Renal periarterial nerve stimulation-induced vasoconstriction at low frequencies is primarily due to release of a purinergic transmitter in the rat.

In the isolated rat kidney, the vasoconstrictor response elicited by periarterial nerve stimulation at low frequencies (2 Hz) is resistant to alpha adrenergic receptor blockade. It has been proposed that in some blood vessels ATP is coreleased with norepinephrine during nerve stimulation to activate P2-purinergic receptors and is responsible for the component of the vasoconstrictor response that is resistant to alpha adrenergic receptor blockade. To assess the contribution of a purinergic transmitter in the vasoconstriction elicited by periarterial nerve stimulation in the isolated Tyrodes-perfused rat kidney, fractional overflow of [3H]norepinephrine and vasoconstrictor responses to renal nerve stimulation were examined after alpha adrenergic receptor blockade and/or P2-purinergic receptor desensitization. The alpha-1 adrenergic receptor antagonists prazosin (0.1-1.0 microM) and corynanthine (0.1-1.0 microM) and the nonselective alpha adrenergic receptor antagonist phentolamine (0.1-1.0 microM) did not significantly reduce vasoconstrictor responses elicited by low frequency (0.5-4 Hz) but attenuated the responses to high-frequency (6-10 Hz) periarterial nerve stimulation. At low-frequency renal nerve stimulation, selective P2-purinergic receptor desensitization abolished the vasoconstriction at 0.5 Hz and dramatically attenuated the responses up to 4 Hz. In the presence of prazosin, the component of the vasoconstrictor response that was resistant to alpha adrenergic receptor blockade at all frequencies of renal nerve stimulation was abolished after treatment with alpha, beta-methylene ATP. On the other hand, in the isolated perfused rabbit kidney, prazosin (1.0 microM) alone reduced dramatically the vasoconstrictor responses to periarterial nerve stimulation over the same frequencies used in the rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Maintenance of meiotic arrest in mouse oocytes by purines: modulation of cAMP levels and cAMP phosphodiesterase activity.

Hypoxanthine and adenosine are present in preparations of mouse ovarian follicular fluid, and these purines maintain mouse oocytes in meiotic arrest in vitro (Eppig et al.: Biology of Reproduction 33:1041-1049. 1985). The first hypothesis tested in this study is that purines which maintain meiotic arrest act by maintaining meiosis-arresting levels of cyclic adenosine monophosphate (cAMP) in the oocyte. Oocyte-cumulus cell complexes were incubated in control medium (no added purines), or medium containing 0.75 mM adenosine, 4 mM hypoxanthine, or both for 3 hr and the percentage of the oocytes that underwent germinal vesicle breakdown (GVB) and the cAMP content of the intact complexes and the oocytes were determined. Adenosine alone had little inhibitory effect on GVB at this time point but sustained higher levels of cAMP in the oocytes. Hypoxanthine maintained 80% of cumulus cell-enclosed oocytes in meiotic arrest and also sustained higher cAMP levels in the oocytes. The addition of adenosine to hypoxanthine-containing medium increased the percentage of oocytes maintained in meiotic arrest, and increased the amount of cAMP in the oocytes above that maintained by either hypoxanthine or adenosine alone. Neither hypoxanthine, adenosine, nor hypoxanthine plus adenosine altered the cAMP content of intact complexes when assayed after 3 hr culture. Microinjection of an inhibitor of the catalytic subunit of cAMP-dependent protein kinase induced GVB in denuded oocytes cultured in medium containing hypoxanthine. This purine, therefore, maintained meiotic arrest by sustaining elevated cAMP levels within the oocytes. The second hypothesis tested in this study is that purines maintain meiosis-arresting levels of cAMP, at least in part, by inhibiting cAMP phosphodiesterase activity. In descending order of potency, 3-isobutyl-1-methylxanthine (IBMX), guanosine, hypoxanthine, adenosine, and xanthosine inhibited cAMP phosphodiesterase in oocyte lysates. Moreover, like the potent phosphodiesterase inhibitor IBMX, hypoxanthine augmented the meiotic arrest and cAMP accumulation mediated by follicle-stimulating hormone (FSH) in intact complexes. Therefore, inhibition of oocyte phosphodiesterase appears to be one mechanism by which the purines could maintain meiosis-arresting levels of cAMP.

Modulation of rapid eye movement sleep in humans by drugs that modify monoaminergic and purinergic transmission.

