Pheromone transduction in the vomeronasal organ

Single-cell physiology and cloning efforts have extended studies of the vomeronasal organ to cellular and molecular levels. Recent work has shown that transduction in the vomeronasal organ is probably mediated by signalling pathways distinct from those that mediate transduction in the main olfactory system. An advance in understanding transduction has come with the cloning from rat vomeronasal organ of a family of putative pheromone receptor genes that bear no sequence similarity to previously cloned receptors. Other work has examined the expression of putative signalling components and found a zonal organization of the epithelium. Patch-clamp studies have described the basic electrical properties of vomeronasal neurons and explored second-messenger pathways.     

Early embryonic formation of the human vomeronasal organ

We studied the origin and development of the vomeronasal system in early human embryos of 10-18 mm crown-rump length under normal and pathological conditions. The formation of the vomeronasal organ from the vomeronasal groove and placode in a 10-mm embryo is described. We propose that the vomeronasal cavity originates in the schizocoel way. We studied the development of the vomeronasal and olfactory nerves. Attention is paid to the development of the nasolacrimal duct and the epithelial plug. The possible use of the vomeronasal system by embryos during the first seven weeks of development is discussed.     

Electrophysiological characterization of chemosensory neurons from the mouse vomeronasal organ

The mechanism of sensory transduction in chemosensory neurons of the vomeronasal organ (VNO) is not known. Based on molecular data, it is likely to be different from that mediating sensory transduction in the main olfactory system. To begin to understand this system, we have characterized the electrophysiological properties of dissociated mouse VNO neurons with patch-clamp recording. Sensory neurons were distinguished from nonsensory neurons by the presence of a dendrite, by immunoreactivity for olfactory marker protein, and by the firing of action potentials. The resting potential of VNO neurons was approximately -60 mV, and the average input resistance was 3 Gomega. Current injections as small as 1-2 pA elicited steady trains of action potentials that showed no sign of elicited steady trains of action potentials that showed no sign of adaptation during a 2 sec stimulus duration. The voltage-gated conductances in VNO neurons are distinct from those in olfactory neurons. The Na+ current is composed of two components; the major component was TTX-sensitive (Ki = 3.6 nM). The outward K+ current activates at -30 mV with kinetics 10 times slower than for K+ currents in olfactory neurons. The Ca2+ current is composed of at least two components: an L-type current and a T-type current that activates at -60 mV and is not found in olfactory neurons. We find no evidence for cyclic nucleotide-gated channels in VNO neurons under a variety of experimental conditions, including those that produced large responses in mouse olfactory neurons, which is further evidence for a novel transduction pathway.     

Molecular aspects of pheromonal communication via the vomeronasal organ of mammals

Recently, two large multigene families of putative G-protein-linked receptors that are expressed in distinct subpopulations of neurones in the vomeronasal organ have been identified. These receptors probably mediate pheromone detection. The most surprising aspects of these findings are that there are so many receptors of two very different classes and that the receptors are unrelated to their counterparts in the main olfactory epithelium. This suggests that many active ligands are likely to exert effects through the vomeronasal organ. Parallel experiments addressing the nature of these ligands indicate a role for some proteins, as well as small molecules, as functional mammalian pheromones. In combination, these results begin to suggest a molecular basis for mammalian pheromone signalling.     

Lectin-binding patterns in the olfactory epithelium and vomeronasal organ of the common marmoset

