AMP Accumulation Research Articles

Extracellular matrix modulates beta2-adrenergic receptor signaling in human airway smooth muscle cells.

The airways of patients with chronic asthma commonly develop an element of fixed airway obstruction, which fails to reverse with inhaled beta2-adrenoceptor agonists. Airway remodeling refers to the structural changes of the bronchi in longstanding asthma and is characterized by increased deposition and altered ratios of extracellular matrix (ECM) proteins. We therefore assessed whether ECM proteins alter beta2-adrenoceptor signaling in human airway smooth muscle cells. We report that a fibronectin environment increases responses to beta2-adrenoceptor stimulation, whereas exposure to collagen V or laminin decreases accumulation of the second messenger cyclic AMP when compared with collagens I or IV. These differences are likely to be physiologically significant as they translate into altered phosphorylation of the downstream target VASP. The altered cAMP levels are due to differences in adenylyl cyclase activity, although expression of the relevant isoforms of enzyme appears unaltered. However, inhibition of Galphai abrogates the differences in beta2-adrenoceptor-mediated cAMP accumulation in cells exposed to different matrix factors. The difference in Galphai signaling is not due to altered Galphai expression. We conclude therefore that ECM modulates Galphai activity in human airway smooth muscle cells, and propose that these changes could contribute to the fixed airway obstruction seen in patients with chronic asthma.

CARDIORESPIRATORY RESPONSES TO EXERCISE AND THE C34T AMPD1 GENE POLYMORPHISM IN THE HERITAGE FAMILY STUDY

An inherited form of AMPD deficiency has been attributed to a non-sense mutation (C to T transition in nucleotide 34) in exon 2 of AMPD1 gene. AMPD deficiency causes AMP accumulation, which can lead to the production of adenosine by AMP 5'-nucleotidase. It is known that adenosine accumulation exerts a vasodilatory response, which could have physiological consequences during exercise. PURPOSE To examine associations of the C34T polymorphism of the AMPD1 gene with cardiorespiratory and performance phenotypes during cycling exercise at absolute and relative power outputs progressing to exhaustion before and after endurance training for 20 weeks in the HERITAGE Family Study cohort (n = 779). Since no Blacks were mutant homozygotes (TT), only Caucasians were considered for analysis (400 normal homozygotes, CC; 97 heterozygotes, CT; and 6 TT). RESULTS Sedentary State. Cycling at the absolute workload of 50 W resulted in higher rate of perceived exertion in TT (p < 0.0001). At the relative intensity of 60% of VO2 max, stroke volume was lower in TT (p < 0.05). Maximal values for power output, SBP, HR, VCO2, and RER were lower in TT (p < 0.05). Response to Training. The cardiorespiratory training response at 50 W and at 60% of VO2 max was similar across C34T-AMPD1 genotypes. However, max ventilation, max VO2, and max VCO2 during exercise increased less in TT (p < 0.01). CONCLUSION The results indicate that subjects with the TT genotype at the C34T AMPD1 gene have diminished exercise capacity and cardiovascular and ventilatory responses to exercise. Endurance training improves submaximal exercise cardiorespiratory and performance phenotypes in carriers of the C34T mutation as much as in non-carriers, but the training response of ventilatory phenotypes during maximal exercise is more limited in TT. Supported by multiple grants from NIH

Purinergic regulation of glucose and glutamine synthesis in isolated rabbit kidney–cortex tubules

The effects of extracellular purinergic agonists and their breakdown products on glucose and glutamine synthesis in rabbit kidney–cortex tubules incubated with aspartate+glycerol or alanine+glycerol+octanoate were investigated. A rapid extracellular degradation of ATP was accompanied by an accumulation of AMP, inosine, and hypoxanthine. Extracellular ATP and its breakdown products accelerated glucose synthesis in renal tubules, while ammonium released from adenine-containing compounds enhanced glutamine synthesis and diminished the degree of gluconeogenesis stimulation. In contrast to AMP and inosine, ATP evoked calcium signals, while both ATP and inosine decreased intracellular cAMP content and accelerated the flux through fructose-1,6-bisphosphatase as concluded from changes in gluconeogenic intermediates. Since (i) the activity of partially purified renal fructose-1,6-bisphosphatase was increased upon protein phosphatase-1 treatment and decreased following treatment of previously dephosphorylated enzyme with protein kinase A catalytic subunit and (ii) both 8-bromoadenosine 3 ′,5 ′-cyclic monophosphate and 8-(4-chlorophenyltio)-cAMP inhibited renal glucose synthesis, it seems likely that in rabbit renal tubules ATP and inosine stimulate gluconeogenesis via cAMP decrease, which favors the appearance of a more active, dephosphorylated form of fructose-1,6-bisphosphatase, a key gluconeogenic enzyme.

