8-cyclopentyltheophylline Research Articles

Cortical spreading depression-induced preconditioning in mouse neocortex is lamina specific

Cortical spreading depression (CSD) is able to confer neuroprotection when delivered at least 1 day in advance of an ischemic event. However, its ability to confer neuroprotection in a more immediate time frame has not previously been investigated. Here we have used mouse neocortical brain slices to study the effects of repeated episodes of CSD in layer V and layer II/III pyramidal neurons. In layer V, CSD evoked at 15-min intervals caused successively smaller membrane depolarizations and increases in intracellular calcium compared with the response to the first CSD. With an inter-CSD interval of 30 min this preconditioning effect was much less marked, indicating that preconditioning lasts between 15 and 30 min. A single episode of CSD also provided a degree of protection in oxygen-glucose deprivation (OGD) by significantly lengthening the time a cell could withstand OGD before anoxic depolarization occurred. In layer II/III pyramidal neurons no preconditioning by CSD on subsequent episodes of CSD was observed, demonstrating that the response of pyramidal neurons to repeated CSD is lamina specific. The A1 receptor antagonist 8-cyclopentyl theophylline (8-CPT) reduced the layer V preconditioning in a concentration-related manner. Inhibition of extracellular formation of adenosine by blocking ecto-5'-nucleotidase with α,β-methyleneadenosine 5'-diphosphate prevented preconditioning in most but not all cells. Block of equilibrative nucleoside transporters 1 and 2 with dipyramidole alone or in combination with 6-[(4-nitrobenzyl)thio]-9-β-d-ribofuranosylpurine also prevented preconditioning in some but not all cells. These data provide evidence that rapid preconditioning of one CSD by another is primarily mediated by adenosine.

Roles of purines in synaptic modulation evoked by hypercapnia in isolated spinal cord of neonatal rat in vitro

The purine compounds, adenosine 5'-triphosphate (ATP) and adenosine, are known to accumulate in the extracellular space and to elicit various cellular responses during hypoxia/ischemia, whereas the roles of purines during hypercapnia are poorly understood. In this study, we examined the effects of various drugs affecting purine turnover on the responses to hypercapnia in the spinal cord. Electrically evoked reflex potentials were measured in an in vitro preparation of the isolated spinal cord of the neonatal rat by extracellular recording. Extracellular adenosine concentrations were assayed by high performance liquid chromatography (HPLC) methods. Hypercapnia (20% CO2) depressed the reflex potentials, which were partially reversed by an adenosine A1 receptor antagonist, 8-cyclopentyl theophylline, but not by a P2 receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid. Exogenous adenosine and ATP also depressed the reflex potentials via adenosine A1 receptors. The hypercapnia-evoked depression was not reversed by inhibitors of gap junction hemichannels, anion channels, P2X7 receptors or equilibrative nucleoside transporters, all of which might be involved in purine efflux pathways. The adenosine accumulation evoked by hypercapnia was not inhibited by tetrodotoxin, ethylene glycol-bis(beta-amino ethyl ether) tetraacetic acid (EGTA) or an ecto-ATPase inhibitor, ARL 67156. Homocysteine thiolactone, used to trap intracellular adenosine, significantly reduced extracellular adenosine accumulation during hypercapnia. These results suggest that hypercapnia released adenosine itself from intracellular sources, using pathways different from the conventional exocytotic mechanism, and that this adenosine depressed spinal synaptic transmission via adenosine A1 receptors.

Effects of chronic caffeine exposure on adenosinergic modulation of the discriminative-stimulus effects of nicotine, methamphetamine, and cocaine in rats

