Adenosine analogues stimulate cyclic AMP formation in rabbit cerebral microvessels via adenosine A2-receptors

This study has examined the accumulation of cyclic AMP in microvessels from rabbit and feline cerebral cortex induced by a series of adenosine analogues to determine the type of receptor involved. The conversion of tritium labelled adenine nucleotides to [3H]cyclic AMP was determined in [3H]adenine labelled microvessels in the presence of an inhibitor of cyclic AMP hydrolysis, rolipram (30 microM). In microvessels from both cats and rabbits two adenosine analogues, N-5'-ethylcarboxamido adenosine (NECA) and L (S)-phenylisopropyladenosine (PIA), stimulated cyclic AMP accumulation. The response was larger and more reproducible in rabbits than in cats. In rabbit cerebral microvessels the order of potency as stimulator of cyclic AMP accumulation was NECA greater than 2-chloroadenosine greater than L-PIA greater than cyclohexyladenosine (CHA) greater than D-PIA. This order of potency defines the receptor involved as being of the A2 subtype. Although the maximal response to CHA appeared to be lower than that to NECA, CHA did not inhibit the response to NECA, suggesting that it is not a classical partial agonist. In the presence of the adenylate cyclase stimulating compound forskolin (I microM) NECA was more active than in its absence (close to 30-fold increase in EC50) and also produced a maximal effect six times higher. The maximal responses to PIA and CHA increased proportionally in the presence of forskolin. These results show that rabbit cerebral microvessels possess adenosine receptors of the A2 subtype capable of stimulating the formation of cyclic AMP. The functional significance of such receptors is not known, but may be related to regulation of vascular permeability.     

Presynaptic inhibitory actions of adenine nucleotides and adenosine on neurotransmission in the rat vas deferens.

Adenine nucleotides and adenosine inhibited the isometric contractions of the rat vas deferens in vitro in response to field stimulation but had no effect on the responses to exogenous noradrenaline. The inhibitions were potentiated by dipyridamole and compound 555, antagonized by theophylline and unchanged by indomethacin, 2-2′-pyridylistogen, phenoxybenzamine and atropine. Adenosine and adenosine 5′-triphosphate inhibited the release of [³H]noradrenaline produced by field stimulation.These results indicate that adenine nucleotides, probably acting via the common metabolite adenosine, inhibit adrenergic neurotransmission at a presynaptic site. Their antagonism by theophylline suggests that a presynaptic ‘purinergic’ receptor system could be involved.     

Myocardial adenosine stimulates release of cyclic adenosine monophosphate from capillary endothelial cells in guinea pig heart

Activation of coronary endothelial cell adenylate cyclase was studied in the isolated guinea pig heart by prelabelling endothelial adenine nucleotides using intracoronary infusion of [³H]-adenosine, and measuring the coronary efflux of [³H]-cyclic adenosine monophosphate (cAMP). Hypoxia (30 % O₂) caused a 4-fold increase in coronary release of [³H]-cAMP, which was decreased by 63 % by infusion of the adenosine receptor antagonist, theophylline (50 M). During normoxic control conditions, degrading adenosine to non-vasoactive inosine by intracoronary infusion of adenosine deaminase (1.7 U/ml) caused a 20 % decrease in the release of [³H]-cAMP. The effect of adenosine deaminase was reversed by a specific enzyme inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride. Coronary efflux of [³H]-cAMP during intracoronary infusion of 1 M adenosine triphosphate (ATP), adenosine diphosphate or adenosine monophosphate (AMP) (plus adenosine deaminase 8 U/ml) was only 13 % of that due to 1 M adenosine. Adenosine receptor blockers theophylline and CGS 15943A caused equivalent inhibition of the coronary vasodilator actions of adenosine and ATP. Intracoronary infusion of prostaglandin E₁ and the ₂-adrenergic agonist procaterol caused parallel, dose-dependent increases in coronary conductance and the venous release of [³H] cAMP. It is concluded that (1) under both normoxic and hypoxic conditions, adenosine formed by the heart may activate endothelial cell adenylate cyclase via membrane adenosine receptors, (2) coronary receptors for adenosine and ATP share common ligand affinities but ATP receptors are not coupled to adenylate cyclase, and (3) other vasodilators known to activate endothelial adenylate cyclase in vitro cause parallel increases in coronary conductance and adenylate cyclase activity in the beating heart.     

