Identification of a 5-HT1A receptor positively coupled to planarian adenylate cyclase

The adenylate cyclase system present in particulate fractions prepared from two planarian species was tested for sensitivity to various neurotransmitters. While dopamine and other catecholamines were ineffective, serotonin was capable of stimulating the enzyme. Among the various serotonin agonists tested, only 8-OH-DPAT resulted effective on the adenylate cyclase activity, thus suggesting the presence in planarians of a serotonin receptor of the type 5-HT_1A.     

Guanosine 5'-triphosphate reqiuirement for activation of adenylate cyclase by serotonin in a somatic cell hybrid.

Activation of adenylate cyclase [ATP: pyrophosphate-lyase (cyclising); EC 4.6.1.1] by serotonin in the NCB-20 mouse neuro-blastoma x foetal Chinese hamster brain hybrid cell line has been shown to require GTP. GTP increases slightly in the initial rate of cyclic AMP synthesis, and serotonin further increases the GTP-dependent increases in enzyme activity. In the presence of a saturating concentration of serotonin (10 μm), half-maximum activation of adenylate cyclase is produced by 0.1 μM GTP.     

Evidence for the Presence of Serotonin Receptors Negatively Coupled to Adenylate Cyclase in the Rabbit Iris-Ciliary Body

Serotonin has no obvious effect on basal cyclic AMP levels but reduces the forskolin-, isoproterenol-, and vasoactive intestinal peptide-induced stimulation of cyclic AMP levels in a dose-dependent manner. Serotonergic, cholinergic, muscarinic, alpha-adrenergic, and dopaminergic antagonists have no effect on the serotonin response. Topical application of a serotonin/pargyline solution to the living eye causes desensitisation of the serotonin response in the iris-ciliary body, an observation confirming the presence of specific serotonergic receptors linked to adenylate cyclase. The 5-HT1A [5-hydroxytryptamine (serotonin) type 1A] receptor agonists 8-hydroxy-2-(di-n-propylamino)tetralin and buspirone mimic the serotonin response in reducing the forskolin-stimulated cyclic AMP levels, as do the indole derivatives 5-methoxytryptamine, 5-hydroxtryptophan, and tryptamine. However, the ineffectiveness of the 5-HT1A agonist ipsapirone and the inability of spiroxatrine to block the serotonin response show that classical 5-HT1A receptors are not involved. The serotonin response is blocked by pertussis toxin and is insensitive to the phosphodiesterase inhibitor theophylline, which indicates the involvement of an inhibitory guanine regulatory protein in the coupling of the serotonin receptor to the adenylate cyclase catalytic unit.     

The adenylate cyclase system of planaria Polycelis tenuis: activation by serotonin and guanine nucleotides

The particulate fraction prepared after homogenization of planaria Polycelis tenuis in a buffer containing 3 mM EDTA and 15 mM 2-mercaptoethanol possesses an adenylate cyclase activity which was enhanced two-fold by serotonin and 20-fold by the nucleotide analog guanosine 5'-(beta-gamma-imino)triphosphate, Gpp(NH)p; when present together, the two activators exhibited a marked synergistic effect. The effect of serotonin was dose dependent, with a KA of 2 micrometer and a Hill coefficient of 0.4. In the presence of 10 micrometer Gpp(NH)p, these values became 45 nM and 1.5, respectively. The effect of serotonin was due to an increase in the maximal velocity of the enzyme and was specifically inhibited by methiotepin. The effect of methiotepin was half-maximal at 0.2 micrometer in the absence of Gpp(NH)p and at 5.0 micrometer in its presence. Planaria thus appear to be the lowest organisms in which guanine nucleotides are active upon adenylate cyclase. As serotonin is normally present in planaria, it is postulated that a serotonin-dependent regulation of adenylate cyclase activity plays a physiological role in this species.     

Adenylate cyclase activity from Hirudo medicinalis segmental ganglia: modulation by calcium and calmodulin

An adenylate cyclase activity stimulated by serotonin and calmodulin is present in the segmental ganglia of the leech Hirudo medicinalis. Removal of the endogenous calcium binding protein does not alter the responsiveness of the enzyme to serotonin. The calmodulin antagonist, trifluoperazine, drastically reduces the amine stimulatory effect on both intact and calmodulin-depleted membranes. We suggest that calmodulin-sensitive and serotonin-stimulated adenylate cyclase are, at least functionally, independent.     