Adenosine receptors are involved in cocaine and methamphetamine discrimination and exposure to caffeine can affect behavioral effects of nicotine in rats. Here we investigated the relative involvement of adenosine A(1) and A(2A) receptors in nicotine, cocaine, and methamphetamine discrimination, before and/or during chronic caffeine exposure. The nonselective adenosine receptor antagonist caffeine, the A(1)-receptor antagonist cyclopentyltheophylline (CPT), and the A(2A)-receptor antagonist MSX-3 were evaluated in rats trained to discriminate 0.4 mg/kg nicotine from saline under a fixed-ratio schedule of food delivery. Effects of adenosine receptor antagonists were then compared in rats discriminating nicotine, methamphetamine, or cocaine from saline during chronic caffeine exposure in their drinking water. Caffeine, CPT, and MSX-3 partially generalized to nicotine and shifted nicotine dose-response curves leftwards. During chronic caffeine exposure, however, all three ligands failed to generalize to nicotine and failed to shift nicotine dose-response curves. In previous experiments, CPT and MSX-3 partially generalized to methamphetamine and cocaine and shifted dose-response curves leftwards. In the present experiments, CPT neither generalized nor shifted dose-response curves for methamphetamine or cocaine during chronic caffeine exposure. However, MSX-3 partially generalized to both psychostimulants and shifted their dose-response curves leftwards. Caffeine partially generalized to cocaine, but not methamphetamine, and shifted both dose-response curves leftwards. Both adenosine A(1) and A(2A) receptors are capable of modulating the discriminative-stimulus effects of nicotine. Chronic caffeine exposure produces complete tolerance to both A(1)- and A(2A)-mediated effects in nicotine-trained rats. In contrast, chronic caffeine exposure produces tolerance to adenosine A(1)-mediated, but not A(2A)-mediated, effects in methamphetamine- and cocaine-trained rats.

Comparison of the ability of adenosine kinase inhibitors and adenosine receptor agonists to attenuate thermal hyperalgesia and reduce motor performance in rats

Inhibitors of adenosine kinase (AK) enhance extracellular concentrations of the inhibitory neuromodulator adenosine (ADO) at sites of tissue hyperexcitability and produce antinociceptive effects in animal models of pain and inflammation. The present study compared the ability of several novel and selective AK inhibitors and ADO receptor-selective agonists to attenuate carrageenan-induced thermal hyperalgesia and to impair motor performance as measured by effects on exploratory motor activity and rotorod performance. The prototypical nucleoside AK inhibitor, 5′deoxy-5-iodotubercidin (5′d-5IT), dose-dependently blocked thermal hyperalgesia (ED 50=0.2 μmol/kg ip) and was 4- and 75-fold less potent in reducing exploratory motor activity and rotorod performance, respectively. The antihyperalgesic effects of 5′d-5IT were fully blocked by the A 1 antagonist, cyclopentyltheophylline (CPT) and the A 2A antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX). Novel nucleoside and non-nucleoside AK inhibitors (A-134974, A-286501 and ABT-702) also potently (ED 50=0.7–2 μmol/kg ip) blocked carrageenan-induced thermal hyperalgesia and were significantly less potent than 5′d-5IT in impairing motor performance. The systemic administration of N 6-cyclopentyladenosine (CPA), an A 1 receptor-selective agonist, CGS 21680, an A 2A receptor-selective agonist, and N 6-ethylcarboxamidoadenosine (NECA), a nonselective ADO receptor agonist potently reduced (ED 50=0.3–1.0 μmol/kg ip) thermal hyperalgesia. Unlike the AK inhibitors, however, these ADO receptor agonists produced significant antinociception only at doses that also decreased motor performance. These data demonstrate that AK inhibitors produce specific antihyperalgesic effects via an interaction with ADO A 1 and A 2A receptors at doses that lack detectable effects on exploratory motor activity and rotorod performance and offer an improved separation between antinociceptive and motor impairing effects as compared to ADO receptor agonists.

Adenosinergic modulation of basal forebrain and preoptic/anterior hypothalamic neuronal activity in the control of behavioral state

