Roles of 5-hydroxytryptamine and dopamine as neurotransmitters eliciting release of erythrophorotropic hormones in the fiddler crab,Uca pugilator

This article reviews the endocrinological, pharmacological and biochemical evidence ascribing neurotransmitter roles for 5-hydroxytryptamine (5-HT, serotonin) in eliciting the release of red pigment-dispersing hormone (RPDH) and for dopamine (DA) in stimulating the release of red pigment-concentrating hormone (RPCH) in the fiddler crab, $\text{Uca pugilator}$. 5-HT produces red pigment dispersion in intact crabs, but only indirectly. Likewise, DA evokes red pigment concentration $\text{in vivo}$ but it has no effect on red chromatophores (erythrophores) in isolated legs. The data obtained with 5-HT and DA agonists and antagonists on red pigment translocation $\text{in vivo}$ and $\text{in vitro}$, are consistent with their neurotransmitter candidacies in evoking the release of these erythrophorotropic hormones.     

Effects of the cilioexcitatory neurohumors dopamine and 5-hydroxytryptamine on cyclic AMP levels in the gill of the mussel Mytilus edulis.

Cyclic 3′, 5′-adenosine monophosphate (cAMP) has been identified in the ciliated gill epithelium of the marine mussel $\text{Mytilus}$$\text{edulis}$. In concentrations which stimulate the rate of particle transport by frontal gill cilia, DA and 5HT stimulate levels of cAMP within the gill. The stimulation occurs in as early as 15 sec and is graded from 10^?6M to 10^?4M. DA plus 5HT is not additive at maximal effective concentrations of both amines. ACH does not mimic the DA or 5HT stimulation of cAMP. Theophylline alone has a weak effect on cAMP levels; however, the effect of theophylline is potentiated in the presence of DA or 5HT. Dibutyryl cAMP produces a gradual stimulation in the rate of particle transport. It is suggested that the dopaminergic and serotonergic excitatory control of particle transport by frontal gill cilia of $\text{Mytilus}$$\text{edulis}$ is mediated through a cAMP second messenger system.     

LSD and related drugs as DA antagonists: Receptor-mediated effects on the synthesis and turnover of DA

We have earlier shown that d-lysergic acid diethylamide, LSD and its 2-bromo derivative, BOL like the dopamine (DA) antagonists haloperidol increased the rate of the $\text{in vivo}$ tyrosine hydroxylation in the striatum measured as the accumulation of DOPA after decarboxylase inhibition.Now we have found that several agents structurally similar to LSD increase the $\text{in vivo}$ tyrosine hydroxylation in the striatum. Psilocybin (50 mg/kg i.p.) and N,N-dimethyltryptamine (50 mg/kg i.p.) caused a short-lasting increase of DOPA accumulation, while mescaline (10 – 100 mg/kg i.p.) did not increase the DOPA accumulation. A marked increase of DOPA accumulation was observed after the 5-hydroxytryptamine (5-HT) antagonist cyproheptadine. The effects of LSD and structurally related drugs on the DOPA accumulation in the striatum appear to be mediated via DA antagonism at receptor level. However, these agents may control the DOPA accumulation via other receptors than DA receptors e.g. 5-HT receptors. A control of DOPA accumulation via receptors other than DA receptors appears to be predominant after treatment with N,N-dimethyltryptamine or psilocybin.     

Deamination of 5-methoxytryptamine, serotonin and phenylethylamine by rat MAO in vitro and in vivo.

Pretreatment of rats with clorgyline, a selective inhibitor of MAO-A, significantly inhibited the $\text{in vivo}$ deamination of intraventricularly administered serotonin (5-HT) and 5-methoxytryptamine (5-MT), but not phenylethylamine (PEA). Pretreatment with d, l-deprenyl, a selective inhibitor of MAO-B, significantly inhibited the $\text{in vivo}$ deamination of all three substrates. Brain and liver homogenates from rats pretreated with clorgyline showed a decreased ability to deaminate ($\text{in vitro}$) 5-MT and 5-HT, but not PEA. Homogenates from animals pretreated with d,l-deprenyl showed a decreased capacity to deaminate PEA, but not 5-MT or 5-HT. Clorgyline, when added to brain and liver homogenates, selectively blocked the deamination of 5-MT and 5-HT, but not PEA, whereas, d,l-deprenyl blocked the deamination of PEA without affecting that of 5-MT or 5-HT. In addition, 5-MT was found to be 100 X more potent than PEA at inhibiting the $\text{in vitro}$ deamination of 5-HT. These findings suggest that 5-MT and 5-HT are favored substrates for MAO-A $\text{in vitro}$ and $\text{in vivo}$. However, $\text{in vivo}$, significant amounts of 5-MT and 5-HT can also be deaminated by MAO-B.     

