Neurokinin receptor modulation of respiratory activity in the rabbit

The respiratory role of neurokinin (NK) receptors was investigated in α-chloralose-urethane-anaesthetized, vagotomized, paralysed and artificially ventilated rabbits by using bilateral microinjections (30–50 nL) of NK receptor agonists and antagonists. Microinjections were performed in a region located just caudal to the rostral expiratory neurons. This region displayed features similar to those of the pre-Bötzinger complex (pre-BötC) of adult cats and rats, and proved to produce excitatory respiratory effects in response to microinjections of d,l -homocysteic acid. We used as agonists (0.1, 0.5 and 5 m m ) substance P (SP), the NK1 receptor agonists [Sar⁹, Met(O₂)¹¹]-SP and GR 73632, the NK2 receptor agonist NKA, the NK3 receptor agonist senktide, and as antagonists (5 m m ) the NK1 receptor antagonist CP-99,994 and the NK2 receptor antagonist MEN 10376. SP always increased respiratory frequency, but NK1 receptor agonists did not change respiratory variables. NKA and senktide at 5 m m increased respiratory frequency. CP-99,994 caused increases in respiratory frequency and did not antagonize the effects of SP. MEN 10376 prevented the respiratory responses induced by NKA and reduced those provoked by SP. SP or the NK1 receptor agonists (5 m m ) injected (1 μL) into the IV ventricle caused marked excitatory effects on respiration. The results suggest that NK2 and NK3, but not NK1, receptors are involved in the excitatory modulation of inspiratory activity within the investigated region and are consistent with the notion that the pre-BötC neurons are important components of the inspiratory rhythm-generating mechanisms.     

Inhibition of the bradycardic component of the von Bezold–Jarisch reflex and carotid chemoreceptor reflex by periaqueductal gray stimulation: involvement of medullary receptors

Stimulation of the dorsolateral periaqueductal gray matter (dlPAG) and the B3 cell group inhibits the cardiovagal component of the baroreflex in rats. Our aim was to determine whether the defence reaction induces similar modulatory effects on the cardiac response of the von Bezold–Jarisch reflex and the carotid chemoreceptor reflex. We examined the effects of dlPAG stimulation on the reflex bradycardia triggered by systemic administration of phenylbiguanide or potassium cyanide. Electrical and chemical stimulation of the dlPAG produced marked inhibition of the cardiovagal components of the von Bezold–Jarisch and the carotid chemoreceptor reflexes. In addition, as 5-HT₃, NK₁ and GABA_A receptor activation blocks cardiac reflex responses, we studied whether these receptors were involved in the dlPAG-induced inhibitory effects. We found that, after microinjection of granisetron (a 5-HT₃ receptor antagonist), bicuculline (a GABA_A receptor antagonist) and GR-205171 (an NK₁ receptor antagonist) into the nucleus of the solitary tract (NTS), reflex bradycardic responses were preserved during dlPAG stimulation. Finally, activation of the B3 region also inhibited both reflex bradycardic responses, and these effects were prevented by prior blockade of 5-HT₃ receptors in the NTS. The inhibitory effect of dlPAG stimulation on the cardiac reflex responses was prevented by inhibition of neurons in the medullary B3 region. In conclusion, 5-HT₃, GABA_A and NK₁ receptors in the NTS appear to be involved in the inhibition of the von Bezold–Jarisch reflex and the carotid chemoreceptor reflex bradycardia evoked by activation of neurons in the dlPAG and the raphé magnus.     

5-Hydroxytryptamine selectively activates the vagal nodose C-fibre subtype in the guinea-pig oesophagus

