Pertussis toxin-dependent and -independent hormonal effects on cultured renal epithelioid cells

The present study has been performed to test for the involvement of pertussis toxin-sensitive GTP-binding proteins (G-proteins) in the cellular transduction of hormone-induced activation of potassium channels. In Madin Darby canine kidney (MDCK) cells, a permanent cell line from dog kidney, epinephrine, acetylcholine, bradykinin, serotonin and ATP hyperpolarize the cell membrane by activation of potassium channels. In cells pretreated with pertussis toxin the hyperpolarizations elicited by either acetylcholine or serotonin are completely abolished; that following epinephrine is blunted and only transient. The hyperpolarizing effects of ATP or bradykinin are not affected by pertussis toxin. Thus, in MDCK cells both pertussis toxin-dependent and -independent mechanisms operate in parallel to enhance the potassium conductance of the cell membrane.     

Effects of cAMP on calcium currents in mollusk neurons differing in the sensitivity of their calcium conductance to serotonin action

The effects of cAMP and serotonin (5-HT) on calcium current (I_Ca) were investigated inHelix pomatia neurons using voltage clamp and intracellular perfusion techniques. Three types of neuronal response to extracellular application of 5-HT (1–10 µM) were found: reversible blockage of calcium conductance, absence of response, and increase in I_Ca amplitude. Intracellular application of exogenous cAMP was also found to produce an increase in I_Ca in cells stimulated by 5-HT action. Effects of 5-HT and cAMP were non-additive under these circumstances and were potentiated equally by cyclic nucleotide phosphodiesterase inhibitor. Applying cAMP led to no noticeable increase in I_Ca amplitude in cells with calcium conductance unchanged or blocked by 5-HT. Findings would indicate that the stimulating action of 5-HT is mediated by a rise in intracellular level of cAMP. It is postulated that two types of calcium channels differing in their dependence on cAMP metabolism exist; the presence of cAMP-dependent calcium channels at the neuronal membrane fits in with a certain type of 5-HT receptor also present in the cell, moreover. A new approach is suggested for research on isolated neurons, i.e., that of functional identification.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 5, pp. 605–512, October–September, 1990.     

Anxiety and increased 5‐HT1A receptor response in NCAM null mutant mice

Mice deficient in the neural cell adhesion molecule (NCAM) show behavioral abnormalities as adults, including altered exploratory behavior, deficits in spatial learning, and increased intermale aggression. Here, we report increased anxiety‐like behavior of homozygous (NCAM^−/−) and heterozygous (NCAM^+/−) mutant mice in a light/dark avoidance test, independent of genetic background and gender. Anxiety‐like behavior was reduced in both NCAM^+/+ and NCAM^−/− mice by systemic administration of the benzodiazepine agonist diazepam and the 5‐HT_1A receptor agonists buspirone and 8‐OH‐DPAT. However, NCAM^−/− mice showed anxiolytic‐like effects at lower doses of buspirone and 8‐OH‐DPAT than NCAM^+/+ mice. Such increased response to 5‐HT_1A receptor stimulation suggests a functional change in the serotonergic system of NCAM^−/− mice, likely involved in the control of anxiety and aggression. However, 5‐HT_1A receptor binding and tissue content of serotonin and its metabolite 5‐hydroxyindolacetic acid were found unaltered in every brain area of NCAM^−/− mice investigated, indicating that expression of 5‐HT_1A receptors as well as synthesis and release of serotonin are largely unchanged in NCAM^−/− mice. We hypothesize a critical involvement of endogenous NCAM in serotonergic transmission via 5‐HT_1A receptors and inwardly rectifying K⁺ channels as the respective effector systems. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 343–355, 1999     

Severe deficits in 5‐HT2A‐mediated neurotransmission in BDNF conditional mutant mice