This review describes a series of animal experiments that investigate the role of endogenous adenosine (AD) in sleep. We propose that AD is a modulator of the sleepiness associated with prolonged wakefulness. More specifically, we suggest that, during prolonged wakefulness, extracellular AD accumulates selectively in the basal forebrain (BF) and cortex and promotes the transition from wakefulness to slow wave sleep (SWS) by inhibiting cholinergic and non-cholinergic wakefulness-promoting BF neurons at the AD A1 receptor. New in vitro data are also compatible with the hypothesis that, via presynaptic inhibition of GABAergic inhibitory input, AD may disinhibit neurons in the preoptic/anterior hypothalamus (POAH) that have SWS-selective activity and Fos expression. Our in vitro recordings initially showed that endogenous AD suppressed the discharge activity of neurons in the BF cholinergic zone via the AD A1 receptor. Moreover, in identified mesopontine cholinergic neurons, AD was shown to act post-synaptically by hyperpolarizng the membrane via an inwardly rectifying potassium current and inhibition of the hyperpolarization-activated current, I h. In vivo microdialysis in the cat has shown that AD in the BF cholinergic zone accumulates during prolonged wakefulness, and declines slowly during subsequent sleep, findings confirmed in the rat. Moreover, increasing BF AD concentrations to approximately the level as during sleep deprivation by a nucleoside transport blocker mimicked the effect of sleep deprivation on both the EEG power spectrum and behavioral state distribution: wakefulness was decreased, and there were increases in SWS and REM sleep. As predicted, microdialyis application of the specific A1 receptor antagonist cyclopentyltheophylline (CPT) in the BF produced the opposite effects on behavioral state, increasing wakefulness and decreasing SWS and REM. Combined unit recording and microdialysis studies have shown neurons selectively active in wakefulness, compared with SWS, have discharge activity suppressed by both AD and the A1-specific agonist cyclohexyladenosine (CHA), while discharge activity is increased by the A1 receptor antagonist, CPT. We next addressed the question of whether AD exerts its effects locally or globally. Adenosine accumulation during prolonged wakefulness occurred in the BF and neocortex, although, unlike in the BF, cortical AD levels declined in the 6th h of sleep deprivation and declined further during subsequent recovery sleep. Somewhat to our surprise, AD concentrations did not increase during prolonged wakefulness (6 h) even in regions important in behavioral state control, such as the POAH, dorsal raphe nucleus, and pedunculopontine tegmental nucleus, nor did it increase in the ventrolateral/ventroanterior thalamic nucleii. These data suggest the presence of brain region-specific differences in AD transporters and/or degradation that become evident with prolonged wakefulness, even though AD concentrations are higher in all brain sites sampled during the naturally occurring (and shorter duration) episodes of wakefulness as compared to sleep episodes in the freely moving and behaving cat. Might AD also produce modulation of activity of neurons that have sleep selective transcriptional (Fos) and discharge activity in the preoptic/anterior hypothalamus zone? Whole cell patch clamp recordings in the in vitro horizontal slice showed fast and likely GABAergic inhibitory post-synaptic potentials and currents that were greatly decreased by bath application of AD. Adenosine may thus disinhibit and promote expression of sleep-related neuronal activity in the POAH. In summary, a growing body of evidence supports the role of AD as a mediator of the sleepiness following prolonged wakefulness, a role in which its inhibitory actions on the BF wakefulness-promoting neurons may be especially important.

Adenosine inhibits voltage-dependent Ca2+ currents in rat dissociated supraoptic neurones via A1 receptors.

1. The modulation of voltage-dependent Ca2+ currents (ICa) by adenosine was investigated in magnocellular neurones acutely dissociated from the rat hypothalamic supraoptic nucleus (SON) by using the whole-cell patch-clamp technique. 2. Adenosine dose dependently and reversibly inhibited ICa elicited by depolarizing voltage steps from a holding potential of -80 mV to potentials ranging from -30 to +20 mV. The mean (+/- s.e.m.) maximum inhibition rate was 36.1 +/- 4.1 % (n = 6) at -20 mV and the EC50 was 9.8 x 10-7 M (n = 6). 3. The inhibition of ICa by adenosine was completely reversed by the selective A1 receptor antagonist 8-cyclopentyl theophylline (CPT), and was mimicked by the selective A1 receptor agonist N 6-cyclohexyladenosine (CHA). 4. The inhibition by CHA was strongly reduced when ICa was inhibited by omega-conotoxin GVIA, a blocker of N-type Ca2+ channels. 5. The adenosine-induced inhibition of ICa was largely reversed by a depolarizing prepulse to +150 mV for 100 ms, which is known to reverse the inhibition of Ca2+ channels mediated by G-protein betagamma subunits. 6. The adenosine receptor-mediated inhibition of ICa was not abolished by intracellularly applied preactivated pertussis toxin (PTX). 7. Using immunohistochemistry, Gzalpha-like immunoreactivity (a PTX-resistant inhibitory G-protein) was observed throughout the SON. 8. These results suggest that adenosine modulates the neuronal activity of SON neurones by inhibiting N-type voltage-dependent Ca2+ channels via A1 receptors which are coupled to PTX-resistant G-proteins.