Purinergic stimulation of cell division and differentiation: mechanisms and pharmacological implications.

Extracellular purine nucleosides and nucleotides in micromolar concentrations stimulate proliferation of a variety of cell types in vitro and in vivo. As well they act synergistically with NGF to stimulate neurite outgrowth from PC12 cells. A variety of purine nucleosides and deoxyribonucleosides promote cell proliferation and increase intracellular cAMP. Their activities are inhibited by adenosine A2 receptor antagonists. Only adenosine interacts with the A2 receptor. We propose that the other nucleosides and deoxyribonucleosides inhibit extracellular adenosine deaminase, thereby increasing the extracellular concentration of adenosine. The nucleotides apparently act by stimulating P2y receptors coupled to inositol phosphate metabolism. We propose that the nucleosides and nucleotides act synergistically with other growth factors because each has distinct but complementary second messenger systems. If our hypotheses are correct, it should prove possible to modulate the growth and morphogenesis in several cell types using drugs that inhibit or stimulate adenosine A2 or purine P2y receptor agonists or the second messenger systems coupled to these receptors.     

The response of lymphatic smooth muscles to vasoactive substances

A study was made of the isotonic response of bovine mesenteric lymphatics to several physiological vasoactive substances. Contractions of lymphatic smooth muscles were induced by serotonin (5-HT), prostaglandin F₂ (PGF₂), noradrenaline (NA), histamine, dopamine and acetylcholine (ACh). The smooth muscles were particularly sensitive to 5-HT. Excepting PGF₂ no other substances could equal 5-HT in the magnitude of the maximum response. The majority of 5-HT receptors seemed to be the D receptors. The decreasing order of the contractile responses was as follows: 5-HT>PGF₂>NA>histamine>dopamine>ACh. The contractile response to ACh was observed only in specimens involving valvular region. It was very likely that, in the lymphatics, there were 2 kinds of receptors for catecholamines, i.e. and receptors, and the stimulation of the former induced smooth muscle contraction and that of the latter relaxation. A difference was noticed between the responses of valvular and intervalvular segments to NA. Relaxations of lymphatic smooth muscles were induced not only by isoproterenol but also by adenosine and adenine nucleotides. The decreasing order of the relaxant responses was as follows: ISP>adenosine >ATP>ADP>cyclic AMPAMP. The relaxant responses to adenine nucleotides tended to reduce with decrease in the number of high energy phosphates.     

Suramin-sensitive suppression of paired-pulse inhibition by adenine nucleotides in rat hippocampal slices

In order to assess the possible presence of presynaptic P2 receptors for nucleotides in the hippocampus, adenosine triphosphate and betagamma-methyleneATP have been examined on paired-pulse inhibition in rat hippocampal slices. Both compounds reproduced the effects of adenosine and reduced the amount of paired-pulse inhibition at an interpulse interval of 10 ms and increased the amount of facilitation at intervals of 20 and 50 ms. These effects were prevented by 8-phenyltheophylline and adenosine deaminase, indicating their mediation by adenosine. The effects were also reduced by suramin at 50 microM, suggesting the possible activation of P2 receptors. It is suggested that a population of P2 receptors may exist which promote the release of endogenous adenosine in the hippocampus.     

Roles of adenosine and serotonin receptors on the antinociception of sildenafil in the spinal cord of rats

Purpose

The phosphodiesterase 5 inhibitor sildenafil has antinociceptive effects, mediated by an increase in cGMP. This study examined the role of spinal adenosine and serotonin receptors played in the antinociceptive effects of intrathecal sildenafil.

Materials and Methods

Intrathecal catheters were inserted into the subarachnoid space of Sprague-Dawley male rats as a drug delivery device. Pain was induced by injecting formalin into the plantar surface of rats and observing nociceptive behavior (flinching response) for 60 mininutes. Then, the effects of intrathecal adenosine and serotonin receptor antagonists on the antinociceptive activity of intrathecal sildenafil were examined.

Results

Intrathecal sildenafil suppressed the flinching response in a dose-dependent manner during phases 1 and 2 in the formalin test. Both CGS 15943 and dihydroergocristine decreased the antinociceptive effects of sildenafil during phases 1 and 2 in the formalin test.

Conclusion

Intrathecal sildenafil effectively attenuated the pain evoked by formalin injection. Both adenosine and serotonin receptors may be involved in the antinociceptive action of sildenafil at the spinal level.     

