin did not block the adenine nucleotide effects. Further, ATP, but not adenosine or other nucleotides, markedly increased intracellular cAMP in a dose-dependent manner. The ATP-induced increase in cAMP was specific to cytolytic cells, because CD19+ B cells and CD4+ T cells did not increase their intracellular cAMP. These studies demonstrate that NK proliferation is regulated through purine receptors by adenine nucleotides, which may play a role in decreased NK cell activity. The response to adenine nucleotides is lineage-specific.     

The interaction of pH and cyclic adenosine 3',5'-monophosphate on activation of motility in Triton X-100 extracted bull sperm

The motility of demembranated bull sperm was found to be governed by the concentrations of cyclic adenosine 3', 5'-monophosphate (cAMP) and Ca2+ at low pH (6.6-7.1), and was less sensitive to these variables at higher pH (7.4-7.8). Although motility was generally found to increase with increasing pH in the range from 6.6 to 7.8, the addition of exogenous cAMP markedly and selectively improved the motility at the lower end of the range (pH 6.6-7.1). In the presence of 10 microM cAMP, low Ca2+ (8.0 X 10(-8) M), and a high concentration of Mg-adenosine 5'-triphosphate (ATP, 8 mM), demembranated sperm at pH 6.8 and 7.1 exhibited swimming similar to that of live ejaculated sperm. At a free Ca2+ concentration of 4.4 X 10(-5) M, the motility was rapidly inhibited at pH 6.8-7.1, whereas at pH 7.4-7.8, the activity was not greatly affected. Since calcium is known to antagonize the cAMP pathway by activating Ca2+-dependent phosphodiesterase and Ca2+-dependent phosphatase, this further supports the idea that cAMP-dependent activation is crucial for motility at low pH. Our results demonstrate that the flagellar axoneme can function normally at relatively acidic pH, and produce vigorous swimming at high levels of ATP. The ATP content of live sperm was measured and found to be high enough (approximately 8 mM) to support the vigorous motility seen at pH 6.6-7.1 in the models.(ABSTRACT TRUNCATED AT 250 WORDS)     

The lack of effect of cyclic adenosine 3':5'-monophosphate on Avena coleoptile growth

The effects of cyclic adenosine 3':5'-monophosphate (cAMP) on the growth of Avena coleoptile segments over 4 to 10 hours were monitored with a position sensing transducer. At pH 6, cAMP (0.1 mm with and without 2.5 mm glucose; or 2 mm alone) or dibutyryl cAMP (0.1 mm) was added at the beginning of the experiment, or after about 1 hour or after about 6 or 7 hours. Under all conditions tested, cAMP compounds had little or no effect on coleoptile segment elongation. Inasmuch as cAMP does not duplicate the rapid and vigorous elongation obtained with 2 mum auxin, the hypothesis that cAMP is a mediator of auxin activity is not supported by experimental evidence in this system. This conclusion is dependent upon the assumption that the cAMP compounds penetrated the tissue.     

Respiratory burst and NAD(P)H oxidase activity are involved in capacitation of cryopreserved bovine spermatozoa