BDNF is thought to provide critical trophic support for serotonin neurons. In order to determine postnatal effects of BDNF on the serotonin system, we examined a line of conditional mutant mice that have normal brain content of BDNF during prenatal development but later depletion of this neurotrophin in the postnatal period. These mice show a behavioral phenotype that suggests serotonin dysregulation. However, as shown here, the presynaptic serotonin system in the adult conditional mutant mice appeared surprisingly normal from histological, biochemical, and electrophysiological perspectives. By contrast, a dramatic and unexpected postsynaptic 5‐HT_2A deficit in the mutant mice was found. Electrophysiologically, serotonin neurons appeared near normal except, most notably, for an almost complete absence of expected 5‐HT_2A‐mediated glutamate and GABA postsynaptic potentials normally displayed by these neurons. Further analysis showed that BDNF mutants had much reduced 5‐HT_2A receptor protein in dorsal raphe nucleus and a similar deficit in prefrontal cortex, a region that normally shows a high level of 5‐HT_2A receptor expression. Recordings in prefrontal slice showed a marked deficit in 5‐HT_2A‐mediated excitatory postsynaptic currents, similar to that seen in the dorsal raphe. These findings suggest that postnatal levels of BDNF play a relatively limited role in maintaining presynaptic aspects of the serotonin system and a much greater role in maintaining postsynaptic 5‐HT_2A and possibly other receptors than previously suspected. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Antagonism of 5-HT1A receptors uncovers an excitatory effect of SSRIs on 5-HT neuronal activity, an action probably mediated by 5-HT7 receptors

Both microdialysis and electrophysiology were used to investigate whether another serotonin (5‐HT) receptor subtype next to the 5‐HT_1A autoreceptor is involved in the acute effects of a selective serotonin reuptake inhibitor on 5‐HT neuronal activity. On the basis of a previous study, we decided to investigate the involvement of the 5‐HT₇ receptors. Experiments were performed with the specific 5‐HT₇ antagonist SB 258741 and the putative 5‐HT₇ agonist AS19. In this study WAY 100.635 was used to block 5‐HT_1A receptors. Systemic administration of SB 258741 significantly reduced the effect of combined selective serotonin reuptake inhibitor and WAY 100.635 administration on extracellular 5‐HT in the ventral hippocampus as well as 5‐HT neuronal firing in the dorsal raphe nucleus. In the microdialysis study, co‐administration of AS19 and WAY 100.635 showed a biphasic effect on extracellular 5‐HT in ventral hippocampus, hinting at opposed 5‐HT₇ receptor mediated effects. In the electrophysiological experiments, systemic administration of AS19 alone displayed a bell‐shaped dose–effect curve: moderately increasing 5‐HT neuronal firing at lower doses while decreasing it at higher doses. SB 258741 was capable of blocking the effect of AS19 at a low dose. This is consistent with the pharmacological profile of AS19, displaying high affinity for 5‐HT₇ receptors and moderate affinity for 5‐HT_1A receptors. The data are in support of an excitatory effect of selective serotonin reuptake inhibitors on 5‐HT neuronal activity mediated by 5‐HT₇ receptors. It can be speculated, that the restoration of 5‐HT neuronal firing upon chronic antidepressant treatment, which is generally attributed to desensitization of 5‐HT_1A receptors alone, in fact results from a shift in balance between 5‐HT_1A and 5‐HT₇ receptor function.     

5‐HT1A Activation Counteracts Cardiovascular But Not Hypophagic Effects of Sibutramine in Rats

The noradrenaline (NA) and serotonin reuptake inhibitor, sibutramine, gives effective weight loss, but full efficacy cannot be attained at approved doses due to cardiovascular side effects. We assessed in rats the contributions of NA and serotonin transporters to sibutramine's hypophagic and cardiovascular effects, and whether selective 5‐hydroxytryptamine (5‐HT_1A) receptor activation could counteract the latter without affecting the former. Food intake was assessed in freely feeding rats and cardiovascular parameters in conscious telemetered rats. Ex vivo radioligand binding was used to estimate brain monoamine transporter occupancy. Sibutramine (1–10 mg/kg p.o.) dose‐dependently reduced food intake; however, 10 mg/kg p.o. markedly elevated blood pressure and heart rate. Sibutramine gave greater occupancy of NA than serotonin reuptake sites. Coadministration of the selective 5‐HT_1A agonist F‐11440 (2.5 mg/kg p.o.) attenuated sibutramine‐induced hypertension and tachycardia without altering its food intake effects. The selective NA reuptake inhibitors, nisoxetine or reboxetine, did not alter food intake alone, but each reduced food intake when combined with F‐11440. These results suggest that sibutramine‐induced hypophagic and cardiovascular effects are largely due to increased brain synaptic NA via NA reuptake inhibition, and that 5‐HT_1A activation can counter the undesirable cardiovascular effects resulting from increased sympathetic activity. Selective NA reuptake inhibitors did not reduce food intake alone but did when combined with 5‐HT_1A activation. Hence increased synaptic serotonin, via serotonin reuptake inhibition or 5‐HT_1A activation, together with increased NA, would appear to produce hypophagia. Thus weight loss with minimal cardiovascular risk could be achieved by 5‐HT_1A activation combined with NA transporter blockade.     