The metabolism of dopamine in rat caudate nucleus can be increased by in vivo “dialysis” perfusion cannulae

The development of dialysis cannula techniques coupled with high performance liquid chromatography and electrochemical detection (HPLC-EC) has provided a means to continuously sample extracellular fluid from deep brain structures $\text{in vivo}$. The present studies show that with HPLC-EC analysis of the acid metabolites of dopamine (DA) and 5-hydroxytryptamine (5-HT) in samples from dialysis cannulae implanted in the caudate nucleus of anaesthetized rats, it is possible to determine the time course of the response of dopamine- and 5-HT containing neurones to administration of monoamine oxidase inhibitors and haloperidol. The tissue concentrations of the DA and 5-HT metabolites were also determined at the conclusion of each individual experiment in both the caudate nucleus containing a cannula and in that without a cannula. In perfusion experiments where no drug was administered the content of the DA metabolites, but not that of the 5-HT metabolite, were significantly elevated in the caudate nucleus containing the cannula as compared with the contralateral tissue. These increases occurred whether the cannula was perfused or not, suggesting that the presence of the cannula may have been causing a lesion which altered the activity of the DA neurones. These results emphasize the importance of tissue analysis in conjunction with the dialysis experiments, especially where perfusion sample contents of the monoamine metabolites are being measured as a reflection of the effects of behavioural manipulation or drug treatment on endogenous neuronal activity.     

State-dependent disruption of short-term facilitation due to overexpression of the apPDE4 supershort form in <Emphasis Type="Italic">Aplysia</Emphasis>

Phosphodiesterases (PDEs) play important roles in synaptic plasticity by regulating cAMP signaling in various organisms. The supershort, short, and long forms of Aplysia PDE4 (apPDE4) have been cloned, and the long form has been shown to play a crucial role in 5- hydroxytryptamine (5-HT)-induced synaptic plasticity in Aplysia. To address the role of the supershort form in 5-HT-induced synaptic plasticity in Aplysia, we overexpressed the apPDE4 supershort form in Aplysia sensory neurons. Consequently, 5-HT-induced hyperexcitability and short-term facilitation in nondepressed synapses were blocked. However, the supershort form did not inhibit 5-HT-induced short-term facilitation in highly depressed synapses. These results show that the supershort form plays an important role in 5-HT-induced synaptic plasticity and disrupts it mainly by impairing cAMP signaling in Aplysia.     

Increase of cyclic AMP-dependent protein kinase type II as an early event in hormone-dependent mammary tumor regression.

Primary, 7,12-dimethylbenz(α)anthracene (DMBA)-induced mammary carcinoma in the rat contains cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent and -independent forms of protein kinase. When growth of DMBA-induced tumors was arrested by either ovariectomy or N⁶,O²′-dibutyryl cAMP treatment of the host, the activity of cAMP-dependent protein kinase type II markedly increased in the tumor cytosol, as shown by DEAE-cellulose chromatography and autophosphorylation. The increase in activity of cAMP-dependent protein kinase was also demonstrable in the tumor cytosol and nuclei following $\text{in}$$\text{vitro}$ incubation of tumor slices with cAMP. These results suggest that protein kinase type II is involved in the regression of hormone-dependent mammary tumors.     

The effects of thyrotropin-releasing hormone on cyclic AMP accumulation in rabbit cerebral cortical tissue in the presence and absence of CNS depressants

The $\text{in vitro}$ effects of thyrotropin-releasing hormone on cAMP accumulation in cortical brain slices from rabbits is reported. Incubation of cortical tissue at three concentrations of thyrotropin-releasing hormone (1,2,5nM) had no discernible effects on baseline cAMP levels. When cortical tissue was incubated in the presence of pentobarbital (.5mM) or if cortical tissue was taken from animals pretreated with α-methyl-p-tyrosine (α-MPT), the baseline cAMP accumulation was depressed. This depression could be eliminated by the addition of TRH to the incubation media. Where cortical tissue from atropine-pretreated animals was used or when atropine was added to the incubation media, there was an increase in baseline cAMP accumulation which was unaffected by addition of TRH. These results show that TRH can modify cAMP accumulation in mammalian cortical brain tissue but this ability may only become evident in situations where normal cAMP concentration has been depressed.     

Long-term narcotic exposure reduces caudate cyclic nucleotide levels, protein phosphorylation and tyrosine hydroxylase activities.

