Involvement of P2 purinoceptors and the nitric oxide pathway in [3H]purine outflow evoked by short-term hypoxia and hypoglycemia in rat hippocampal slices

The objective of this study was to study how the outflow of [3H]purines is altered during a brief period of ischemic-like conditions in superfused hippocampal slices and to show whether it is regulated by P2 purinoceptors and the nitric oxide (NO) pathway. The outflow of [3H]purines increased in response to 5 min of combined hypoxia/hypoglycemia. High performance liquid chromatography analysis verified the efflux of [3H]adenosine-triphosphate, [3H]adenosine-diphosphate, [3H]adenosine-monophosphate, [3H]adenosine, [3H]inosine, and [3H]hypoxanthine in response to ischemic-like conditions. The P2 receptor antagonists suramin and pyridoxal-phosphate-6-azophenyl-2'-4'-disulphonic-acid-tetrasodium (PPADS) reduced significantly the [3H]purine efflux evoked by ischemic-like conditions, showing that P2 purinoceptors are involved in the initiation of purine outflow. The NO synthase inhibitor N-nitro-l-arginine-methyl-ester (l-NAME) attenuated significantly the [3H]purine outflow, evoked by ischemic-like conditions, while 7-nitroindazole (7-NI) caused only a mild decrease in the outflow. The NO donor sodium nitroprusside increased significantly the basal efflux of [3H]purines. In summary, a brief period of combined hypoxia/hypoglycemia induced the efflux of ATP in addition to the outflow of other purines. Since P2 receptor antagonists decreased the [3H]purine outflow evoked by ischemic-like conditions we propose that ATP, acting on P2 purinoceptors, is responsible for further efflux of purines after ischemic-like period. It seems likely that NO is also involved in the regulation of purine outflow, since inhibition of NO production attenuated the [3H]purine outflow, evoked by ischemic-like conditions, while exogenous NO facilitated the basal outflow.     

Removal of extracellular sodium prevents anoxia-induced injury in freshly dissociated rat CA1 hippocampal neurons

Anoxia is believed to cause nerve injury and death in part, by inducing sustained, elevated levels of intracellular Ca²⁺. The increased concentration of intracellular Ca²⁺ is capable, by itself, of inducing nerve injury and death, even without the added stress of anoxia. However, we have recently shown that an increased level of intracellular Ca²⁺ is not necessary for anoxia-induce CA1 nerve injury. Since we have observed that extracellular Na⁺ decreases during anoxia, we studied the role of extracellular Na⁺ in anoxia-induced nerve injury. Removal of extracellular Na⁺ and its replacement with the impermeant cation N-methyl-d-glucamine (NMDG⁺) completely protected freshly dissociated CA1 neurons during and after severe anoxia, for up to 90 min. Intracellular Ca²⁺ decreased during anoxia, recovering during reoxygenation. Propidium iodide was excluded from the neurons for as long as Na⁺ was absent. Addition of Na⁺ (by replacing NMDG⁺) following anoxia resulted in rapid bleb formation, swelling and intracellular Ca²⁺ rise. Removal of Na⁺ before the rupture of blebs caused either shrinkage or pinching off of blebs so that the neuron apparently returned to its previous undisturbed state. We conclude that: (1) replacement of Na_o⁺ with NMDG⁺ totally prevents anoxia-induced nerve injury as manifested by morphological changes, e.g., swelling, bleb formation and membrane injury; (2) upon re-exposure to Na_o⁺ following anoxia in the absence of Na_o⁺, neurons swell and bleb; (3) the appearance of blebs following anoxic exposure in the absence of Na_o⁺ is reversible and Na_o⁺ dependent. (4) Bleb formation itself is not necessarily immediately lethal to the neuron.     

