cyclic-gmp and 8-phenyltheophylline

Previously, it had been observed that nitric oxide (NO) contributes to hypoxia-induced pial artery dilation in the newborn pig. Additionally, it was also noted that activation of ATP-sensitive K+ channels (KATP) contribute to cGMP-mediated as well as to hypoxia-induced pial dilation. Although somewhat controversial, adenosine is also thought to contribute to hypoxic cerebrovasodilation. The present study was designed to investigate the role of NO, cyclic nucleotides, and activation of KATP channels in the elicitation of adenosine's vascular response and relate these mechanisms to the contribution of adenosine to hypoxia-induced pial artery dilation. The closed cranial window technique was used to measure pial diameter in newborn pigs. Hypoxia-induced artery dilation was attenuated during moderate (PaO2 approximately 35 mm Hg) and severe hypoxia (PaO2 approximately 25 mm Hg) by the adenosine receptor antagonist 8-phenyltheophylline (8-PT) (10(-5) M) (26 +/- 2 vs. 19 +/- 2 and 34 +/- 2 vs. 22 +/- 2% for moderate and severe hypoxia in the absence vs. presence of 8-PT, respectively). This concentration of 8-PT blocked pial dilation in response to adenosine (8 +/- 2, 16 +/- 2, and 23 +/- 2 vs. 2 +/- 2, 4 +/- 2, and 6 +/- 2% for 10(-8), 10(-6), and 10(-4) M adenosine before and after 8-PT, respectively). Similar data were also obtained using adenosine deaminase as a probe for the role of adenosine in hypoxic pial dilation. Adenosine-induced dilation was associated with increased CSF cGMP concentration (390 +/- 11 and 811 +/- 119 fmol/ml for control and 10(-4) M adenosine, respectively). The NO synthase inhibitor, L-NNA, and the cGMP antagonist, Rp 8-bromo cGMPs, blunted adenosine-induced pial dilation (8 +/- 1, 14 +/- 1, and 20 +/- 3 vs. 3 +/- 1, 5 +/- 1, and 8 +/- 3% for 10(-8), 10(-6), and 10(-4) M adenosine before and after L-NNA, respectively). Adenosine dilation was also blunted by glibenclamide, a KATP antagonist (9 +/- 2, 14 +/- 3, 21 +/- 4 vs. 4 +/- 1, 8 +/- 2, and 11 +/- 2% for 10(-8), 10(-6), and 10(-4) M adenosine before and after glibenclamide, respectively). Finally, it was also observed that adenosine-induced dilation was associated with increased CSF cAMP concentration and the cAMP antagonist, Rp 8-bromo cAMPs, blunted adenosine pial dilation. These data show that adenosine contributes to hypoxic pial dilation. These data also show that NO, cGMP, cAMP, and activation of KATP channels all contribute to adenosine induced pial dilation. Finally, the

Topics: Adenosine; Adenosine Deaminase; Adenosine Triphosphate; Animals; Arteries; Blood Pressure; Cyclic AMP; Cyclic GMP; Enkephalin, Leucine; Enkephalin, Methionine; Female; Glyburide; Hypoxia; Male; Nitric Oxide; Nucleotides, Cyclic; Pia Mater; Potassium Channels; Swine; Theophylline; Vasodilation; Vasodilator Agents

1. We have used the isolated buffer-perfused mesenteric arterial bed of the rat to assess the modulation of vasorelaxation to potassium channel openers (KCOs) by basal nitric oxide. 2. The dose-response curves to the KCOs, levcromakalim and pinacidil, in preconstricted preparations were significantly shifted to the left in the presence of the nitric oxide synthase inhibitor (100 microM) NG-nitro-L-arginine methyl ester (levcromakalim, ED50 = 4.47 +/- 0.70 nmol vs. 1.73 +/- 0.26 nmol, P < 0.001; pinacidil, ED50 = 16.1 +/- 4.8 nmol vs. 5.43 +/- 1.10 nmol, P < 0.001). The vasorelaxant responses to papaverine, a vasodilator which acts independently of potassium channels was unaffected by NG-nitro-L-arginine methyl ester (L-NAME). 3. Removal of the endothelium, by perfusion with the detergent CHAPS (0.3%), significantly (P < 0.001) increased the potency of levcromakalim as a vasodilator (ED50 4.47 +/- 0.70 nmol vs. 2.59 +/- 0.31 nmol). The subsequent administration of L-NAME following perfusion with CHAPS did not lead to any additional enhancement of responses to levcromakalim. 4. The presence of the non-selective adenosine antagonist, 8-phenyltheophylline (8-PT, 10 microM) significantly (P < 0.001) shifted the dose-response curve to levcromakalim to the left (ED50 4.47 +/- 0.70 nmol vs. 1.11 +/- 0.32 nmol). In the presence of both L-NAME and 8-PT, the dose-response curve to levcromakalim was also significantly (P < 0.01) shifted to the left compared with control (ED50 in the presence of both L-NAME and 8-PT was 0.42 +/- 0.08 nmol). 5. The presence of 8-bromo cyclic GMP (10 microM) reversed the increase potency of levcromakalim, observed following inhibition of nitric oxide synthase (ED50 in the presence of L-NAME was 0.59 +/- 0.01 nmol and in the presence of 8-bromo cyclic GMP plus L-NAME the ED50 was 3.17 +/- 0.80 nmol). However in the absence of L-NAME, the cell permeable analogue of cyclic GMP, 8-bromo cyclic GMP, did not affect the dose-response curve to levcromakalim compared with control (control ED50 value was 4.16 +/- 0.52 nmol vs. 3.85 +/- 1.13 nmol in the presence of 8-bromo cyclic GMP). 6. The present investigation demonstrates that both basal nitric oxide and adenosine modulate vasorelaxation to the KCOs levcromakalim and pinacidil. The modulatory effect of nitric oxide may be mediated via cyclic GMP.

