Excretion and degradation of exogenous adenosine 3',5'-monophosphate by isolated perfused rat kidney

To determine the role played by the kidney in the metabolism and excretion of plasma adenosine 3′,5′-monophosphate (cAMP) we have studied the fate of this nucleotide (0.01–1.0mM) when it is perfused in a recirculating medium through the isolated rat kidney. cAMP was rapidly taken up and degraded by the kidney, the rate of its disappearance from the perfusate being at least twice its rate of excretion in the urine. Nevertheless, the cAMP excretory rate exceeded the filtration rate by 1.5 to 2 fold, and thus net secretion (transtubular transport) was demonstrated. The rates of filtration, perfusate clearance, and degradation of cAMP were proportional to its perfusate concentration. Methyl xanthines (caffeine and aminophylline) at 10mM, and probenecid at 0.9mM abolished transtubular transport of cAMP and greatly retarded disappearance of the nucleotide from the perfusate. It is concluded that there is a ready penetration of cAMP into renal cells from peritubular capillaries. Depending on the perfusate concentration of cAMP, transtubular transport may or may not exceed the simultaneous intra-renal breakdown of the compound. A low rate of cAMP excretion in the urine may accompany a considerably higher rate of cAMP clearance from the perfusate by the kidney.     

Cyclic adenosine 3',5'-monophosphate and coryneform bacteria

Abstract We have detected cyclic AMP in the culture medium of amino acid-producing coryneform bacteria. Extracellular concentrations of cyclic AMP vary depending on the nutritional medium, the growth phase and the carbon source. It is also shown that the activity of Brevibacterium flavum adenylate cyclase in intact cells is stimulated in the presence of glucose. Furthermore, addition of cyclic AMP to B. flavum cultures decreased amino acid production similarly to the effect produced by the addition of inorganic phosphate.     

Cyclic adenosine 3',5'-monophosphate analogues modulate rat placental cell growth and differentiation

Cyclic adenosine 3',5'-monophosphate (cAMP) has been implicated in the control of placental function. The present investigation was designed to evaluate the actions of cAMP analogues on the control of rat placental development. Two model systems were used to assess the actions of cAMP in the placenta: 1) a rat placental cell line and 2) rat labyrinth placental explants. Elevation of intracellular cAMP via treatment with cAMP analogues, 3-isobutyl-1-methylxanthine, forskolin, or cholera toxin inhibited placental cell DNA synthesis whereas treatment with an analogue to cyclic guanosine 3',5'-monophosphate was without effect. The inhibitory actions of dibutyryl cAMP on DNA synthesis were at least partially reversible and were not the result of metabolic toxicity. Dibutyryl cAMP had dramatic effects on the organization and morphology of placental cells growing in vitro and diminished the ability of the placental cells to grow following transplantation into allogeneic hosts. Differentiation-associated characteristics of rat placental cells were also affected by cAMP. cAMP analogues stimulated placental cell progesterone release and inhibited placental cell alkaline phosphatase activity. Dibutyryl cAMP had effects on placental labyrinth explants similar to its effects on the placental cell line. Dibutyryl cAMP inhibited explant outgrowth while stimulating explant release of progesterone. In summary, cAMP effectively modulates the growth and differentiation of rat placental cells in vitro.     

Cyclic adenosine 3', 5'-monophosphate in Neisseria gonorrhoeae.

Intracellular cyclic adenosine 3', 5'-monophosphate (cAMP) was measured in two laboratory strains of Neisseria gonorrhoeae. Decreasing the glucose content of a defined media from 33 mM to 5.5 mM glucose resulted in an 11-to 25-fold increase of intracellular cAMP.     

Histamine induces ovulation in the isolated perfused rat ovary

Ovulatory effects of histamine and specific antagonists were studied in isolated perfused ovaries from immature rats treated with 10 i.u. PMSG to stimulate follicular growth and maturation. Histamine alone, like LH, induced ovulation in all ovaries tested, but the number of follicular ruptures was lower after histamine (7.0 and 2.2 ruptures, respectively, per ovary). The histamine-induced ovulations could be inhibited dose-dependently by the H1-receptor antagonist, pyrilamine, or the H2-antagonists, cimetidine and ranitidine. At the concentrations tested, these antagonists did not, when given separately, reduce the LH-induced ovulations significantly, but pyrilamine and cimetidine in combination lowered the ovulation frequency by 65%. The prostaglandin synthesis inhibitor, indomethacin, was not able to block the histamine-induced ovulations.     