Cyclic guanosine 3′,5′-monophosphate-mediated neuroprotection by nitric oxide in dissociated cultures of rat dorsal root ganglion neurones

In dissociated cultures of dorsal root ganglia (DRG) derived from 15-day-old rats, many neurones expressed nitric oxide synthase (NOS) and this expression was found to be reduced by nerve growth factor. The application of blockers of NOS caused selective death of those neurones expressing NOS. The soluble guanylate cyclase (sGC) blocker ODQ also caused neuronal death. The appearance of the neurones undergoing cell death was typical of apoptosis. This suggests that NO has a neuroprotective action in DRG neurones which is probably mediated by its activation of cyclic guanosine 3′,5′-monophosphate. These observations are discussed in relation to the developmental and neuropathic changes in NOS expression by DRG neurones.     

Neurotoxicity in organotypic hippocampal slices mediated by adenosine analogues and nitric oxide

Adenosine (ADO) and nitric oxide (NO) have been implicated in a variety of neurophysiological actions, including induction of long-term potentiation, regulation of cerebral blood flow, and neurotoxicity/neuroprotection. ADO has been shown to promote NO release from astrocytes by a direct effect on A₁ and A₂ receptors, thus providing a link between actions of NO and adenosine in the brain. However, while adenosine acts as an endogenous neuroprotectant, NO is believed to be the effector of glutamate neurotoxicity. To resolve this apparent paradox, we have further investigated the effects of adenosine and NO on neuronal viability in cultured organotypic hippocampal slices exposed to sub-lethal (20′) in vitro ischemia. Up to a concentration of 500 μM ADO did not cause toxicity while exposures to 100 μM of the stable ADO analogue chloroadenosine (CADO) caused widespread neuronal damage when paired to anoxia/hypoglycemia. CADO effects were significantly prevented by the ADO receptor antagonist theophylline and blockade of NO production by l-NA (100 μM). Moreover, CADO effects were mimicked by the NO donor SIN-1 (100 μM). Application of 100 μM ADO following blockade of adenosine deaminase (with 10 μM EHNA) replicated the effects of CADO. CADO, ADO+EHNA but not ADO alone caused a prolonged and sustained release of nitric oxide as measured by direct amperometric detection. We conclude that at high concentrations and/or following blockade of its enzymatic catabolism, ADO may cause neurotoxicity by triggering NO release from astrocytes. These results demonstrate for the first time that activation of pathways other than those involving neuronal glutamate receptors can trigger NO-mediated neuronal cell death in the hippocampus.     

3,4-diaminopyridine-evoked noradrenaline release in rat hippocampal slices: facilitation by endogenous or exogenous nitric oxide

The involvement of nitric oxide (NO) in the evoked release of noradrenaline (NA) was studied in rat hippocampal slices preincubated with [³H]NA and stimulated with 3,4-diaminopyridine (3,4-DAP; 200 μM) for 2 min. The 3,4-DAP-evoked [³H]overflow was enhanced by the NO synthase substratel-arginine, but not byd-arginine; it was reduced by the NO synthase inhibitorN^G-mitro-l-arginine, whichl-arginine. Also drugs known to produce NO in-vitro, like sodium nitroprusside (SNP), 3-morpholino-sydnonimine (SIN-1) andS-nitroso-N-acetylpenicillamine (SNAP) enhanced the 3,4-DAP-evoked NA release. The NO scavenger hemoglobin showed no significant effects when given alone, but reduced or abolished, respectively, the facilitatory effects of SNP, or SNAP andl-arginine. The cyclic GMP derivatives 8-Br-cGMP and Sp-8-p-chlorophenylthioguanosine-3′,5′-cyclic monophosphorothioate (Sp-8-pCPT-cGMPS) also acted facilitatory, whereas the corresponding Rp-enantiomer of the latter compound was inactive, but antagonized the effect of Sp-8-pCPT-cGMPS. NA release evoked by 3,4-DAP (10 μM) from rat hippocampus synaptosomes was not affected byl-arginine orN^G-nitro-l-arginine but slightly increased by SNAP and Sp-8-pCPT-cGMPS. Antagonists at NMDA, non-NMDA and metabotropic glutamate receptors neither affected the 3,4-DAP-evoked NA release nor the facilitatory effect ofl-arginine. From these findings we conclude that endogenously formed NO facilitates 3,4-DAP-evoked NA release in rat hippocampus, possibly by a cyclic GMP dependent mechanism; NMDA receptor stimulation and glutamate release seem not to be involved in this phenomenon.     