Photoaffinity Labeling of Adenylate Cyclase-Linked Serotonin Receptors in Aplysia Neurons

Serotonin stimulated adenylate cyclase in Aplysia neurons with a Kact of 0.7 microM. Under the same conditions, 1-[2-(4-aminophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine stimulated adenylate cyclase with a Kact of 20 microM. The azido derivative of this compound, 1-[2-(4-azidophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine, or of serotonin, (4-amino, 3-nitrophenylazido-serotonin), also stimulated the cyclase in the dark, but with lower efficiency (Kact greater than 10(-4) M). Irradiation of the membranes in the presence of 100 microM 1-[2-(4-azidophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine abolished 75% of the cyclase activity stimulated by 5 microM serotonin. Under the same conditions, 100 microM 4-amino, 3-nitrophenylazido-serotonin did not inhibit serotonin-stimulated adenylate cyclase activity. When [3H]1-[2-(4-azidophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine (20 microM) was irradiated with membranes for 5 min at 4 degrees C, a dozen peptides were labeled, as revealed by a fluorogram of sodium dodecyl sulfate-polyacrylamide gels. Among them, the labeling of five polypeptides (molecular weights of 45,000, 55,000, 63,000, 80,000, and 94,000) was protected by the presence of 0.2 mM serotonin during photolysis. These peptides may be related to serotonin receptors.     

A new mechanism of inhibiting adenylate cyclase involving a beta-adrenergic receptor]

The previously unknown mechanism of adenylate cyclase activity inhibition by catecholamines has been found. It is realized through a beta-adrenoreceptor in the smooth muscle of fresh-water mollusc Anodonta cygnea. As to its ligand-binding characteristics (one class of binding sites with Kd = 0.35 + 0.06 nM, a competitive series of ligands substitution: isoproterenol greater than adrenalin greater than propranolol greater than noradrenaline greater than serotonin = dopamine greater than phentolamine) as well as to negative regulation of the GTP affinity this receptor is similar to beta-adrenoreceptors of higher vertebrates. The dose-dependent inhibiting effect (to 50-60%) of isoproterenol and noradrenaline on the basal, GTP- and serotonin-stimulated activity of adenylate cyclase and cAMP level which is removed only by beta-adrenergic blockers is shown in vitro and in vivo. It is concluded that inhibition of adenylate cyclase activity by catecholamines in the muscular tissue of the mollusc is realized via beta-adrenoreceptor.     

A family of Drosophila serotonin receptors with distinct intracellular signalling properties and expression patterns.

Biogenic amines such as serotonin elicit or modulate a wide range of behaviours by interacting with multiple receptor subtypes. We have isolated cDNA clones encoding three distinct Drosophila serotonin receptors which belong to the G protein-coupled receptor family. When expressed in mammalian cells, these receptors activate different intracellular effector systems. The 5HT-dro1 receptor stimulates adenylate cyclase while the 5HT-dro2A and the 5HT-dro2B receptors inhibit adenylate cyclase and activate phospholipase C. Expression of all three receptors starts in late embryos and is restricted to distinct populations of cells in the central nervous system. The 5HT-dro2A receptor is predominantly expressed in midline motor neurons (VUM neurons) that innervate larval muscles thus suggesting a role for this receptor in motor control.     

GTP-dependent anion-sensitive adenylate cyclase in snail ganglia potentiation of neurotransmitter effects

Snail ganglia possess an anion-sensitive adenylate cyclase. This enzyme was stimulated 100% by chloride in a strictly GTP-dependent manner. The apparent affinity of chloride for adenylate cyclase was 2 X 10(-4) M. Halogens were found to be the most active anions. Some inorganic anions such as SO4(2-) and H2PO4- were inactive, as were all the organic anions tested. Stimulation was not cumulative for any maximal concentration of the active anions except fluoride. Chloride potentiated the effect of fluoride, indicating that the anion effect is not fluoride-like. Another striking result is that chloride enhanced adenylate cyclase sensitivity to the neurotransmitters serotonin and dopamine. The absence of chloride stimulation when Mg2+ was replaced by Mn2+ further indicates a role of the GTP-binding protein (the G/F unit). Chloride could reversibly stimulate the adenylate cyclase activity already maximally stimulated by guanyl 5'-imidodiphosphate. We therefore suggest that, in snail ganglia, chloride raises the activity of the G/F unit-catalytic unit complex at some stage after its formation. The same specific anion-sensitive adenylate cyclase was also found in some of the rat tissues tested.     