Cyclic AMP-dependent secretion of Ca 19-9 by LS174T human colon carcinoma cells

Prolonged increase of cyclic adenosine-monophosphate (cAMP) level in culture medium of a human colon cancer cell (LS174T) inhibits cellular growth and stimulates Ca 19-9 expression. The raise in cAMP level was produced by dibutyryl cyclic AMP (DBcAMP) or by forskolin an agent acting at the level of cAMP generation. Both these agents in a range of concentration between 10(-3)-10(-5) M have an inhibitory effect on the growth which is dose and time dependent. The inhibition was reversible as demonstrated by complete restoration of cell growth soon after the withdrawal of the substances from the culture medium. When cAMP levels in culture medium was raised, an increase in Ca 19-9 expression was observed and it appears that cyclic nucleotides have at least two effects: the first to cause rapid release of already synthesized Ca 19-9 and second to stimulate new antigen synthesis. The findings of the present study demonstrated that LS174T cells are unable to proliferate upon sustained accumulation of intracellular cyclic AMP suggesting the use of strategies able to increase cAMP levels for therapy of colon cancer. Furthermore, the finding that cAMP may also be a regulator of Ca 19-9 synthesis and release indicates the utility of cell line LS174T as a model for studies on the mechanism of synthesis and secretion of specific tumoral markers in colon cancer.

Postnatal Handling Induces Long-term Modifications in Central β-noradrenergic Signalling in Rats

Neonatal handling has been shown to induce a short-term reduction in the binding properties of g -adrenoceptors and in their primary biochemical responses in the young rat brain, which may account for the reduced responsiveness to stress observed in the handled rats. We have studied the persistence and duration of these changes in cerebral cortex, cerebellum and hippocampus at successive stages of life in neonatally handled rats. Binding properties of the g -adrenoceptors in handled animals were essentially similar to those of the control rats from 3 to 24 months of age. However, handling disturbed the efficacy of the g -adrenoceptor intracellular signalling since the isoprenaline-induced accumulation of cyclic AMP was reduced in cerebral cortex at 1, 12 and 24 months; in cerebellum at 12 and 18 months; and in hippocampus at 3 and 12 months. This effect might be partially dependent on a regulatory action of handling on the adenylyl cyclase enzyme itself since both basal and forskolin-induced accumulation of cyclic AMP were persistently reduced. Therefore, postnatal handling can be considered as an effective intervention that modifies sensitivity to various hormonal and neurochemical signals; these changes may be involved over a long period of time in protection against an excessive response to stress. These results suggest that the long-lasting adaptation of the adenylyl cyclase transduction system, which affects the primary biochemical response of the g -adrenoceptor, may participate in the effects of this manipulation.

Adenosine Diphosphate Ribose Dilates Bovine Coronary Small Arteries through Apyrase- and 5′-Nucleotidase-Mediated Metabolism

Cyclic adenosine diphosphate ribose and adenosine diphosphate ribose (ADPR) play an important role in the regulation of intracellular Ca²⁺ release and K⁺ channel activity in the coronary arterial smooth muscle. The role of these signaling nucleotides in the control of vascular tone has yet to be determined. The present study was designed to determine whether ADPR produces vasodilation in coronary arteries and to explore the mechanism of action of ADPR. ADPR (10–60 µmol/l) was found to produce endothelium-independent relaxation in a concentration-dependent manner in isolated and pressurized small bovine coronary arteries. The ADPR-induced vasodilation was substantially attenuated by adenosine deaminase (0.2 U/ml), and the P₁ purinoceptor antagonist 8-(p-sulfophenyl)theophylline (50 µmol/l), with maximal inhibitions of 60 and 80%, respectively. When the coronary arterial homogenates were incubated with ADPR, the production of adenosine and 5′-AMP was detected. The adenosine production was blocked by the 5′-nucleotidase inhibitor, α,β-methylene adenosine 5′-diphosphate (MADP, 1 mmol/l), which was accompanied by a corresponding accumulation of 5′-AMP. This 5′-AMP accumulation was substantially inhibited by the apyrase inhibitor sodium azide (10 mmol/l). Moreover, ADPR was hydrolyzed into 5′-AMP by purified apyrase. In agreement with their inhibitory effect on the adenosine production, MADP and sodium azide significantly attenuated the vasodilator response to ADPR. The metabolism of ADPR to adenosine was only detected in cultured coronary arterial smooth muscle cells but not in endothelial cells. We concluded that ADPR produces vasodilation in small coronary arteries and that the action of ADPR is associated with the adenosine production via an apyrase- and 5′-nucleotidase-mediated metabolism.