Serotonergic activation of 5HT1A and 5HT2 receptors modulates sexually dimorphic communication signals in the weakly electric fish Apteronotus leptorhynchus

Serotonin modulates agonistic and reproductive behavior across vertebrate species. 5HT_1A and 5HT_1B receptors mediate many serotonergic effects on social behavior, but other receptors, including 5HT₂ receptors, may also contribute. We investigated serotonergic regulation of electrocommunication signals in the weakly electric fish Apteronotus leptorhynchus. During social interactions, these fish modulate their electric organ discharges (EODs) to produce signals known as chirps. Males chirp more than females and produce two chirp types. Males produce high-frequency chirps as courtship signals; whereas both sexes produce low-frequency chirps during same-sex interactions. Serotonergic innervation of the prepacemaker nucleus, which controls chirping, is more robust in females than males. Serotonin inhibits chirping and may contribute to sexual dimorphism and individual variation in chirping. We elicited chirps with EOD playbacks and pharmacologically manipulated serotonin receptors to determine which receptors regulated chirping. We also asked whether serotonin receptor activation generally modulated chirping or more specifically targeted particular chirp types. Agonists and antagonists of 5HT_1B/1D receptors (CP-94253 and GR-125743) did not affect chirping. The 5HT_1A receptor agonist 8OH-DPAT specifically increased production of high-frequency chirps. The 5HT₂ receptor agonist DOI decreased chirping. Receptor antagonists (WAY-100635 and MDL-11939) opposed the effects of their corresponding agonists. These results suggest that serotonergic inhibition of chirping may be mediated by 5HT₂ receptors, but that serotonergic activation of 5HT_1A receptors specifically increases the production of high-frequency chirps. The enhancement of chirping by 5HT_1A receptors may result from interactions with cortisol and/or arginine vasotocin, which similarly enhance chirping and are influenced by 5HT_1A activity in other systems.     

Gender‐dependent regulation of G‐protein‐gated inwardly rectifying potassium current in dorsal raphe neurons in knock‐out mice devoid of the 5‐hydroxytryptamine transporter

Agonists at G‐protein‐coupled receptors in neurons of the dorsal raphe nucleus (DRN) of knock‐out mice devoid of the serotonin transporter (5‐HTT^?/?) exhibit lower efficacy to inhibit cellular discharge than in wild‐type counterparts. Using patch‐clamp whole‐cell recordings, we found that a G‐protein‐gated inwardly rectifying potassium (GIRK) current is involved in the inhibition of spike discharge induced by 5‐HT_1A agonists (5‐carboxamidotryptamine (5‐CT) and (±)‐2‐dipropylamino‐8‐hydroxy‐1,2,3,4‐tetrahydronaphthalene hydrobromide (8‐OH‐DPAT); 50 nM–30 μM) in both wild‐type and 5‐HTT^?/? female and male mice. These effects were mimicked by 5′‐guanylyl‐imido‐diphosphate (Gpp(NH)p; 400 μM) dialysis into cells with differences between genders. The 5‐HTT^?/? knock‐out mutation reduced the current density induced by Gpp(NH)p in females but not in males. These data suggest that the decreased response of 5‐HT_1A receptors to agonists in 5‐HTT^?/? mutants reflects notably alteration in the coupling between G‐proteins and GIRK channels in females but not in males. Accordingly, gender differences in central 5‐HT neurotransmission appear to depend—at least in part—on sex‐related variations in corresponding receptor‐G protein signaling mechanisms. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Muscarinic acetylcholine receptor produced in recombinant baculovirus infected Sf9 insect cells couples with endogenous G-proteins to activate ion channels.