Regulation of prostaglandin endoperoxide synthase by cyclic adenosine 3',5'-monophosphate in the in vitro-perfused rat ovary

The preovulatory regulation of two enzymes in the prostaglandin biosynthetic pathway, prostaglandin endoperoxide synthase (PGS) and prostacyclin synthase (ISN), was examined in granulosa cells and residual tissue of rat ovaries perfused in vitro. Ovaries from rats primed with pregnant mare's serum gonadotropin (20 IU) were perfused for up to 20 h starting the morning of induced proestrus. The amounts of PGS and ISN present were analyzed with immunoblotting techniques. Soluble extracts from granulosa cells and residual ovarian tissues were obtained at different times (0 h, 3 h, 7 h, 12 h) after treatment in vitro with luteinizing hormone (LH, 0.1 microgram/ml) and 3-isobutyl-1-methylxanthine (IBMX; 0.2 mM) and at 7 h in untreated control ovaries or after treatment with forskolin (30 microM) or LH (0.1 microgram/ml). The levels in the perfusion medium of cyclic adenosine 3',5'-monophosphate (cAMP), progesterone, testosterone, and estradiol were measured and the number of ovulations were examined. The levels of PGS after treatment with LH + IBMX increased up to 7 h and remained high at 12 h, a time that is close to the time of ovulation. The increase was more pronounced in the granulosa cells than in the residual tissue. Treatment with forskolin induced synthesis of PGS in granulosa cells, and the levels at 7 h were similar to those after stimulation with LH + IBMX. The levels of PGS were lower in granulosa cells of the group stimulated with LH alone than in granulosa cells from ovaries stimulated with LH + IBMX or forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic adenosine 3',5' monophosphate, calcium and protein phosphorylation in flagellar motility

cAMP and calcium are two important regulators of sperm flagellar motility. cAMP stimulates sperm motility by activating cAMP-dependent protein kinase and catalyzing the phosphorylation of sperm proteins. The stimulation of sperm motility by cAMP appears to be at two different levels. Evidence has been presented to suggest that cAMP-dependent phosphorylations may be required in order for motility to be initiated. In addition, cAMP-dependent phosphorylation appears to modulate specific parameters of motility resulting in higher beat frequency or greater wave amplitude. Calcium, on the other hand, when elevated intracellularly to 10(-6) M or higher, inhibits flagellar motility. The calcium-binding protein, calmodulin, appears to mediate a large number of effects of calcium on motility. Evidence suggests that calcium-calmodulin may be involved at the level of the membrane to pump calcium out of the flagellum. In addition, calcium-calmodulin may be involved in the control of axonemal function by regulating dynein ATPase and myosin light chain kinase activities. The identification of cAMP-dependent protein kinase, calmodulin and myosin light chain kinase in the sperm head suggests that cAMP and calcium-dependent phosphorylations are also involved in the control of the fertilization process, i.e., the acrosome reaction, in a manner similar to that known for the control of stimulus/secretion coupling. Finally, the effects of cAMP on flagellar motility are mediated by protein phosphorylation while the effects of calcium on motility are also in part, mediated by effects on protein phosphorylation.     