Chronic morphine implantation was repeated at three-day intervals with increasing doses of morphine base. Caudate tissues from brains of these animals evidenced significantly lower levels of 3', 5'-AMP and lower activities of endogenous protein phosphorylation and tyrosine hydroxylase $\text{in}$$\text{vitro}$. 3', 5'-GMP levels were no longer altered from control levels. The effects of the three week incrementing morphine dose treatment were still evident in the biochemical measures of caudate tissues from brains of animals withdrawn and abstinent for three successive weeks.     

Lithosperm inhibition of anterior pituitary hormones.

The enzyme system for the synthesis of the pteridine pigment, sepiapterin, from $\text{2-amino-4-hydroxy-6-(D-}\text{erythro}\text{-1',2',3'-trihydroxyprophyl)}$ triphosphate (dihydroneopterin triphosphate) has been found in extracts of $\text{Drosophila melanogaster}$. NADP⁺ or NADPH and Mg²⁺ are required for this enzymatic transformation. No sepiapterin is produced when dihydroneopterin is supplied as substrate in place of dihydroneopterin triphosphate.     

Effects of 4-methyl-α-ethyl-tyramine, 4, α-dimethyl-tyramine and tetrabenazine on the release and uptake of 5-hydroxytryptamine by human blood platelets invitro

4-Methyl-α-ethyl-tyramine and its 4, α-dimethyl analogue release 5-HT from human blood platelets $\text{in}$$\text{vitro}$. At lower concentrations they inhibit the uptake of 5-HT into platelets. Tricyclic antidepressant drugs do not block 5-HT release by these compounds. On removal of the depletor, platelets recover their ability to take up 5-HT; platelets preloaded with exogenous 5-HT lose the same proportion of amine as those containing only endogenous 5-HT. Tetrabenazine behaves similarly, but its actions are partial, whereas those of the tyramines are more complete. The temperature dependence of spontaneous and drug-induced 5-HT release has been measured. The results are discussed in terms of the action of these drugs and with special reference to the use of human blood platelet as a model of a 5-HT-containing nerve ending.     

A demonstration that 5-hydroxytryptamine administered peripherally can affect sexual behavior in male rats

In the male rat, subcutaneous injections for 7 days of 20 mg/Kg B.W./day of 5-hydroxytryptamine creatinin sulphate (5-HT), caused remarkable i$\text{n}$hibitory effects on sexual behavior.The mount and intromission latencies were incre$\text{a}$sed in rats treated with 5-HT, whereas ejacul$\text{a}$tion latency in the few rats treated with 5-HT that it achieved, was similar to that obtained in control rats.The mount and intromission frequencies were decreased in the rats treated with 5-HT.The mean inter-intromission interval (MII) and post-ejaculatory interval were prolonged in rats treated with 5-HT.These data provide evidence for the role of peripheral 5-HT in regulating sexual behavior of - male rats.     

Mechanism of glycogenolytic action of cycloheximide in rat liver

Cycloheximide (0.1 to 0.2 m$\text{M}$) increases cAMP concentration 3 to 4-fold in isolated rat liver slices $\text{in vitro}$. This increase in cAMP concentration parallels an increase in phosphorylase activity. When cycloheximide, at a concentration used to inhibit protein synthesis (1 to 2 μg/g body weight), is administered to whole animals, phosphorylase is activated up to 13-fold by 6 hours. This leads to almost complete depletion of liver glycogen (from about 40 mg/g liver to 0.4 mg/g liver).     

Release of endogenous dopamine, 3,4-dihydroxyphenylacetic acid,and amino acid transmitters from rat striatal slices

The release of endogenous dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) was measured in superfused striatal slices of the rat and the results compared with data obtained for the release of endogenous (a) DA and DOPAC in the cerebral cortex, nucleus accumbens and thalamus; (b) 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), GABA, and glutamate in the striatum; and (c) GABA, glutamate and 5-HT in the cerebral cortex. In superfused slices of all four CNS regions, there appeared to be a Ca²⁺-dependent, K⁺-stimulated release of endogenous DA. In addition, in slices of the striatum and nucleus accumbens there also appeared to be a Ca²⁺-dependent, 60 mM K⁺ stimulated release of endogenous DOPAC. In the striatum, 16 mM Mg²⁺ was as effective as 2.5 mM Ca²⁺ in promoting the 60 mM K⁺-stimulated release of DOPAC. In addition, 16 mM Mg²⁺ appeared to function as a weak Ca²⁺ agonist since it also promoted the release of DA to approximately 40% of the level attained with Ca²⁺ in the presence of 60 mM K⁺. On the other hand, in the striatum, 16 mM Mg²⁺ inhibited the Ca²⁺-dependent, 60 mM K⁺-stimulated release of GABA and glutamate. Similar Mg²⁺-inhibition was observed in the cerebral cortex not only for GABA and glutamate but also for DA and 5-HT. With the use of -methyl -tyrosine (tyrosine hydroxylase inhibitor), cocaine (uptake inhibitor) and pargyline (monoamine oxidase inhibitor), it was determined that (a) most of the released DA and DOPAC was synthesized in the slices during the superfusion; (b) DOPAC was not formed from DA which had been released and taken up; and (c) DA and DOPAC were released from DA nerve terminals. In addition, the data indicate a difference in the release process between the amino acids and the monoamines from striatal slices since Mg²⁺ inhibited the Ca²⁺-dependent, K⁺-stimulated release of GABA and glutamate and appeared to promote the release of DA and 5-HT.     