Modulation of rapid eye movement (REM) sleep is a well-established effect of many centrally acting drugs. However, there is uncertainty concerning the nature of the changes and their significance, and it is in this context that we have analyzed the effects of several groups of drugs that alter monoaminergic or purinergic transmission on sleep in humans. The analysis shows that drugs that modulate noradrenergic and serotonergic transmission lead to marked suppression of REM sleep, irrespective of any increase or decrease in sleep duration. There is no evidence that the timing of the ultradian cycle of REM sleep relative to sleep onset is altered by these drugs. On the other hand, reduced REM sleep with dopamimetic drugs is due solely to increased wakefulness. However, there can be more subtle effects of some drugs on REM sleep. Benzodiazepine receptor agonists and drugs that modify purinergic transmission modulate the appearance of early REM activity. There may, therefore, be two discrete systems that control entry into REM sleep, and that are responsive to drugs. The exact appearance and timing of REM periods may be modulated by a feedback mechanism involving GABAergic, or possibly purinergic, transmission, while monoaminergic and cholinergic influences exert a reciprocal and overriding control of REM sleep.

Characterization of a purF operon mutation which affects colicin V production.

A mini-Tn10-kan insertion mutation identified a gene in the chromosome of Escherichia coli required for colicin V production from plasmid pColV-K30. With the complete restriction map of E. coli, the mutation was rapidly mapped to 50.0 min, within the purF operon. Sequence analysis showed that the insertion occurred in a gene with no previously known function which is located directly upstream of purF. We designated this gene cvpA for colicin V production. The mutant requires adenine for growth, probably because of a polar effect on purF expression. However, an adenine auxotroph showed no defect in colicin V production, suggesting that the cvpA mutation is responsible for the effect on colicin V production. Two possible models of cvpA1 allele function are discussed.

Effects of dilazep (Comelian) on the central purinergic system: inhibitory effects on clonidine-induced aggressive behavior.

Mice given clonidine (20-50 mg/kg i.p. and 5 micrograms i.c.v.) exhibited aggressive behavior. Dilazep as well as N6-(L-phenylisopropyl) adenosine (adenosine agonist) inhibited this behavior. Dilazep combined with N6-(L-phenylisopropyl) adenosine markedly inhibited the behavior at low doses that were without effect when given alone whereas the inhibitory effect of dilazep on the behavior was reversed by caffeine and 8-phenyltheophylline, which are adenosine antagonists. The results suggest that the inhibitory action of dilazep on clonidine-induced aggressive behavior can be substantially attributed to central purinoceptor stimulation.

Purinergic supersensitivity following sympathectomy adds further support to co-transmission in the rat vas deferens.

Adrenergic and purinergic compounds contract the longitudinal muscles of the rat vas deferens. Whereas ATP and related purinergic analogs produced contractions of greater magnitude in the prostatic half as compared to that of the epididymal end, the magnitude of the alpha 1-adrenoceptor-induced responses was larger in the epididymal than in the prostatic half of the rat ductus. Chemical sympathectomy following a 48 hr 6-hydroxydopamine-treatment (6-OHDA) caused a leftward displacement of the concentration-response curves for adrenergic and purinergic drugs, this effect being more evident in the prostatic segment. Sympathectomy caused a significant increase in the maximal response induced by ATP and adrenergic compounds which was more evident in the prostatic half of the rat ductus. The denervation-induced supersensitivity was stimulus-specific since angiotensin II and acetylcholine showed no significant change in potency. In the case of bradykinin, there was a manifest increase in the maximal response of the prostatic segment of the ductus of the chemically denervated tissues. In addition, denervation also caused an increase in the potency of prazosin and phentolamine as alpha 1-adrenoceptor blocking agents; denervation did not change the potency of yohimbine as an alpha 2-adrenoceptor blocker.

Endogenous purines may modulate adrenergic neurotransmission in the human fallopian tube.

In the present investigation we have examined the possibility that endogenous purines may act as neuromodulatory agents in the human fallopian tube, using the selective adenosine receptor antagonist 8-p-sulfophenyltheophylline (PSOT). PSOT applied to untreated preparations enhanced the contractile responses to transmural nerve stimulation in the human fallopian tube, both in the secretory and proliferative hormonal phases. Furthermore, PSOT enhanced nerve-induced [3H]noradrenaline release during both hormonal phases. This can be taken as indirect evidence that endogenous purines inhibited the adrenergic neuroeffector transmission under the experimental conditions. The inhibitory effect presumably was exerted at both pre- and postjunctional adenosine receptors.