Effects of cholecystokinin tetrapeptide (CCK4) and anxiolytic drugs on the electrically evoked [H]5-hydroxytryptamine outflow from rat cortical slices

The outflow of [³H]5-hydroxytryptamine ([³H]5-HT) from electrically stimulated rat cortical slices was measured to ascertain the modulatory role of endogenous cholecystokinin (CCK) on the amine outflow and to test the hypothesis that different anxiolytic compounds inhibit 5-HT secretion. The [³H]5-HT outflow evoked at 10 Hz was increased up to +30% by CCK₄ 300–1000 nM, the effect being prevented by the CCK_B receptor antagonist GV 150013, 3 nM. The limited sensitivity to CCK₄ seemed to depend on 5-HT auto-receptor feedback because pre-treatment with 100 nM methiothepin enhanced the [³H]5-HT outflow and lowered the CCK₄ threshold concentration from 300 to 30 nM. In addition, pre-treatment with 1 μM bacitracin to inhibit CCK metabolism increased [³H]5-HT efflux. This effect was concentration-dependently counteracted by GV150013 suggesting the presence of an endogenous CCK positive modulation. GV150013 30 nM, the 5-HT_1A partial agonist buspirone 300 nM and the GABA_A receptor modulator diazepam 10 nM, known to have anxiolytic properties, all significantly reduced the [³H] amine outflow from cortical slices by about 20%. This inhibition depended on their interaction with their respective receptors, which seemed to restrain the activity of functionally interconnected glutamatergic interneurones. In fact, APV (50 μM) and NBQX (10 μM) prevented the effect of the anxiolytic compounds. Thus, anxiolytic drugs with different receptor targets can reduce 5-HT outflow by dampening the glutamatergic signal, and in turn, the secretory process of the serotonergic nerve ending.     

The effect of high-frequency electrical stimulation and norepinephrine on cyclic AMP levels in normal versus norepinephrine-depleted rat hippocampal slices

Cyclic 3',5'-adenosine monophosphate (cAMP) generation by neuronal activity and norepinephrine (NE) was studied in rat hippocampal slices. High-frequency perforant path stimulation increased cAMP levels 2.5-fold in the dentate gyrus 1 min, but not 30 min, post-stimulation. This increase was abolished by depletion of NE with 6-hydroxydopamine. NE (50 microM) also caused a 3-fold rise in cAMP in whole slices and this stimulation was not altered by NE depletion. These results are consistent with our previous data suggesting that cAMP production is involved in the expression of long-term potentiation and NE-induced long-lasting potentiation in the dentate gyrus.     

Enhancement of dendritic branching in cultured hippocampal neurons by 17β-estradiol is mediated by nitric oxide

Both 17beta-estradiol (E2) and nitric oxide (NO) are important in neuronal development, learning and memory, and age-related memory changes. There is growing evidence that a number of estrogen receptor-mediated effects of estradiol utilize nitric oxide as an intermediary. The role of estradiol in hippocampal neuronal differentiation and function has particular implications for learning and memory.Low levels of estradiol (10nM) significantly increase dendritic branching in cultured embryonic rat hippocampal neurons (158% of control). This study investigates the hypothesis that the estrogen-stimulated increase in dendritic branching is mediated by nitric oxide. We found that nitric oxide donors also produce significantly increased dendritic branching S-nitroso-N-acetylpenicillamine (SNAP: 119%; 2,2'-(hydroxynitrosohydrazino)bis-ethanamine (NOC-18): 128% of control). We then determined that the increases in dendritic branching stimulated by estradiol or by a nitric oxide donor were both blocked by an inhibitor of guanylyl cyclase. Dendritic branching was also stimulated by a cell permeable analog of cyclic guanosine monophosphate (dibutyryl-cGMP: 173% of control). Estradiol-stimulated dendritic branching was reversed by the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide (carboxy-PTIO). This study provides evidence that estradiol influences the development of embryonic hippocampal neurons in culture by increasing the production of nitric oxide or by increasing the sensitivity of the neurons to nitric oxide. Nitric oxide in turn stimulates dendritic branching via activation of guanylyl cyclase.     