Trout CC chemokines: comparison of their sequences and expression patterns

Several thousand EST sequences were recently made available in the EMBL sequence database from the rainbow trout Oncorhynchus mykiss. BLAST based searches were utilised to identify sequences resembling mammalian CC chemokines within these ESTs. Fifteen new and unique CC chemokine-like sequences were identified for trout, bringing the total of known CC chemokine sequences in trout to 18 when including those already published. Some of these trout chemokines appeared highly related (in pairs) suggesting recent duplication events or tight evolutionary constraints. Phylogenetically, the trout chemokine sequences grouped with both inducible and constitutive mammalian CC chemokine subtypes, suggesting early divergence of these functional groups. Expression analyses on gill and head kidney show constitutive expression of many of these trout CC chemokines in these lymphoid-rich tissues. However, induction of some of the chemokines structurally related to 'inducible' CC chemokines was observed in a trout macrophage-like cell line (RTS-11) in response to stimulation with recombinant TNFalpha.     

Calcium response to adenosine and ATP in rabbit afferent arterioles.

The effects of purine compounds in the renal vasculature are almost exclusively restricted to pre-glomerular vessels. Although their physiological role as extracellular messengers is not clear, there are extensive data indicating the importance of adenosine and ATP in the regulation of renal haemodynamics. This study was undertaken to characterize the calcium response of rabbit afferent arteriole to adenosine, ATP and other nucleotides. Experiments were performed in isolated afferent arterioles, microdissected from rabbit kidneys and loaded with fura-2. Intracellular calcium concentration ([Ca2+]i) was measured by video in proximal and distal parts of the afferent arteriole. Application of 100 microM adenosine or ATP increased [Ca2+]i in both arteriolar regions. In all cases the response had two well distinguishable phases: a quick peak increase and a plateau phase that equilibrated at a [Ca2+]i significantly higher than the basal level. UTP (100 microM) had no effect on the arteriole. Removal of extracellular calcium (2.5 mM EGTA) abolished only the plateau phase in response to adenosine, without significantly changing the peak increase. In contrast, the response to ATP was completely abolished in both arteriolar regions, where [Ca2+]i decreased upon application of the agonist and rapidly increased after restoration of calcium concentration to plasma level. We conclude that P1 and P2X receptors are present along the rabbit afferent arteriole and mediate calcium mobilization, with the same distribution in the proximal and distal segments.     

Influences of adenosine on the fetus and newborn

Few signaling molecules have the potential to influence the developing mammal as the nucleoside adenosine. In contrast to most neurotransmitters, adenosine is released by all cells and is present in all tissues. The adenosinergic system is therefore not dependent on the presence of mature synaptic structures or an intact autonomic nervous system for its release. However, similar to other signaling molecules, adenosine levels are dynamically regulated and increase with increased tissue activity, hypoxia, or stress. Local adenosine concentrations thus provide a "humoral barometer" of acute changes in cellular physiology. The receptors that transduce adenosine action include A1, A2a, A2b, and A3 adenosine receptors. These receptors differ in their affinities for adenosine and in patterns of tissues expression. During development A1 adenosine receptors (A1ARs) are especially important, and A1ARs are among the earliest receptors expressed in the embryonic brain and heart. In the developing heart, the adenosinergic system is the dominant regulator of fetal cardiac function and A1AR activation inhibits cardiac cell division leading to cardiac hypoplasia. In the forming central nervous system, A1AR activation potently inhibits the development of axons and can lead to leukomalacia. These recent data suggest that adenosine is an important modulator of mammalian development.     

Coupling of airway ciliary activity and mucin secretion to mechanical stresses by purinergic signaling

The mucociliary clearance system is comprised of three components, ion transport activities controlling the height of airway surface liquid (ASL), mucin secretion, and ciliary activity. These activities in humans are controlled principally by local agonists, extracellular nucleotides and nucleosides released from the epithelium. Importantly, mechanical stresses stimulate goblet cell mucin secretion, ciliary beating, and Cl⁻ and fluid secretion through mechanically induced nucleotide release. Emerging evidence also implicates co-secretion of nucleotides and mucin from goblet cells as a source of extracellular agonist. At rest, ATP is released onto airway surfaces at 370 fmol/min cm², but only 3% of released ATP is recovered in ASL. Secreted UTP meets with a similar fate. A wide variety of hydrolytic and transphosphorylating ecto-enzymes convert the triphosphate nucleotides into ADP, AMP, and adenosine, UDP, UMP, and uridine. Of these, ATP, adenosine, UTP, and UDP act as agonists at apical P2Y₂ (ATP, UTP), P2Y₆ (UDP), and A_2B (adenosine) receptors on ciliated and/or goblet cells to regulate mucociliary clearance.     

