Involvement of tachykinin NK1 and NK2 receptors in substance P-induced microvascular leakage hypersensitivity and airway hyperresponsiveness in guinea-pigs

Tachykinins, such as substance P, might be involved in the development of airway hyperresponsiveness (AHR) and airway inflammation. However, it is unknown which tachykinin receptors mediate these biological activities. The effects of two antagonists of tachykinin neurokinin-1 (NK1) and tachykinin neurokinin-2 (NK2) receptors, SR 140333 and SR 48968, respectively, were investigated on substance P (SP)-induced airway hyperresponsiveness and potentiation of the histamine-induced increase in microvascular leakage, in phosphoramidon-pretreated guinea-pigs. Guinea-pigs were pretreated with phosphoramidon (0.1 mM aerosol for 15 min) and exposed 15 min later to saline solution alone or to saline solution containing SP (0.1 mg.mL-1 for 30 min). Twenty four hours later, the animals were anaesthetized and prepared for the recording of the pulmonary inflation pressure (PIP) to acetylcholine or for the investigation of microvascular leakage to histamine. Pretreatment of the guinea-pigs with a single dose of SR 48968 (1 mg.kg-1, i.p.) 30 min before SP exposure, significantly prevented the development of AHR, whereas SR 140333 (1 mg.kg-1, i.p.) did not. In a second set of experiments, phosphoramidon-pretreated guinea-pigs exposed to SP presented a significant potentiation of the histamine-induced increase in microvascular leakage in pulmonary airways. When the guinea-pigs were pretreated with SR 140333, an inhibition of the increased microvascular leakage to histamine was observed. In contrast, no significant inhibitory activity was noted when the guinea-pigs were pretreated with SR 48968. The present data demonstrate the importance of tachykinin NK2 receptor stimulation in the development of airway hyperresponsiveness and that of tachykinin NK1 receptor stimulation in microvascular leakage hypersensitivity in phosphoramidon-pretreated and substance P-exposed guinea-pigs. The results also suggest a dissociation between the presence of microvascular leakage and the occurrence of airway hyperresponsiveness.     

Tachykinin NK1 but not NK2 receptors mediate non-cholinergic excitatory junction potentials in the circular muscle of guinea-pig colon

1. The effect of tachykinin NK1 and NK2 receptor antagonists on noncholinergic excitatory junction potentials (e.j.ps) evoked by electric field stimulation (EFS) in the circular muscle of the guinea-pig proximal colon was investigated by means of a sucrose-gap technique. 2. In the presence of 1 microM atropine, submaximal EFS (10 Hz, 20-30 V, 0.5 ms pulse width, 1 s train duration) evoked an inhibitory junction potential (i.j.p.) followed by e.j.p. with superimposed action potentials (APs) and contraction. Addition of either NG-nitro-L-arginine (L-NOARG, 0.1 mM) or apamin (0.1 microM) inhibited the evoked i.j.p. and the combined administration of the two agents almost abolished it. In the presence of both L-NOARG and apamin, an atropine-resistant e.j.p. was the only electrical response evoked by EFS in 50% of cases and a small i.j.p. (10% of original amplitude) followed by e.j.p. was evident in the remainder. 3. In the presence of L-NOARG and apamin, the tachykinin NK1 receptor antagonists, (+/-)-CP 96,345 and GR 82,334 (10 nM-3 microM) concentration-dependently inhibited the atropine-resistant e.j.p. and accompanying contraction evoked by EFS. EC50 values were: 0.77 microM (e.j.p. inhibition) and 0.22 microM (inhibition of contraction) for (+/-)-CP 96,345; 0.61 microM (e.j.p. inhibition) and 0.20 microM (inhibition of contraction) for GR 82,334. The tachykinin NK2 receptor antagonists, MEN 10,376 (up to 3 microM) and SR 48,968 (up to 1 microM) had no effect on the atropine-resistant e.j.p. MEN 10,376 (3 microM) but not SR 48,968 produced a slight inhibition of the evoked contraction. 4. (+/- )-CP 96,345 (3 microM) and GR 82,334 (3 microM) markedly reduced (81 and 89% inhibition, respectively)the atropine-resistant ej.p. in the absence of L-NOARG and apamin, without affecting the ij.p. MEN 10,376 (3 microM) and SR 48,968 (1 microM) had no significant effect on noncholinergic ij.p. and ej.p. evoked in the absence of apamin and L-NOARG.5. The electrical and mechanical responses to the NK, receptor agonist [Sar9]substance P (SP) sulfone were blocked by (+/-)-CP 96,345 (3 1M) or GR 82,334 (3 microM) which, at the same concentration, failed to affect the responses to the NK2 receptor agonist [PAla8] neurokinin A (NKA) (4-10). In contrast, MEN10,376 (3 microM) or SR 48,968 (1 microM) blocked the response to [beta Ala8]NKA(4-10) without affecting the response to [Sar9]SP sulfone.6. In the presence of L-NOARG and apamin, and in the absence of atropine, EFS of low pulse width(0.02-0.03 ms, other parameters as above) produced cholinergic ej.ps and contraction which were unaffected by GR 82,334 (3 microM). (+/-)-CP 96,345 (3 JAM) produced 24% reduction in the area of the atropine-sensitive ej.p. without affecting the peak amplitude of ej.p. or contraction.7. These findings demonstrate that the noncholinergic ej.ps and accompanying contraction of the circular muscle of the guinea-pig colon are produced through activation of intramural tachykininergic nerves and that the resultant smooth muscle response is almost entirely mediated through NK1 receptors.     