Heparin (a glycosaminoglycan) and quercetin (a calcium-ATPase plasma membrane specific inhibitor) induce bovine sperm capacitation. Mitochondria from frozen semen are capable of generating oxidative energy. The aim of the study was to determine oxygen uptake variation and the participation of diphenileneiodonium (DPI)-sensitive oxidases from spermatozoa capacitated with heparin or quercetin. Oxygen uptake was measured polarographically and 2 microM diphenileneiodonium (DPI) was used as a specific inhibitor of NAD(P)H-oxidases. Sperm capacitation was determined by the chlorotetracycline technique. Heparin produced a respiratory burst (17.0+/-3.2 microL O2/h/10(8) spermatozoa; mean+/-S.D.) versus control (11.3+/-0.9 microL O2/h/10(8) spermatozoa; P<0.05). Oxygen uptake and sperm hypermotility were inhibited by cyanide. Treatment with DPI blocked heparin capacitation and oxygen uptake (cyanide-sensitive) decreased to control levels. Respiration of quercetin-treated samples (cyanide-sensitive; 9.7+/-0.7 microL O2/h/10(8) spermatozoa) was not significantly different from the controls; oxygen uptake was not modified by DPI, but quercetin capacitation was inhibited (P<0.05). The effect of DPI with heparin confirmed that oxidases participate in capacitation induction. The addition of superoxide dismutase and/or catalase to heparin- or quercetin-treated samples, failed to modify oxygen uptake and blocked capacitation (P<0.05), suggesting that the superoxide anion (O2*-) participates in the capacitation induction. High mitochondrial activity from heparin-treated samples indicated that energy requirements, especially for hypermotility, were supported by the respiratory chain. Although a respiratory burst was not produced by quercetin, DPI-sensitive-oxidases (O2*- source) were necessary for capacitation. In cryopreserved bovine spermatozoa, heparin- or quercetin-induced capacitation required different levels of mitochondrial energy and DPI-sensitive oxidase activity.     

Metabolic effects of hyperbaric oxygen in postischemic muscle

In traumatic injuries to the extremities, with a circulatory insufficiency, the resultant ischemia leads to decreasing levels of the energy-rich compounds adenosine triphosphate (ATP) and phosphocreatine (PCr) and increasing levels of lactate in muscle. A tourniquet model for temporary ischemia was used to determine if hyperbaric oxygen treatment could enhance the cellular metabolic restitution when the circulation was restored. The circulation of the rat hindlimb was interrupted for 1.5 and 3 hours. After 1.5 hours of ischemia, the levels of adenosine triphosphate, phosphocreatine, and lactate were restored to normal in muscle biopsies taken 5 hours after the ischemia. After 3 hours of ischemia, there were marked reductions of adenosine triphosphate and phosphocreatine and elevated lactate values in the postischemic muscle, indicating severe ischemic damage. Hyperbaric oxygen treatment at 2.5 atm for 45 minutes reduced these changes significantly. A certain number of hyperbaric oxygen treatments were necessary to maintain this effect. It is concluded that repeated hyperbaric oxygen treatments in the postischemic phase stimulate aerobic metabolism.     

Role of membrane-bound 5''-nucleotidase in nucleotide uptake by the moderate halophile Vibrio costicola

Intact cells of Vibrio costicola hydrolyzed ATP, ADP, and AMP. The membrane-bound 5'-nucleotidase (C. Bengis-Garber and D. J. Kushner, J. Bacteriol. 146:24-32, 1981) was solely responsible for these activities, as shown by experiments with anti-5'-nucleotidase serum and with the ATP analog, adenosine 5'-(beta gamma-imido)-diphosphate. Fresh cell suspensions rapidly accumulated 8-14C-labeled adenine 5'-nucleotides and adenosine. The uptake of ATP, ADP, and AMP (but not the adenosine uptake) was inhibited by adenosine 5'-(beta gamma-imido)-diphosphate similarly to the inhibition of the 5'-nucleotidase. Furthermore, the uptake of nucleotides had Mg2+ requirements similar to those of the 5'-nucleotidase. The uptake of ATP was competitively inhibited by unlabeled adenosine and vice versa; inhibition of the adenosine uptake by ATP occurred only in the presence of Mg2+. These experiments indicated that nucleotides were dephosphorylated to adenosine before uptake. The hydrolysis of [alpha-32P]ATP as well as the uptake of free adenosine followed Michaelis-Menten kinetics. The kinetics of uptake of ATP, ADP, and AMP also each appeared to be a saturable carrier-mediated transport. The kinetic properties of the uptake of ATP were compared with those of the ATP hydrolysis and the uptake of adenosine. It was concluded that the adenosine moiety of ATP was taken up via a specific adenosine transport system after dephosphorylation by the 5'-nucleotidase.     

