Adenosine 3',5'-monophosphate in nervous tissue: increase associated with synaptic transmission.

Brief periods of stimulation of the preganglionic nerve fibers produced a severalfold increase in the content of adenosine 3',5'-monophosphate in superior cervical sympathetic ganglia, whereas postganglionic stimulation did not. These and other experiments indicated that the increased concentrations of adenosine 3'5'-monophosphate were closely associated with the process of synaptic transmission. This increase occurred primarily in postsynaptic cells.

Deregulation of MADS-box transcription factor genes in a mutant defective in the WUSCHEL-LIKE HOMEOBOX gene EVERGREEN of Petunia hybrida.

Angiosperm inflorescences develop in two fundamentally different ways. In monopodial plants, for example in Arabidopsis thaliana, the flowers are initiated as lateral appendages of a central indeterminate inflorescence meristem. In sympodial plants, flowers arise by terminal differentiation of the inflorescence meristem, while further inflorescence development proceeds from new sympodial meristems that are generated at the flank of the terminal flower. We have used the sympodial model species Petunia hybrida to investigate inflorescence development. Here, we describe a mutant, bonsai (bns), which is defective in flower formation, inflorescence branching, and control of meristem size. Detailed microscopic analysis revealed that bns meristems retain vegetative charateristics including spiral phyllotaxis. Consistent with a block in flower formation, bns mutants exhibit a deregulated expression of various MADS-box genes. Molecular analysis revealed that the bns mutant carries a transposon insertion in the previously described EVERGREEN (EVG) gene, which belongs to the WUSCHEL-LIKE HOMEOBOX (WOX) transcription factor gene family. EVG falls in the WOX9 subfamily, which has diverse developmental functions in angiosperms. The comparison of WOX9 orthologues in five model species for flowering shows that these genes play functionally divergent roles in monopodial and sympodial plants, indicating that the WOX9 regulatory node may have played an important role in the evolution of shoot architecture.

Modulation of VIP-Stimulated cAMP Formation by Excitatory Amino Acids in Mouse Cerebral Cortex.

We have investigated the modulatory action of excitatory amino acids (EAA) on vasoactive intestinal polypeptide (VIP)-stimulated cAMP formation in mouse cerebral cortical slices. Glutamate and aspartate potentiate in a concentration-dependent manner the effect of VIP. In order to characterize the type of receptor involved, we have used three prototypical EAA receptor agonists, that is, kainate, N-methyl-d-aspartate (NMDA) and quisqualate. Kainate mimicked the effect of glutamate, NMDA was inactive and quisqualate displayed an inhibitory action. Furthermore, ibotenate also potentiated the effect of VIP on cAMP formation, while l-homocysteate exhibited an inhibitory action. Ibotenate was 4-fold more potent and 2.5 times more effective than glutamate. However, the effects of kainate and ibotenate were not additive, suggesting the activation of a common receptor. Thus, based on this metabotropic action, EAA can be categorized into the following classes: (i) those that potentiate the effect of VIP, such as glutamate, aspartate, kainate and ibotenate; (ii) those that inhibit the effect of VIP, such as l-homocysteate and quisqualate; and (iii) those that are ineffective, such as NMDA and d-homocysteate. The effects of glutamate or ibotenate on VIP-stimulated cAMP formation were completely inhibited by l-phosphoserine and only partially by kynurenate. In a low chloride medium, or in the presence of 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate-hydrochloride (TMB-8), an inhibitor of calcium release from internal stores, EAA did not potentiate the effect of VIP, thus stressing the importance of these ions for the transduction of the glutamatergic signal. Our results indicate the existence of marked interactions between EAA and VIP on cAMP formation; the pharmacology of these interactions is, however, clearly distinct from the classical pharmacology of EAA which is mainly based on electrophysiological and binding studies.

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on dopamine synthesis and release in the rabbit retina in vitro.

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on the synthesis and release of dopamine (DA) was investigated in the retina of the rabbit in vitro. The results indicated that MPTP did not affect the synthesis of DA, but increased its release. Blockade of the activity of monoamine oxidase (MAO) with pargyline (100 microM) did not abolish the capacity of MPTP to release DA. Similarly, these pharmacological effects of MPTP were not dependent on the presence or absence of melanin.