Abstract  The afferent neurons innervating the oesophagus originate from two embryonic sources: neurons located in vagal nodose ganglia originate from embryonic placodes and neurons located in vagal jugular and spinal dorsal root ganglia (DRG) originate from the neural crest. Here, we address the hypothesis that 5-hydroxytryptamine (5-HT) differentially stimulates afferent nerve subtypes in the oesophagus. Extracellular recordings of single unit activity originating from nerve terminals were made in the isolated innervated guinea-pig oesophagus. Whole cell patch clamp recordings (35 °C) were made from the primary afferent neurons retrogradely labelled from the oesophagus. 5-Hydroxytryptamine (10  μ mol L⁻¹) activated vagal nodose C-fibres (70%) in the oesophagus but failed to activate overtly vagal jugular nerve fibres and oesophagus-specific spinal DRG neurons. The response to 5-HT in nodose C-fibre nerve terminals was mimicked by the selective 5-HT₃ receptor agonist 2-methyl-5-HT (10  μ mol L⁻¹) and nearly abolished by the 5-HT₃ receptor antagonists ondansetron (10  μ mol L⁻¹) and Y-25130 (10  μ mol L⁻¹). In patch clamp studies, 2-methyl-5-HT (10  μ mol L⁻¹) activated a proportion of isolated oesophagus-specific nodose capsaicin-sensitive neurons (putative cell bodies of nodose C-fibres). We conclude that the responsiveness to 5-HT discriminates placode-derived (vagal nodose) C-fibres from the neural crest-derived (vagal jugular and spinal DRG) afferent nerves in the oesophagus. The response to 5-HT in nodose C-fibres is mediated by the 5-HT₃ receptor in their neuronal membrane.     

Glutamate Receptors on type I Vestibular Hair Cells of Guinea-pig

Afferent nerve calyces which surround type I vestibular hair cells (VHCI) have recently been shown to contain synaptic-like vesicles and to be immunoreactive to glutamate antibodies. In order to understand the physiological significance of these observations, the presence of glutamate receptors on type I vestibular sensory cells has been investigated. The effect of excitatory amino acids applied by iontophoresis was examined by spectrofluorimetry using fura-2 sensitive dye. Glutamate application caused a rapid and transient increase in intracellular calcium concentration ([Ca²⁺]ⁱ), in a dose-dependent manner. The ionotropic glutamate receptors agonists N -methyl- d -aspartic acid (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and quisqualic acid (QA) induced an increase of [Ca²⁺]_i. The NMDA receptor antagonist 2-amino-5-phosphonovaleric acid and the AMPA receptor antagonist 6,7-dinitro-quinoxaline-2,3-dione partially blocked the glutamate response, by 39 ± 10 and 53 ± 11% respectively. Metabotropic receptors were also revealed by the specific agonist trans -1-amino-cyclopentyl-1,3-dicarboxylate. The presence of different glutamate receptors on the VHCI membrane suggests two kinds of feedback, (i) At the base of the sensory cell, autoreceptors may locally control the synaptic transmission, (ii) At the apex, postsynaptic receptors may modulate sensory transduction from glutamate release at the upper part of the afferent nerve calyx. These feedbacks suggest presynaptic modulation of the vestibular hair cell response which could affect its sensitivity.     

Serotonin and neuroprotection in functional bowel disorders

Abstract  The 5-HT₄ partial agonist tegaserod is effective in the treatment of chronic constipation and constipation predominant irritable bowel syndrome. 5-HT₄ receptors are located on presynaptic terminals in the enteric nervous system. Stimulation of 5-HT₄ receptors enhances the release of acetylcholine and calcitonin gene related peptide from stimulated nerve terminals. This action strengthens neurotransmission in prokinetic pathways, enhancing gastrointestinal motility. The knockout of 5-HT₄ receptors in mice not only slows gastrointestinal activity but also, after 1 month of age, increases the age-related loss of enteric neurons and decreases the size of neurons that survive. 5-HT₄ receptor agonists, tegaserod and RS67506, increase numbers of enteric neurons developing from precursor cells and/or surviving in culture; they also increase neurite outgrowth and decrease apoptosis. The 5-HT₄ receptor antagonist, GR113808, blocks all of these effects, which are thus specific and 5-HT₄-mediated. 5-HT₄ receptor agonists, therefore, are neuroprotective and neurotrophic for enteric neurons. Because the age-related decline in numbers of enteric neurons may contribute to the dysmotilities of the elderly, the possibility that the neuroprotective actions of 5-HT agonists can be utilized to prevent the occurrence or worsening of these conditions should be investigated.     