The role of different cytochrome P450 isozymes (CYP) in the N-demethylation of chlorimipramine and chlorpromazine has been investigated in liver microsomes from rats by studying the effects of multiple subchronic doses of chlorimipramine, chlorpromazine, phenobarbital and beta-naphthoflavone on the N-demethylation of ethylmorphine, mono-N-demethyl-chlorimipramine and chlorpromazine and on the hydroxylation of aniline. With control microsomes, CYP-dependent metabolism of chlorimipramine and chlorpromazine (100 nmol; 30 min incubation) resulted in the formation of predominantly chlorimipramine (46.5 +/- 4.9 nmol) whereas chlorpromazine (14.1 +/- 0.9 nmol) accounted for only part of the overall metabolism of chlorpromazine. Multiple doses of chlorimipramine increased the capacity of microsomes to N-demethylate ethylmorphine (9.8 +/- 0.73 and 6.08 +/- 0.06 nmol min(-1) (mg protein)(-1) for chlorimipramine-treated and control rats, respectively) as well as itself (4.65 +/- 0.25 and 3.10 +/- 0.33 nmol min(-1) (mg protein)(-1), respectively). Multiple doses of chlorpromazine induced aniline-hydroxylase activity (1.11 +/- 0.16 and 0.94 +/- 0.06 nmol min(-1) (mg protein)(-1) for chlorimipramine and control microsomes, respectively) but the capacity to N-demethylate itself was unchanged. Phenobarbital treatment induced ethylmorphine N-demethylation activity, but did not affect N-demethylation activity, towards chlorimipramine and chlorpromazine. In control microsomes the N-demethylation capacity of chlorimipramine or chlorpromazine (0.160 +/- 0.025 and 0.015 +/- 0.003 nmol min(-1) (mg protein)(-1), respectively) was one order of magnitude lower than that of chlorimipramine or chlorpromazine. The capacity to N-demethylate either chlorimipramine or chlorpromazine was increased by treatment with either phenobarbital or beta-naphthoflavone. In control microsomes, sulphaphenazole markedly inhibited both chlorimipramine-N-mono- and di-N-demethylation, whereas quinidine markedly inhibited the rate of formation of chlorpromazine. The CYP2C and CYP2D subfamilies seem to be involved in the mono N-demethylation of chlorimipramine and chlorpromazine, respectively. Moreover the CYP1A and CYP2B subfamilies might participate in the N-demethylation of either chlorimipramine or chlorpromazine. This could have important implications in the clinical use of chlorimipramine and chlorpromazine in view of the genetic polymorphism of CYP2C and CYP2D isozymes in man.     

Modulation of serum testosterone and autonomic function through stimulation of the male human vomeronasal organ (VNO) with pregna-4,20-diene-3,6-dione

In mammals, external chemosensory signals from conspecifics of the opposite sex acting on vomeronasal organ receptors can modulate the release of gonadotropins. There is developmental, anatomical and functional evidence showing that the human vomeronasal organ (VNO) has the characteristics of a chemosensory organ. We have been using naturally occurring human pheromones to serve as models for designing novel synthetic compounds that we call vomeropherins. In previous publications we reported that vomeropherin pregna-4,20-diene-3,6-dione (PDD) delivered to the VNO of normal female and male human volunteers significantly affected male subjects only, decreasing respiration and cardiac frequency, augmenting alpha brain waves, and significantly decreasing serum luteinizing hormone (LH) and follicle stimulating hormone (FSH). Results of the present work confirm that PDD produces a local dose-dependent effect in the male human VNO. This is followed by a mild parasympathomimetic effect characterized by 10% increase of vagal tone, together with decreased frequency of electrodermal activity events. Furthermore, PDD locally delivered to the male human VNO significantly decreases serum LH and testosterone (p < 0.01). The present results contribute additional evidence supporting the functionality of the human VNO and its repercussions in autonomic and psychophysiological functions, as well as in neuroendocrine secretions.     

Genetically fixed enhanced G protein activation in essential hypertension

Recent studies have shown that enhanced Na/H exchanger activity, a frequently observed abnormality in essential hypertension, persists in Epstein-Barr-virus-immortalised lymphoblasts from afflicted patients. This phenomenon is associated with an enhanced proliferation of these cell lines. Studies have ruled out structural changes in the Na/H exchanger protein, but demonstrated a distinct enhancement of intracellular signal transduction in these cell lines. The ultimate cause can be assigned to an obviously genetically fixed increased reactivity of pertussis-toxin-sensitive G proteins, which can easily explain the increased formation of intracellular- second messengers, enhanced Ca2+ signals, and enhanced proliferation of these cell lines. Thus, hypertension in the subset of patients with enhanced Na/H exchanger activity could be caused by increased G protein reactivity.     