Evidence for metabotropic glutamate receptor activation in the induction of depolarization-induced suppression of inhibition in hippocampal CA1

Depolarization-induced suppression of inhibition (DSI) is a transient reduction of GABAA receptor-mediated IPSCs that is mediated by a retrograde signal from principal cells to interneurons. Using whole-cell recordings, we tested the hypothesis that mGluRs are involved in the DSI process in hippocampal CA1, as has been proposed for cerebellar DSI. Group II mGluR agonists failed to affect either evoked monosynaptic IPSCs or DSI, and forskolin, which blocks cerebellar DSI, did not affect CA1 DSI. Group I and group III mGluR agonists reduced IPSCs, but only group I agonists occluded DSI. (S)-MCPG blocked (1S,3R)-ACPD-induced IPSC suppression and markedly reduced DSI, whereas group III antagonists had no effect on DSI. Many other similarities between DSI and the (1S,3R)-ACPD-induced suppression of IPSCs also were found. Our data suggest that a glutamate-like substance released from pyramidal cells could mediate CA1 DSI by reducing GABA release from interneurons via the activation of group I mGluRs.     

Organization of glutamate receptors at the synapse

Glutamate receptors can be divided in several groups with distinct functional properties. An additional level of complexity has emerged from recent high resolution immunogold analyses which have provided evidence for a differential targeting of glutamate receptors to specific subsynaptic membrane domains. Notably, different types of glutamate receptor may differ in their distance to the release site and in their spatial relation to glutamate transporters. These data imply that the subsynaptic expression of a given glutamate receptor may bias its response to a released quantum of transmitter and suggest that receptor targeting may be implicated in the modulation of glutamatergic neurotransmission.     

Group 1 metabotropic glutamate receptors contribute to slow-onset potentiation in the rat CA1 region in vivo

It has been demonstrated in the CA1 region of the hippocampus in vitro, and in the dentate gyrus and CA1 region in vivo, that application of the metabotropic glutamate receptor (mGluR) agonist, 1S, 3R-amino cyclopentane 2,3-dicarboxylic acid triggers a slow-onset potentiation of synaptic transmission in the hippocampus. This study examined the involvement of group 1 and 2 mGluRs in this phenomenon in the CA1 region of freely moving rats. Drugs were applied via the lateral cerebral ventricle, and measurements were obtained from the CA1 region via permanently implanted electrodes. The group 1 mGluR agonists, 3,5-dihydroxyphenylglycine (DHPG, 20-100 nmol/5 microl) and trans-azetidine-2,4-dicarboxylic acid (ADA, 100 nmol-1 micromol/5 microl) induced a dose-dependent potentiation of basal synaptic transmission. The mGluR antagonist R,S-alpha-methyl-carboxyphenylglycine (MCPG, 1 micromol), and the group 1 mGluR antagonist, S-4-carboxyphenylglycine (4CPG, 100 nmol) completely inhibited the effects of both DHPG and ADA. The group 2 mGluR agonist, (S)-4-carboxy-3-hydroxy phenylglycine (4C3H-PG, 50-200 nmol/5 microl) induced a dose-dependent decrease of basal synaptic transmission. These results suggest that in the CA1 region in vivo, slow-onset potentiation may be mediated by group 1 mGluRs.     