Cyclic AMP metabolism by swine adipocyte microsomal and plasma membranes

Extracellular cyclic AMP is source of extracellular adenosine in brain and kidney. Whether this occurs in adipose tissue is unknown. The present study evaluated the capacity of swine adipocyte plasma membranes to metabolize cyclic AMP to AMP and adenosine, via phosphodiesterase (PDE) and 5′-nucleotidase (5′-NT), respectively. Plasma membranes (PM) and microsomal membranes (MM) were isolated from over-the-shoulder subcutaneous adipose tissue of 3 month-old male miniature swine. The purity of the membrane fractions was determined and PDE and 5′-NT activities in PM and MM fractions were corrected for cross-contamination. The maximal activity of MM-PDE was 7-fold greater than that of PM-PDE. MM-PDE was 100% inhibited by 5 μM cilostamide, while PM-PDE was unaffected by this PDE3B inhibitor. Inhibitors of PDE1, PDE2, PDE4 and PDE5 also failed to inhibit PM-PDE. However, 1 mM DPSPX inhibited PM-PDE activity by 72%. When PM were incubated with 0.8 μM cyclic AMP for 20 min, AMP accumulation was four times that of adenosine. These data demonstrate that cyclic AMP can be converted to AMP and adenosine by the PM-bound enzymes 5′-NT and PDE, and suggest that the PM-PDE responsible for extracellular cyclic AMP metabolism to AMP is distinct from the intracellular MM-PDE.

Cis-urocanic acid attenuates histamine receptor-mediated activation of adenylate cyclase and increase in intracellular Ca2+.

UVB irradiation causes suppression of delayed hypersensitivity. Various photoreceptors and mediators of these changes have been proposed, one of which is cis-urocanic acid formed from the naturally occurring trans-urocanic acid in the epidermis on exposure to UV irradiation. The mechanism by which cis-urocanic acid alters the immune system is not fully clarified, but it acts through different mechanisms, perhaps via histamine or histamine-like receptors. Histamine stimulation of keratinocytes induces activation of adenylate cyclase leading to an accumulation of cyclic AMP and an increase in intracellular Ca2+. Thus we investigated the effects of cis-urocanic acid on these transmembrane signaling systems in keratinocytes. Normal human keratinocytes were cultured in serum-free KGM medium. Cyclic AMP was measured by radioimmunoassay. Alterations in intracellular Ca2+ in single living keratinocytes were measured using an inverted fluorescence microscope and an ARGUS-200/CA digital imaging system. Cis-urocanic acid itself did not induce adenylate cyclase activation in cultured normal human keratinocytes. Cis-urocanic acid inhibited histamine-induced cyclic AMP accumulation. It did not affect keratinocyte growth, and did not induce an increase in intracellular Ca2+, but did attenuate the histamine-induced increase in intracellular Ca2+ but not that induced by epinephrine. Cis-urocanic acid acts on keratinocytes via modulation of the effects of histamine.