Following the infection of insect ovarian cells (Sf9) with recombinant bearing the cDNA coding for the rat muscarinic acetylcholine (ACh) receptor subtype m3, ionic flux across the membrane in response to the application of ACh was examined electrophysiologically. We show that ACh activates potassium currents. The response is abolished when cells are treated with pertussis toxin. No ACh-induced currents are observed from uninfected cells or cells infected with virus which do not contain the cDNA coding for ACh receptors in its genome. The characteristics of single channel currents show time-dependent changes following the application of ACh. Initially, ACh activates brief channel currents with a conductance of about 5 pS. The conductance level of channels gradually increases in steps to 10 pS and then to 20 pS and 40 pS. At the same time, channel open probability also increases. Thereafter, additional channels appear, opening and closing independently of, or at times in synchrony with, the original channel.     

Analgesia induced by 2- or 100-Hz electroacupuncture in the rat tail-flick test depends on the anterior pretectal nucleus

Aims

The anterior pretectal nucleus (APtN) and electroacupuncture (EA) activate descending mechanisms to modulate nociceptive inputs in the spinal dorsal horn. This study examines qualitatively whether mechanisms in the APtN participate in the EA-induced analgesia in rats.

Main methods

The tail-flick test was utilized to examine the changes produced by non-selective antagonists of serotonergic (methysergide, 37 pg), muscarinic (atropine, 10 ng) and opioid (naloxone, 10 ng) receptors; selective antagonists against μ (CTOP, 6.4 μg), δ (ICI174,864, 6.9 μg) or κ (nor-BNI, 7.3 μg); 5HT₁ (methiothepin, 0.47 μg), 5HT₂ (ketanserin, 5.4 μg), or 5HT₃ (MDL 72222, 15.7 μg); and GABA_A (bicuculline, 150 ng) receptors injected into the dorsal (d) or ventral (v) APtN on the antinociception induced by a 20-min EA applied at 2- or 100-Hz frequency to the Zusanli and Sanyinjiao acupoints.

Key findings

The 2-Hz EA-induced analgesia was blocked by naloxone, CTOP or atropine, was less intense after bicuculline, was shorter after methysergide or methiothepin in dAPtN, and was less intense after methysergide, methiothepin and bicuculline in vAPtN. The 100-Hz EA-induced analgesia was less intense after methysergide, methiothepin and CTOP in vAPtN, and remained unchanged after injection of the antagonists into the dAPtN.

Significance

The 2-Hz EA-induced analgesia utilizes cholinergic muscarinic, μ-opioid, GABA_A and 5-HT₁ mechanisms in the dAPtN and μ-opioid and 5-HT₁ mechanisms in the vAPtN, while 100-Hz EA-induced analgesia utilizes μ-opioid and 5-HT₁ mechanisms in the vAPtN but does not utilize them in the dAPtN.     

Mass spectrometric characterization of recombinant rat 5‐hydroxytryptamine receptor 1A (5‐HT1AR) expressed in tsA201 human embryonic kidney cells

The 5‐hydroxytryptamine 1A receptor (serotonin 1A receptor; 5‐HT_1AR) is involved in a large series of brain functions, and roles in anxiety, depression, and cognition have been reported. So far, published information on mass spectrometrical characterization of 5‐HT_1AR is limited to the presence of two 5‐HT_1AR peptides in rat's whole brain as observed by in‐solution digestion followed by LC‐MS/MS. Knowledge about the protein sequence and PTMs, however, would have implications for generation of specific antibodies and designing studies on the 5‐HT_1AR at the protein level. A rat recombinant 5‐HT_1AR was extracted from the tsA201 cell line, run using several gel‐based principles with subsequent in‐gel digestion with several proteases, chymotrypsin, trypsin, AspN, proteinase K, and pepsin followed by nano‐LC‐ESI‐MS/MS analysis on a high capacity ion trap and an LTQ Orbitrap Velos. Using two search engines, Mascot and Modiro?, the recombinant 5‐HT_1AR was identified showing 94.55% sequence coverage. A single phosphorylation at S301 was identified and verified by phosphatase treatment and a series of amino acid substitutions were detected. Characterization of 5‐HT_1AR, a key player of brain functions and neurotransmission, was shown and may enable generation of specific antibodies, design of future, and interpretation of previous studies in the rat at the protein level.     