Metabotropic glutamate receptors in the rat nucleus accumbens

The effects of glutamate metabotropic receptors (mGluRs) on excitatory transmission in the nucleus accumbens were investigated using electrophysiological techniques in rat nucleus accumbens slices. The broad-spectrum mGluR agonist (1S,3R)-1-aminocyclopentyl-1,3-dicarboxylate, the mGluR group 2 selective agonists (S)-4-carboxy-3-hydroxyphenylglycine, (1S,3S)-ACPD) and (2S,1'S,2'S)-2-(2'-carboxycyclopropyl)glycine (L-CCG1), and the mGluR group 3 specific agonist L-2-amino-4-phosphonobutyrate (L-AP4) all reversibly inhibited evoked excitatory synaptic responses. The specific group 1 mGluR agonist (R,S)-3,5-dihydroxyphenylglycine [(R,S)-DHPG] did not depress transmission. Dose-response curves showed that the rank order of agonist potencies was: L-CCG1 > L-AP4 > (1S,3S)-ACPD. Group 2 and 3 mGluRs inhibited transmission via a presynaptic mechanism, as they increased paired-pulse facilitation, decreased the frequency of miniature excitatory postsynaptic currents and had no effect on their amplitude. The mGluRs did not inhibit transmitter release by reducing voltage-dependent Ca2+ currents through N- or P-type Ca2+ channels, as inhibition persisted in the presence of omega-conotoxin-GVIA or omega-Aga-IVA. The depression induced by mGluRs was not affected by specific antagonists of dopamine D1, GABA-B or adenosine A1 receptors, indicating direct effects. Finally, (R,S)-DHPG specifically blocked the postsynaptic afterhyperpolarization current (I(AHP)). Our results represent the first direct demonstration of functional mGluRs in the nucleus accumbens of the rat.     

Evidence for a direct interaction between insulin receptor substrate-1 and Shc

Insulin receptor substrate-1 (IRS-1) and Shc are two proteins implicated in intracellular signal transduction. They are activated by an increasing number of extracellular signals, mediated by receptor tyrosine kinases, cytokine receptors, and G protein-coupled receptors. In this study we demonstrate that Shc interacts directly with IRS-1, using the yeast two-hybrid system and an in vitro interaction assay. Deletion analysis of the proteins to map the domains implicated in this interaction shows that the phosphotyrosine binding domain of Shc binds to the region of IRS-1 comprising amino acids 583-661. An in vitro association assay, performed with or without activation of tyrosine kinases, gives evidence that tyrosine phosphorylation of IRS-1 and Shc drastically improves the interaction. Site-directed mutagenesis on IRS-1 583-693 shows that the asparagine, but not the tyrosine residue of the N625GDY628motif domain, is implicated in the IRS-1-Shc-phosphotyrosine binding interaction. Mutation of another tyrosine residue, Tyr608, also induced a 40% decrease in the interaction. This study, describing a phosphotyrosine-dependent interaction between IRS-1 and Shc, suggests that this association might be important in signal transduction.     