Migration and activation of antigen-specific CD8+ T cells upon in vivo stimulation with allogeneic tumor

The response of CD8+ T cells to allogeneic tumor was studied by adoptive transfer of cells from TCR transgenic 2C mice specific for Ld alloantigen. Transferred cells were monitored during the course of a response to i.p. challenge with live P815 (H-2d) using the 1B2 mAb specific for the 2C TCR. Tumor was present in the draining LN and spleen within 3 to 4 days of challenge. The first changes in 1B2+ cells occurred in the spleen on day 4; VLA-4 expression increased in an Ag-specific manner and L-selectin expression decreased in an Ag-nonspecific manner. The number of 1B2+ cells in the spleen declined over days 4 to 6. The first detectable increase in CD25 expression and blast transformation was in the peritoneal cavity beginning days 5 and 6. Clonal expansion was largely limited to this site and was maximal on day 8. As expansion occurred in the peritoneal cavity; the number of 1B2+ cells in the draining LN and spleen also increased. These cells had an activated phenotype (CD44(high), VLA-4(high)) but most did not express CD25 and were not blasts. These results suggest that initial Ag recognition in the spleen results in altered expression of adhesion receptors so that cells gain access to the peritoneal cavity where they undergo clonal expansion and differentiation. Following the response, 1B2+ cells decline in number but a memory population (CD44(high), L-selectin(high and low)) persists for long times in the spleen and LN.     

Palmitoylation of human endothelinB. Its critical role in G protein coupling and a differential requirement for the cytoplasmic tail by G protein subtypes

By site-directed mutagenesis, three cysteine residues (amino acids 402, 403, and 405) in the carboxyl terminus of human endothelinB (ETB) were identified as potential palmitoylation sites. Substitutions of all of the three cysteine residues with serine gave an unpalmitoylated mutant, C2S/C3S/C5S. When expressed in Chinese hamster ovary cells, C2S/C3S/C5S was localized on the cell surface, retained high affinities to ET-1 and ET-3, and was rapidly internalized when bound to the ligand. However, unlike the wild-type ETB, C2S/C3S/C5S transmitted neither an inhibitory effect on adenylate cyclase nor a stimulatory effect on phospholipase C, indicating a critical role of palmitoylation in the coupling with G proteins, regardless of the G protein subtypes. Truncation of the carboxyl terminus including Cys403/Cys405 gave a deletion mutant Delta403 that was palmitoylated on Cys402 and lacked the carboxyl terminus downstream to the palmitoylation site. Delta403 did transmit a stimulatory effect on phospholipase C via a pertussis toxin-insensitive G protein but it failed to transmit an inhibitory effect on adenylate cyclase. These results indicated a differential requirement for the carboxyl terminus downstream to the palmitoylation site in the coupling with G protein subtypes, i.e. it is required for the coupling with Gi but not for that with Gq.     

Changes in the protein metabolism of the CNS of a teleost following stimulation of the lateral-line organ

Unilateral stimulation of the lateral-line organ (LLO) of the teleost Scardinius erythrophthalmus by current water caused an increase in the afferent fibre activity in the stimulated organ of about 3.5 times as compared with the non-stimulated LLO. There was an increase of [3H] histidine incorporation as compared to controls following stimulation applied either at the beginning (1 h) or at the end (1.5-6 h) of various incorporation times. Following a 1 h stimulation period, the different areas of the LLO-system (lateral nerve, medulla oblongata, cerebellum and valvulae cerebelli), as well as the optic tectum, subtectum and spinal cord showed a significant increase of protein labelling; whereas after 12 h post-incorporation times only the lateral nerve showed highly significant differences as compared to controls. In animals stimulated at the end of a 12 h pre-incorporation period (1.5-6 h) there was a significant increase of protein labelling in all investigated structures of the brain as compared to controls.     

