An antagonistic interaction between A2B adenosine and D2 dopamine receptors modulates the function of rat carotid body chemoreceptor cells

We have previously demonstrated that adenosine controls the release of catecholamines (CA) from carotid body (CB) acting on A2B receptors. Here, we have tested the hypothesis that the control is exerted via an interaction between adenosine A2B and dopamine D2 receptors present in chemoreceptor cells. Experiments were performed in vitro in CB from 3 months rats. The effect of A2B adenosine and D2 dopamine agonists and antagonists applied alone or in combination were studied on basal (20%O2) and hypoxia (10%O2)-evoked release of CA and cAMP content of CB. We have found that adenosine A2 agonists and D2 antagonists dose-dependently increased basal and evoked release CA from the CB while A2 antagonists and D2 agonists had an inhibitory action. The existence of A2B-D2 receptor interaction was established because the inhibitory action of A2 antagonists was abolished by D2 antagonists, and the stimulatory action of A2 agonists was abolished by D2 agonists. Further, A2 agonists increased and D2 agonist decreased cAMP content in the CB; their co-application eliminated the response. The present results provide direct pharmacological evidence that an antagonistic interaction between A2B adenosine and D2 dopamine receptors exist in rat CB and would explain the dopamine-adenosine interactions on ventilation previously observed.     

Function of the rat carotid body chemoreceptors in ageing

Some age-related deficits in the ventilatory responses have been attributed to a decline in the functionality of the carotid body (CB) arterial chemoreceptors, but a systematic study of the CB function in ageing is lacking. In rats aged 3-24 months, we have performed quantitative morphometry on specific chemoreceptor tissue, assessed the function of chemoreceptor cells by measuring the content, synthesis and release of catecholamines (a chemoreceptor cell neurotransmitter) in normoxia and hypoxia, and determined the functional activity of the intact organ by measuring chemosensory activity in the carotid sinus nerve (CSN) in normoxia, hypoxia and hypercapnic acidosis. We found that with age CBs enlarge, but at the same time there is a concomitant decrease in the percentage of chemoreceptor tissue. CB content and turnover time for their catecholamines increase with age. Hypoxic stimulation of chemoreceptor cells elicits a smaller release of catecholamines in rats after 12 months of age, but a non-specific depolarizing stimulus elicits a comparable release at all ages. In parallel, there was a marked decrease in the responsiveness to hypoxia, but not to an acidic-hypercapnic stimulus, assessed as chemosensory activity in the CSN. We conclude that in aged mammals chemoreceptor cells become hypofunctional, leading to a decreased peripheral drive of ventilation.     

Hypoxia induces adenosine release from the rat carotid body

The effect of hypoxia on the release of adenosine was studied in vitro in the rat whole carotid body (CB) and compared with the effect of hypoxia (2%, 5% and 10% O(2)) on adenosine concentrations in superior cervical ganglia (SCG) and carotid arteries. Moderate hypoxia (10% O(2)) increased adenosine concentrations released from the CBs by 44%, but was not a strong enough stimulus to evoke adenosine release from SCG and arterial tissue. The extracellular pathways of adenosine production in rat CBs in normoxia and hypoxia were also investigated. S-(p-nitrobenzyl)-6-thioinosine (NBTI) and dipyridamole were used as pharmacological tools to inhibit adenosine equilibrative transporters (ENT) and alpha,beta-methylene ADP (AOPCP) to inhibit ecto-5'-nucleotidase. Approximately 40% of extracellular adenosine in the CB came from the extracellular catabolism of ATP, under both normoxic and hypoxic conditions. Low pO(2) triggers adenosine efflux through activation of NBTI-sensitive ENT. This effect was only apparent in hypoxia and when adenosine extracellular concentrations were reduced by the blockade of ecto-5'-nucleotidase. We concluded that CB chemoreceptor sensitivity could be related to its low threshold for the release of adenosine in response to hypoxia here quantified for the first time.     