Cyclic AMP levels in isolated frog skin epithelium: effects of phosphodiesterase inhibitors, oxytocin and catecholamines.

Direct measurements of cyclic AMP were performed in the isolated epithelium of frog skin. Phosphodiesterase inhibitors (methylxanthines, papaverine) and activators of adenylyl cyclase (oxytocin, catecholamines) significantly increased the cyclic AMP content. Propranolol completely blocked the generation of cAMP induced by beta-adrenergic agonists but had little or no effect on that induced by oxytocin. Phentolamine enhanced the cAMP production by adrenalin and noradrenalin. At supramaximal concentrations, oxytocin and isoproterenol produced similar increments in cAMP, while exposure to both agents roughly doubled the increase in cAMP. The results suggest the presence of independent receptors for oxytocin and catecholamines in frog skin, with additive effects on cAMP generation.

Receptors coupled to adenylate cyclase in isolated rabbit retina.

Intact pieces or homogenates of rabbit retina were exposed to various established or putative retinal neurotransmitters for the study of receptors (or receptor-subtypes) linked to the production of cAMP. Since a dopamine-sensitive adenylate cyclase has been previously characterized in the retina of several species, the novel D(1)-agonist SKF 38393 was applied under similar experimental conditions. This compound was found to be more potent (although less efficacious) than dopamine, confirming the existence of a population of D(1)-receptors. On the other hand, the novel D(1)-antagonist SCH 23390 was able to inhibit the stimulating effects of dopamine and of SKF 38393 in a concentration-dependent manner. Attempts to detect D(2)-receptors (negatively coupled with adenylate cyclase) were not conclusive, when a selective D(2)-agonist (quinpirole) was applied in the absence or presence of a D(2)-antagonist (sulpiride). A stimulation of cAMP production (mediated by A(2)-receptors) was also detected in response to adenosine or adenosine-analogs, which was blocked by IBMX in a concentration-dependent manner. The effects of adenosine were potentiated in the presence of dipyridamole, an adenosine uptake inhibitor. Compared to the effects of dopamine and adenosine, the stimulation induced by VIP, a retinal neuropeptide, was found to be much more pronounced. These results indicate that retinal cAMP can be generated by three different neurotransmitters in an independent way via the stimulation of specific receptors.

The isolated retina of mammals: A useful preparation for enzymatic-(adenylyl cyclase) and/or binding studies of dopamine receptors.

Rabbit and bovine retinae were used for investigations in vitro related to dopamine receptors. Dose-dependent stimulations of cyclic AMP formation induced by dopamine (or dopamine-like drugs including ADTN and some ergot alkaloid derivatives), which can be blocked by dopamine antagonists (neuroleptics), lithium or d-LSD, were found either in intact retinae or in homogenates. The neuroleptic sulpiride was the only exception since it was devoid of inhibitory effects. Binding studies of retinal dopamine receptors were also achieved with homogenates. By using (3)H-spiroperidol as a ligand, saturability (in the amolar range), stereospecificity (by using butaclamol- or thioxanthene-isomers), tissue linearity, and pH- or temperature-dependence were all demonstrated. Furthermore, displacement curves of (3)H-spiroperidol stereospecifically bound generated by various dopamineand/or catecholamine-related drugs seem to indicate that dopamine receptors are the only monoamine receptors in this tissue and that a coupling possibly exists between binding- and catalytic-sites. These results suggest that by studying both dopamine-sensitive adenylyl cyclase and drug-induced displacement of (3)H-spiroperidol binding the mammalian retina is appropriate to selectively characterize the type of action of various drugs with CNS dopamine receptors.

Prostanoids and their role in cell-cell interactions in the central nervous system.