Effects of reserpine and 6-hydroxydopamine on the adrenergic and purinergic components of sympathetic nerve responses of the rabbit saphenous artery.

1 The effects of reserpine and of 6-hydroxydopamine on the contractions of the rabbit isolated saphenous artery produced by stimulation of the sympathetic nerves were studied. 2 In vessels exposed to reserpine, substantial contractions to nerve stimulation were recorded despite a 95.7% reduction in the noradrenaline content of the tissue. These responses of the vessel were not significantly affected by the alpha 1-antagonist, prazosin, whereas after desensitization of the P2-purinoceptor with alpha, beta-methylene ATP, no response to nerve stimulation remained. 3 In vessels exposed to 6-hydroxydopamine, no nerve-mediated responses were observed. 4 Noradrenaline-containing nerves were observed by fluorescence histochemistry in control tissues, but were not observed in tissues treated with reserpine or 6-hydroxydopamine. 5 The potencies of ATP and histamine were not significantly affected by reserpine or 6-hydroxydopamine treatment. However, there was a slight supersensitivity to noradrenaline in reserpine-treated and 6-hydroxydopamine-treated vessels compared with that of control vessels. Prazosin was selective for alpha-adrenoceptors, while alpha, beta-methylene ATP was selective for P2-purinoceptors. 6 These results substantiate the finding that ATP and noradrenaline are sympathetic cotransmitters in the rabbit isolated saphenous artery, and demonstrate that ATP can act as a transmitter independently of noradrenaline in this vessel.

Modulatory action of purinergic drugs on high potassium-induced epileptiform bursting in rat hippocampal slices.

Increase of the potassium concentration up to 8 mM in the superfused solution of rat hippocampal slices leads to the development of an epileptiform bursting. The derivative agonist L-phenyl isopropyl adenosine (L-PIA) (0.05-0.5 microM) is able to block the potassium induced epileptiform activity. The adenosine antagonist caffeine (100 microM) reverts the antiepileptic effect of L-PIA. Our data show a modulatory action of the purinergic transmission in a model of experimental in vitro epilepsy, and point out about a control of endogenous adenosine in the development of focal epileptiform activity. The relationships between the purinergic influence on the release of neurotransmitters, and the convulsant-anticonvulsant effects of the drugs are discussed.

Effect of different purine auxotrophic mutations on mouse-virulence of a Vi-positive strain of Salmonella dublin and of two strains of Salmonella typhimurium.

Ten cfu of a Vi-positive strain of Salmonella dublin, given i.p. to BALB/c mice, caused death. Of transductional derivatives with nutritional requirements expected to reduce virulence, those with complete blocks at aroA (therefore aromatic-dependent) or at purA (therefore adenine-dependent) were non-virulent; no deaths from i.p. inocula of 10(6) or 10(7) cfu, respectively. Transductants with identified blocks at purF, purG, purC or in the purJHD operon, all with purine requirement satisfied by hypoxanthine or any other purine, were of reduced virulence, with some deaths from 10(4) cfu or, for most of the derivatives, from only 10(2) cfu. Blocks at guaA or guaB, causing guanine requirement, likewise resulted only in reduced virulence, with some deaths from inocula of 10(2). Transductional derivatives of two mouse-virulent (LD50, i.p., less than 25 cfu) strains of S. typhimurium were similarly tested, with generally similar results, in that purA and purB (adenine-requiring) transductants were non-virulent, no deaths from 2.5 x 10(7) cfu, and those with purF, purG or purC defects (hypoxanthine-responding) retained partial virulence. However, the guaA and guaB (guanine-requiring) derivatives were of much reduced virulence; all mice survived 2.5 x 10(5) cfu but only one survived 2.5 x 10(7) cfu. In both the S. dublin and the S. typhimurium tests autopsy cultures showed that fatal infections resulted from multiplication of the auxotrophic strain, not from that of non-exacting revertants.

Is the purinergic system involved in the control of pathological movements?

The EEG and behavioral effects of caffeine, carbamazepine and haloperidol were assessed in adult male rabbits. Caffeine (50 mg/kg i.v.) induced a long-lasting EEG desynchronization (45.6/60 min in mean) which was not modified by pretreatment of the rabbits with carbamazepine or haloperidol. All these drugs, administered alone, failed to induce stereotypy in rabbits. The administration of caffeine in combination with carbamazepine and/or haloperidol induced stereotyped behavior.