A(2b) receptor mediates adenosine inhibition of taurine efflux from pituicytes

BACKGROUND INFORMATION: Recent work suggests that part of the control of vasopressin output is mediated by taurine released from pituicytes, the astroglial cells of the neurohypophysis. Taurine release, in turn, is stimulated by hypotonic conditions and by vasopressin itself. As adenosine is generated from ATP co-released with vasopressin, it appeared important to study its effects on taurine efflux from pituicytes. RESULTS: We measured radioactive efflux from cultured pituicytes and whole neurohypophyses pre-loaded with [(3)H]taurine. Cultured pituicytes were also used to study adenosine-receptor mRNA expression. Taurine efflux elicited by hypotonic shocks is approximately 30-50% smaller in the presence of 10 microM adenosine or 1 microM NECA (5'-N-ethylcarboxamidoadenosine). Both compounds also inhibited basal efflux in a manner that was not immediately reversible. Agonists of the adenosine A1-, A2a- or A3-receptor subtypes have no relevant effect on basal taurine release, and the A1-receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine) has no effect on the inhibition of release by NECA. In turn, the A2b-receptor antagonists MRS 1706 {N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide} or alloxazine partially reverse the inhibition of basal or hypotonicity-evoked efflux by NECA. Both A1- and A2b-receptor mRNAs are expressed in pituicytes, which is consistent with an A1-receptor-mediated effect on cell morphology and an A2b-receptor-mediated effect on taurine release. Forskolin and dibutyryl cAMP mimic the inhibitory effects of purinergics on basal taurine efflux, and the adenylate cyclase inhibitor DDA (2',5'-dideoxyadenosine) partially reverses the inhibition of the hypotonic response by NECA.Conclusions. Our results suggest that purinergic inhibition of taurine efflux from pituicytes operates through A2b receptors coupled to intracellular cAMP increase. They point to a possible modulation of neurohypophysial hormone output by endogenous adenosine released in either physiological or pathological situations.     

Inhibition of hippocampal synaptic activity by ATP, hypoxia or oxygen-glucose deprivation does not require CD73

Adenosine, through activation of its A(1) receptors, has neuroprotective effects during hypoxia and ischemia. Recently, using transgenic mice with neuronal expression of human equilibrative nucleoside transporter 1 (hENT1), we reported that nucleoside transporter-mediated release of adenosine from neurons was not a key mechanism facilitating the actions of adenosine at A(1) receptors during hypoxia/ischemia. The present study was performed to test the importance of CD73 (ecto-5'-nucleotidase) for basal and hypoxic/ischemic adenosine production. Hippocampal slice electrophysiology was performed with CD73(+/+) and CD73(-/-) mice. Adenosine and ATP had similar inhibitory effects in both genotypes, with IC(50) values of approximately 25 μM. In contrast, ATP was a less potent inhibitor (IC(50) = 100 μM) in slices from mice expressing hENT1 in neurons. The inhibitory effects of ATP in CD73(+/+) and CD73(-/-) slices were blocked by the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and were enhanced by the nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI), consistent with effects that are mediated by adenosine after metabolism of ATP. AMP showed a similar inhibitory effect to ATP and adenosine, indicating that the response to ATP was not mediated by P2 receptors. In comparing CD73(-/-) and CD73(+/+) slices, hypoxia and oxygen-glucose deprivation produced similar depression of synaptic transmission in both genotypes. An inhibitor of tissue non-specific alkaline phosphatase (TNAP) was found to attenuate the inhibitory effects of AMP and ATP, increase basal synaptic activity and reduce responses to oxygen-glucose deprivation selectively in slices from CD73(-/-) mice. These results do not support an important role for CD73 in the formation of adenosine in the CA1 area of the hippocampus during basal, hypoxic or ischemic conditions, but instead point to TNAP as a potential source of extracellular adenosine when CD73 is absent.     