Effects of the HIV-1 Envelope Glycoprotein gp120 in Cerebellar Cultures. [Ca2+]i Increases in a Glial Cell Subpopulation

The various types of cells present in cultures prepared from the postnatal rat cerebellum, identified by their gross morphology and immunocytochemistry, were loaded with the specific dye fura-2 and analysed individually for [Ca²⁺]_i changes induced by the HIV-1 envelope glycoprotein gp120 and a variety of other treatments. In granule neurons [Ca²⁺]_i increases were induced by high KCl and glutamate (mainly through the NMDA receptor) while in type-1 astrocytes this effect was observed after serotonin, carbachol and also quisqualate. In contrast, administration of gp120 was always without effect in these cells. Type-2 astrocytes (an arborized cell type responsive to agonists targeted to the glutamatergic AMPA and cholinergic receptors) were also most often unresponsive to the viral glycoprotein. However, among the cells exhibiting the arborized phenotype, a subpopulation (-13%) responded to gp120 with conspicuous [Ca²⁺]_i increases sustained by both release from intracellular stores and influx across the plasma membrane. These responses to the viral protein did not involve activation of either voltage-gated Ca²⁺ channels or glutamatergic receptors. Although not yet conclusively identified by specific cytochemical markers, the gp120-responsive cells resemble type-2 astrocytes and differ from neurons and type-1 astrocytes both in gross phenotype and in a number of receptor/channel properties: positivity to AMPA and cholinergic agonists; negativity to NMDA, serotonin and high KCl. From these results it is concluded that a subpopulation of glial cells is affected by gp120. The role of these cells in HIV brain infection and damage requires further studies to be precisely established.     

Cholinergic Modulation of the Action Potential in Rat Hippocampal Neurons

The cholinergic input to the hippocampus from the medial septum is important for modulating hippocampal activity and functions, including theta rhythm and spatial learning. Neuromodulation by transmitters in central nervous system neurons usually affects cell excitability by modifying the membrane potential, discharge pattern and spike frequency. Here we describe another type of neuromodulation: changing the action potential waveform. During intracellular recordings from CA1 pyramidal cells in hippocampal slices from rats, the cholinergic agonist carbachol caused several reversible changes in the action potential: low doses (2 μM) caused an increase in spike duration; high doses (10–40 μM) or long-lasting applications also reduced the spike amplitude and rate of rise, and raised the spike threshold. These effects are similar to those of metabotropic glutamate receptor agonists or phorbol esters, both of which activate protein kinase C. The effects were blocked by the muscarinic antagonist atropine, and were prevented by Ca²⁺-free medium and by Ca²⁺-channel blockers. However, the cholinergic spike modulation was not occluded or mimicked by blocking the Ca²⁺-dependent K⁺ currents I_c or I_AHP, suggesting that these K⁺ currents are not involved in the modulation.     

Pro9]SP and [pGlu6, Pro9]SP(6-11) interact with two different receptors in the guinea-pig ileum as demonstrated with new SP antagonists.

Structural considerations led us to postulate that the introduction of the dipeptides DPro9-Pro10 and DPro9-MeLeu10 should lock the C-terminal tetrapeptide of SP in a type II' beta-turn structure, a prerequisite for antagonist activity. Indeed, as the GR 71251, [DPro9, Pro10, Trp11]SP was more potent in inhibiting the septide, (pA2 = 6.5), than the [Pro9]SP, (pA2 < or = 5), spasmogenic activity in the guinea-pig ileum bioassay. This result confirms that septide, [pGlu6, Pro9]SP(6-11), a peptide active in the guinea-pig ileum bioassay and practically devoid of binding potencies for the three specific NK-1, NK-2 and NK-3 tachykinin binding sites interacts with a tachykinin receptor different from the NK-1 receptor sensitive to [Pro9]SP. Interestingly enough, the reintroduction of the leucine side-chain in position 10 yielded [DPro9, MeLeu10, Trp11]SP, an antagonist, equipotent in inhibiting both the septide- and the [Pro9]SP-evoked contractile response in the guinea-pig ileum bioassay, (pA2 = 6.6).     

SB-207266: 5-HT4 receptor antagonism in human isolated gut and prevention of 5-HT-evoked sensitization of peristalsis and increased defaecation in animal models