Characterization of rat pulmonary monoamine oxidase.

The substrate specificities and kinetics of rat lung monoamine oxidase (MAO) have been studied. Utilizing the irreversible MAO inhibitors, clorgyline and deprenyl, rat lung was shown to possess at least two types of MAO, A and B. Tyramine was a substrate for both forms of the enzyme, whereas 5-hydroxytryptamine (5-HT) was a preferred substrate for the A-form. In contrast to most other tissues, 2-phenylethylamine was not solely a B-type substrate for the rat lung MAO and some metabolism by the A-type was apparent $\text{(}\text{B}\text{A}\text{=}\text{80}\text{20}\text{)}$. Using tyramine as substrate the ratio A/B was shown to be $\text{55}\text{45}$. Rat pulmonary MAO-B was inhibited by deprenyl and the kinetics of MAO-A studied. The Km values for the A-form for tyramine, phenylethylamine and 5-HT were relatively similar and were 270, 244 and 170 μM respectively. Whereas, when the A-form was inhibited by clorgyline, the Km values for the B-form were found to differ considerably: 330, 42 and 850 μM for tyramine, phenylethylamine and 5-HT respectively.     

Proctolin: a peptide transmitter candidate in insects.

The slow, striated muscles of the proctodeum (hindgut) of the cockroach, $\text{Periplaneta americana}$ (L.), were examined pharmacologically with reference to the responses evoked by nerve stimulation, glutamate, 5-HT, and proctolin, a myotropic peptide from $\text{Periplaneta}$ recently isolated and identified. The graded contractions evoked by repetitive nerve stimulation were simulated by 5-HT and proctolin at threshold concentrations of about 10⁻⁷ and 10⁻⁹ M respectively; responses to glutamate (∼10⁻⁴ M) were not similarly graded. The 5-HT receptors are distinct from other receptors, including the post-synaptic receptors, since they were specifically blocked by bromolysergic acid diethylamide. Proctolin was fully active on TTX-treated or surgically denervated muscle indicating that the proctolin receptors are located on the muscle fibre membrane. Tyramine, at threshold levels 5×10⁻⁸ M, reversibly antagonized the responses evoked by proctolin and by nerve stimulation but was without effect on the 5-HT and glutamate responses. Neurally evoked responses were potentiated by subthreshold concentrations of proctolin but not by glutamate. Pharmacologically, the proctolin and post-synaptic receptors appear to be identical and distinct from the glutamate and 5-HT receptors. Since proctolin is known to be a constituent of an efferent pathway of the proctodeal nerves, the evidence suggests that it may function as an excitatory transmitter substance. Peptidergic transmission is discussed in relation to the ultrastructural organization of the proctodeal nerve terminals which contain neurosectory granules in addition to electron-lucent, synaptic vesicles.     

The effect of 2,4-dinitrophenol on Dictyostelium discoideum oscillations.

During aggregation the cellular slime mold $\text{Dictyostelium discoideum}$ emits pulses of cAMP about every 5 minutes. Only a small fraction of the aggregating cells produces the pulses autonomously, while most cells synthesize and release the nucleotide in a chain-reaction response to the autonomous signals (1). We report here that 2,4-dinitrophenol, KCN, and caffeine all inhibit the autonomous cAMP oscillations but do not interfere with the triggered response. Because of this, and other data (2), we question current models of the oscillatory synthesis of cAMP.     

Dopamine-sensitive adenylate cyclase of the caudate nucleus of rats treated with morphine.