Blockade of N- and Q-type Ca channels inhibit K-evoked [H]acetylcholine release in rat hippocampal slices

In the present study, we examined the contribution of specific Ca²⁺ channels to K⁺-evoked hippocampal acetylcholine (ACh) release using [³H]choline loaded hippocampal slices. [³H]ACh release was Ca²⁺-dependent, blocked by the nonspecific Ca²⁺ channel blocker verapamil, but not by blockade of L-type Ca²⁺ channels. The N-type Ca²⁺ channel blocker, ω-conotoxin GVIA (ω-CgTx GVIA; 250 nM) inhibited [³H]ACh release by 44% and the P/Q-type Ca²⁺ channel blocker ω-agatoxin IVA (ω-Aga IVA; 400 nM) inhibited [³H]ACh release by 27%, with the combination resulting in a nearly additive 79% inhibition. Four hundred or one thousand nM ω-Aga IVa was necessary to inhibit [³H]ACh release, ω-Conotoxin MVIIC (ω-CTx-MVIIC) was used after first blocking N-type Ca²⁺ channels with ω-CgTx GVIA (1 μM). Under these conditions, 500 nM ω-CTx-MVIIC led to a nearly maximal inhibition of the ω-CgTx GVIA-insensitive [³H]ACh release. Based on earlier reports about the relative sensitivity of cloned and native Ca²⁺ channels to these toxins, this study indicates that N- and Q-type Ca²⁺ channels primarily mediate K⁺-evoked hippocampal [³H]ACh release.     

Astrocytes have both M1 and M2 muscarinic receptor subtypes

In astrocytes, carbachol evoked the turnover of membrane inositol phospholipids prelabelled with [3H]inositol, as revealed by [3H]inositol phosphate accumulation in the presence of 5 mM lithium. This effect was blocked by atropine and by pirenzepine (IC50 2.2 nM and 56 nM, respectively). Carbachol partially attenuated the isoproterenol-stimulated cyclic adenosine 3',5'-monophosphate production in astrocytes by a direct effect on adenylate cyclase, an effect blocked by atropine and pirenzepine. These results suggest that astrocytes express muscarinic receptor subtypes.     

Reduction by sevoflurane of adenosine 5′-triphosphate-activated inward current of locus coeruleus neurons in pontine slices of rats

Increasing evidence indicates that volatile general anesthetics exert their effects by affecting various types of membrane conductance expressed in the central nervous system (CNS), such as ligand-gated receptor-channels. The most recently identified family of the receptor-channels in the CNS are the extracellular ATP-gated channels (P2X purinoceptors). In the present study, we tested whether volatile anesthetics can affect P2X receptor function in the CNS network. We recorded whole-cell currents of locus coeruleus (LC) neurons in pontine slices from young rats. Adenosine 5′-triphosphate (ATP) sodium (0.03–3 mM) evoked a rapidly rising and moderately desensitizing inward current (50–200 pA) in a dose-dependent manner in LC neurons at a holding potential of −80 mV. Perfusion with clinically relevant concentration of sevoflurane (0.1–0.5 mM) reduced the ATP-induced inward current in a dose-dependent manner (to 56.8±5.9% of control with 0.5 mM sevoflurane; mean±S.E.M., n=13). Estimated IC₅₀ of sevoflurane was 0.59 mM. We conclude that the attenuation of extracellular ATP-mediated signaling in the central nervous system might be one of the multiple actions of volatile anesthetics.     

The involvement of nitric oxide in the secretion of β-endorphin from the pituitary intermediate lobe of the rat