Caffeine has a dual influence on NMDA receptor–mediated glutamatergic transmission at the hippocampus

Caffeine, a stimulant largely consumed around the world, is a non-selective adenosine receptor antagonist, and therefore caffeine actions at synapses usually, but not always, mirror those of adenosine. Importantly, different adenosine receptors with opposing regulatory actions co-exist at synapses. Through both inhibitory and excitatory high-affinity receptors (A₁R and A₂R, respectively), adenosine affects NMDA receptor (NMDAR) function at the hippocampus, but surprisingly, there is a lack of knowledge on the effects of caffeine upon this ionotropic glutamatergic receptor deeply involved in both positive (plasticity) and negative (excitotoxicity) synaptic actions. We thus aimed to elucidate the effects of caffeine upon NMDAR-mediated excitatory post-synaptic currents (NMDAR-EPSCs), and its implications upon neuronal Ca²⁺ homeostasis. We found that caffeine (30–200 μM) facilitates NMDAR-EPSCs on pyramidal CA1 neurons from Balbc/ByJ male mice, an action mimicked, as well as occluded, by 1,3-dipropyl-cyclopentylxantine (DPCPX, 50 nM), thus likely mediated by blockade of inhibitory A₁Rs. This action of caffeine cannot be attributed to a pre-synaptic facilitation of transmission because caffeine even increased paired-pulse facilitation of NMDA-EPSCs, indicative of an inhibition of neurotransmitter release. Adenosine A_2ARs are involved in this likely pre-synaptic action since the effect of caffeine was mimicked by the A_2AR antagonist, SCH58261 (50 nM). Furthermore, caffeine increased the frequency of Ca²⁺ transients in neuronal cell culture, an action mimicked by the A₁R antagonist, DPCPX, and prevented by NMDAR blockade with AP5 (50 μM). Altogether, these results show for the first time an influence of caffeine on NMDA receptor activity at the hippocampus, with impact in neuronal Ca²⁺ homeostasis.

     

Opposite modulatory roles for adenosine A1 and A2A receptors on glutamate and dopamine release in the shell of the nucleus accumbens. Effects of chronic caffeine exposure

Previous studies have demonstrated opposing roles for adenosine A1 and A2A receptors in the modulation of extracellular levels of glutamate and dopamine in the striatum. In the present study, acute systemic administration of motor-activating doses of the A2A receptor antagonist MSX-3 significantly decreased extracellular levels of dopamine and glutamate in the shell of the rat nucleus accumbens (NAc) and counteracted both dopamine and glutamate release induced by systemic administration of motor-activating doses of either the A1 receptor antagonist CPT or caffeine. Furthermore, exposure to caffeine in the drinking water (1 mg/mL, 14 days) resulted in tolerance to the effects of systemic injection of CPT or caffeine, but not MSX-3, on extracellular levels of dopamine and glutamate in the NAc shell. The present results show: first, the existence of opposite tonic effects of adenosine on extracellular levels of dopamine and glutamate in the shell of the NAc mediated by A1 and A2A receptors; second, that complete tolerance to caffeine's dopamine- and glutamate-releasing effects which develops after chronic caffeine exposure is attributable to an A1 receptor-mediated mechanism. Development of tolerance to the dopamine-releasing effects of caffeine in the shell of the NAc may explain its weak addictive properties and atypical psychostimulant profile.     