Diaphragm activation with intramuscular stimulation in dogs

We studied in 10 supine anesthetized dogs diaphragm contraction produced by electrical activation with intramuscular electrodes surgically implanted in the ventral surface of the diaphragm and compared this with activation of the ipsilateral phrenic nerve (C5, 6, and 7) before it entered the thorax. Repetitive 40-Hz pulse trains with supramaximal current stimulus were used after hyperventilation of the animals to apnea. A single intramuscular electrode within 1 to 2 cm of the site of phrenic nerve entry into the diaphragm produced a mean transdiaphragmatic pressure of 12.0 cm H2O +/- 0.97 SE and mean tidal volume of 0.27 L +/- 0.04 SE. Mean values observed with phrenic nerve stimulation were not statistically different, and both electrode systems produced equivalent outward abdominal motion and upper rib cage paradox, as monitored by inductive plethysmography. There was no difference in gas exchange during stimulation with a single hemidiaphragm electrode and mechanical ventilation compared at the same tidal volume and respiratory rate. Blockade of neuromuscular transmission with curare eliminated intramuscular and phrenic nerve stimulation proportionately, suggesting that activation of the diaphragm is dependent in both cases on the phrenic nerve. This technique does not entail manipulation of the phrenic nerve and may have clinical application as an alternative technique for diaphragm pacing.     

Toxin-induced activation of the G protein p21 Rho by deamidation of glutamine

Pathogenic Escherichia coli are responsible for a variety of diseases, including diarrhoea, haemolytic uraemic syndrome, kidney infection, septicaemia, pneumonia and meningitis. Toxins called cytotoxic necrotizing factors (CNFs) are among the virulence factors produced by uropathogenic (CNF1) or enteropathogenic (CNF2) E. coli strains that cause diseases in humans and animals, respectively. CNFs induce an increase in the content of actin stress fibres and focal contacts in cultured cells. Effects of CNFs on the actin cytoskeleton correlated with a decrease in the electrophoretic mobility of the GTP-binding protein Rho and indirect evidence indicates that CNF1 might constitutively activate Rho. Here we show that CNF1 catalyses the deamidation of a glutamine residue at position 63 of Rho, turning it into glutamic acid, which inhibits both intrinsic GTP hydrolysis and that stimulated by its GTPase-activating protein (GAP). Thus, this deamidation of glutamine 63 by CNF1 leads to the constitutive activation of Rho, and induces the reorganization of actin stress fibres. To our knowledge, CNF1 is the first example of a bacterial toxin acting by deamidation of a specific target protein.     

Domains involved in the specificity of G protein activation in phospholipase C-coupled metabotropic glutamate receptors

G protein-coupled glutamate receptors (mGluR) have recently been characterized. These receptors have seven putative transmembrane domains, but display no sequence homology with the large family of G protein-coupled receptors. They constitute therefore a new family of receptors. Whereas mGluR1 and mGluR5 activate phospholipase C (PLC), mGluR2, mGluR3, mGluR4 and mGluR6 inhibit adenylyl cyclase (AC) activity. The third putative intracellular loop, which determines the G protein specificity in many G protein-coupled receptors, is highly conserved among mGluRs, and may therefore not be involved in the specific recognition of G proteins in this receptor family. By constructing chimeric receptors between the AC-coupled mGluR3 and the PLC-coupled mGluR1c, we report here that both the C-terminal end of the second intracellular loop and the segment located downstream of the seventh transmembrane domain are necessary for the specific activation of PLC by mGluR1c. These two segments are rich in basic residues and are likely to be amphipathic alpha-helices, two characteristics of the G protein interacting domains of all G protein-coupled receptors. This indicates that whereas no amino acid sequence homology between mGluRs and the other G protein-coupled receptors can be found, their G protein interacting domains have similar structural features.     