Activation of phospholipase C and guanylyl cyclase by endothelins in human trabecular meshwork cells

PURPOSE. To characterize effects of endothelins on activities of phospholipase C (PLC) and nucleotide cyclases in human trabecular meshwork (TM) cells. METHODS. Cultured simian virus 40-transformed human TM (HTM-3) or non-transformed (HTM-16) cells were used. Changes in the PLC activity were determined by assaying the production of [ 3 H] inositol phosphates. Accumulation of cyclic GMP or cyclic AMP in cell lysate was measured by radioimmunoassay. RESULTS. Endothelin-1 (ET-1; 1 µM) stimulated PLC in HTM-16 cells, but Sarafotoxin S6c (SRTX), an ET B receptor subtype- selective agonist (1 µM), did not. Similar results were obtained in HTM-3 cells: ET-1, but not ET-3 or SRTX, activated PLC in a dose-dependent manner, with a calculated EC 50 of 646 pM. The peptide also stimulated the accumulation of cGMP in a concentration-dependent manner with an EC 50 of 37.2 pM. ET-3 or SRTX was not effective except at much higher concentrations. Both the PLC and guanylyl cyclase stimulation induced by ET-1 (10 nM) were completely inhibited by pretreating the cells with BQ-123 (<10 µM), an ET A receptor selective antagonist, but not by BQ-788 (10 µM), an ET B receptor subtype-specific antagonist. Neither ET-1 nor ET-3 stimulated adenylyl cyclase activity in HTM-3 cells at concentration as high as 1 µM. COCLUSIONS. ET-1 activates PLC and guanylyl cyclase in TM cells. Potency profiles of ET receptor agonists and antagonists suggest that the ET A receptor subtype is involved in both actions of ET-1. The effects of the ET peptides in TM cells are interesting and could be part of the mechanism of their IOP-lowering effect.

Induction mechanism of nerve growth factor synthesis by Kami-untan-to; Role of cyclic AMP and c-fos mRNA accumulation

Kami-untan-to, a Japanese traditional herbal medicine, induces NGF secretion/synthesis on astroglial cells. However, the intracellular signal transduction and genetic mechanisms associated with KUT action have not been clarified. In this paper, the effects of various protein kinase inhibitors on KUT induced NGF secretion of astroglial cells were examined. Pretreatment of astroglial cells with either K-252a, a nonselective protein kinase inhibitor, or H-89, a selective protein kinase A (PKA) inhibitor blocked the KUT-induced NGF secretion in a dose-dependent manner. Treatment of astroglial cells with KUT or forskolin, an adenylate cyclase activator, led to immediate induction of intracellular cyclic AMP (cAMP). Addition of KUT in astroglial cell cultures also induced expression of c-fos mRNA, and was followed by induction of NGF mRNA. Furthermore, pretreatment with c-fos antisense oligonucleotides significantly inhibited the KUT-induced NGF secretion in astroglial cells. These findings suggest that the activation of cAMP-PKA pathway and the induction of c-fos mRNA may play important roles for an enhancing effect of KUT on NGF secretion in astroglial cells.

Regulation of muscle glycogen phosphorylase activity following short-term endurance training.

The purpose of this study was to examine the regulation (hormonal, substrate, and allosteric) of muscle glycogen phosphorylase (Phos) activity and glycogenolysis after short-term endurance training. Eight untrained males completed 6 days of cycle exercise (2 h/day) at 65% of maximal O2 uptake (Vo2max). Before and after training subjects cycled for 15 min at 80% of Vo2max, and muscle biopsies and blood samples were obtained at 0 and 30 s, 7.5 and 15 min, and 0, 5, 10, and 15 min of exercise. Vo2max was unchanged with training but citrate synthase (CS) activity increased by 20%. Muscle glycogenolysis was reduced by 42% during the 15-min exercise challenge following training (198.8 +/- 36.9 vs. 115.4 +/- 25.1 mmol/kg dry muscle), and plasma epinephrine was blunted at 15 min of exercise. The Phos a mole fraction was unaffected by training. Muscle phosphocreatine utilization and free Pi and AMP accumulations were reduced with training at 7.5 and 15 min of exercise. It is concluded that posttransformational control of Phos, exerted by reductions in substrate (free Pi) and allosteric modulator (free AMP) contents, is responsible for a blunted muscle glycogenolysis after 6 days of endurance training. The increase in CS activity suggests that the reduction of muscle glycogenolysis was due in part to an enhanced mitochondrial potential.