Adenosine receptor-mediated modulation of dopamine release in the nucleus accumbens depends on glutamate neurotransmission and N-methyl-D-aspartate receptor stimulation

Adenosine, by acting on adenosine A(1) and A(2A) receptors, exerts opposite modulatory roles on striatal extracellular levels of glutamate and dopamine, with activation of A(1) inhibiting and activation of A(2A) receptors stimulating glutamate and dopamine release. Adenosine-mediated modulation of striatal dopaminergic neurotransmission could be secondary to changes in glutamate neurotransmission, in view of evidence for a preferential colocalization of A(1) and A(2A) receptors in glutamatergic nerve terminals. By using in vivo microdialysis techniques, local perfusion of NMDA (3, 10 microm), the selective A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 3, 10 microm), the selective A(1) receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 300, 1000 microm), or the non-selective A(1)-A(2A) receptor antagonist in vitro caffeine (300, 1000 microm) elicited significant increases in extracellular levels of dopamine in the shell of the nucleus accumbens (NAc). Significant glutamate release was also observed with local perfusion of CGS 21680, CPT and caffeine, but not NMDA. Co-perfusion with the competitive NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (APV; 100 microm) counteracted dopamine release induced by NMDA, CGS 21680, CPT and caffeine. Co-perfusion with the selective A(2A) receptor antagonist MSX-3 (1 microm) counteracted dopamine and glutamate release induced by CGS 21680, CPT and caffeine and did not modify dopamine release induced by NMDA. These results indicate that modulation of dopamine release in the shell of the NAc by A(1) and A(2A) receptors is mostly secondary to their opposite modulatory role on glutamatergic neurotransmission and depends on stimulation of NMDA receptors. Furthermore, these results underscore the role of A(1) vs. A(2A) receptor antagonism in the central effects of caffeine.     

Disruption of adenosinergic modulation of ventilation at rest and during hypercapnia by neonatal caffeine in young rats: role of adenosine A(1) and A(2A) receptors

Caffeine is commonly used to treat respiratory instabilities related to prematurity. However, the role of adenosinergic modulation and the potential long-term effects of neonatal caffeine treatment (NCT) on respiratory control are poorly understood. To address these shortcomings, we tested the following hypotheses: 1) adenosine A(1)- and A(2A)-receptor antagonists modulate respiratory activity at rest and during hypercapnia; 2) NCT has long-term consequences on adenosinergic modulation of respiratory control. Rat pups received by gavage either caffeine (15 mg/kg) or water (control) once a day from postnatal days 3 to 12. At day 20, rats received intraperitoneal injection with vehicle, DPCPX (A(1) antagonist, 4 mg/kg), or ZM-241385 (A(2A) antagonist, 1 mg/kg) before plethysmographic measurements of resting ventilation, hypercapnic ventilatory response (5% CO(2)), and occurrence of apneas in freely behaving rats. In controls, data show that A(2A), but not A(1), antagonist decreased resting ventilation by 31% (P = 0.003). A(1) antagonist increased the hypercapnic response by 60% (P < 0.001), whereas A(2A) antagonist increased the hypercapnic response by 42% (P = 0.033). In NCT rats, A(1) antagonist increased resting ventilation by 27% (P = 0.02), but the increase of the hypercapnic response was blunted compared with controls. A(1) antagonist enhanced the occurrence of spontaneous apneas in NCT rats only (P = 0.005). Finally, A(2A) antagonist injected in NCT rats had no effect on ventilation. These data show that hypercapnia activates adenosinergic pathways, which attenuate responsiveness (and/or sensitivity) to CO(2) via A(1) receptors. NCT elicits developmental plasticity of adenosinergic modulation, since neonatal caffeine persistently decreases ventilatory sensitivity to adenosine blockers.     

New pyrrolopyrimidin-6-yl benzenesulfonamides: potent A2B adenosine receptor antagonists

A new series of 4-(1,3-dialkyl-2,4-dioxo-2,3,4,5-tetrahydro-1H-pyrrolo[3,2-d]pyrimidin-6-yl)benzenesulfonamides has been identified as potent A2B adenosine receptor antagonists. The products have been evaluated for their binding affinities for the human A2B, A1 and A3 adenosine receptors. 6-(4-{[4-(4-Bromobenzyl)piperazin-1-yl]sulfonyl}phenyl)-1,3-dimethyl-1H-pyrrolo[3,2-d]pyrimidine-2,4(3H,5H)-dione (16) showed a high affinity for the A2B adenosine receptor (IC50=1 nM) and selectivity (A1: 183x; A3: 12660x). Synthesis and SAR of this novel class of compounds showing improved absorption properties is presented herein.     