Regulation of ion transport via apical purinergic receptors in intact rabbit airway epithelium

We investigated purinergic receptors involved in ion transport regulation in the intact rabbit nasal airway epithelium. Stimulation of apical membrane P2Y receptors with ATP or UTP (200 M) induced transient increases in short-circuit current (I_sc) of 13 and 6% followed by sustained inhibitions to 8 and 17% below control level, respectively. Serosal application of nucleotides had no effect. The ATP-induced response appeared to involve additional activation of apical adenosine (P1) and P2X receptors. The inhibitory effect of ATP and UTP on I_sc was eliminated by pretreatment with amiloride (100 M), while the stimulatory effect was potentiated, indicating that ATP and UTP inhibit Na⁺ and stimulate Cl^– current. Ionomycin (1 M) induced responses similar to UTP and ATP and desensitized the epithelium to the nucleotides, indicating involvement of intracellular Ca²⁺ (Ca²⁺_i). Furthermore, ATP, UTP and ionomycin induced 21, 24, and 21% decreases, respectively, in transepithelial conductance. Measurements of unidirectional isotope fluxes showed a 39% decrease in the dominant net Na⁺ absorption in response to ATP, while the smaller net Cl^– secretion increased only insignificantly and unidirectional Cl^– fluxes decreased significantly. The results suggest that nucleotides released to the airway surface liquid exert an autocrine regulation of epithelial NaCl absorption mainly by inhibiting the amiloride-sensitive epithelial Na⁺ channel (ENaC) and paracellular anion conductance via a P2Y receptor-dependent increase in Ca²⁺_i, while stimulation of Cl^– secretion is of minor importance.     

The eicosanoid generating capacity of isolated cell populations from the gills of the rainbow trout, Oncorhynchus mykiss.

Rainbow trout gill filaments generated a wide range of eicosanoid products following calcium ionophore challenge. The putative lipoxygenase products were separated by reverse phase high performance liquid chromatography (RP-HPLC), while prostanoids were quantified by enzyme immunoassay. Three main monohydroxy compounds containing conjugated dienes were observed after RP-HPLC namely 12-(S) hydroxyeicosatetraenoic acid (12-HETE), 12-(S) hydroxyeicosapentaenoic acid (12-HEPE) and 14-(S) hydroxydocosahexaenoic acid (14-HDHE), derived from endogenous arachidonic, eicosapentaenoic and docosahexaenoic acids, respectively. Their identification was confirmed by mass spectrometry. A further five compounds containing conjugated trienes were also observed but in lesser amounts. One of these products was identified as 8,15-dihydroxyeicosatetraenoic acid (8,15-DiHETE) based on its UV spectrum, co-elution with authentic standard on RP-HPLC and mass spectrometry. Overall, the generation of these products suggests the presence of 12- and possibly 15-lipoxygenase activities in trout gill acting on endogenous sources of fatty acid. To determine if the various cell types in trout gill had differing eicosanoid generating potential, gills were disrupted and the resultant cell suspensions separated by density gradient centrifugation. Following this three bands were formed on the gradients and the cell populations from these were characterised using periodic acid Schiff's (PAS) reactivity for mucosubstances, haematoxylin and eosin staining, and immunoreactivity with both monoclonal and polyclonal antibodies. The first band consisted of polygonal cells and other more minor cell types, the second cell band contained mainly polygonal and PAS-positive goblet epithelial cells, while the third band consisted of mainly erythrocytes. There were significant differences in the eicosanoid generating potential of the isolated cells, with cells from the second band generating significantly more 12-HETE and 8,15-DiHETE than those from both the first band and unfractionated populations. The eicosanoid generating activity of the trout gill epithelial cell line, RTG-W1, was also elucidated. It proved to be a modest generator of eicosanoids in that only low levels of thromboxane B2 and prostaglandin E2 were detected while no lipoxygenase products were observed.