Kisspeptin1 modulates odorant‐evoked fear response via two serotonin receptor subtypes (5‐HT1A and 5‐HT2) in zebrafish

Kiss1, a neuropeptide predominantly expressed in the habenula, modulates the serotonin (5‐HT) system to decrease odorant cue [alarm substance (AS)]‐evoked fear behaviour in the zebrafish. The purpose of this study was to assess the interaction of Kiss1 with the 5‐HT system as well as to determine the involvement of the 5‐HT receptor subtypes in AS‐evoked fear. We utilized 0. 28 mg/kg WAY 100635 (WAY), a selective 5‐HT_1A receptor antagonist, to observe the effects of Kiss1 administration on AS‐evoked fear. We found WAY significantly inhibited the anxiolytic effects of Kiss1 (p < 0.001) with an exception of freezing behaviour. Based on this, we utilized 92.79 mg/kg methysergide, a 5‐HT₁ and 5‐HT₂ receptor antagonist, and found that methysergide significantly blocked the anxiolytic effects of Kiss1 in the presence of the AS (p < 0.001). From this, we conclude that Kiss1 modulates AS‐evoked fear responses mediated by the 5‐HT_1A and 5‐HT₂ receptors.

     

Responses of rat spinal ganglion neurons mediated by activation of serotonin receptors of the third type

Application of serotonin (5-hydroxytryptamine; 5-HT) to rat dorsal root ganglion neurons under conditions in which potassium conductance was blocked by cesium ions elicited depolarizing responses followed by an increase in membrane conductance. The responses did not exhibit desensitization and were due to activation of 5-HT receptors of the third type (5-HT₃Rs), since they were insensitive to methysergide, the 5-HT₂R antagonist, but were inhibited by tropicetrone (ISC 205–930) and metoclopramide, the 5-HT₃R antagonists. The reversal potential of the 5-HT-induced depolarizing responses was –11.9 mV; their amplitude decreased following a decrease in extracellular Na⁺ concentration but remained constant after intracellular injection of GTP. The amplitude of the responses increased following elevation of intracellular cAMP concentration caused by theophylline or sodium fluoride whose potentiating effect was reduced by butamide, a protein kinase A inhibitor. Potentiation of the 5-HT-induced responses was also produced by increased intracellular Ca²⁺ concentration following either direct intracellular injections or a burst of action potentials. The potentiation could be prevented by trifluoroperazine, the calmodulin inhibitor. The 5-HT effects were also potentiated by methylfurmetide, an activator of muscarinic acetylcholine receptors. The effect of methylfurmetide was slightly decreased by trifluoroperazine and was markedly decreased by polymixin B, a protein kinase C inhibitor. The effects of 5-HT were also enhanced by ethanol.Neirofiziologiya/Neurophysiology, Vol. 25, pp. 258–263, July–August, 1993.     

Pharmacological Characterization of 5-HT1A Autoreceptor-Coupled GIRK Channels in Rat Dorsal Raphe 5-HT Neurons