Although the presence of prostaglandin PGF(2?) has been demonstrated in the central nervous system in the mid sixties, it has taken a rather long time to pinpoint a role of certain metabolites of arachidonic acid in the regulation of neural activity. The modern family of bioactive compounds known as "prostanoids" or "eicosanoids" includes the classical end-products of the cyclooxygenase pathway (prostaglandins, prostacyclin and thromboxane), as well as the molecules formed after the activation of 5- and/or 15-lipoxygenases (leucotrienes and lipoxines), 12-lipoxygenase (hepoxilins) or of epoxygenase (epoxides). Although the brain levels of arachidonic acid-the precursor generating prostaglandins from the series 2-are very low, a plethora of stimuli appears to trigger its release from membrane phospholipids mainly by activation of phospholipase A(2) or subordinately phospholipase C; furthermore, its reesterification can also be subtly regulated by endogenous metabolic processes. Numerous prostanoids have now been detected in the nervous system, namely in neurons, astrocytes, cerebrospinal fluid and cerebral vascular endothelium. Efforts have been oriented at the elucidation of the roles of prostanoids in some physiological conditions (for example sleep regulation) or pathological situations (fever, migraine, epilepsy, schizophrenia). Moreover, several investigators have examined the localization of neuronal membrane receptors for prostanoids and searched for the mechanisms of signal transduction or the identity of second messengers. Those embody cyclic nucleotides (cAMP and cGMP) and calcium. There is also compelling evidence for a modulation by prostanoids of the release of noradrenaline, serotonin and vasoactive intestinal peptide (VIP) as well as of several hormones of the hypothalamic-hypophyseal tract. In addition, neurotransmitters can influence prostanoid synthesis; this has been demonstrated in particular for noradrenaline and more recently for acetylcholine. Prostanoids can also amplify neurotransmitter-mediated signals. Thus, ?(1)-adrenergic agonists, H(1)-histaminergic agonists as well as adenosine potentiate cAMP formation elicited by the VIP, through a concomitant generation of prostaglandins mediated by a direct coupling with phospholipase A(2). Baclofen (a GABA(B)-receptor agonist) exerts a similar potentiation mediated in part by the increased activity of 5-lipoxygenase. Furthermore, eicosanoids generated by 12-lipoxygenase are involved in the histamine- or FMRFamide-induced hyperpolarization (opening of K(+) channels) that has been demonstrated in identified sensory neurons of Aplysia. Finally, the stimulation of N- methyl - d - aspartate receptors (a subclass of glutamate receptors) leads to a release of arachidonic acid as well as of 11- and 12-hydroxyeicosatetraenoic acids in cultured striatal neurons. Arachidonic acid and a large number of its classical or recently discovered metabolites therefore display various effects in the central nervous system, both at the level of integrated processes and of the fine synaptic circuitry, where they can act as intracellular or extracellular local messengers triggering new cascades of short term or long term cellular events.

The increase in cyclic-AMP levels elicited by vasoactive intestinal peptide (VIP) in mouse cerebral cortical slices is potentiated by ergot alkaloids.

We have recently observed that noradrenaline potentiates, via the activation of specific ?-receptors, the stimulatory effects of vasoactive intestinal peptide on cyclic-AMP levels. We report here that certain ergot derivatives of the ergopeptine class, such as bromocriptine, ergotamine and codergocrine known to interact with alpha-adrenergic receptors, will also potentiate the effects of VIP on cyclic-AMP levels, without increasing directly the levels of the cyclic nucleotide. To our knowledge, these results demonstrate for the first time the existence of an interaction between a neuropeptide and ergot alkaloids within the cerebral cortex.

Protective effect of neurotrophic factors, neuropoietic cytokines and dibutyryl cyclic AMP on hydrogen peroxide-induced cytotoxicity on PC12 cells: a possible link with the state of differentiation.

We present evidence that the survival of PC12 cells exposed to hydroxyl radicals generated by hydrogen peroxide applied for 30 min at 1 mM was effective when they were differentiated in response to Nerve Growth Factor (NGF) and/or other inducers of neurite outgrowth such as basic-fibroblast growth factor and dibutyryl cyclic AMP. The time- and dose-dependent differentiation triggered by NGF was (1) markedly increased by basic fibroblast growth factor, interleukin-6 or dibutyryl cyclic AMP; (2) diminished by leukemia inhibitory factor or ciliary neurotrophic factor; (3) not potentiated by insulin-like growth factor I or progesterone. The influence of these various factors and agents on PC12 cells was evaluated by the estimation of neurite outgrowth, whereas their possible protective effects were assessed by the measurement of cell survival. Our results would indicate that the factors and agents that induced differentiation were also able to protect the cells against an oxidative stress.

Nitric oxide modulates endogenous dopamine release in bovine retina.