Ecto-nucleotide pyrophosphatase modulates the purinoceptor-mediated signal transduction and is inhibited by purinoceptor antagonists.

1. The effect of ecto-nucleotide pyrophosphatase (ecto-NPPase; EC 3.6.1. 9) on the ATP- and ADP-mediated receptor activation was studied in rat C6 glioma cells. The P2-purinoceptor antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and reactive blue (RB2) are potent inhibitors (IC(50)=12+/-3 microM) of the latter enzyme. 4,4'-diisothiocyanatostilbene-2,2' disulfonic acid (DIDS), 5'-phosphoadenosine 3'-phosphate (PAP) and suramin were less potent inhibitors with an IC(50) of 22+/-4, 36+/-7 and 72+/-11 microM respectively. 2. P1-purinoceptor antagonists CGS 15943, cyclo-pentyl theophylline (CTP) and theophylline did not affect the activity of the ecto-NPPase. 3. ATP- and ADP-mediated P2Y(1)-like receptor activation inhibited the (-)-isoproterenol-induced increase of intracellular cyclic AMP concentration. PPADS, an ineffective P2Y-antagonist in C6, potentiated the ATP and ADP effect approximately 3 fold due to inhibition of nucleotide hydrolysis by the ecto-NPPase. 4. We conclude that ecto-NPPase has a modulator effect on purinoceptor-mediated signalling in C6 glioma cell cultures.

Actions of adenosine A1 and A2 receptor antagonists on CFTR antibody-inhibited beta-adrenergic mucin secretion response.

1. The cystic fibrosis gene protein, the cystic fibrosis transmembrane conductance regulator (CFTR) acts as a chloride channel and is a key regulator of mucin secretion. The mechanism by which 3-isobutyl-1-methylxanthine (IBMX) corrects the defect in CFTR mediated beta-adrenergic stimulation of mucin secretion has not been determined. The present study has investigated the actions of adenosine A1 and A2 receptor antagonists to determine whether ability to stimulate mucin secretion correlates with correction of CFTR antibody inhibited beta-adrenergic response and whether excessive cyclic AMP rise is required. 2. CFTR antibodies were introduced into living rat submandibular acini by hypotonic swelling. Following recovery, mucin secretion in response to isoproterenol was measured. 3. The adenosine A1 receptor antagonist, 8 cyclopentyltheophylline (CPT) was a less potent stimulator of mucin secretion than was the A2 receptor antagonist dimethylpropargylxanthine (DMPX). A concentration of CPT close to the Ki for A1 receptor antagonism (10 nM) did not stimulate mucin secretion. 4. DMPX, although a potent stimulator of mucin secretion, did not correct CFTR antibody inhibited mucin secretion. 5. CPT corrected defective CFTR antibody inhibited mucin secretion at a high (1 mM) concentration, suggesting a mechanism other than adenosine receptor antagonism. 6. DMPX potentiated the isoproterenol induced cyclic AMP rise, whereas CPT did not. 7. Correction of the defective CFTR mucin secretion response did not correlate with ability to stimulate mucin secretion and did not require potentiation of beta-adrenergic induced increases in cyclic AMP. This affords real promise for the development of a selective drug treatment for cystic fibrosis.

CPT (8-cyclopentyl theophylline) corrects CFTR antibody inhibited mucin secretion.

CPT (8-cyclopentyl theophylline) corrects CFTR antibody inhibited mucin secretion.