Asymmetry in the distributions of the four nucleotides at mRNA initiation and 3' termini sites: some geometrical implications.

All mammalian and non-mammalian vertebrate sequences in the database have been aligned by their transcription initiation sites, and separately, by their mRNA 3' termini. A simple analysis of the distribution of single nucleotide composition in the 1000 nucleotides around these sites yields surprising results. An asymmetric pattern in the behaviour of complementary nucleotides, i.e., C vs. G and A vs. T, in both these sites is observed. The four nucleotides also behave in an opposite manner around mRNA transcription initiation and 3' termini. This may suggest that these are signals affecting the intrinsic dynamics of the DNA structure and as such facilitate the first stages of the recognition process of the RNA polymerase in the neighbourhood of transcription initiation. These signals may also play a role in transcription termination rather than serve as signals for the mRNA cleavage/processing machinery.

Hoogsteen base pairs proximal and distal to echinomycin binding sites on DNA.

Forms of the DNA double helix containing non-Watson-Crick base-pairing have been discovered recently based on x-ray diffraction analysis of quinoxaline antibiotic-oligonucleotide complexes. In an effort to find evidence for Hoogsteen base-pairing at quinoxaline-binding sites in solution, chemical "footprinting" (differential cleavage reactivity) of echinomycin bound to DNA restriction fragments was examined. We report that purines (A greater than G) in the first and/or fourth base-pair positions of occupied echinomycin-binding sites are hyperreactive to diethyl pyrocarbonate. The correspondence of the solid-state data and the sites of diethyl pyrocarbonate hyperreactivity suggests that diethyl pyrocarbonate may be a sensitive reagent for the detection of Hoogsteen base-pairing in solution. Moreover, a 12-base-pair segment of alternating A-T DNA, which is 6 base pairs away from the nearest strong echinomycin-binding site, is also hyperreactive to diethyl pyrocarbonate in the presence of echinomycin. This hyperreactive segment may be an altered form of right-handed DNA that is entirely Hoogsteen base-paired.

Local control of blood pressure by purines.

Dual control of local blood flow by purines is described: adenosine 5'-triphosphate (ATP) released as a cotransmitter with noradrenaline from perivascular sympathetic nerves acts on P2X-purinoceptors on smooth muscle cells to produce vasoconstriction; ATP released from endothelial cells during hypoxia (and ADP released from aggregating platelets) acts on P2Y-purinoceptors on endothelial cells which results in production of endothelium-derived relaxing factor and subsequent vasodilatation. It is suggested that the endothelial-mediated vasodilatation is a pathophysiological mechanism to protect the host tissue (e.g. brain or heart) from damage produced by hypoxia following ischaemia. The ATP released from the endothelial cells is rapidly broken down to adenosine which augments this protective mechanism by acting directly on P1-purinoceptors on vascular smooth muscle to produce a longer lasting component of vasodilatation and on perivascular sympathetic nerve terminals to inhibit release of excitatory neurotransmitters. The possibility that impairment of normal endothelial-mediated responses in atherosclerosis and hypertension can lead to local vasospasm is considered.

The role of purines in the maintenance of meiotic arrest in mouse oocytes.

Meiotic arrest of mammalian oocytes within ovarian follicles is maintained by a specific factor(s) within the follicle. There is strong evidence that cAMP plays an important role in the control of meiosis. Purines have also been implicated in the maintenance of meiotic arrest in vivo. Hypoxanthine and/or adenosine have been identified in pig and mouse follicular fluid and exert a meiosis-arresting action on mouse oocytes in culture. While adenosine apparently need not be metabolized to exert its action on oocyte maturation, the action of hypoxanthine is apparently due to the production of guanyl and/or xanthyl compounds by the oocyte-cumulus cell complex. The inosine monophosphate dehydrogenase inhibitors, mycophenolic acid and bredinin, induced maturation in cumulus cell-enclosed oocytes maintained in meiotic arrest by hypoxanthine. Hypoxanthine and adenosine are not toxic to oocytes, because oocytes undergo normal fertilization and pre- and post-implantation development following exposure to these molecules in vitro. It is not known how gonadotropins stimulate the resumption of meiosis within the follicle, but there are several possibilities: (1) the intrafollicular level of an oocyte maturation inhibitor is decreased; (2) the oocyte is uncoupled from surrounding follicle cells; (3) an inhibitory molecule is secreted or metabolized by the oocyte; and/or (4) a positive stimulus is produced by the follicle that overrides the presence of inhibitory molecules. Preliminary evidence suggests that cumulus cells may produce a positive stimulus that induces the maturation of cultured cumulus cell-enclosed oocytes. Whether germinal vesicle breakdown in vivo results from a positive induction, a loss of inhibitory input, or a combination of these two mechanisms remains to be determined.