Estrogen Uncouples Steroidogenesis from 3′,5′-Cyclic Adenosine Monophosphate Regulation in the Rabbit Corpus Luteum1

The hypothesis was investigated that estradiol, the luteotrophic hormone in the rabbit, uncouples luteal progesterone production from regulation by LH/cyclic AMP. Progesterone production by corpus luteum (CL) incubated with vehicle, 3-isobutyl-1-methyl-xanthine (IBMX), hCG, or hCG+IBMX was compared in pseudopregnant rabbits treated continuously with estradiol (estradiol-maintained), withdrawn from estradiol for 24-48 h (estradiol-withdrawn), or withdrawn and then replaced with estradiol for 6 or 24 h (estradiol-replaced). Progesterone production in estradiol-maintained rabbits was not altered by hCG and/or IBMX, but was stimulated significantly in estradiol-withdrawn rabbits. This response was reversed (i.e., abolished) in estradiol-replaced (24 h) rabbits. The loss of responsiveness to hCG was not attributable to impaired accumulation of cyclic AMP: basal and hCG-stimulated cyclic AMP concentrations were similar in luteal tissues of estradiol-maintained and estradiol-withdrawn rabbits. The loss of responsiveness to hCG was also not a consequence of maximal progesterone production: CL of estradiol-replaced (6 h) rabbits were also insensitive to hCG, and this occurred before progesterone production attained a maximal rate. We conclude that a striking feature of the luteotrophic action of estrogen is to uncouple the regulation of progesterone production from cyclic AMP.

Endothelin synthesis and receptors in human endometrium throughout the normal menstrual cycle

This study was undertaken to investigate the presence of messenger RNA (mRNA) for prepro-endothelin-I (ET-1) and the known receptor subtypes (ETA and ETB) in human endometrium at different stages of the menstrual cycle obtained at hysterectomy. Northern blot analysis revealed expression of ET-1 mRNA in human endometrium during the normal menstrual cycle. The concentration of ET-1 mRNA in endometrial tissue was greater during the menstrual and proliferative phases than during the ovulatory and secretory phases. Immunoreactive ET-1 was secreted into the medium of isolated endometrial stromal cells. Oestradiol and progesterone significantly attenuated ET-1 release in endometrial stromal cells cultured for 6 days. ETA and ETB mRNA were also present in endometrial tissue of the normal cycle. The concentration of ETA receptor mRNA was greater in the proliferative phase than in the secretory phase, whereas expression of ETB mRNA increased in menstrual phase. ET-1 significantly increased extracellular accumulation of cyclic AMP (cAMP), intracellular generation of inositol phosphates and significantly enhanced DNA synthesis in cultured endometrial stromal cells from the proliferative phase. Our results showed that human endometrial cells synthesized and released ET-1, and contained ETA and ETB receptors which were functionally coupled to phosphoinositide breakdown and to adenylate cyclase with the increase of cAMP by ET-1 stimulation. Our findings suggest that ET-1 may have a potential autocrine and/or paracrine function in human endometrial stromal cells.

Effects of dopamine on adenylyl cyclase activity and amylase secretion in rat parotid tissue.

Several previous studies have shown that dopamine causes amylase secretion from rat parotid tissue. However, the mechanism of this dopamine action is still unclear. The present study was designed to characterize dopamine action in rat parotid gland tissue by examining the effects of dopamine on cyclic AMP accumulation, adenylyl cyclase activity, and amylase release. Dopamine significantly enhanced accumulation of cyclic AMP in parotid slices and stimulated adenylyl cyclase activity in parotid membrane preparations. It also significantly stimulated amylase release from parotid slices. The stimulatory effects of dopamine on cyclic AMP accumulation, adenylyl cyclase activity, and amylase release were effectively blocked with propranolol, a beta-adrenergic antagonist, but not by either SCH 23390, a preferential D1 antagonist, or butaclamol, a preferential D2 antagonist. No substantial specific binding sites for D1 receptors were detectable by [3H]SCH 23390 binding in parotid membranes. These results suggest that the stimulatory effect of dopamine on amylase secretion in rat parotid tissue is not mediated through specific D1 dopamine receptors but rather through beta-adrenergic receptors.