Late developmental changes in the ability of adenosine A1 receptors to regulate synaptic transmission in the hippocampus

Paired-pulse facilitation (PPF) of CA3–CA1 excitatory postsynaptic potentials (EPSP) was compared in hippocampal slices from juvenile (postnatal day (P) 15–21) and young adult rats (P28–P35) following application of adenosine. Relative to juveniles, young adults expressed an increase in baseline synaptic strength that was accompanied by a decrease in PPF suggesting a developmental increase in transmitter release. While adenosine depressed the EPSP slope to a similar extent in juveniles and young adults, PPF increased during adenosine application only for young adults. The differential effect of adenosine on PPF was not due to differences in receptor function or in extracellular ligand levels, since the A 1 antagonist cyclopentyltheophylline (CPT) did not differentially affect PPF across age. Adenosine could increase PPF in juvenile slices under conditions of enhanced transmitter release, through an increase in the bath Ca 2+ concentration, or addition of forskolin to the bath. These data indicate that the ability to modify synaptic transmission through presynaptic adenosine A 1 receptors increases across postnatal development with the maturation of release mechanisms.

Upregulated renal adenosine A1 receptors augment PKC and glucose transport but inhibit proliferation.

Adenosine A1 receptor densities were increased in cultured LLC-PK1 and OK cells by chronic treatment with the adenosine receptor antagonists 1,3,7-trimethylxanthine (caffeine, 1 mM) and 1,3-dimethyl-8-cyclopentylxanthine [cyclopentyltheophylline (CPT), < or = 0.4 mM], respectively. The A1 receptor number per cell was increased twofold by 10-day treatments with 1 mM caffeine or 0.1 mM CPT, and the sodium-coupled glucose uptake was augmented twofold by 1 mM caffeine and sevenfold by 0.1 microM CPT (higher doses of CPT were progressively less stimulatory). Glucose uptake was blocked by acute (2-h) treatment with CPT, adenosine deaminase, or calphostin C. Caffeine (1 mM) or CPT (> or = 0.1 mM) inhibited cell proliferation for the first 10 days, then cell growth assumed a normal proliferative rate despite continued presence of antagonist. Cytosolic protein kinase C (PKC) beta-isoform immunoactivity and PKC-beta II mRNA were elevated at least twofold during 10 days of 0.1 mM CPT or 1 mM caffeine treatment. The sustained elevation in sodium-glucose symport and PKC activity observed with adenosine receptor antagonists was similar to acute (2-h) effects of the adenosine A1 agonist R(-)-N6-phenylisopropyladenosine (R-PIA, 0.1-1 microM). Moreover, cell proliferation was increased by adenosine (0.1 microM R-PIA), whereas Na-K-adenosinetriphosphatase activity was unaltered with chronic antagonist or acute adenosine treatments. Caffeine treatment may have some non-adenosine A1 receptor-mediated actions, because it slightly (30%) augmented protein kinase A activity. It is concluded that chronic exposure of proximal tubule cells to caffeine or CPT augments PKC and sodium-glucose transport but retards cell proliferation mainly via adenosine A1 receptor-mediated mechanisms.

Purine suppression of proliferation of Sertoli-like TM4 cells in culture.

The effect of adenosine and related compounds on the proliferation of cultured TM4 cells, a Sertoli-like cell line, has been examined. Adenosine, as well as A1 and A2 adenosine receptor agonists (cyclohexyladenosine and N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine) inhibited cell proliferation. These effects were prevented by 8-cyclopentyl theophylline, 1,3-dimethyl-propargylxanthine and caffeine, antagonists at the A1, A2 and both receptors, respectively. The xanthines had no effect by themselves and, consistent with this, the bathing medium was found not to contain detectable levels of adenosine. It is concluded that TM4 cell proliferation can be regulated by receptors for adenosine.

Effect of an intracellular calcium chelator on the regulation of electrically evoked [3H]‐noradrenaline release from rat hippocampal slices