A study of the possible modulation by nitric oxide (NO) of endogenous dopamine (DA) release was performed in bovine retina in vitro. NO synthase activity was measured in retinal homogenates and totally blocked by L-nitroarginine methyl ester (L-NA). Intact retinas were also exposed to hydroxylamine, an NO-generator which significantly decreased both basal and potassium-induced liberation of DA. In contrast, dibutyryl cGMP was ineffective. Furthermore, L-NA was able to increase basal DA release, its effects being potentiated in calcium free medium. Taken together, these results suggest that endogenous NO regulates DA release via cGMP independent mechanisms.

Dark-induced supersensitivity of dopamine D-1 and D-2 receptors in rat retina.

Light deprivation of rat retina leads to a rapid (within 6 h) development of a state of supersensitivity (upregulation) of dopamine D-1 receptors (positively coupled to adenylate cyclase), which are essentially involved in the modulation by light of the electrical activity and communication between horizontal cells. In contrast, the supersensitivity of D-2 receptors (negatively coupled to cAMP generating system) appears to develop only after 2 days (better after 4 days) of dark adaptation, although these receptors are linked to multiple light-dependent retinal functions. These results suggest the existence of different mechanisms of sensory adaptation for these two subtypes of dopamine receptors.

The effects of VIP on cyclic AMP and glycogen levels in vertebrate retina.

The effects of VIP and related-peptides (PHI, secretin, glucagon) on cyclic AMP formation were investigated in intact pieces of rabbit retina. VIP and PHI increased cyclic AMP levels with EC50 of 160 nM and 300 nM respectively. At 5 microM the peptides increased cyclic AMP 46 fold (VIP) and 38 fold (PHI). Secretin was much less potent and glucagon was totally inactive. VIP was also tested for its effects on glycogen levels under similar experimental conditions. In contrast to its pronounced glycogenolytic action in mouse cerebral cortical slices, VIP at 1 microM decreased only moderately (38.3%) 3H-glycogen newly synthesized from 3H-glucose by pieces of rabbit retina. Furthermore a discrepancy between the efficacy of VIP in increasing cyclic AMP and in promoting glycogenolysis appears to exist. A similar dissociation between these two cellular events was also observed with other neuroactive substances. Thus the pronounced increase in cyclic AMP induced by dopamine and forskolin was accompanied by only a moderate decrease in 3H-glycogen levels. Conversely 50 mM potassium induced a 79.9% decrease in 3H-glycogen levels without any significant increase in cyclic AMP.

VIP-and glucagon-induced formation of cyclic AMP in intact retinae in vitro.

VIP, glucagon or several other neuropeptides were applied to intact retinae in vitro. VIP was found to be a very potent stimulator of cyclic AMP production in rabbit retina, whereas glucagon was efficient in pigeon retina. Attempts to modify the VIP-induced formation of cyclic AMP by somatostatin or dopamine-related drugs (neuroleptics) were not successful. These biochemical data may be correlated to some extent with the recent immunohistochemical localisation of VIP and glucagon in mammalian or avian retinae.

Direct evidence for the stimulation of rabbit retina dopamine receptors by ergot alkaloids.

The effects of three ergot alkaloids on cyclic AMP concentrations in isolated retinae of the rabbit were investigated. Significant increases in cyclic AMP concentration were found after a 10 min exposure to 1 microM ergometrine or agroclavine. The increases in cyclic AMP elicited by ergometrine, agroclavine or 2-bromo-alpha-ergocryptine (CB-154), at 100 microM concentrations, were comparable to those seen after dopamine or the dopamine-like drug, apomorphine. Furthermore, the effects of the ergot alkaloids were blocked by the specific dopamine receptor blocking agent, fluphenazine. It is concluded that some ergot alkaloids or their derivatives are potential dopamine-mimetic drugs.

Differential effects of benzamides and thioxanthenes on dopamine-elicited accumulation of cyclic AMP in isolated rabbit retina.

Benzamides or thioxanthenes were tested as potential antagonists of the cyclic AMP accumulation induced by 10(-4) M dopamine in intact rabbit retinae in vitro in the presence of 5 to 7 mM theophylline. The neuroleptic sulpiride (10(-4) M) was found to be totally inactive whereas a substituted benzamide (clebopride) had small but significant antagonist effect at the same concentration. Among thioxanthenes, isomers of cisconfiguration, which are potent neuroleptics, completely inhibit the cyclic AMP accumulation induced by dopamine, in contrast to trans-isomers which had no inhibitory effects. These data would confirm that retina in vitro is another suitable model for screening antipsychotic activity of classical neuroleptics and that the mechanism of action of sulpiride still needs further investigation.