Nitric oxide (NO) generated by the enzyme nitric oxide synthase (NOS) has been implicated in the regulation of a variety of endocrine functions. A number of biochemical and anatomical studies have demonstrated the presence of neuronal NOS (nNOS) in the neuroendocrine axis and have shown significant effects of NO on the release of hypothalamic and pituitary hormones. Using a C-terminal directed peptide antibody that is specific for nNOS we have found a predominance of nNOS in the neural lobe of the pituitary and in a single layer of epithelial cells, possibly a remnant of Rathke's pouch that form a border between the intermediate lobe and the anterior lobe. Furthermore, we have examined the effect of sodium nitroprusside (SNP), a donor of NO on the secretion of β-endorphin (β-EP) from the isolated neuro-intermediate lobe (NIL) of the rat and cultured rat melanotrophs. It was shown that in explant cultures of intact neuro-intermediate lobes, SNP (100 μM) was able to cause an inhibition of β-EP secretion. In the presence of sulpiride (10 μM), a dopamine D₂-receptor antagonist, there was a partial reversal of the SNP effect. On the other hand SNP did not affect β-EP secretion in primary cultures of melanotrophs that no longer possessed any innervation. Taken together these data suggest that NO has an indirect inhibitory effect on the secretion of β-EP by the intermediate lobe via the release of dopamine.     

Characteristics of CA1 activation through the hippocampal trisynaptic pathway in the unanaesthetized rat

The hippocampal CA1 field is activated by the entorhinal cortex mainly through the hippocampal excitatory trisynaptic circuit. Field responses of the CA1 region were evoked by ipsilateral CA3 or perforant path volley (mono- or trisynaptic activation, respectively) in paralyzed, locally anaesthetized rats and studied as a function of the stimulus patterns presented. The relationship of these responses with the concomitant EEG was also explored. Results showed that mono- and especially trisynaptically evoked responses were progressively enhanced by increasing the stimulus frequency from 0.1 to 1.0 Hz. At specific intensities the trisynaptically evoked population spike (PS) was present only with a rather fixed frequency of stimulation (approximately 0.5 Hz). PS was produced in 100% of the responses using 0.7 Hz, indicating the existence of a threshold-like level for this stimulus parameter. The frequency of presented paired pulses differentially affected pair-pulse facilitation of mono- and trisynaptically evoked excitatory postsynaptic potentials (EPSP): higher frequency decreased the former and increased the latter. All evoked responses studied (i.e. EPSP and PS) showed steep increments and decrements in amplitude, clearly developing several clusters. Moreover, the amplitude distribution of trisynaptic PS often varied spontaneously from maximal to negligible values, showing an all-or-none distribution. Clustering was interpreted as evidence of the existence in the hippocampus of functional neuronal aggregates. All-or-none distribution of trisynaptic PS was found to be associated with the EEG pattern, PS amplitude being maximal during irregular EEG activity and minimal during theta rhythm. Present results suggest that (1) the entorhinal cortex may exert modulatory actions on CA1 by a mechanism widely based on the frequency of the input; (2) information transfer from the entorhinal cortex to other brain areas throughout the hippocampus is biased by hippocampal EEG; and (3) electronic coupling may be functionally predominant in the hippocampus.     

Integrin Mac-1 and β-amyloid in microglial release of nitric oxide

The β-amyloid protein associated with Alzheimer's disease (AD) has been well characterized biochemically; however, its primary biological function and mode of action in AD has not been determined. We have shown previously that β-amyloid (β25–35), in combination with interferon-γ (IFN-γ), can induce nitric oxide release from cultured hippocampal microglial cells. In the present study, binding of β-amyloid with the leukocyte integrin Mac-1, a cell surface receptor on microglia, was studied by observing (1) inhibition of β-amyloid (β25–35)-mediated release of nitric oxide from cultured microglial cells following exposure to monoclonal antibodies against Mac-1 (anti-CD18 and anti-CD11b) and (2) competitive binding of fluorochrome-labeled β25–35 with anti-CD18 or anti-CD11b using fluorescent flow cytometry. Wt.3 (anti-CD18 antibody) and OX42 (anti-CD11b antibody) were as effective as opsonized zymosan at inducing the release of nitric oxide from microglia. Furthermore, Wt.3 and OX42 acted synergistically to induce maximum nitric oxide release. An interaction between β-amyloid and CD18 of Mac-1 was evidenced by the suppressive action of β25–35 on Wt.3-mediated release of nitric oxide and the synergistic action between OX42 and β25–35 in inducing nitric oxide release from microglia. The tissue culture study was supported by competitive binding assays of fluorochrome-labeled β25–35 and Mac-1 antibodies (Wt.3 or OX42). The majority of microglial cells (71%) did bind biotinylated β-amyloid in the presence of cytochalasin B, suggesting that β-amyloid binding to microglia is a receptor-mediated event. Furthermore, pre-exposure to Wt.3, but not OX42, significantly decreased binding of biotinylated β25–35 to microglia. These findings suggest that CD18 of Mac-1 may play a role in β-amyloid-mediated release of nitric oxide.     