SB-207266 is a new 5-HT₄ receptor antagonist which in a pilot study reduced the symptoms of irritable bowel syndrome. To help validate this and further studies, we examined the ability of SB-207266 to antagonize at the human 5-HT₄ receptor (human isolated intestine) and to affect the mechanisms of peristalsis (guinea-pig isolated ileum) and defaecation (conscious, fed mice). In the human intestine, the potency of 5-HT₄ receptor antagonism (pK_B 9.98) was similar to that previously demonstrated using a guinea-pig model of the receptor, validating the use of SB-207266 in clinical trials. In each of the animal models, SB-207266 did not affect normal patterns of intestinal motility measured in the absence of exogenous 5-HT. However, SB-207266 10–1000 p M concentration-dependently antagonized the ability of 5-HT (0.1 μ M ) to sensitize the peristaltic reflex and lower the distension threshold at which peristalsis was evoked. In mice, oral or subcutaneous (s.c.) doses of SB-207266 dose-dependently prevented the ability of the 5-HT precursor, 5-hydroxytryptophan (5-HTP, 10 mg kg⁻¹ s.c.) to increase both the rate of defaecation of formed faecal pellets and their fluid content. SB-207266 was maximally active at 10 μg kg⁻¹ s.c. and 1000 μg kg⁻¹ p.o. SB-207266 may therefore represent a new class of therapeutic agent, capable of preventing the actions of an important sensitizer of gut function.     

Modulation by substance P of synaptic transmission in the mouse hippocampal slice

The modulatory action of substance P on synaptic transmission of CA1 neurons was studied using intra‐ or extracellular recording from the mouse hippocampal slice preparation. Bath‐applied substance P (2–4 μ m ) or the selective NK₁ receptor agonist substance P methylester (SPME, 10 n m –5 μ m ) depressed field potentials (recorded from stratum pyramidale) evoked by focal stimulation of Schaffer collaterals. This effect was apparently mediated via NK₁ receptors since it was completely blocked by the selective NK₁ antagonist SR 140333. The field potential depression by SPME was significantly reduced in the presence of bicuculline. Intracellular recording from CA1 pyramidal neurons showed that evoked excitatory postsynaptic potentials (EPSPs) and evoked inhibitory postsynaptic potentials (IPSPs) were similarly depressed by SPME, which at the same time increased the frequency of spontaneous GABAergic events and reduced that of spontaneous glutamatergic events. The effects of SPME on spontaneous and evoked IPSPs were prevented by the ionotropic glutamate receptor blocker kynurenic acid. In tetrodotoxin (TTX) solution, no change in either the frequency of spontaneous GABAergic and glutamatergic events or in the amplitude of responses of pyramidal neurons to 4 μ m α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) or 10 μ m N ‐methyl‐ d ‐aspartate (NMDA) was observed. On the same cells, SPME produced minimal changes in passive membrane properties unable to account for the main effects on synaptic transmission. The present data indicate that SPME exerted its action on CA1 pyramidal neurons via a complex network mechanism, which is hypothesized to involve facilitation of a subset of GABAergic neurons with widely distributed connections to excitatory and inhibitory cells in the CA1 area.     

GABAergic signaling induces divergent neuronal Ca2+ responses in the suprachiasmatic nucleus network

Intercellular communication between γ-aminobutyric acid (GABA)ergic suprachiasmatic nucleus (SCN) neurons facilitates light-induced phase changes and synchronization of individual neural oscillators within the SCN network. We used ratiometric Ca²⁺ imaging techniques to record changes in the intracellular calcium concentration ([Ca²⁺]_i) to study the role of GABA in interneuronal communication and the response of the SCN neuronal network to optic nerve stimulations that mimic entraining light signals. Stimulation of the retinohypothalamic tract (RHT) evoked divergent Ca²⁺ responses in neurons that varied regionally within the SCN with a pattern that correlated with those evoked by pharmacological GABA applications. GABA_A and GABA_B receptor agonists and antagonists were used to evaluate components of the GABA-induced changes in [Ca²⁺]_i. Application of the GABA_A receptor antagonist gabazine induced changes in baseline [Ca²⁺]_i in a direction opposite to that evoked by GABA, and similarly altered the RHT stimulation-induced Ca²⁺ response. GABA application induced Ca²⁺ responses varied in time and region within the SCN network. The NKCC1 cotransporter blocker, bumetanide, and L-type calcium channel blocker, nimodipine, attenuated the GABA-induced rise of [Ca²⁺]_i. These results suggest that physiological GABA induces opposing effects on [Ca²⁺]_i based on the chloride equilibrium potential, and may play an important role in neuronal Ca²⁺ balance, synchronization and modulation of light input signaling in the SCN network.     