(S)-α-Chlorohydrin Inhibits Protein Tyrosine Phosphorylation through Blocking Cyclic AMP - Protein Kinase A Pathway in Spermatozoa

α-Chlorohydrin is a common contaminant in food. Its (S)-isomer, (S)-α-chlorohydrin (SACH), is known for causing infertility in animals by inhibiting glycolysis of spermatozoa. The aim of present work was to examine the relationship between SACH and protein tyrosine phosphorylation (PTP), which plays a critical role in regulating mammalian sperm capacitation. In vitro exposure of SACH 50 μM to isolated rat epididymal sperm inhibited PTP. Sperm-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDS) activities, the intracellular adenosine 5'-triphosphate (ATP) levels, 3'-5'-cyclic adenosine monophosphate (cAMP) levels and phosphorylation of protein kinase A (PKA) substrates in rat sperm were diminished dramatically, indicating that both glycolysis and the cAMP/PKA signaling pathway were impaired by SACH. The inhibition of both PTP and phosphorylation of PKA substrates by SACH could be restored by addition of cAMP analog dibutyryl-cAMP (dbcAMP) and phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Moreover, addition of glycerol protected glycolysis, ATP levels, phosphorylation of PKA substrates and PTP against the influence of SACH. These results suggested SACH inhibited PTP through blocking cAMP/PKA pathway in sperm, and PTP inhibition may play a role in infertility associated with SACH.     

Effect of adenosine compounds (ATP, cAMP) on renin release in vitro.

Renin release by surviving canine renal cortical slices incubated media with ATP or cAMP at concentrations of 5 X 10(-5)--5 X 10(-3) M has been studied. Both adenosine compounds were significantly increasing renin release. A linear correlation was observed between their dose and the renin activity of the medium. The difference between the effects of ATP and cAMP appeared to be caused by phosphodiesterase, since the difference was eliminated if to the medium containing cAMP 5 X 10(-2) M theophylline, a phosphodiesterase inhibitor was added.     

Serotonin produces a configurational change of cultured smooth muscle cells that is associated with elevation of intracellular cAMP.

Early passaged bovine pulmonary artery smooth muscle cells (SMC) respond to serotonin (5-HT) by developing a reversible change in configuration. (Lee et al. J. Cell. Physiol. 138:145, 1989). This configurational change does not occur in pulmonary artery endothelial cells (EC) subjected to 5-HT and is adenosine triphosphate (ATP) dependent, lost with passage of SMC, and inhibited by various agents that block high-affinity 5-HT uptake. We now report a second configurational change (also dendritic formation) of SMC produced by 5-HT only in the presence of isobutylmethylxanthine (IBMX), an inhibitor of phosphodiesterase. This configurational change was also ATP dependent, but unlike the first response, (Lee et al., 1989), it occurred in both first and later passaged SMC and was not inhibited by blockade of 5-HT uptake. Also, unlike the response with 5-HT alone that failed to elevate cAMP, this one was associated with a large elevation of cAMP (eight fold above control values), similar to the response to the beta-agonist isoproterenol, plus IBMX. The second response was not blocked by a variety of 5-HT receptor antagonists but was reproduced by (+/-)-8-hydroxy-DPAT HBr (8-OH-DPAT), a reputed 5-HT1A agonist. The response was not dependent upon Ca2+ and was blocked by 1-2 mM n-phenylanthranilic acid or anthracene-9-carboxylic acid, electrically conductive Cl- channel inhibitors. Hence, 5-HT in the presence of IBMX causes a marked elevation of cAMP of SMC and this elevation in cAMP likely results in a cellular configurational change through a Cl- channel-dependent mechanism similar to that we previously described for EC in the presence of beta-adrenergic agonist stimulation (Ueda et al. Circ. Res. 66:951, 1990). EC do not show a similar response to 5-HT possibly because cAMP is not adequately elevated, even in the presence of IBMX, to enhance Cl- channel activity. We propose that our observations indicate the presence of two sites of action of 5-HT on the smooth muscle cell, one intracellularly and another at a cell surface receptor.     