Metabotropic glutamate receptors in the ventrolateral medulla of rats

We investigated the hypothesis that stimulation of metabotropic excitatory amino acid receptors in the ventrolateral medulla evokes cardiovascular responses. Thus, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD], a selective agonist of metabotropic excitatory amino acid receptors, was microinjected into the rostral or caudal ventrolateral medulla of halothane-anesthetized Sprague-Dawley rats. Microinjections of (1S,3R)-ACPD (100 pmol-1 nmol) into the rostral ventrolateral medulla produced dose-dependent increases in mean arterial pressure (+20 +/- 4 mm Hg by 100 pmol and +35 +/- 2 mm Hg by 1 nmol, p < 0.01 versus artificial cerebrospinal fluid) and integrated splanchnic sympathetic nerve activity (+17 +/- 3% and +46 +/- 4%, respectively, p < 0.01), whereas (1S,3+)-ACPD microinjected into the caudal ventrolateral medulla decreased mean arterial pressure (-28 +/- 2 mm Hg by 100 pmol and -48 +/- 6 mm Hg by 1 nmol, p < 0.01 versus artificial cerebrospinal fluid) and splanchnic sympathetic nerve activity (-24 +/- 4% and -49 +/- 5%, p < 0.01). The blockade of ionotropic excitatory amino acid receptors by the combined injection of 2-amino-7-phosphonoheptanoic acid (200 pmol) and 6,7-dinitroquinoxaline-2,3-dione (200 pmol), which effectively blocked the responses elicited by either N-methyl-D-aspartate (20 pmol) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (5 pmol), failed to affect the responses evoked by either (1S,3R)-ACPD (100 pmol) or L-glutamate (2 nmol) microinjected in the rostral and caudal ventrolateral medulla. These results suggest that metabotropic receptors are present and mediate cardiovascular responses evoked by L-glutamate injections into the rostral and caudal ventrolateral medulla.     

Evidence for ryanodine receptors in Schistosoma mansoni

The present study investigated the presence of ryanodine receptors in the trematode Schistosoma mansoni. [3H]Ryanodine specific binding sites were found in the four subcellular fractions of S. mansoni; however, more binding sites were recovered in the heterogeneous fraction P1 and the microsomal fraction P4, as was thapsigargin-sensitive (Ca2+-Mg2+)ATPase activity, marking the sarco/endoplasmic reticulum calcium ATPase (SERCA) pumps. This binding had an equilibrium dissociation constant (Kd) in the nanomolar range, an apparent maximal number of receptors (Bmax) of about 80 fmol/mg of protein, and was modulated by ions (Ca2+, Mg2+) and some pharmacological tools such as caffeine. Ryanodine was able to accelerate the rate of 45Ca2+ release from actively loaded vesicles, and also to induce a transient contraction of the whole worm. We conclude that ryanodine-sensitive Ca2+ release channels are present in S. mansoni, with properties very similar to the ones present in higher animals.     

The metabotropic glutamate receptors of the vestibular organs

This research sought to test the presence and function of metabotropic excitatory amino acid receptors (mGluR) in the frog semicircular canal (SCC). The mGluR agonist +/- 1-aminocyclopentane-trans-1,3-dicarboxylate (ACPD) produced an increase in afferent firing rates of the ampullar nerve of the intact posterior canal. This increase was not due to a stimulation of cholinergic efferent terminals or the acetylcholine (ACh) receptor, since atropine, in concentrations which blocked the response to exogenous acetylcholine, did not affect the response to ACPD. Likewise, ACPD effects were not due to stimulation of postsynaptic NMDA receptors, since the NMDA antagonist D(-)-2-amino-5-phosphonopentanoate (AP-5) did not affect the response to ACPD, reinforcing the reported selectivity of ACPD for mGluRs. When the SCC was superfused with artificial perilymph known to inhibit hair cell transmitter release (i.e. low Ca-high Mg), ACPD failed to increase afferent firing. This suggests that the receptor activated by ACPD is located on the hair cell. Pharmacological evidence suggested that the mGluRs involved in afferent facilitation belong to Group I (i.e. subtypes 1 and 5). In fact, the Group III agonist AP-4 had no effect, and the ACPD facilitatory effect was blocked by the Group I mGluR antagonists (S)-4-carboxyphenylglycine (CPG) and (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA). Additional pharmacological evidence supported the presence of Group I mGluRs. Interestingly, the mGluR antagonists, AIDA and 4CPG, by themselves did not affect the resting firing rates of ampullar afferents. This may suggest that the mGluRs are not involved in resting activity but perhaps only in evoked activity (as suggested in Guth et al. (1991) Hear. Res. 56, 69-78). In addition, the mRNA for the mGluR1 has been detected in hair cells of both SCC, utricle, and saccule. In summary, the evidence points to an mGluR localized to the hair cell (i.e. an autoreceptor) which may be activated to produce a positive feedback augmentation of evoked but not resting transmitter release and thus affect afferent activity.     