Tachykinins protect cholinergic neurons from quinolinic acid excitotoxicity in striatal cultures

The neuroprotective effect of tachykinins against excitotoxic death of cholinergic neurons was studied in rat striatal cell cultures. Quinolinic acid (QUIN) and kainic acid (KA) produced a dose dependent decrease in choline acetyttransferase activity, but KA was more potent. Our results show that substance P (SP) totally reversed the toxicity induced by 125 μM QUIN but not by 40 μM KA. This effect was also observed using protease inhibitors or a SP-analog resistant to degradation, [Sar⁹]-Substance P. The survival of neuron specific enolase- and acetylcholinesterase (AChE)-positive cells after treatment with QUIN alone or in the presence of SP was also examined. We observed that, while a decrease in total cell number produced by QUIN was not prevented by SP treatment, AChE-positive cells were rescued from the toxic damage. To characterize the SP protective effect we used more selective agonists of the three classes of neurokinin (NK) receptors. [Sar⁹, Met(O₂)¹¹]-Substance P (NK₁ receptor agonist), [Nle¹⁰]-Neurokinin A (NK₂ receptor agonist) or [Me-Phe⁷]-Neurokinin B (NK₃ receptor agonist) were all able to block the toxic effect of QUIN on cholinergic activity. These results show that tachykinins provide an important protective support for striatal neurons, suggesting a possible therapeutical benefit in neurodegenerative disorders affecting cholinergic neurons.     

D1 Dopamine Receptor Agonist (SKF-38393) Induction of FosImmunoreactivity in Progestin Receptor-Containing Areasof Female Rat Brain

Injection of dopamine or dopamine receptor subtype agonists facilitates the expression of lordosis in estrogen-primed female rats. The D₁ receptor specific agonist, SKF-38393, facilitates lordosis in estradiol-primed female rats via a process that requires progestin receptors. Based on these data, neuronal response to the D₁ receptor agonist SKF-38393 was assessed by expression of the immediate early gene protein, Fos. In the first experiment we examined the modulation of Fos expression by D₁ agonists in progestin receptor-containing areas of estradiol-primed female rat brain. In the second experiment we examined if there are progestin receptor-containing cells that respond to stimulation of D₁ receptors with increased Fos expression. Ten to 14 days following ovariectomy and stereotaxic surgery, animals were injected with 5  μg estradiol benzoate. Forty eight  h later they were injected intracerebroventricularly with 100  ng of SKF-38393 or saline. One  h following injection animals were perfused, and brain sections immunostained for Fos protein. Results from the first experiment suggest that SKF-38393 increased the total number of Fos immunoreactive cells in the mid-ventromedial hypothalamic nucleus/ventrolateral portion (VMHVL), the caudal VMHVL, the paraventricular hypothalamic nucleus and the caudate putamen. In the medial preoptic area, the rostral VMHVL and the arcuate hypothalamic nucleus, there was a significant increase in the number of darkly stained Fos-immunoreactive cells following the SKF-38393 treatment. In the second study, SKF-38393 increased the number of progestin receptor-containing cells which contained Fos immunoreactivity in the caudal VMHVL. The results suggest potential sites of action for the facilitation of sexual behavior by centrally administered D₁ agonists.     

Mechanisms for Synchronous Calcium Oscillations in Cultured Rat Cerebellar Neurons

Removal of Mg²⁺ caused oscillations of the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and the membrane potential in cultured cerebellar granule neurons. Oscillations of [Ca²⁺]_i were synchronous in all the cells, and were restricted to the neurons (immunocytochemically identified) that responded to exogenous N -methyl-D-aspartate (NMDA). Oscillations were blocked by Ca²⁺ removal, nickel, NMDA receptor antagonists, ω-agatoxin IVA, tetrodotoxin, sodium removal and γ-aminobutyric acid, but not by dihydropyridines, ω-conotoxin M VIIA or by emptying the intracellular Ca²⁺ stores with thapsigargin or ionomycin. The upstroke of the [Ca²⁺]_i oscillations coincided in time with an increase in manganese permeability of the plasma membrane. Propagation of the [Ca²⁺]_i wave followed more than one pathway and the spatiotemporal pattern changed with time. Membrane potential oscillations consisted of transient slow depolarizations of ˜20 mV with faster phasic activity superimposed. We propose that the synchronous [Ca²⁺]_i oscillations are the expression of irradiation of random excitation through a neuronal network requiring generation of action potentials and functional glutamatergic synapses. Oscillations of [Ca²⁺]_i are due to cyclic Ca²⁺ entry through NMDA receptor channels activated by synaptic release of glutamate, which requires Ca²⁺ entry through P-type Ca²⁺ channels activated by action potentials at the presynaptic terminal.     