Modulation by Monoamines of Somatostatin-Sensitive Adenylate Cyclase on Neuronal and Glial Cells from the Mouse Brain in Primary Cultures

Primary cultures of mouse embryonic neuronal or glial cells from the cerebral cortex, striatum, and mesencephalon were used to identify and determine the cellular localization of somatostatin receptors coupled to an adenylate cyclase. Somatostatin inhibited basal adenylate cyclase activity on neuronal but not on glial crude membranes in the three structures examined. The somatostatin-inhibitory effect on neuronal crude membranes was still observed in the presence of (-)-isoproterenol, 3,4-dihydroxyphenylethylamine (dopamine, DA), or 5-hydroxytryptamine (5-HT, serotonin) used at a concentration (10(-5) M) inducing maximal adenylate cyclase activation. In addition, in most cases biogenic amines modified the pattern of the somatostatin-inhibitory effect, triggering either an increase in the peptide apparent affinity for its receptors or an increase in the maximal reduction of adenylate cyclase activity or both. However, 5-HT did not modify the somatostatin-inhibitory response on striatal and cortical neuronal crude membranes. The changes in somatostatin-inhibitory responses were interpreted as a colocalization of the amine and the peptide receptors on subtypes of neuronal cell populations. Finally, somatostatin was shown to inhibit adenylate cyclase activity following its activation by (-)-isoproterenol on glial crude membranes of the striatum and the mesencephalon but not on those of the cerebral cortex.     

Adenylate cyclase from Hirudo medicinalis segmental ganglia: Modulation by physiological and non-physiological agents

1. In Hirudo medicinalis segmental ganglia GTP is essential for the full expression of the stimulatory action of serotonin on the adenylate cyclase activity. The amine, in turn, increases the overall affinity of the enzymatic system for GTP.2. GTPγS and Gpp(NH)p, non-hydrolysable analogues of GTP, dose-dependently enhance the basal enzyme activity, but impair the stimulatory effect of serotonin.3. Fluoride ions biphasically modulate the leech adenylate cyclase both in the absence and in the presence of GTP. The ion effect is also influenced by non-physiological guanine nucleotides     

Effect of adenylate cyclase activators and Mg2+ on the binding and the electron spin resonance spectra of N-methylmaleimide nitroxide in membrane particles from the liver fluke Fasciola hepatica

Optimal conditions for activation of adenylate cyclase in membrane particles were studied. Enzyme activation with serotonin (5-hydroxytryptamine). NaF, and guanosine 5'-(3-O-thio)-triphosphate (GTP gamma S) was time-and temperature-dependent. Mg2+ was required for enzyme activation. Adenylate cyclase that was activated by NaF or GTP gamma S was gradually inhibited by N-methylmaleimide while enzyme activated with serotonin and GTP responded faster to inhibition by the same sulfhydryl reagent. Th enzyme responded in a similar fashion to a spin-labeled N-methylmaleimide analog 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrolidinyloxyl (i.e., N-methylmaleimide nitroxide). Binding of the spin label was enhanced following enzyme activation by serotonin, NaF, or GTP gamma S in the presence of Mg2+. Activation of the enzyme was accompanied by an increase in the strong immobilization peaks in the EPR spectra. Both effects, the increase in binding and in the strong immobilization peaks, can be induced by Mg2+ alone. The results indicate that a general conformational induced by Mg2+ may be essential for adenylate cyclase activation.     

Catecholamine-Sensitive Adenylate Cyclase in the White Perch (Roccus americanus) Retina: Evidence for β-Adrenergic and Dopamine Receptors Linked to Adenylate Cyclase

We have examined the catecholamine-sensitive adenylate cyclase in the retina of the white perch (Roccus americanus). Both dopamine and the beta-adrenergic agonist isoproterenol stimulate cyclic AMP accumulation in this retina, but serotonin, an indoleamine, and phenylephrine, an alpha-adrenergic agonist, had no effect. The stimulation of adenylate cyclase by isoproterenol is more potent and effective than that of dopamine. The effects of dopamine and isoproterenol are mediated via independent dopamine and beta-adrenergic receptors. Haloperidol, a dopamine antagonist, blocks the stimulatory effect of dopamine but not of isoproterenol. Conversely, propranolol, a beta-adrenergic antagonist, blocks the stimulatory effect of isoproterenol but not of dopamine. The effects of dopamine and isoproterenol are not additive. In fractions of purified horizontal cells we found evidence for dopamine receptors linked to adenylate cyclase but did not find evidence for the presence of cyclase coupled beta-adrenergic receptors. The cellular location of the beta-adrenergic receptors is unknown. Our findings demonstrate the existence of both beta-adrenergic and dopamine receptors coupled to adenylate cyclase in the white perch retina. However, we did not find either epinephrine or norepinephrine, endogenous ligands of the beta-receptor, to be present in retinal extracts subjected to HPLC.     