Identification of the populations of enteric neurons that have NK1 tachykinin receptors in the guinea-pig small intestine

In the present study we have compared the effects of the classical antipsychotic drug haloperidol and four different atypical antipsychotics (clozapine, risperidone, olanzapine, ziprasidone) on extracellular levels of dopamine and noradrenaline in the medial prefrontal cortex (MPFC) of conscious rats. Haloperidol (10, 100 and 800 nmol/kg), clozapine (0.3, 1, 10 and 30 micromol/kg), risperidone (100, 500 and 5000 nmol/kg), olanzapine (10, 100 and 500 nmol/kg) and ziprasidone (10, 100 and 1000 nmol/kg) were administered subcutaneously to rats. All compounds induced increases in dialysate levels of dopamine and noradrenaline in the medial prefrontal cortex. The increases induced by the four antipsychotic agents in extracellular levels of dopamine and noradrenaline displayed a striking co-variation both in dose and time. A similar co-variation was seen in the decrease of dopamine and noradrenaline, after administration of a low dose (30 nmol/kg, s.c.) of the dopamine D2/3 receptor agonist (+)-7-hydroxy-2-(N,N-di-n-propylamino) tetralin ((+)-7-OH-DPAT). It is concluded that there is a close coupling between the release of dopamine and noradrenaline in the medial prefrontal cortex. The mechanism of action of this interaction, that might be of importance for a better understanding of the mechanism of action of antipsychotic drugs, is discussed.     

Metabotropic glutamate receptors and neuronal degenerative disorders

Spirituality plays an integral role in the care of the terminally ill. Hospice philosophy promotes patient/family centred care that is palliative, holistic and interdisciplinary. Historically, spiritual care has been a major component of hospice care that is consistent with these values. Some issues related to the role of spirituality in medicine and hospice care include the difference between spirituality and religion, the patient-physician relationship, provision of spiritual care, and who provides this care. Guidelines for spiritual caregiving include self-knowledge of one's own spiritual needs, authenticity and honesty and respect for the beliefs and practices of the patient and family.     

Evidence for direct interaction of ketamine with alpha 1- and beta 2-adrenoceptors

1. Ketamine has a number of effects that suggest that it may interact with alpha- and beta-adrenoceptors. To date, the experimental evidence for this has been indirect and has been based on physiological studies using competitive blocking agents. In the present study we sought to determine from receptor binding studies whether ketamine binds directly to alpha- and beta-adrenoceptors. 2. Membrane preparations of alpha 1- and beta 2-adrenergic binding sites were obtained from urinary bladder and urethrae of sheep. These binding sites were characterized by saturation analyses using [3H]-prazosin for alpha 1-adrenoceptor binding sites and [125I]-cyanopindolol (CYP) for the beta 2-adrenoceptor binding sites. The receptors were further characterized by displacement studies using selective and non-selective antagonists. 3. Studies in which ketamine was used to displace [3H]-prazosin revealed a Kd of 3.40 +/- 1.23 x 10(-3) mol/L for ketamine binding to alpha 1-adrenoceptors. Displacement studies of [125I]-CYP by ketamine showed a Kd of 0.35 +/- 0.03 x 10(-3) mol/L for ketamine binding to beta 2-adrenoceptors. 4. We conclude that ketamine interacts directly with both alpha 1- and beta 2-adrenoceptors and that such interactions probably explain the reported effects of this agent on the vasculature and the bronchial tree.     

Metabotropic glutamate receptors contribute to the induction of long-term depression in the CA1 region of the hippocampus

Long-term depression of synaptic transmission was induced following the prior induction of long-term potentiation in the CA1 region of rat hippocampal slices. We show that the induction of this form of synaptic depression can be prevented by (+)-alpha-methyl-4-carboxyphenylglycine, a selective antagonist of metabotropic glutamate receptors.     