Neuropeptide Y and the Calcitonin Gene-related Peptide Attenuate Learning Impairments Induced by MK-801 via a Sigma Receptor-related mechanism

It has been shown recently that low doses of sigma (σ) receptor ligands like 1,3-di-(2-tolyl)guanidine (DTG), (+) N -allylnormetazocine [(+)SKF 10 047] and (+)pentazocine can antagonize learning impairments induced by dizocilpine (MK-801), a non-competitive antagonist at the NMDA receptor channel. This antagonism has been proposed to involve σ receptor sites since it is blocked by the administration of purported o antagonists such as NE-100 and BMY-14802. It has also been demonstrated that peptides of the neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP) families modulate, in vivo , é labelling and electrophysiological effects in the hippocampal formation. Accordingly, we investigated if NPY- and CGRP-related peptides modulate cognitive processes by interacting with σ sites in mice. In order to test this hypothesis, a step-down passive avoidance task was used. Interestingly, similarly to various σ agonists, NPY, peptide YY (PYY) and the Y₁ agonist [Leu³¹Pro³⁴]NPY (but not NPY13–36, a purported Y₂ agonist), as well as hCGRPα and the purported CGRP₂ agonist [CyS(ACM)^2–7]hCGRPα (but not CGRP_8–37, a CGRP₁ receptor antagonist), significantly attenuated learning impairments induced by MK-801. Furthermore, the effects of NPY, [Leu³¹ Pro³⁴]NPY, hCGRPα and [CYS(ACM)^2–7]hCGRPα were blocked by the administration of the σ antagonist, BMY-14802. The present data suggest that NPY- and CGRP-related peptides can indirectly interact in vivo with σ receptors to modulate cognitive processes associated with NMDA receptor function.     

Pharmacological characterization of grooming induced by a selective NK-1 tachykinin receptor agonist

Bilateral intranigral administration of the selective NK-1 tachykinin receptor agonist [AcArg⁶, Sar⁹, Met(O₂)¹¹]SP_6–11 (0–11 nmol total bilateral dose) selectively induced grooming in rats. This response was blocked by concurrent intranigral administration of the NK-1 tachykinin receptor antagonist RP 67580 (2 nmol), but not by NK-2 (L-659, 877) or NK-3 ([Trp⁷, β-Ala⁸]NKA_4–10) antagonists. Pretreatment with systemic opioid (naloxone 1.5 mg/kg) and D₁ dopamine (SCH 23390 100 μg/kg) receptor antagonists also attenuated tachykinin-induced grooming, which was unaffected by D₂ dopamine (sulpiride 30 mg/kg) or 5-HT_2A+C (ritanserin 2 mg/kg) antagonists. Grooming induced by intranigral [AcArg⁶, Sar⁹, Met(O₂)¹¹]SP_6–11 was also attenuated by bilateral 6-hydroxydopamine lesions of te substantia nigra. These findings indicate that grooming induced by intranigral tachykinins reflects activation of NK-1 receptors and is dependent upon endogenous dopamine and consequent selective stimulation of D₁ dopamine receptors.     

Rapid Action of Oestrogen in Luteinising Hormone-Releasing Hormone Neurones: The Role of GPR30

Previously, we have shown that 17β-oestradiol (E₂) induces an increase in firing activity and modifies the pattern of intracellular calcium ([Ca²⁺]_i) oscillations with a latency < 1 min in primate luteinising hormone-releasing hormone (LHRH) neurones. A recent study also indicates that E₂, the nuclear membrane impermeable oestrogen, oestrogen-dendrimer conjugate, and the plasma membrane impermeable oestrogen, E₂-BSA conjugate, all similarly stimulated LHRH release within 10 min of exposure in primate LHRH neurones, indicating that the rapid action of E₂ is caused by membrane signalling. The results from a series of studies further suggest that the rapid action of E₂ in primate LHRH neurones appears to be mediated by GPR30. Although the oestrogen receptor antagonist, ICI 182, 780, neither blocked the E₂-induced LHRH release nor the E₂-induced changes in [Ca²⁺]_i oscillations, E₂ application to cells treated with pertussis toxin failed to result in these changes in primate LHRH neurones. Moreover, knockdown of GPR30 in primate LHRH neurones by transfection with human small interference RNA for GPR30 completely abrogated the E₂-induced changes in [Ca²⁺]_i oscillations, whereas transfection with control siRNA did not. Finally, the GPR30 agonist, G1, resulted in changes in [Ca²⁺]_i oscillations similar to those observed with E₂. In this review, we discuss the possible role of G-protein coupled receptors in the rapid action of oestrogen in neuronal cells.     