Purinergic modulation of T-lymphocyte activation: differential susceptibility of distinct activation steps and correlation with intracellular 3',5'-cyclic adenosine monophosphate accumulation.

The mechanism by which purinergic agonists modulate murine T-lymphocyte activation and proliferation was investigated. Adenosine and other compounds such as ATP and 2-chloroadenosine (ClAdo) were found to block T-cell mitogenesis induced by concanavalin A (Con A) in a dose-dependent fashion. The nonmetabolizable adenosine analog ClAdo was the most potent agent capable of inhibiting T-cell mitogenesis. Extracellular addition of the permeable cAMP analog dibutyryl cyclic AMP (dbcAMP) also led to a dose-dependent blockade of T-cell mitogenesis, although with less efficiency when compared to ClAdo. Addition of IL-2-enriched fluids failed to reverse blockade of T-cell mitogenesis by ClAdo or dbcAMP. ClAdo blocked T-cell enlargement induced after 20 hr of culture with Con A. We analyzed the effect of micromolar concentrations of ClAdo on interleukin-2 (IL-2) production, expression of IL-2 receptors (7D4 and 3C7 surface antigens), and induction of IL-2 responsiveness after in vitro cultivation with Con A. ClAdo inhibited both IL-2 secretion and induction of IL-2 responsiveness up to control levels in the same dose range it inhibited T-cell mitogenesis. However, cell surface expression of IL-2 receptors was not affected. Short incubations of resting splenic T cells with ClAdo led to a dose-dependent accumulation of cyclic AMP in responding cells. This effect was markedly reduced by the purinergic antagonist 3-isobutyl-1-methylxanthine (IBMX) but was not prevented by the adenosine uptake blocker dipyridamole. ClAdo elicited cAMP accumulation in the same dose range it inhibited T-cell activation events. Extracellular administration of dbcAMP to splenic T cells stimulated by Con A mimicked the effects of ClAdo on T-cell activation parameters, as revealed by a dose-dependent blockade of both IL-2 secretion and IL-2 responsiveness induction, without affecting IL-2 receptor expression. Short incubations of Con A-activated T-cell blasts with ClAdo also led to a dose-dependent accumulation of cAMP. We then analyzed the effect of purines and dbcAMP on IL-2-mediated activated T-cell growth. Purines caused a dose-dependent inhibition of IL-2-mediated T-cell proliferation and ClAdo was the most potent purinergic agonist tested. The effect of ClAdo on Con A-induced T blasts was shifted to the right, if compared to earlier T-cell activation steps.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of hypoxanthine on methotrexate-induced differentiation of cultured human choriocarcinoma (BeWo) cells.

When cultured human choriocarcinoma (BeWo) cells are exposed to methotrexate, proliferation ceases and cells undergo a complex differentiative response that resembles development of normal trophoblast. Although thymidylate starvation has been shown to be causative in methotrexate-induced expression of syncytiotrophoblastic markers by BeWo cells, the role of purine deprivation is uncertain since previous studies utilized growth media containing exogenous purines. This work investigated the effects of hypoxanthine on methotrexate-induced cell enlargement, expression of placental alkaline phosphatase, and morphological differentiation to the syncytiotrophoblast-like phenotype. When methotrexate exposures (1 microM, 48 h) were conducted in a purine-free basal medium supplemented with dialyzed fetal bovine serum, RNA synthesis was greatly reduced and cell enlargement did not occur. Specific methods for removing purines (charcoal extraction and xanthine oxidase treatment) decreased the ability of serum to support cell enlargement during methotrexate exposures, whereas addition of hypoxanthine to culture fluids restored its ability to support maximal increases in cell mass, confirming that purines were the factors lost during dialysis. In contrast, morphologically differentiation to the syncytiotrophoblast-like phenotype and increased expression of placental alkaline phosphatase were unaffected by the availability of purines during exposure to methotrexate.