Solubilization of stable adenosine A1 receptors from rat brain.

Despite numerous reports of solubilization of adenosine A1 receptors, little progress has been made in isolating or purifying the receptor, owing to the extreme lability of the preparations. The present solubilization strategies recognized the possible role of endogenous adenosine to produce adenosine-receptor-N-protein complexes, which are intrinsically unstable, and instead attempted to use caffeine to solubilize free adenosine receptors, which might be more stable. Endogenous adenosine was removed from membranes by using adenosine deaminase along with GTP to accelerate the release of receptor-bound adenosine. The receptors were then occupied with caffeine and solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS) in the presence of glycerol. These soluble preparations exhibited the characteristics of free adenosine receptors. They bound the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPDPX) with high affinity to a single class of binding sites, which were insensitive to GTP. The binding activity was extremely stable, with a half-life of about 5 days at 4 degrees C; there was little change in either receptor number or affinity during 3 days at 4 degrees C. This methodology should greatly facilitate the characterization, isolation and purification of the adenosine A1 receptor.     

Differential glutamate-dependent and glutamate-independent adenosine A1 receptor-mediated modulation of dopamine release in different striatal compartments

Adenosine and dopamine are two important modulators of glutamatergic neurotransmission in the striatum. However, conflicting reports exist about the role of adenosine and adenosine receptors in the modulation of striatal dopamine release. It has been previously suggested that adenosine A(1) receptors localized in glutamatergic nerve terminals indirectly modulate dopamine release, by their ability to modulate glutamate release. In the present study, using in vivo microdialysis, we provide evidence for the existence of a significant glutamate-independent tonic modulation of dopamine release in most of the analyzed striatal compartments. In the dorsal, but not in the ventral, part of the shell of the nucleus accumbens (NAc), blockade of A(1) receptors by local perfusion with the selective A(1) receptor antagonist 8-cyclopentyl-1,3-dimethyl-xanthine or by systemic administration of the non-selective adenosine antagonist caffeine induced a glutamate-dependent release of dopamine. On the contrary, A(1) receptor blockade induced a glutamate-independent dopamine release in the core of the NAc and the nucleus caudate-putamen. Furthermore, using immunocytochemical and functional studies in rat striatal synaptosomes, we demonstrate that a fraction of striatal dopaminergic terminals contains adenosine A(1) receptors, which directly inhibit dopamine release independently of glutamatergic transmission.     

Adenosine and dopamine receptor interactions in striatum and caffeine-induced behavioral activation

This review will examine how dopamine, a monoamine neurotransmitter, and adenosine, a neuromodulator, regulate behavioral activation, primarily as reflected by locomotor activity, in rodents. Complex interactions among 2 major types of adenosine receptors (A1AR and A2AAR) and 2 dopamine receptors (D1R and D2R) occur due to physical interactions that alter their ligand-binding properties and subsequent effects on common postreceptor signaling molecules. The output from these interactions in striatum modulates neurotransmission and subsequently influences spontaneous locomotor activity. Caffeine is a nonselective adenosine receptor antagonist that blocks 2 major types of adenosine receptors, A1AR and A2AAR, in the brain. Pharmacologic manipulation of these receptors with drugs such as caffeine offers potential therapeutic benefit for treatment of Parkinson disease.     