Copper Modulation of NMDA Responses in Mouse and Rat Cultured Hippocampal Neurons

The effect of Cu²⁺ on NMDA receptors was studied in cultured mouse and rat hippocampal neurons using whole-cell patch-clamp and a fast perfusion system. Analysis of the Cu²⁺ concentration-response curve for inhibition of NMDA-induced currents suggests that free Cu²⁺ directly inhibits NMDA receptors with an IC₅₀ of 0.27 μM. Cu²⁺ was ineffective in blocking NMDA receptor activity when complexed with NMDA or glycine; NMDA-Cu²⁺ and glycine-Cu²⁺ complexes acted as agonists of similar potency to the free amino acids. The inhibition by Cu²⁺ (10–100 μM) of responses to 10 μM NMDA was essentially voltage-independent. The onset of inhibition by 100 μM Cu²⁺ of responses to 2 FM glutamate acting at NMDA receptors was significantly faster than NMDA receptor deactivation evoked by a sudden decrease in the concentration of glycine or glutamate, or of both agonists. This suggests that CU²⁺ acts as a non-competitive antagonist, and does not directly interfere with the binding of glutamate or glycine to their recognition sites on the NMDA receptor complex. In the absence of NMDA the apparent association rate constant for binding of Cu²⁺ to NMDA receptors, calculated from the rate of onset of block by Cu²⁺ of test responses to NMDA, was 19 times slower than in the presence of 30 μM NMDA, suggesting that Cu^z+ interacts preferentially with agonist-bound receptors. Our results show that Cu²⁺ is a potent inhibitor of NMDA receptor-mediated responses.     

Pharmacology of MEN 11467: a potent new selective and orally- effective peptidomimetic tachykinin NK(1) receptor antagonist

We have investigated the pharmacological properties of MEN 11467, a novel partially retro-inverse peptidomimetic antagonist of tachykinin NK(1) receptors. MEN 11467 potently inhibits the binding of [(3)H] substance P (SP) to tachykinin NK(1) receptors in the IM9 limphoblastoid cell line (pK(i) = 9.4 +/- 0.1). MEN 11467 is highly specific for the human tachykinin NK(1) receptors, since it has negligible effects (pK(i) <6) on the binding of specific ligands to tachykinin NK(2) or NK(3) receptors and to a panel of 30 receptors ion channels unrelated to tachykinin receptors. The antagonism exerted by MEN 11467 at tachykinin NK(1) receptors is insurmountable in saturation binding experiments, both K(D) and B(max) of SP were significantly reduced by MEN 11467 (0.3-10 nM). In the guinea-pig isolated ileum, MEN 11467 (0.03-1 nM) produced a nonparallel rightward shift of the concentration-response curve to SP methylester with a concomitant reduction of the Emax to the agonist (pK(B) = 10.7 +/- 0.1). Moreover the antagonist activity of MEN 11467 was hardly reversible despite prolonged washout. In vivo, MEN 11467 produced a long lasting (> 2-3h) dose-dependent antagonism of bronchoconstriction induced by the selective tachykinin NK(1) receptor agonist, [Sar(9), Met(O(2))(11)]SP in anaesthetized guinea-pigs (ID(50)s' = 29+/-5, 31+/-12 and 670+/-270 microg/kg, after intravenous, intranasal and intraduodenal administration, respectively), without affecting bronchoconstriction induced by methacholine. After oral administration MEN 11467 produced a dose-dependent inhibition of plasma protein extravasation induced in guinea-pig bronchi by [Sar(9), Met(O(2))(11)] (ID(50) = 6.7 +/- 2 mg/kg) or by antigen challenge in sensitized animals (ID(50) = 1.3 mg/kg). After i.v. administration MEN 11467 weakly inhibited the GR 73632-induced foot tapping behaviour in gerbil (ED(50) = 2.96 +/- 2 mg/kg), indicating a poor ability to block central tachykinin NK(1) receptors. These results demonstrate that MEN 11467 is a potent, highly selective and orally effective insurmountable pseudopeptide antagonist of peripheral tachykinin NK(1) receptors with a long duration of action.