Endothelin synthesis and receptors in human endometrium throughout the normal menstrual cycle

This study was undertaken to investigate the presence of messenger RNA (mRNA) for prepro-endothelin-I (ET-1) and the known receptor subtypes (ETA and ETB) in human endometrium at different stages of the menstrual cycle obtained at hysterectomy. Northern blot analysis revealed expression of ET-1 mRNA in human endometrium during the normal menstrual cycle. The concentration of ET-1 mRNA in endometrial tissue was greater during the menstrual and proliferative phases than during the ovulatory and secretory phases. Immunoreactive ET-1 was secreted into the medium of isolated endometrial stromal cells. Oestradiol and progesterone significantly attenuated ET-1 release in endometrial stromal cells cultured for 6 days. ETA and ETB mRNA were also present in endometrial tissue of the normal cycle. The concentration of ETA receptor mRNA was greater in the proliferative phase than in the secretory phase, whereas expression of ETB mRNA increased in menstrual phase. ET-1 significantly increased extracellular accumulation of cyclic AMP (cAMP), intracellular generation of inositol phosphates and significantly enhanced DNA synthesis in cultured endometrial stromal cells from the proliferative phase. Our results showed that human endometrial cells synthesized and released ET-1, and contained ETA and ETB receptors which were functionally coupled to phosphoinositide breakdown and to adenylate cyclase with the increase of cAMP by ET-1 stimulation. Our findings suggest that ET-1 may have a potential autocrine and/or paracrine function in human endometrial stromal cells.

Ovarian hormones regulate alpha 1- and beta-adrenoceptor interactions in female rat pinealocytes.

We have examined the effects of alpha 1- and beta-receptor activation on cyclic AMP (cAMP) accumulation in cultured pinealocytes from female ovariectomized rats, and from intact rats at proestrus treated with the antiestrogen Tamoxifen, the antiprogestagen RU486, or with both. Isoproterenol (a beta-agonist) significantly increased cAMP levels in pinealocytes from intact and ovariectomized rats. This response was considerably enhanced in pinealocytes from rats treated with RU486 or Tamoxifen alone, but not with both. Moreover, incubation of pinealocytes with isoproterenol together with phenylephrine (an alpha 1-agonist) produced a synergistic effect in animals that had been ovariectomized, or that had received RU486, either alone or in combination with Tamoxifen. These results suggest that, in female rats, ovarian steroids may regulate the interactions between alpha 1- and beta-adrenergic receptors, and thus modulate pineal melatonin synthesis during the oestrous cycle through these mechanisms.

The recovery of ovine pars tuberalis cells from melatonin-induced sensitization is a slow, protein synthesis-dependent phenomenon.

The pars tuberalis (PT) of the anterior pituitary is characterized by the presence of a high concentration of melatonin receptors, and acute exposure of cells from this tissue to melatonin inhibits the accumulation of cyclic AMP (cAMP) stimulated by forskolin. Conversely, exposure of ovine PT (oPT) cells to melatonin for periods of up to 16 h causes a progressive increase in subsequent basal and forskolin-stimulated production of cAMP. These observations are consistent with the possibility that the PT is involved in the mediation of melatonin-dependent phenomena in mammals. If the chronic effects of exposure to melatonin are indeed functionally significant, then one would anticipate that those responses of oPT cells known to be dependent upon levels of cAMP would also show an enhanced response to stimulation following prolonged exposure to the hormone. In the present study, the activation of cAMP-dependent protein kinase and the synthesis of secretory protein by oPT cells were found to be sensitized by prolonged exposure to physiological concentrations of melatonin. In the case of the synthesis of secretory protein this effect of melatonin was confined to those proteins whose synthesis has been shown to be sensitive to melatonin in acute experiments. These observations support the hypothesis that melatonin-induced sensitization modulates the putative biosynthetic and secretory function of the PT. The present study also examined the mechanism of sensitization of oPT cells by melatonin. The development of sensitization was not affected by simultaneous exposure of oPT cells to forskolin (1 microM) during pretreatment with melatonin. This observation suggests that melatonin-induced sensitization occurs independently of the established acute effects of the hormone on cAMP levels in oPT cells. Since no effects of melatonin upon any other signalling cascade have been observed in these cells, the most plausible explanation for this finding is that sensitization is a direct consequence of prolonged activation of melatonin receptors. Such a mechanism might be linked to the partial down-regulation of melatonin receptors known to occur in oPT cells in response to prolonged exposure to the hormone. In order to test this hypothesis further, the process of recovery from the sensitizing effects of melatonin was examined. The recovery of oPT cells from the sensitizing effects of exposure to melatonin (100 pM, 16 h) took place gradually and, even after an interval of 16 h, cells that had previously been exposed to melatonin for 16 h remained sensitized to approximately 20% of the extent seen immediately following pretreatment with melatonin for 16 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Acidosis during ischemia promotes adenosine triphosphate resynthesis in postischemic rat heart. In vivo regulation of 5'-nucleotidase.