Effect of gas atmosphere on the development of one-cell bovine embryos in two culture systems

The effect of two concentrations of oxygen on the development of bovine embryos was compared using two separate co-culture systems. In Experiment I, bovine oocytes were matured and fertilized in vitro and were then co-cultured for 7 days in 20 mul drops of M199 with 10% fetal calf serum containing oviduct cells. When cultures were performed in an atmosphere of 5% CO(2) in air (20% O(2)) or in a mixture of 5% CO(2), 5% O(2) and 90% N(2) (5% O(2)), 22 of 179 (12%) and 56 of 179 (31%) zygotes developed to or beyond the late morula stage (P<0.0001), respectively. After freezing, thawing and 48 hours of additional culture, 2 of 21 (10%) and 18 of 53 (34%) embryos were judged viable (P<0.001) within the respective treatment groups. In Experiment II, zygotes produced by the same means were co-cultured in 0.5 ml of M199 containing 10% fetal calf serum with monolayers of buffalo rat liver (BRL) cells. In 20% O(2), 51 of 177 (29%) zygotes developed into viable embryos, while in 5% O(2) only 9 of 177 (5%) were judged viable after 7 days of culture (P<0.0001). Post-freezing survival rates were 53% and 67% for embryos from the two respective oxygen concentration treatment groups. The transfer of 20 Grade 1 frozen/thawed embryos produced by co-culture with BRL cells produced six pregnancies (30%). These experiments show that the critical effect of oxygen concentration on embryo development in vitro and the ability of embryos produced by in vitro procedures to survive freezing can be influenced by the type of culture system employed.     

Relationship between intracellular ATP and the sodium pump activity in dog renal tubules

To examine the potential effect of the cellular ATP concentration and of the phosphate potential on the function of the sodium pump in intact renal cells, the ATP content of dog cortical tubules was first modified by a 30-min preincubation with one of the following effectors: 5 or 10 mM fructose, 2.5 mM adenosine 5'-monophosphate (AMP), or 2.5 mM adenosine in the presence of substrates (10 mM glutamine + 1 mM glutamate with either 10 mM lactate (low ATP) or 10 mM pyruvate (high ATP)). The tubules were then incubated in Krebs-Henseleit saline using two different phosphate concentrations and the same substrate mixture. The ATP content in tubular cells was modified by these treatments, ranging from 2.2 to 5.7 mM. The oxygen uptake by the tubules was measured before and after application of a small amount of nystatin (0.05 mM, 6 mumol/g wet wt.), added to impose an identical and submaximal increment of work to the Na(+)-K+ ATPase in tubules, irrespective of their ATP condition. This manoeuvre was followed by the addition of 1 mM ouabain to inhibit the sodium pump and quantify the respiration related to the activity of the Na+ pump. No significant effect of the ATP content on the respiratory cost of the Na(+)-K+ ATPase activity was noted when the [ATP] was above the normal concentration of approximately 3.0 mM before or after introduction of nystatin. In a second group of experiments, tubules were treated with 0.1 mM digitonin (13 mumol/g wet wt.) and resuspended in intracellular-like and sodium-free medium. The respiration was measured before and after the addition of increasing Mg-ATP concentrations (0-12 mM). A fixed quantity of Na+ (20 mM) was then introduced before ouabain was applied. The oxygen uptake was measured in these three conditions. We observed a fixed increment of ouabain-sensitive respiration upon stimulation of the pump activity by sodium at ATP concentrations ranging from 2 to 7 mM. The same observation applied when the free energy released from ATP hydrolysis ranged from -50 to -56 kJ.mol-1 and when the [ATP]/[ADP].[Pi] ratio ranged from 1.5 to 7.5 mM-1. These results suggest that the Na+:ATP stoichiometry of the Na(+)-K+ ATPase is not modified by [ATP] in dog cortical tubules when the ATP content is at or above the physiological value. Furthermore, the stoichiometry of the pump does not appear to change when the phosphate potential and (or) the free energy released from ATP hydrolysis are altered.     