Serotonin sensitive adenylate cyclase in horse brain synaptosomal membranes.

In different membranal preparations isolated from horse brain stritum we have shown the existence of an adenylate cyclase system sensitive to serotonin (5-HT). Activation of the adenylate cyclase was determined by measuring cAMP using a radioimmunoassay. This serotoninergic sensitive enzyme is characterized by a high apparent affinity constant (in the nanomolar range), located on synaptosomal membranes. It is inhibited by antiserotoninergic drugs (cyproheptadine, cinanserin, methysergide, LSD), and synergistically activated by GTP. This serotoninergic activation is clearly additive to the activation induced by dopamine. It appears different from the adenylate cyclase system previously described in the literature which is also activated by 5-HT, but which has a low apparent affinity constant (in the micromolar range); the latter is apparently located in non-synaptosomal membranes, and its activation by 5-HT is non-additive to the activation induced by dopamine.The serotoninergic sensitive adenylate cyclase reported in this study, might be related to the serotoninergic binding system which we have previously described which has similar affinity constant, a similar subcellular distribution and which is inhibited in the same concentration ranges by antiserotoninergic drugs. These two systems might represent a synaptosomal serotoninergic receptor complex.     

Participation of the adenylate cyclase system in the postsynaptic mechanism of heterosynaptic facilitation

In an analysis of the postsynaptic mechanism of heterosynaptic facilitation, changes in the amplitude of the excitatory postsynaptic current (EPSC) and the current evoked by application of acetylcholine (ACh current), acting on the adenylate cyclase system of the LC-1 and RC-1 neurons of the molluskPlanorbis corneus, were compared. Both responses are n-cholinergic and depend on the membrane conductivity for Na⁺ and K⁺. Application of serotonin led to a 100–300% increase in the amplitude of the EPSC and (in most cases) the ACh current. However, in 30% of the cases, the increase in the EPSC was accompanied by a decrease in the ACh current. This is probably due to the different contributions of Na⁺ and K⁺ to the mechanism of activation of the conductivity of th channel-receptor complex of the nonsynaptic cell membrane. The influence of serotonin on the EPSC and ACh current was simulated by the action of phosphodiesterase blockers and adenylate cyclase activators. Phosphodiesterase activators and protein kinase blockers reversibly inhibited the EPSC and ACh current. Thus, activation of the adenylate cyclase system, mediated by the action of serotonin, promotes the development of a postsynaptic mechanism of formation of heterosynaptic facilitation of the EPSC in the command neurons of the mollusk.A. A. Bogomolets Institute of Physiology, Ukrainian Academy of Sciences, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 6, pp. 676–683, November–December, 1991.     

Cyclic nucleotides regulate the morphologic alterations required for chemotaxis in monocytes

The initial morphologic response of human monocytes to chemoattractants is a change in shape from round to a triangular "motile" configuration (polarization). At doses chemotactic in vitro, chemoattractants induced rapid (t 1/2 = 45 sec), sustained (greater than 40 min) polarization of monocytes in suspension. Extracellular Ca++ was not required for polarization induced by chemoattractants, but in the absence of Ca++ kinetics were slowed (t 1/2 = 6.5 min). Phenylephrine, carbamycholine, serotonin, and ascorbate also caused rapid polarization of monocytes. Unlike chemoattractants, polarization by the pharmacologic agents was unsustained (less than 15 min), absolutely required extracellular Ca++, and affected about 50% of the cells responsive to chemoattractants. Based on relative sensitivities to alpha 1- and alpha 2-adrenergic agonists and antagonists, polarization caused by adrenergic agents was mediated by alpha 2-receptors. Muscarinic and alpha 2-adrenergic agonists, serotonin, and ascorbate enhanced the rate and number of monocytes polarizing to suboptimal doses of chemoattractants. Thus, the initial morphologic changes induced by chemoattractants appear to utilize an activation pathway shared with a variety of agents that enhance cGMP levels and inhibit adenylate cyclase. In contrast, theophylline, histamine, and isoproterenol, all agents that activate adenylate cyclase and elevate cAMP levels, inhibited monocyte polarization to chemoattractants. As in PMN, pharmacologic agents that increase cAMP levels inhibited monocyte chemotaxis in vitro, whereas those that inhibit adenylate cyclase and increase cGMP enhanced monocyte chemotactic responses. Thus, the initial morphologic response of monocytes to chemoattractants as well as the processes required for sustained directional motility are modulated by cyclic nucleotides.     