Cyclic response of hypophysectomized rats to ovulation induced by LHRH agonist: mediation by prostaglandins

Further confirmation that the LHRH/LHRH agonist-induced ovulation in the hypophysectomized (hypx) rat is due to a direct ovarian effect and not mediated by LH release from residual pituitary tissue or other CNS sites is provided by the persistence of this effect despite concomitant median eminence lesion or passive immunization to LH. Adrenalectomy did not affect the ovulatory activity of the LHRH agonist, D-Trp6-N alpha MeLeu7-DesGly10-Pro9-NHEt-LHRH (Wy-40,972), in the hypx rat. Prior administration of a potent LHRH antagonist blocked ovulation induced in hypx proestrous rats by Wy-40,972 but not by LH-S19. Ovulation can be induced by Wy-40,972 one day earlier (e.g. metestrus) in the intact rat than it can in the hypx rat. Results in the hypx metestrous rat indicate that the ovulatory responsiveness of the intact rat at this stage of the cycle may occur by complementary action of Wy-40,972-stimulated endogenous LH release and a direct ovarian effect of the agonist. Prostaglandins (PG) are involved in the ovulatory mechanism of Wy-40,972 in the hypx proestrous rat as evidenced by the dose-dependent inhibition of this effect by PG synthetase inhibitors, indomethacin and Fentiazac. Moreover, there were significant increases in ovarian concentrations of PGF2 alpha and PGE2-PGE1 in response to Wy-40,972 that could be prevented by indomethacin. However, exogenous administration of either of these PG's was not effective in inducing ovulation in the hypx rat.     

Accumulation of adenosine 3',5'-monophosphate in rat brain slices: effects of prostaglandins.

Prostaglandin E₁ and E₂ and 15(S)-15-methyl PGE₂ methyl ester stimulate the accumulation of radioactive cyclic AMP in brain slices from Sprague-Dawley rats, labelled during a prior incubation with [¹⁴C] adenine. Prostaglandins A₁ and B₁ have marginal effects and prostaglandin F_1α has no effect. Relatively high concentrations of about 80 μM PGE₁, PGE₂ and 15(S)-15-methyl PGE₂ are required to elicit a maximal 2–5 fold increase in accumulation of cyclic AMP in slices from cerebrum, but significant increases are elicited by 3.5 μM prostaglandin. Similar increases are elicited in slices from neocortex, striatum or midbrain-thalamus-hypothalamus, while lesser increases pertain in slices from cerebellum, medulla-pons or hippocampus. The accumulation of cyclic AMP elicited by PGE₁ in slices from cerebrum was not blocked by naloxone, propranololphentolamine, tetracaine, theophylline, or by nearly equimolar concentrations of either of two prostaglandin antagonists, 7-oxa-13-prostynoic acid and the dibenzoxazepine hydrazide, SC 19220. Morphine potentiated the effects of PGE₁. The combination of 85 μM PGE₁ with either isoproterenol, norepinephrine, adenosine or veratridin did not increase the accumulation of cycli AMP significantly above those elicited by the isoproterenol, norepinephrine, adenosine or veratridine alone. The combined effect of PGE₁ and norepinephrine in the presence of a β-adrenergic antagonist, sotalol, was, however, additive. The results indicate that PGE₁ stimulates cyclic AMP formation in rat brain slices, but that it either has antagonist activity with respect to accumulations of cyclic AMP-elicited by other agents or has no detectable agonist activity when cyclases are maximally stimulated by other agents.     

Inhibition of follicle-stimulating hormone-induced ovulation by indomethacin in the perfused rat ovary

In isolated, perfused ovaries of rats treated with pregnant mare's serum gonadotropin (PMSG), purified preparations of ovine follicle-stimulating hormone (FSH) (oFSH-211B) and rat FSH (rFSH-I-6), 100 ng/ml, were found to induce ovulations (4.8 +/- 0.9, n = 4, and 6.4 +/- 2.0, n = 5, ovulations per ovary, respectively). Indomethacin (5 micrograms/ml) added to the perfusate inhibited this ovulatory effect and exogenous prostaglandin F2 alpha (PGF2 alpha) (1 microgram/ml), or prostaglandin E2 (PGE2) (0.5 microgram/ml), reversed the blockade. Ovine FSH and rFSH had only a weak stimulatory effect on estradiol release, and only rFSH caused a significant increase in progesterone accumulation. Indomethacin reduced the stimulatory effect of rFSH on progesterone release, and this effect was reversed by PGE2 but not by PGF2 alpha. In a 6-h incubation experiment with preovulatory rat follicles, we tested the biological activity of gonadotropins used to induce oocyte maturation. The concentration of FSH used in the perfusion experiments induced oocyte maturation in more than 88% of the oocytes studied. The data confirm earlier findings that FSH can induce ovulations and show that prostaglandins are involved in this process. The data also indicate that prostaglandins might be involved in the FSH-induced increase of progesterone levels.     