Functional switch from facilitation to inhibition in the control of glutamate release by metabotropic glutamate receptors

We have investigated the role of metabotropic glutamate receptors linked to phosphoinositide hydrolysis in the control of glutamate release in cerebrocortical nerve terminals. The activation of these receptors with the agonist 3,5-dihydroxyphenylglycine enhanced intra-synaptosomal diacylglycerol and facilitated both the depolarization-induced increase in the cytosolic free Ca2+ concentration and the release of glutamate. However, 5 min after receptor activation, a second stimulation of the pathway with the agonist failed to produce diacylglycerol and to facilitate glutamate release. Interestingly, during the period in which the diacylglycerol response was desensitized, a strong agonist-induced inhibition of Ca2+ entry and glutamate release was observed. This change in the presynaptic effects of 3,5-dihydroxyphenylglycine is reversible since 30 min after the first stimulation, the agonist-induced inhibition of release disappeared, whereas both the production of diacylglycerol and the facilitation of glutamate release were recovered. The tonic elevation of the extracellular glutamate concentration from basal levels (0.8 microM) up to 5 microM also produced the switch from facilitation to inhibition in the receptor response. The existence of this activity-dependent switch in the presynaptic control of glutamate release suggests that release facilitation is limited to conditions under which an appropriate clearance of synaptic glutamate exists, probably to prevent the neurotoxic accumulation of glutamate in the synapse.     

Changes in mRNA for metabotropic glutamate receptors after transient cerebral ischaemia

Using a rat 4-vessel occlusion model of cerebral ischaemia we studied the changes in the mRNA level for the metabotropic receptor subtypes mGluR1 alpha, mGluR1 beta, mGluR2, mGluR3, mGluR4, and mGluR5 by means of in situ hybridization with oligonucleotides. After 24 hours of reperfusion the mRNA levels were significantly increased for mGluR2 and mGluR4 while it was significantly decreased for mGluR5. These results suggest that vulnerable neurones react to an increased extracellular glutamate concentration by differential regulation of the mRNA for metabotropic glutamate receptor subtypes which perhaps reflects the different pre- or postsynaptic location and different involvement in ischaemic neurodegeneration.     

Structure and function of glutamate and nicotinic acetylcholine receptors

The past year has seen remarkable progress in defining the structure of various ligand-gated ion channels. Images of opened and closed nicotinic acetylcholine receptors at 9 A resolution have now made it easier to identify the conformational changes underlying gating. In addition, recent studies on glutamate receptors have led to a radical revision of their postulated transmembrane topology: models for agonist-binding and allosteric domains now use sites previously thought to lie in cytoplasmic loops. Other areas that are being actively pursued include identification of the amino acids lining the ion channels, accurate measurements of Ca2+ fluxes, and tests of transmembrane topology in kainate receptor subunits.     

Dopaminergic modulation of inhibitory glutamate receptors in the lobster stomatogastric ganglion

The intrinsic rhythmicity of the spiny lobster stomatogastric ganglion (STG) is strongly influenced by the strengths of the graded synapses between identified cells within the neural network. These synaptic strengths can be powerfully influenced by chemical neuromodulators such as dopamine and serotonin. Most of the intraganglionic chemical synapses in the STG are mediated by postsynaptic inhibitory glutamate receptors (IGluRs). To determine whether or not direct effects on these IGluRs contribute to the modulation of synaptic strength, unidentified STG neurons were extracted into primary culture and the effects of these aminergic neuromodulators on the glutamate-evoked membrane current were assessed. Dopamine (100 microM) reliably and significantly reduced the whole cell slope conductance of all IGluRs tested. Serotonin (20 microM) never affected the IGlu response, although it clearly altered other cellular membrane properties. Although all identified STG neurons may not conform to these observations, the data reveal a specific dopamine-activated modulatory pathway within cultured neurons that reduces IGluR slope conductance. The relationship between IGluR modulation and net synaptic modulation in situ contributes to an emerging model in which synaptic strengths can be multiply modulated at different functional sites, yielding a complex, distributed, and state-dependent regulatory structure.