Early effects on restoration of evoked field potentials in the hippocampal CA1 region after reversible hypoxia/hypoglycemia by the radical scavenger N-tert.-butyl-α-phenylnitron

In transverse hippocampus slices a short period of hypoxia/hypoglycemia induced by perfusion with an O₂/glucose-free medium caused early loss and incomplete restoration of evoked field potentials in the CA₁ region. In the present study a search was made for whether the formation of free oxoradicals immediately after starting the hypoxic phase could be part of the breakdown and incomplete restoration of the excitatory potentials (EPs). It was shown that preincubation and postischemic incubation with the radical scavenger PBN did not prevent the potential breakdown but significantly enhances potential restoration, even when PBN was added to the perfusion medium 40 min after hypoxia. Thus, free oxoradicals may damage membrane constituents such as receptors or channel proteins at a very early phase, before neuronal death is pronounced. The results also show that treatment with radical scavengers has a beneficial effect on early hypoxic damage.     

Adenosine activates ATP-sensitive K currents in pericytes of rat retinal microvessels: role of A1 and A2a receptors

In the CNS, contractile pericytes are positioned on the endothelial walls of microvessels where they are thought to play a role in adjusting blood flow to meet local metabolic needs. This function may be particularly important in the retina where pericytes are more numerous than at any other site. Despite the putative importance of pericytes, knowledge of the mechanisms by which vasoactive molecules, such as adenosine, regulate their function is limited. Using the perforated-patch configuration of the patch-clamp technique to monitor the whole-cell currents of pericytes located on microvessels freshly isolated from the adult rat retina, we found that adenosine reversibly activated a hyperpolarizing current in 98% of the sampled pericytes. This adenosine-induced current is likely to be due to the opening of ATP-sensitive potassium (K_ATP) channels since it had a reversal potential near the equilibrium potential for K⁺, was inhibited by the K_ATP channel blocker, glibenclamide, and was mimicked by pinacidil, which is a K_ATP channel opener. Experiments with specific agonists and antagonists indicated that both the high affinity A₁ and the lower affinity A_2a adenosine receptors provided effective pathways for activating K_ATP currents in pericytes recorded under normal metabolic conditions. However, during chemical ischemia, the A₁ receptor pathway rapidly became ineffective. In contrast, activation of A_2a adenosine receptors continued to open K_ATP channels in ischemic pericytes. These results suggest that the regulation of K_ATP channels via A₁ and A_2a receptors allows adenosine to serve over a broad range of metabolic conditions as a vasoactive signal in the retinal microvasculature.     

The activity of constitutive nitric oxide synthase is increased by the pathway cAMP/cAMP-activated protein kinase in human platelets. New insights into the antiaggregating effects of cAMP-elevating agents