Transport and metabolism of N6- and C8-substituted analogs of adenosine 3',5'-cyclic monophosphate and adenosine 3'5'-cyclic phosphorothioate by the isolated perfused rat kidney

The clearance and metabolism of N6-substituted (N6-dimethyl-), C8-substituted (8-bromo-, 8-p-chlorophenylthio- (PCPT-)), and exocyclic oxygen substituted phosphorothioate diastereomers (cAMPS(Sp)) and cAMPS (Rp)) of adenosine 3':5'-monophosphate (cyclic AMP, cAMP) has been studied in an isolated perfused rat kidney. The N6- and C8-substituted analogs of cyclic AMP (10-100 microM) were not cleared as rapidly as exogenous cyclic AMP and were metabolized: N6- and C8-substituted analogs of adenosine accumulated in perfusate and urine. All analogs exhibited net transtubular secretion, i.e. their urinary excretion rate greater than glomerular filtration rate. Probenecid (0.9 mM) included in the perfusate abolished transtubular secretion and inhibited the metabolism of PCPT-cyclic AMP, suggesting that cyclic AMP analogs, like cyclic AMP itself, penetrate the renal cell at the peritubular membrane by an organic acid transport system. The phosphorothioate diastereomers of cyclic AMP: cAMPS(Sp) and cAMPS(Rp) were cleared as rapidly from the perfusate as cyclic AMP, were extensively secreted (urinary excretion/ glomerular filtration greater than or equal to 10) and exhibited no metabolism. The latter analog would seem most suitable as an intracellular agonist for cyclic AMP-mediated phenomena in the rat kidney.     

Inhibition of ovulation by tyrosine kinase inhibitors in the in vitro perfused rat ovary

Protein tyrosine kinase activity, leading to tyrosine phosphorylation of the intracellular domains of receptors or non-receptor proteins, is an important feature of downstream signalling after receptor binding of a variety factors, such as growth factors and cytokines. Since several members of these classes of paracrine-autocrine mediator may be involved in the intraovarian events of ovulation, the present study was designed to evaluate the effect of protein tyrosine kinase inhibition on the in vitro perfused rat ovary. Immature rats were primed with 20 iu pregnant mares' serum gonadotrophin 48 h before surgical isolation of the right ovary with connecting vasculature. The ovary was placed in a perfusion system for either 10 h, to examine ovarian concentrations of the established ovulatory mediators plasminogen activator, prostaglandins E2 and F2 alpha, or for 20 h, enabling a complete ovulatory process to occur in vitro. Ovulation was induced by ovine LH (0.2 microgram ml-1) in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.2 mmol l-1) and the effects of two different protein tyrosine kinase inhibitors, genistein and tyrphostin A25, were studied. Unstimulated control ovaries did not ovulate and showed low secretion of progesterone and oestradiol. Addition of LH + 3-isobutyl-1-methylxanthine resulted in a marked stimulation of steroid release, and ovulations occurred in all ovaries (9.0 +/- 0.9; mean +/- SEM). The protein tyrosine kinase inhibitors, genistein and tyrphostin A25, significantly inhibited ovulation at the higher concentrations tested (3.0 +/- 0.3 at 100 mumol genistein l-1; 5.8 +/- 1.0 at 500 mumol tyrphostin A25 l-1) but no effect was seen at lower concentrations. The presence of genistein and tyrphostin A25 at any concentration used did not significantly decrease the LH + 3-isobutyl-1-methylxanthine-induced progesterone or oestradiol concentrations. The intraovarian concentrations of plasminogen activator activity, and prostaglandin E2 and F2 alpha were not altered by the presence genistein (100 mumol l-1). In conclusion, the results of the present study indicate that protein tyrosine kinase signalling pathways are integral parts of the mammalian ovulatory process but do not involve actions on the synthesis of steroids, plasminogen activator or prostaglandins.