G protein-activated inwardly rectifying potassium (GIRK) channels in 5-HT neurons are assumed to be principal effectors of 5-hydroxytryptamine 1A (5-HT_1A) autoreceptors, but their pharmacology, subunit composition and the role in regulation of 5-HT neuron activity have not been fully elucidated. We sought for a pharmacological tool for assessing the functional role of GIRK channels in 5-HT neurons by characterizing the effects of drugs known to block GIRK channels in the submicromolar range of concentrations. Whole-cell voltage-clamp recording in brainstem slices were used to determine concentration-response relationships for the selected GIRK channel blockers on 5-HT_1A autoreceptor-activated inwardly rectifying K⁺ conductance in rat dorsal raphe 5-HT neurons. 5-HT_1A autoreceptor-activated GIRK conductance was completely blocked by the nonselective inwardly rectifying potassium channels blocker Ba²⁺ (EC₅₀ = 9.4 μM, full block with 100 μM) and by SCH23390 (EC₅₀ = 1.95 μM, full block with 30 μM). GIRK-specific blocker tertiapin-Q blocked 5-HT_1A autoreceptor-activated GIRK conductance with high potency (EC₅₀ = 33.6 nM), but incompletely, i.e. ~16% of total conductance resulted to be tertiapin-Q-resistant. U73343 and SCH28080, reported to block GIRK channels with submicromolar EC₅₀s, were essentially ineffective in 5-HT neurons. Our data show that inwardly rectifying K⁺ channels coupled to 5-HT_1A autoreceptors display pharmacological properties generally expected for neuronal GIRK channels, but different from GIRK1-GIRK2 heteromers, the predominant form of brain GIRK channels. Distinct pharmacological properties of GIRK channels in 5-HT neurons should be explored for the development of new therapeutic agents for mood disorders.     

Activation of A3 Adenosine Receptor Induces Calcium Entry and Chloride Secretion in A6 Cells

We have previously demonstrated that in A₆ renal epithelial cells, a commonly used model of the mammalian distal section of the nephron, adenosine A₁ and A_2A receptor activation modulates sodium and chloride transport and intracellular pH (Casavola et al., 1997). Here we show that apical addition of the A₃ receptor-selective agonist, 2-chloro-N⁶-(3-iodobenzyl)-adenosine-5′-methyluronamide (Cl-IB-MECA) stimulated a chloride secretion that was mediated by calcium- and cAMP-regulated channels. Moreover, in single cell measurements using the fluorescent dye Fura 2-AM, Cl-IB-MECA caused an increase in Ca²⁺ influx. The agonist-induced rise in [Ca²⁺] _i was significantly inhibited by the selective adenosine A₃ receptor antagonists, 2,3-diethyl-4,5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate (MRS 1523) and 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS 1191) but not by antagonists of either A₁ or A₂ receptors supporting the hypothesis that Cl-IB-MECA increases [Ca²⁺] _i by interacting exclusively with A₃ receptors. Cl-IB-MECA-elicited Ca²⁺ entry was not significantly inhibited by pertussis toxin pretreatment while being stimulated by cholera toxin preincubation or by raising cellular cAMP levels with forskolin or rolipram. Preincubation with the protein kinase A inhibitor, H89, blunted the Cl-IB-MECA-elicited [Ca²⁺] _i response. Moreover, Cl-IB-MECA elicited an increase in cAMP production that was inhibited only by an A₃ receptor antagonist. Altogether, these data suggest that in A₆ cells a G _s /protein kinase A pathway is involved in the A₃ receptor-dependent increase in calcium entry. Received: 9 March 2000/Revised: 14 August 2000     

Brainstem 5-Hydroxytrytamine1A Binding Sites are not Down-Regulated by Agonists which Induce Tolerance in the Rat: Myoclonus and other Serotonergic Behaviors

Abstract

To study the regulation of 5-HT_1A receptors in the brainstem, the region most relevant to the serotonin syndrome and to serotonin-responsive human myoclonic disorders, we chronically treated rats with various 5-HT_1A agonists and labeled 5-HT_1A sites with [³H]8-OH-DPAT. Daily injection for 30 consecutive days of 10 mg/kg ip 8-OH-DPAT (pre- and post-synaptic 5-HT_1A agonist) significantly decreased 8-OH-DPAT-evoked flat body posture, forelimb myoclonus, and hypothermia compared to chronic vehicle injection. There was no cross tolerance to 8-OH-DPAT in rats chronically injected with ipsapirone or buspirone (presynaptic 5-HT_1A agonists). However, none of the 5HT_1A agonists significantly altered B_max of brainstem 5-HT_1A binding sites. Chronic injection with other drugs such as 1-propranolol, (±) pindolol and spiperone (5-HT_1A and 5-HT₂ antagonists), methysergide (5-HT₁ and 5-HT₂ antagonist), and agonists and antagonists at various other 5-HT receptors also had no effect on binding parameters. These data demonstrate lack of cross-tolerance between pre- and post-synaptically acting 5-HT_1A agonists and absence of down-regulation of presynaptic 5-HT_1A sites at doses which induced tolerance of 5-HT_1A-mediated behaviors of the serotonin syndrome. They suggest changes in the post-synaptic cell rather than the receptor recognition site as the mechanism of tolerance.     