Human platelets synthesize nitric oxide (NO) through an endothelial-type NO synthase (ecNOS) activated also by substances enhancing 3',5'-cyclic adenosine monophosphate (cAMP) concentrations, such as catecholamines, beta-adrenoceptor agonists and adenosine. To verify whether cAMP directly activates ecNOS through the cAMP-dependent protein kinase A (PKA), we evaluated (i) the influence of 8-Br-cAMP, adenosine and forskolin on ecNOS activity and phosphorylation at Ser(1177) and (ii) the effect of PKA inhibition on ecNOS activity. Platelets from 10 healthy male volunteers were used for aggregation studies and measurement of NOS activity (conversion of L-[(3)H]-arginine to L-[(3)H]-citrulline) following exposure to 8-Br-cAMP, adenosine and forskolin, both in the absence and in the presence of the PKA inhibitor Rp-cAMPS (100 micromol/l). The phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein (VASP) at Ser(157) and Ser(239) and of ecNOS at Ser(1177) was evaluated by Western blot. NOS activity (pmol L-citrulline/10(8) platelets) increased from 0.090+/-0.002 to 0.148+/-0.013 with 500 micromol/l 8-Br-cAMP (p<0.0001), to 0.140+/-0.008 with 30 micromol/l adenosine (p<0.0001) and to 0.140+/-0.009 with 10 micromol/l forskolin (p<0.0001). Rp-cAMPS decreased baseline NOS activity from 0.093+/-0.001 to 0.075+/-0.006 (p<0.02) and prevented the stimulation by 8-Br-cAMP, adenosine and forskolin. Platelet exposure to 8-Br-cAMP and forskolin, beside the phosphorylation of the specific PKA substrate VASP, markedly increased the expression of ecNOS protein phosphorylated at Ser(1177). The study shows that NOS activity of human platelets is increased by the cAMP/PKA pathway which is involved in NO synthesis induced by adenosine, forskolin and potentially by every antiaggregating substance enhancing intraplatelet cAMP via receptor-dependent and -independent mechanisms.     

Effects of prostaglandin E2, forskolin and cholera toxin on cAMP production and in vitro LH-RH release from the rat hypothalamus

The present study attempts to elucidate the possible role of adenosine 3′,5′-monophosphate (cAMP) and prostaglandin E₂ (PGE₂) in the function of the neural luteinizing hormone-releasing hormone (LH-RH) apparatus. To this end, in vitro LH-RH release from superfused hypothalamic fragments and cAMP production by hypothalamic P₂ membrane fractions were measured. Immature female rats (day 28) were ovariectomized and implanted with Silastic capsules containing estradiol (235 μg/ml). Two days later, animals were sacrificed and the mediobasal hypothalamic preoptic area (hypothalamic units or fragments) were removed. To examine in vitro LH-RH release from superfused hypothalamic fragments, effluents were collected into tubes on ice at 10-min intervals and LH-RH concentration was determined by radioimmunoassay (RIA). Following a 50-min control period, a step-wise increment in several doses of PGE₂ (each dose for a 50-min interval) evoked a dose-related increase in LH-RH release. PGE₂ induced significant (P<0.01) increments in LH-RH release at doses of 5.68 × 10⁻⁷, 5.68 × 10⁻⁶, and 5.68 × 10⁻⁵ M, respectively. When adenylate cyclase activators, such as forskolin and cholera toxin were infused in a step-wise manner (each dose for a 50-min interval) following a 50-min control period, a dose-related increase in LH-RH release was also obtained; forskolin and cholera toxin significantly (P<0.01) stimulated LH-RH release at doses of 1 × 10⁻⁴ and 5.4 × 10⁻¹⁰ M, respectively. These two substances were ineffective in stimulating LH-RH release when hypothalamic fragments were superfused in calcium-free plus EGTA (10 mM) containing medium. An intermittent infusion of dibutyryl cAMP (dbcAMP: 1 × 10⁻⁷ M, 10-min on, 20-min off) resulted in rhythmic LH-RH release from median eminences superfused in vitro. In separate experiments, to examine adenylate cyclase activity, P₂ membrane fractions from the mediobasal hypothalamus were preincubated with appropriate test agents. Adenylate cyclase reaction was initiated by adding adenosine triphosphate. After a 15-min incubation, the reaction was terminated by boiling, the supernatant recovered and subjected to cAMP determination by RIA. The following results were obtained: (1) in vivo E priming of ovariectomized animals significantly increased basal adenylate cyclase activity of P₂ membrane preparations as compared to those from unprimed rats; (2) known adenylate cyclase activators, such as forskolin and cholera toxin clearly produced a dose-related increase in cAMP production; and (3) PGE₂ at the concentration of 5.68 × 10⁻⁶ M stimulated cAMP production. It appears that cAMP and PGE₂ may be involved in the activation of the LH-RH neural apparatus. It is tempting to postulate that PGE₂ may stimulate adenylate cyclase to increase intracellular cAMP levels which, in turn, trigger the release of LH-RH from the median eminence nerve terminals.