Acute Caffeine Treatment Increases Extracellular Nucleotide Hydrolysis from Rat Striatal and Hippocampal Synaptosomes

The psychostimulant caffeine promotes behavioral effects such as hyperlocomotion, anxiety, and disruption of sleep by blockade of adenosine receptors. The availability of extracellular adenosine depends on its release by transporters or by the extracellular ATP catabolism performed by the ecto-nucleotidase pathway. This study verified the effect of caffeine on NTP-Dase 1 (ATP diphosphohydrolase) and 5-nucleotidase of synaptosomes from hippocampus and striatum of rats. Caffeine and theophylline tested in vitro were unable to modify nucleotide hydrolysis. Caffeine chronically administered in the drinking water at 0.3 g/L or 1 g/L for 14 days failed to affect nucleotide hydrolysis. However, acute administration of caffeine (30 mg/kg, ip) produced an enhancement of ATP (50%) and ADP (32%) hydrolysis in synaptosomes of hippocampus and striatum, respectively. This activation of ATP and ADP hydrolysis after acute treatment suggests a compensatory effect to increase adenosine levels and counteract the antagonist action of caffeine.     

3H]-MRE 2029-F20, a selective antagonist radioligand for the human A2B adenosine receptors

MRE 2029-F20 [N-benzo[1,3]dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]-acetamide] is a selective antagonist ligand of A2B adenosine receptors. For use as a radioligand, 1,3-diallyl-xanthine, the precursor of [3H]-MRE 2029-F20, was synthesized, and tritiated on the allyl groups. [3H]-MRE 2029-F20 bound to human A2B receptors expressed in CHO cells showed a KD value of 1.65+/-0.10 nM and Bmax value of 36+/-4 fmol/mg protein. [3H]-MRE2029-F20 represents a useful tool for the pharmacological characterization of human A2B adenosine receptor subtype.     

Analogs of caffeine: antagonists with selectivity for A2 adenosine receptors

Several analogs of caffeine have been investigated as antagonists at A2 adenosine receptors stimulatory to adenylate cyclase in membranes from rat pheochromocytoma PC12 cells and human platelets and at A1 adenosine receptors inhibitory to adenylate cyclase from rat fat cells. Among these analogs, 1-propargyl-3,7-dimethylxanthine was about 4- to 7-fold and 7-propyl-1,3-dimethylxanthine about 3- to 4-fold more potent than caffeine at A2 receptors of PC12 cells and platelets. At A1 receptors of fat cells, both compounds were about 2-fold less potent than caffeine. These caffeine analogs have an A1/A2 selectivity ratio of about 10-20 and are the first selective A2 receptor antagonists yet reported. The results may provide the basis for the further development of highly potent and highly selective A2 adenosine receptor antagonists.     

腺苷对家兔颈动脉化学感受器活动的影响

The response of single carotid chemoreceptor afferent fibers upon adenosine acting on the carotid body (CB) was examined in 39 urethan-anesthetized rabbits. Totally 73 units with spontaneous discharge were recorded in our experiment. The results were as follows: (1) Of 55 units, 51 showed an increase in discharge frequency from 0.76 +/- 0.10 to 1.53 +/- 0.23 imp/s. A few new units were recruited concomitantly in response to intracarotid injection of adenosine (10 micrograms/kg). (2) Adding adenosine in the doses of 0.5, 1.5, 10, 50 and 100 micrograms/kg to the perfusate passing through the isolated carotid sinus led to dose-dependent increase in the discharge from 0.51 +/- 0.06 to 0.58 +/- 0.07, 0.78 +/- 0.13, 0.96 +/- 0.15, 1.11 +/- 0.17, 1.34 +/- 0.21 and 1.38 +/- 0.18 imp/s, respectively (P less than 0.001, n = 9 units). (3) In other 9 units with spontaneous discharge rate of 1.30 +/- 0.40 imp/s, the activity was decreased to 0.56 +/- 0.19 imp/s (P less than 0.01) by intracarotid injection of dopamine (50 micrograms/kg). Intracarotid injection of adenosine to the CB pretreated with dopamine still activated the units with an increase in firing rate to 1.07 +/- 0.28 imp/s (P less than 0.01). However, the increment was less prominent as compared with that of adenosine administration before dopamine injection (P less than 0.001). From the results obtained, it is hypothesized that the exciting effect of adenosine on the CB chemoreceptor may be attributed to its action on the presynaptic component of the chemoreceptor complex in attenuating the release of inhibitory transmitter dopamine, and its direct stimulating action on the chemosensory nerve endings.     