The addition of narcotic analgesics $\text{in vitro}$ to nerve ending preparations from rat caudate nucleus in an assay of adenylate cyclase activity (AC) resulted in an inhibition of basal AC only at drug concentrations of 10−⁴M or higher, and no inhibition of dopamine-stimulated (DA) AC at these drug concentrations. The acute administration of morphine at a moderately high dose (60 mg/kg) produced an increase in striatal cAMP levels, and increases in basal and DA-AC in caudate nerve-endings. In morphine-tolerant rats, striatal cAMP levels and basal AC were similar to control values, while DA-AC was elevated. These results suggest: (1) that opiates do not act directly on DA-AC, the ‘dopamine receptor’, and (2) that the observed behavioural DA sensitivity in tolerant animals may be produced by the DA-AC supersensitivity.     

Which postsynaptic action of dopamine is mediated by cyclic AMP?

The experimental basis, for proposals that adenosine 3' ,5'-cyclic monophosphate (cAMP) acts as intracellular mediator of one or the other postsynaptic actions of dopamine (DA) in mammalian sympathetic ganglia, is analyzed. These synaptic actions of DA are (I) a hyperpolarizing one, as the direct transmitter for the slow-inhibitory postsynaptic potential (s-IPSP); and (II) a modulatory one, inducing an enduring enhancement of the slow-excitatory postsynaptic response (s-EPSP) to another transmitter, acetylcholine (ACh).(A) Stimulation of adenyl cyclase by DA or appropriate neural input appears generally to support either role; however, the comparative characteristics of DA and norepinephrine (NE) in relation to adenyl cyclase do not appear to be in accord with those in relation to hyperpolarizing actions. (B) Postsynaptic actions of cAMP do not support a role in DA action-I, but they are fully appropriate for DA action-II. (C) Phosphodiesterase inhibition by the relatively potent and selective agent RO-20-1724, under conditions reported to protect and increase cAMP in these ganglia, is shown not to augment the s-IPSP or DA-hyperpolarization; although theophylline does augment these responses, this effect is shown not to be attributable to an inhibition of phosphodiesterase, and it does not provide support for a role in DA action-I. Effects of these inhibitors are at least compatible with the proposed modulatory role-II for cAMP. (D) The timing of the putative chemical reactions involved in a mediation by cAMP would appear to be far too slow for the purposes of DA action-I (s-IPSP response), but it is readily accommodated by the slow onset and development of the modulatory change induced by DA action-II.The suggestion that guanosine 3', 5'-cyclic monophosphate (cGMP) may mediate the slow muscarinic depolarizing response to ACh (s-EPSP) has gained definitive experimental support. Suitable cholinergic stimulation of guanyl cyclase has been demonstrated. The postsynaptic action of cGMP in a low concentration range fits with the unique characteristics of the s-EPSP, at least for cells with normal, not already depolarized, resting membrane potentials. cGMP has also been found capable of antagonizing the modulatory action of either DA (action-II) or of cAMP, but only in a remarkably time-dependent manner.It is concluded that cAMP does not mediate the inhibitory synaptic DA action-I (s-IPSP response), but that it probably does mediate the enduring modulatory change in the s-EPSP (DA action-II). cGMP probably does mediate the production of the s-EPSP by ACh. cAMP would thus have a synergistic, rather than opposing, physiological action in relation to cGMP. A re-examination of the functional significance of related DA-activated adenyl cyclase systems in the brain is suggested.     

Conformational preferences of dopamine analogues for inhibition of norepinephrine N-methyltransferase. Conformationally defined adrenergic agents. 7

A series of analogues of dopamine (DA) with varying degrees of conformational flexibility have been examined as potential substrates or competitive inhibitors of the enzyme norepinephrine N-methyltransferase (NMT). A conformationally defined (rigid) analogue of the fully extended conformation of DA, 2-amino-6, 7-dihydroxybenzonorbornene hydrobromide ($\text{3}$; 6, 7-D2HX) proved to be a better substrate than the non-catechol parent 2-aminobenzonorbornene ($\text{4}$; 2HX). However, analogues $\text{3}$ and $\text{4}$ displayed equivalent competitive inhibitory activity toward phenylethanolamine (PEA). Neither 6, 7-ADTN (5), a DA analogue in the 2-aminotetralin (2AT) system, nor 6, 7-DTHIQ ($\text{7}$), a DA analogue in the tetrahydroisoquinoline (THIQ) system, showed substrate activity; 6, 7-ADTN was a poorer competitive inhibitor than the parent 2AT but 6, 7-DTHIQ was a better competitive inhibitor than its parent, THIQ ($\text{8}$). A tricyclic conformationally defined analogue $\text{9}$ of 6, 7-ADTN was devoid of either substrate or inhibitory activity. From these results it may be concluded that a fully extended side chain conformation is required for NMT substrate activity, and the better substrate activity for 6, 7-D2HX compared to $\text{4}$ is consistent with a proper catechol orientation for interaction with the norepinephrine (NE) binding site of NMT.