1. The electrically (3 Hz, 5 min) evoked [3H]-noradrenaline ([3H]-NA) release from rat hippocampal slices was reduced by prior treatment of the slices with 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetomethylester (BAPTA/AM) in a concentration-(10 to 500 microM) dependent manner (40% at 30 microM). Reduction of medium calcium from 1.3 to 0.5 mM caused a larger (70%) decrease. BAPTA free acid (100 mM), a non-permeable Ca(2+)-chelator had no significant effect. 2. Basal [3H]-noradrenaline release was reduced by BAPTA/AM in a concentration-dependent manner (50% at 30 microM), but reduction of external Ca2+ from 1.3 to 0.5 mM did not alter basal release. 3. About 10% of total [3H]-NA in the slices was released at 3 Hz stimulation in 1.3 mM Ca2+ buffer. Addition of the alpha 2-adrenoceptor antagonist, idazoxan (1 microM), increased electrically evoked [3H]-NA release to 26% but stimulated release was not altered by the adenosine A1-receptor antagonist, 8-cyclopentyl theophylline (8-CPT) (1 microM). 4. Evoked release was reduced by the alpha 2-receptor agonist, UK 14,304, in a concentration-dependent manner in the presence of 8-CPT (1 microM). The magnitude of this effect was not altered by the treatment of slices with 30 microM BAPTA/AM. 5. The adenosine A1 receptor agonist, N6-cyclohexyl adenosine (CHA) (1 microM) inhibited electrically evoked [3H]-NA release by about 40% in the presence of idazoxan (1 microM). The effect of CHA was not significantly altered by treatment of slices with BAPTA/AM. 7. The present results show that spontaneous [3H]-NA release is affected by reduction of intracellular Ca2+, but not by reduction of extracellular Ca2+ or by the presynaptic agonists or w-conotoxin. By contrast, electrically evoked release was affected more strongly by alterations of extracellular Ca2+ than by buffering intracellular Ca2+. The reduction of electrically evoked [3H]-NA release by agonists at the adenosine Al-receptor and a2-adrenoceptor is probably mediated through the control of Ca2+ entry via membrane ion channels or at a low affinity Ca2'-site governing evoked release.

Analysis of adenosine actions on Ca2+ currents and synaptic transmission in cultured rat hippocampal pyramidal neurones.

1. The role of adenosine receptors in reducing calcium currents (ICa) and in triggering presynaptic inhibition was studied using whole-cell patch-clamp techniques to record ICa and synaptic currents from the cell bodies of cultured rat hippocampal pyramidal neurones. Recordings of intracellular Ca2+ using the indicator dye Fura-2 were used to obtain further insights into the actions of adenosine agonists. 2. The adenosine analogue 2-chloroadenosine (2-CA) reduced ICa in these neurones. This action was also evident when Ba2+ was used as the charge carrier through Ca2+ channels. Adenosine also reduced the influx of Ca2+ into the cell body during a depolarizing voltage-clamp pulse as measured with Fura-2. The potency of various adenosine receptor agonists was as follows: cyclopentyladenosine greater than cyclohexyl-adenosine greater than or equal to R-phenylisopropyladenosine greater than 2-CA greater than S-phenylisopropyladenosine, consistent with the pharmacological profile of an A1 adenosine receptor. 3. The specific A1 receptor antagonist cyclopentyltheophylline (CPT) blocked the actions of 2-CA on ICa in a competitive fashion. 4. The actions of 2-CA on ICa were abolished by pre-incubation of cultured cells with pertussis toxin (PTX; 250 ng/ml). Intracellular dialysis with the GTP analogue GTP-gamma-S (guanosine-5'-O-(3-thiotriphosphate] enhanced the actions of 2-CA and rendered the response irreversible. 5. Excitatory postsynaptic currents (EPSCs) were recorded from pyramidal neurones under whole-cell voltage clamp by stimulating nearby neurones with an extracellular electrode. 2-CA potently and reversibly reduced the amplitude of EPSCs. This action was shown to be due to presynaptic inhibition of neurotransmitter release. 6. The order of potency of different adenosine agonists in reducing EPSCs was as follows: cyclopentyladenosine greater than cyclohexyladenosine greater than or equal to R-phenylisopropyladenosine greater than 2-CA greater than S-phenylisopropyladenosine. CPT inhibited the action of 2-CA in a competitive fashion. 7. The effects of 2-CA on synaptic transmission were abolished by pre-treatment with 250 ng/ml PTX, indicating that a PTX-sensitive G-protein is involved in this action. 8. These results indicate that activation of adenosine receptors does induce a reduction in ICa in hippocampal pyramidal neurones. Furthermore, this effect and the reduction of excitatory synaptic transmission by adenosine analogues are both mediated by PTX-sensitive G-proteins and have identical pharmacological properties.