Larvae of the small white butterfly,Pieris rapae,express a novel serotonin receptor

The biogenic amine serotonin ( 5‐hydroxytryptamine, 5‐HT) is a neurotransmitter in vertebrates and invertebrates. It acts in regulation and modulation of many physiological and behavioral processes through G‐protein‐coupled receptors. Five 5‐HT receptor subtypes have been reported in Drosophila that share high similarity with mammalian 5‐HT_1A, 5‐HT_1B, 5‐HT_2A, 5‐HT_2B, and 5‐HT₇ receptors. We isolated a cDNA (Pr5‐HT₈) from larval Pieris rapae, which shares relatively low similarity to the known 5‐HT receptor classes. After heterologous expression in HEK293 cells, Pr5‐HT₈ mediated increased [Ca²⁺]_i in response to low concentrations (< 10 nM) of 5‐HT. The receptor did not affect [cAMP]_i even at high concentrations (> 10 μM) of 5‐HT. Dopamine, octopamine, and tyramine did not influence receptor signaling. Pr5‐HT₈ was also activated by various 5‐HT receptor agonists including 5‐methoxytryptamine, (±)‐8‐Hydroxy‐2‐(dipropylamino) tetralin, and 5‐carboxamidotryptamine. Methiothepin, a non‐selective 5‐HT receptor antagonist, activated Pr5‐HT₈. WAY 10635, a 5‐HT_1A antagonist, but not SB‐269970, SB‐216641, or RS‐127445, inhibited 5‐HT‐induced [Ca²⁺]_i increases. We infer that Pr5‐HT₈ represents the first recognized member of a novel 5‐HT receptor class with a unique pharmacological profile. We found orthologs of Pr5‐HT₈ in some insect pests and vectors such as beetles and mosquitoes, but not in the genomes of honeybee or parasitoid wasps. This is likely to be an invertebrate‐specific receptor because there were no similar receptors in mammals.

     

Characterisation of 5-HT3C, 5-HT3D and 5-HT3E receptor subunits: evolution, distribution and function

The 5‐HT₃ receptor is a member of the ‘Cys‐loop’ family of ligand‐gated ion channels that mediate fast excitatory and inhibitory transmission in the nervous system. Current evidence points towards native 5‐HT₃ receptors originating from homomeric assemblies of 5‐HT_3A or heteromeric assembly of 5‐HT_3A and 5‐HT_3B. Novel genes encoding 5‐HT_3C, 5‐HT_3D, and 5‐HT_3E have recently been described but the functional importance of these proteins is unknown. In the present study, in silico analysis (confirmed by partial cloning) indicated that 5‐HT_3C, 5‐HT_3D, and 5‐HT_3E are not human–specific as previously reported: they are conserved in multiple mammalian species but are absent in rodents. Expression profiles of the novel human genes indicated high levels in the gastrointestinal tract but also in the brain, Dorsal Root Ganglion (DRG) and other tissues. Following the demonstration that these subunits are expressed at the cell membrane, the functional properties of the recombinant human subunits were investigated using patch clamp electrophysiology. 5‐HT_3C, 5‐HT_3D, and 5‐HT_3E were all non‐functional when expressed alone. Co‐transfection studies to determine potential novel heteromeric receptor interactions with 5‐HT_3A demonstrated that the expression or function of the receptor was modified by 5‐HT_3C and 5‐HT_3E, but not 5‐HT_3D. The lack of distinct effects on current rectification, kinetics or pharmacology of 5‐HT_3A receptors does not however provide unequivocal evidence to support a direct contribution of 5‐HT_3C or 5‐HT_3E to the lining of the ion channel pore of novel heteromeric receptors. The functional and pharmacological contributions of these novel subunits to human biology and diseases such as irritable bowel syndrome for which 5‐HT₃ receptor antagonists have major clinical usage, therefore remains to be fully determined.