Modulation of the function of boar spermatozoa via adenosine and fertilization promoting peptide receptors reduce the incidence of polyspermic penetration into porcine oocytes

Effects of adenosine and pGlu-Glu-ProNH(2) (FPP) on the function and in vitro penetration of boar spermatozoa were examined. First, the effects of dibutyryl cAMP or agonists and antagonists of adenosine receptors (inhibitory adenosine receptors, A1AdR; stimulatory adenosine receptors, A2AdR) on freshly ejaculated spermatozoa were determined by chlortetracycline fluorescence assessment. Capacitation of spermatozoa was stimulated when they were cultured in a medium with dibutyryl cAMP, adenosine, A2AdR agonist, and adenosine plus A1AdR antagonist (CPT). However, acrosome reaction was inhibited only by adenosine. A1AdR agonist did not affect intact spermatozoa. A2AdR antagonist (DMPX) neutralized all of the effects of adenosine. Second, interaction of adenosine and FPP was examined. Gln-FPP, a competitive inhibitor of FPP, and DMPX inhibited the effects of adenosine and FPP, and CPT neutralized the inhibitory effect of FPP on acrosome reaction. Last, the effects of adenosine, FPP, and caffeine on the rate of sperm penetration were examined using frozen-thawed spermatozoa. Adenosine, FPP, and caffeine significantly enhanced the rate of sperm penetration as compared with the case of no additions. Caffeine treatment resulted in a high rate of polyspermic fertilization. In contrast, adenosine and FPP treatments resulted in an increased proportion of normal fertilization in in vitro-matured oocytes. These results suggest that boar spermatozoa can be modulated by the adenylyl cyclase/cAMP pathway via A2AdR in intact cells to induce capacitation and A1AdR in capacitated cells to inhibit spontaneous acrosome loss and that FPP receptors interact with A2AdR in intact cells and with A1AdR in capacitated cells. Furthermore, adenosine and FPP seem to be useful in reducing the incidence of polyspermic penetration.     

Neonatal caffeine induces sex-specific developmental plasticity of the hypoxic respiratory chemoreflex in adult rats

Caffeine is widely used to treat apneas of prematurity during the neonatal period; however, the potential consequences of administering a neonatal caffeine treatment (NCT) during a critical period for respiratory control development are unknown. The present study therefore determined whether NCT in rats alters the hypoxic respiratory chemoreflex measured at adulthood. Newborn rats received either caffeine (15 mg/kg) or water (control) each day from postnatal day 3 to 12. The ventilatory response to a hypoxic challenge (inspired O(2) fraction = 0.12) was first evaluated in awake adult female and male rats using whole body plethysmography. Results showed that NCT increased the initial phase of the breathing frequency response to hypoxia in males only. This result was confirmed in anesthetized and artificially ventilated adult male rats where NCT also increased the phrenic burst frequency response to hypoxia. RT-PCR assessment of mRNA encoding for adenosine A(1A) and A(2A) receptors, dopamine D(2) receptors, and tyrosine hydroxylase in the rat carotid bodies showed that NCT enhanced mRNA expression levels of adenosine A(2A), dopamine D(2) receptors, and tyrosine hydroxylase of males but not females. Subsequent experiments on awake male rats showed that injection of the adenosine A(2A) receptor antagonist ZM2413855 (1 mg/kg ip) before ventilatory measurements abolished, in NCT rats, the enhanced respiratory frequency response observed during the early phase of hypoxia. We propose that NCT elicits a sex-specific increase in the hypoxic respiratory chemoreflex, which is related, at least partially, to an enhancement in adenosine A(2A) receptors in the rat carotid body.