Capacity for ATP resynthesis during recovery from ischemia or hypoxia is limited to the size of the adenine nucleotide pool, which is determined in part by the activity of cytosolic 5'-nucleotidase (5'-NT): AMP-->adenosine plus inorganic phosphate (Pi). To define in vivo regulation of 5'-NT, we used the tools of 31P nuclear magnetic resonance (NMR), spectroscopy and chemical assay to measure the substrates (AMP), products (Pi, adenosine, and its catabolites), and inhibitors (Pi and H+) of 5'-NT in isolated perfused rat hearts exposed to hypoxia (where pH remains near 7) and no flow, global ischemia (where pH falls to 6.1). We estimated 5'-NT reaction velocity, assessed the relative contributions of Pi and H+ to enzyme inhibition, and defined the consequences of changes in 5'-NT activity on ATP resynthesis after hypoxia and ischemia. We conclude that (a) 5'-NT is activated during hypoxia and early ischemia but is inhibited during prolonged ischemia, (b) H+ (pH < 6.2) is a potent inhibitor of 5'-NT, and (c) differences in AMP accumulation are sufficient to explain the differences in the capacity for net ATP resynthesis in ischemic and hypoxic tissue. These observations have implications for our understanding of heterogeneity of ischemic injury and myocardial protection during ischemia.

Suppression of plasminogen activator inhibitor type 1 release from cultured human umbilical vein endothelial cells by basic fibroblast growth factor

We investigated the release of plasminogen activator inhibitor type 1 (PAI-1) from cultured vascular endothelial cells after exposure to basic fibroblast growth factor (bFGF). Treatment with human recombinant bFGF of confluent cultures of endothelial cells derived from human umbilical vein resulted in a reduction of the accumulation of PAI-1 antigen (PAI-1:Ag) in the conditioned medium. The suppressive effect of bFGF completely disappeared in the presence of anti-bFGF antibody. The reduction of endothelial PAI-1:Ag release induced by bFGF was suggested to be independent of intracellular accumulation of cyclic AMP. On the other hand, nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase pathway of arachidonic acid metabolism, suppressed the spontaneous release of PAI-1:Ag by itself; in the presence of NDGA, bFGF failed to further suppress the PAI-1:Ag release. The effect of bFGF and indomethacin, an inhibitor of the cyclooxygenase pathway, was additive on the PAI-1:Ag release. The present data suggest that bFGF reduces the endothelial PAI-1:Ag release via suppression of the putative lipoxygenase pathway which up-regulates a part of the spontaneous PAI-1:Ag release.

Characterization of adenosine receptor-mediated generation of cyclic AMP in slices of rat cerebral cortex with chronic epileptic activity.

Cyclic AMP accumulations elicited by adenosine analogues 2-chloroadenosine (2-CADO), R-N6-phenylisopropyladenosine (R-PIA), and N6-cyclohexyladenosine (CHA) were investigated in cortical slices of chronic iron-induced epileptic rats. Cyclic AMP accumulation was elicited 9- to 18-fold by 2-CADO and it was elicited 5- to 7-fold by either R-PIA or CHA; 2-CADO was more potent than R-PIA or CHA in eliciting cyclic AMP accumulation. The adenosine analogues elicited cyclic AMP accumulation in a dose-dependent manner, and the elicitation was inhibited by the adenosine antagonist 8-phenyltheophylline. The 2-CADO-elicited accumulation of cyclic AMP was greatly increased in the cortical region on the primary epileptic side, while the R-PIA- or CHA-elicited accumulation did not change in any cortical region. The deviation detected only in the 2-CADO-elicited accumulation of cyclic AMP may be due to the difference in relative potency for adenosine receptors of the adenosine analogues. The results suggest that adenosine receptor-mediated generation of cyclic AMP is altered in the primary region of iron-induced epileptic cortex, in which heterogeneous alterations in different adenosine receptor subtypes may occur in the epileptic process.