Effects of parathyroid hormone and cyclic AMP analogues on the activity of ornithine decarboxylase and expression of the differentiated phenotype of chondrocytes in culture

Parathyroid hormone (PTH) greatly increased the level of adenosine 3', 5' cyclic monophosphate (cAMP) in rabbit costal chondrocytes in culture 2 minutes after its addition. PTH, as well as N6 O2' dibutyryl adenosine 3', 5' cyclic monophosphate (DBcAMP) and 8 Bromo adenosine 3', 5' cyclic monophosphate (8 Br-cAMP) induced ornithine decarboxylase (ODC; L-ornithine carboxylyase; EC 4.1.1.17), which reached a maximum 4 hours after their addition. Neither cAMP, N6 O2' dibutyryl guanosine 3', 5' cyclic monophosphate (DBcGMP), nor sodium butyrate increased the activity of the enzyme. PTH had no effect on DNA synthesis, while DBcAMP and 8 Br-cAMP decreased DNA synthesis. Expression of the differentiated phenotype of chondrocytes in culture was also induced by PTH, DBcAMP, and 8 Br-cAMP, but not by cAMP, DBcGMP, or sodium butyrate, as judged by morphological change. Glycosaminoglycan synthesis, a characteristic of the cartilage phenotype, began to increase 8 hours after addition of PTH or DBcAMP, reaching a plateau 32 hours after their addition. These findings suggest that PTH induces increase of ODC activity and expression of the differentiated phenotype of chondrocytes through increase of cAMP and that induction of OCD is closely related to expression of the differentiated phenotype of chondrocytes.     

Hamster sperm Na+, K+-adenosine triphosphatase: increased activity during capacitation in vitro and its relationship to cyclic nucleotides

These in vitro studies of golden hamster sperm were undertaken to determine whether: Na+, K+-adenosine triphosphatase (ATPase) activity is required for capacitation; Na+, K+-ATPase activity is altered during capacitation; and cyclic nucleotides can control this enzyme activity. Hamster sperm were incubated in a medium in which capacitation occurred in an asynchronous manner and in which acrosome reactions began to occur after approximately 3.5 h of incubation. Inhibition of the hamster sperm acrosome reaction by the Na+, K+-ATPase inhibitor ouabain (1 microM) added at Time (T) = 2 or T = 3 h could be fully reversed by the addition of the ionophore nigericin (0.1 microM) at T = 3.5 h. However, when ouabain was added at T = 0 or T = 1 h, similar nigericin addition could not completely reverse the inhibition. Na+, K+-ATPase activity of hamster sperm increased by 2 h of incubation (compared to that measured initially after 15 min) and this activity remained elevated at 3.5 h. Addition of either monobutyryl cyclic adenosine 3':5'-monophosphate ( BtcAMP ) (12.9 microM) or monobutyryl cyclic guanosine monophosphate ( BtcGMP ) (10.5 microM), or the phosphodiesterase inhibitor SQ20009 (10 microM) at 2 h produced a stimulation of acrosome reactions at 4 and 5 h. However, while BtcGMP and SQ 20009 also induced a further increase in Na+, K+-ATPase activity measured at 3.5 h, BtcAMP had no effect. Intracellular cAMP and cGMP levels measured showed cAMP increased by 2 h and remained elevated when measured at 3.5 h, while cGMP could not be consistently detected at 15 min, 2 h or 3.5 h. However, assays of high numbers of uncapacitated sperm did detect a low level of cGMP. These results suggest that Na+, K+-ATPase activity increases in and is essential for early capacitation [and thereby eventually for the acrosome reaction (AR)] of hamster sperm and that the increase in Na+, K+-ATPase activity occurring during capacitation is probably mediated by intracellular cGMP but not cAMP, although both cyclic nucleotides stimulate the hamster sperm AR.     