Okadaic acid modulates exocytotic and transporter-dependent release of dopamine in bovine retina in vitro.

Bovine retinas were isolated for the study of the modulation of exocytotic and transporter-dependent release of dopamine (DA) in vitro. Endogenous DA was measured in the medium using HPLC with electrochemical detection under successive incubations with transfers in fresh medium every 30 min. As expected, potassium caused a calcium-dependent exocytotic liberation of DA. Amphetamine or tyramine induced a calcium-independent release by reversing DA transport across the plasma membrane. Okadaic acid, a specific inhibitor of phosphatases 1 and 2A, induced a slight but significant DA release in the absence of calcium. Furthermore, the toxin increased potassium-, amphetamine- or tyramine-induced DA release independently of extracellular calcium. In addition, okadaic acid completely annulled the ability of a calcium-free extracellular environment to inhibit the potassium-induced DA release. Finally, the toxin prevented the time-dependent decline in the efficacies of amphetamine or tyramine to release DA. In agreement with proposed schemes described for rat striatum, the results of the present study confirmed the existence of distinct release modes of DA in bovine retina. The results obtained with okadaic acid suggest that phosphatase 1 and/or phosphatase 2A constitute part of a direct or indirect mechanism to inhibit both exocytotic and transporter-dependent DA release.

Dopamine- and apomorphine-sensitive adenylate cyclase in homogenates of rabbit retina.

Dopamine (0.5-100 muM) as well as apomorphine (1-100 muM) were found to be potent stimulators of adenylate cyclase in homogenates of rabbit retina. When compared with the dopamine-effect at 10 muM, half-stimulation was also obtained in response to noradrenaline or adrenaline, whereas isoprenaline or phenylephrine were ineffective. Furthermore, the dopamine-induced production of cyclic AMP wab blocked by chloropromazine and haloperidol. These data would suggest the occurrence of a specific "dopamine receptor" in the retina of the rabbit.

L-dopa mediated accumulation of cyclic AMP in isolated rabbit retinae in vitro. Effects of light and/or pharmacological factors.

The ability of L-dopa to stimulate the formation of cyclic AMP in pieces of intact rabbit retina in vitro has been studied and compared with that of dopamine and of other dopamine-mimetic drugs. Dose-dependent effects were measured in response to 5 microM up to 100 microM L-dopa with a maximal stimulation after 20 min of incubation at 35 degrees C. The L-dopa precursor, tyrosine, was totally ineffective. The L-dopa mediated response was detectable only in pieces of intact tissues (and not in homogenates) and is presumably due to the formation of newly formed dopamine, since it was completely inhibited by a decarboxylase inhibitor (benserazide). The biochemical response (cyclic AMP increase) was facilitated by ambient light, this effect being potentiated by 56 mM K+ or fully inhibited by 1 mM gamma-butyrolactone (GBL). The data suggest that the measurement of cyclic AMP levels in pieces of rabbit retina may provide a useful neurochemical model for the study of physiological and/or pharmacological agents able to interact at pre- and/or post-synaptic dopaminergic sites.

Effects of ethanol on VIP-and/or noradrenaline-stimulated cAMP formation in mouse brain.

Among several effects, ethanol (EtOH) interferes with membrane fluidity and lipid-protein interactions. As proteins are influenced by surrounding lipids, the activity of membrane-bound enzymes such as adenylate cyclase (AC) could be modulated by EtOH, as shown in potentiating, at toxic concentrations, the stimulating effect of hormones or neurotransmitters. We have also found that EtOH potentiates in a dose-dependent manner (EC50 = 100 mM) the cAMP production elicited by vasoactive intestinal peptide (VIP), already noticeably at 70 mM, without affecting basal cAMP levels (up to 400 mM). Propanol produces a similar potentiation, whereas methanol was inactive. Butanol (200 mM) displays toxic effects. The potentiation induced by EtOH is similar for peptide- (VIP) or monoamine- (noradrenaline) stimulated cAMP formation, suggesting a primary action at a interaction between VIP and NA in stimulating cAMP formation.