Topics: Animals; Benzopyrans; Cromakalim; Cyclic GMP; Dose-Response Relationship, Drug; Guanidines; In Vitro Techniques; Male; Mesenteric Artery, Superior; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroprusside; Papaverine; Pinacidil; Potassium Channels; Pyrroles; Rats; Rats, Wistar; Stereoisomerism; Theophylline; Vasodilation; Vasodilator Agents

In the perfused guinea-pig heart reactive hyperaemia (RH) after occlusion of coronary flow (1-60 s) was inhibited by 100-60% with NG-nitro-L-arginine (100 microM) and to a lesser extent (by 35%) after 8-phenyltheophylline (10 microM), but not by indomethacin (5 microM). Inhibition of adenosine deaminase by erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) (5 microM) not only increased the concentration of adenosine in the coronary perfusate, but also prolonged the duration of RH. RH induced cardiac generation of prostacyclin, nitric oxide and adenosine as indicated by the appearance of 6-keto-PGF1 alpha, cyclic GMP, adenosine, inosine, hypoxanthine, xanthine and urate in the perfusate. Only NO and adenosine, but not prostacyclin, were responsible for RH. RH after short-term (1-10 s) coronary occlusion was mediated by NO, whereas adenosine and NO maintained RH that followed after longer (20 s-10 min) periods of cardiac ischaemia. Prostacyclin never participated in the mediation of RH.

Topics: 6-Ketoprostaglandin F1 alpha; Adenosine; Animals; Coronary Circulation; Cyclic GMP; Cyclooxygenase Inhibitors; Electrocardiography; Endothelins; Epoprostenol; Guinea Pigs; Hyperemia; In Vitro Techniques; Indomethacin; Myocardial Ischemia; Myocardium; Nitric Oxide; Nitric Oxide Synthase; Perfusion; Prostaglandin Antagonists; Purinergic P1 Receptor Antagonists; Purines; Theophylline

The inflammatory mediator adenosine caused sustained Cl- secretion across monolayers of T84 cells. The effect was promptly reversed by the adenosine receptor antagonist 8-phenyltheophylline and appeared to be mediated through an adenosine A2-receptor [rank order of potency: 5'-(N-ethyl)-carboxamido-adenosine (NECA) greater than adenosine greater than (-)-N6-(phenylisopropyl)adenosine (PIA) greater than or equal to (+)-PIA]. High doses of adenosine and its analogues increased cellular adenosine 3',5'-cyclic monophosphate (cAMP) but not guanosine 3',5'-cyclic monophosphate (cGMP) or free cytosolic Ca2+. However, lower concentrations of adenosine had maximal effects on Cl- secretion with little or no effect on cAMP. In other respects, Cl- secretion resembled that induced by cAMP-mediated secretagogues such as vasoactive intestinal peptide (VIP). Addition of both low and high doses of NECA activated basolateral K+ and apical Cl- channels, exhibited synergism with Ca2(+)-mediated secretagogues, did not produce additive effects with VIP or Escherichia coli heat-stable enterotoxin, and was associated with cAMP-dependent protein kinase-mediated protein phosphorylation. The results suggest that either adenosine mobilizes an intracellular pool of cAMP that is extremely efficiently coupled to the cAMP-dependent protein kinase and is thereafter rapidly destroyed or that second messenger(s) other than cAMP, cGMP, or Ca2+ are able to activate Cl- secretion in the T84 cell line. In the latter case, such messenger(s), as yet unidentified, might represent a final common pathway for cyclic nucleotide-activated Cl- secretion.

Topics: 2-Chloroadenosine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Calcium; Cell Line; Chlorides; Cyclic AMP; Cyclic GMP; Cytosol; Electrolytes; Epithelium; Histamine; Humans; Inflammation; Kinetics; Phenylisopropyladenosine; Phosphorylation; Protein Kinases; Theophylline

The effect of GABA and related drugs on 3'5' cAMP and 3'5' cGMP was investigated in slices of guinea-pig cerebral cortex, kept at rest or electrically stimulated. GABA 1 X 10(-4) - 1 X 10(-3) M raised the cAMP basal levels, bud reduced its increase due to electrical stimulation. These effects were antagonized by picrotoxin 1.6 X 10(-5) M. On the contrary, endogenous GABA did not influence cAMP and cGMP content. In fact, neither ethanolamine-O-sulphate 2 X 10(-3) M nor picrotoxin 1.6 X 10(-5) M affected the normal nucleotide values at all. Higher picrotoxin concentrations (3.2 - 8 X 10(-5) M), however, increased both cyclic nucleotides. Phentolamine counteracted the actions of both GABA 1 X 10(-4) M and picrotoxin 8 X 10(-5) M: therefore, the observed increase in cyclic nucleotides were due to norepinephrine release. Tetrodotoxin antagonized GABA but not picrotoxin metabolic effects. Thus it is suggested that the exogenous amino acid released norepinephrine through a sodium-dependent process, while picrotoxin released the monoamine directly from its storage sites. Clearly, attention must be paid when putative transmitters or their antagonists are added at high concentrations to isolated tissue to study the effect of endogenous compounds.

Topics: Adenosine; alpha-Methyltyrosine; Animals; Cerebral Cortex; Cyclic AMP; Cyclic GMP; Electric Stimulation; GABA Antagonists; gamma-Aminobutyric Acid; Guinea Pigs; Methyltyrosines; Neurons; Norepinephrine; Phentolamine; Picrotoxin; Reserpine; Theophylline