Uptake of cyclic adenosine-3,5-monophosphate by cultured mammalian cells and frog muscles

Uptake of 3H-cAMP by isolated frog sartorii muscles and cultured mouse 3T3 and 3T6 cells was studied. It was shown that after 1-2 hours of incubation the intracellular level of cAMP in frog muscles reached 10-20% of its concentration in the incubation medium. About 50% of intracellular radioactivity was represented by chromatographically pure cAMP which testifies to the insignificant cAMP metabolism rate. In the experiments with 3T3 and 3T6 cells the influence of possible admixtures and degradation products on 3H-cAMP uptake was revealed. To avoid these influences it is necessary to measure the uptake of cAMP in the presence of theophylline and with abundance of adenosine. In such experimental conditions the intracellular level of cAMP after 1 hour of incubation did not exceed 10% of its extracellular level, which is similar to values obtained on frog muscles. Permeability coefficient of cAMP for membrane of frog muscles and 3T3 and 3T6 cells was about 10(-9)-10(-8) cm X sec-1.     

ATP and related purines stimulate motility,spatial congregation,and coalescence in red algal spores

Adenosine 5′‐triphosphate (ATP) is a versatile extracellular signal along the tree of life, whereas cAMP plays a major role in vertebrates as an intracellular messenger for hormones, transmitters, tastants, and odorants. Since red algal spore coalescence may be considered analogous to the congregation process of social amoeba, which is stimulated by cAMP, we ascertained whether exogenous applications of ATP, cAMP, adenine, or adenosine modified spore survival and motility, spore settlement and coalescence. Concentration‐response studies were performed with carpospores of Mazzaella laminarioides (Gigartinales), incubated with and without added purines. Stirring of algal blades released ADP/ATP to the cell media in a time‐dependent manner. 10–300 μM ATP significantly increased spore survival; however, 1,500 μM ATP, cAMP or adenine induced 100% mortality within less than 24 h; the exception was adenosine, which up to 3,000 μM, did not alter spore survival. ATP exposure elicited spore movement with speeds of 2.2–2.5 μm · s^?1. 14 d after 1,000 μM ATP addition, spore abundance in the central zone of the plaques was increased 2.7‐fold as compared with parallel controls. Likewise, 1–10 μM cAMP or 30–100 μM adenine also increased central zone spore abundance, albeit these purines were less efficacious than ATP; adenosine up to 3,000 μM did not influence settlement. Moreover, 1,000 μM ATP markedly accelerated coalescence, the other purines caused a variable effect. We conclude that exogenous cAMP, adenine, but particularly ATP, markedly influence red algal spore physiology; effects are compatible with the expression of one or more membrane purinoceptor(s), discarding adenosine receptor participation.     

The activation of motility in quiescent hamster sperm from the epididymis by calcium and cyclic nucleotides

Control is exerted on the movement of mammalian spermatozoa at ejaculation and at capacitation. Here the activation of motility in motionless pre-ejaculated sperm was investigated. This was done by isolating quiescent caudal epididymal sperm from the hamster and observing that the addition of either calcium cAMP, cGMP, or cUMP conferred full motility upon them. Other salts, nucleotides, caffeine, sugars, or oxygen did not. Epididymal fluid which contains phosphodiesterase had too little calcium to activate the sperm while seminal plasma had more than enough. The cAMP content of quiescent sperm was low, but ATP levels were high. At the activation of motility, sperm cAMP synthesis became very rapid. It thus appears that sperm are quiescent on the male because they lack cAMP, and that calcium, supplied at ejaculation, initiates rapid cAMP synthesis to produce motility.