Mechanism of the suppression of the spinal pain syndrome by serotonin derivatives

The ability of serotonin derivatives to stimulate cAMP accumulation in isolated nerve terminals and lumbar enlargement of the spinal cord of normal rats was compared. The effect of the compounds on the intensity of spinal pain syndrome was also assessed. It has been established that substitutes injected into NH2-group of serotonin in 5-OH position attenuate the ability to stimulate cAMP accumulation in synaptosomes, with the effect more pronounced with substitutes of larger volume. A certain correlation between the ability of serotonin derivatives to stimulate adenylate cyclase in vivo and in vitro, on the one hand, and their analgetic effect, on the other hand, is suggested.     

Receptors acting through adenylate cyclase in the CNS

The author summarizes the current literature on neurotransmitters (dopamine, noradrenaline, serotonin and histamine) acting through adenlyate cyclase and also those ating direcctly without the intermission of c-AMP. The criteria of receptor binding are also stressed. The characteristics of D1 and D2 dopaminergic, beta1 adrenergic, H1 and H2 histaminergic receptors, their localisation and specific antagonists resp. agonists are emphasized too. Finally a new model is presented where adenylate cyclase and ionophore share a common receptor.     

Separation of soluble adenylate and guanylate cyclases from the mature rat testis.

The mature rat testis contains both a soluble guanylate cyclase and a soluble adenylate cyclase. Both these soluble enzymes prefer manganous ion for activity. It is known that guanylate cyclase can, when activated by a variety of agents, catalyze the formation of cyclic AMP. The following experiments were performed to determine whether the testicular soluble adenylate and guanylate cyclase activities were carried on the same molecule. Analysis of supernatants from homogenized rat testis by gel filtration and sucrose density gradient centrifugation showed that the two activities were clearly separable. The molecular weight of guanylate cyclase is 143 000, while that of adenylate cyclase is 58 000. Treatment of the column fractions with 0.1 mM sodium nitroprusside allowed guanylate cyclase activity to be expressed with Mg(2+) as well as with Mn(2+). Sodium nitroprusside did not affect the metal ion or substrate specificity of adenylate cyclase. These experiments show that adenylate and guanylate cyclase activities are physically separable.     

Serotonin-Elicited Amplification of Adenylate Cyclase Activity in Hippocampal Membranes from Adult Rat

The activity of the adenylate cyclase located in membranes prepared from hippocampus of adult rat can be stimulated by serotonin (5-HT) (Ka = 4 X 10(-7) M). The maximal effect is obtained with 10 microM 5-HT. Freezing of the tissue decreases the 5-HT stimulation; this stimulation is optimal in the presence of 82.5 mM Tris-maleate buffer (pH 7.4) and 50 microM GTP. The adenylate cyclase activity of membranes prepared from cortex, hypothalamus, and colliculi of adult rats is not significantly stimulated by 5-HT. Dopamine (DA) also stimulates adenylate cyclase located in hippocampal membranes; its effect can be blocked by haloperidol (10(-6) M), which fails to inhibit 5-HT stimulation. Moreover, p-chlorophenylalanine treatment for 2 weeks or selective lesion of 5-HT axons afferent to the hippocampus increases the Vmax of 5-HT stimulation, but fails to change that of DA stimulation. The 5-HT stimulation can be inhibited by metergoline, spiroperidol, and pizotyline (10(-6) M), but not by the same concentrations of mianserin, ketanserine, alprenolol, phenoxybenzamine, and mepyramine. The 5-HT stimulation of adenylate cyclase of hippocampal membranes can be mimicked by tryptamine, 5-methoxytryptamine, bufotenine, and to a lesser extent by LSD; N-methyltryptamine, N-methyltryptophan, and 5-hydroxytryptophan are inactive. Studies with kainic acid suggest that the 5-HT recognition site (5-HT1) linked to adenylate cyclase is located on the membrane of intrinsic hippocampal neurons.