Selective in vivo stimulation of stress-activated protein kinase in different rat tissues by immobilization stress

The quaternary structure of Lumbricus terrestris hemoglobin was investigated by small-angle x-ray scattering (SAXS). Based on the SAXS data from several independent experiments, a three-dimensional (3D) consensus model was established to simulate the solution structure of this complex protein at low resolution (about 3 nm) and to yield the particle dimensions. The model is built up from a large number of small spheres of different weights, a result of the two-step procedure used to calculate the SAXS model. It accounts for the arrangement of 12 subunits in a hexagonal bilayer structure and for an additional central unit of clylinder-like shape. This model provides an excellent fit of the experimental scattering curve of the protein up to h = 1 nm-1 and a nearly perfect fit of the experimental distance distribution function p(r) in the whole range. Scattering curves and p(r) functions were also calculated for low-resolution models based on 3D reconstructions obtained by cryoelectron microscopy (EM). The calculated functions of these models also provide a very good fit of the experimental scattering curve (even at h > 1 nm-1) and p(r) function, if hydration is taken into account and the original model coordinates are slightly rescaled. The comparison of models reveals that both the SAXS-based and the EM-based model lead to a similar simulation of the protein structure and to similar particle dimensions. The essential differences between the models concern the hexagonal bilayer arrangement (eclipsed in the SAXS model, one layer slightly rotated in the EM model), and the mass distribution, mainly on the surface and in the central part of the protein complex.     

Pheromone regulated production of inositol-(1, 4, 5)-trisphosphate in the mammalian vomeronasal organ

Social behaviors of most mammals are profoundly affected by chemical signals, pheromones, exchanged between conspecifics. Pheromones interact with dendritic microvilli of bipolar neurons in the vomeronasal organ (VNO). To investigate vomeronasal signal transduction pathways, microvillar membranes from porcine VNO were prepared. Incubation of such membranes from prepubertal females with boar seminal fluid or urine results in an increase in production of inositol-(1, 4, 5)-trisphosphate (IP3). The dose response for IP3 production is biphasic with a GTP-dependent component at low stimulus concentrations and a nonspecific increase in IP3 at higher stimulus concentrations. The GTP-dependent stimulation is mimicked by GTPgammaS and blocked by GDPbetaS. Furthermore, the GTP-dependent component of the stimulation of IP3 production is sex specific and tissue dependent. Studies with monospecific antibodies reveal a G alpha(q/11)-related protein in vomeronasal neurons, concentrated at their microvilli. Our observations indicate that pheromones in boar secretions act on vomeronasal neurons in the female VNO via a receptor mediated, G protein-dependent increase in IP3. These observations set the stage for further investigations on the regulation of stimulus-excitation coupling in vomeronasal neurons. The pheromone-induced IP3 response also provides an assay for future purification of mammalian reproductive pheromones.     

GABAergic neurons in the embryonic olfactory pit/vomeronasal organ: maintenance of functional GABAergic synapses in olfactory explants

In previous work, we showed a robust gamma-aminobutyric acid (GABAergic) synaptic input onto embryonic luteinizing hormone-releasing hormone (LHRH) neurons maintained in olfactory explants. In this study, we identify GABAergic neurons in olfactory pit (OP) of embryonic mice in vivo and study, using patch-pipet whole-cell current and voltage clamp techniques, synaptic interactions of these neurons in explant cultures. In vivo, glutamate decarboxylase (GAD, the enzyme which synthesizes GABA) mRNA was first detected in nasal regions on Embryonic Day (E) 11.5. From E12.5 to E13.5, robust GAD expression was localized to cells primarily in the ventral aspect of the OP. GAD mRNA was not detected over dorsally located cells in olfactory sensory or respiratory epithelium. In addition, GAD mRNA was not observed in cells along olfactory axons. GAD mRNA was dramatically reduced in the OP/vomeronasal organ by E16.5. Using antibodies against both GABA and GAD, immunopositive axonal-like tracts were detected in the nasal septum on E12.5. GABAergic staining decreased by E13.5. To examine synaptic interactions of these GABAergic cells, embryonic olfactory explants were generated and maintained in serum-free media. As explants spread, neuron-like cells migrated into the periphery, sometimes forming ganglion-like clusters. Cells were recorded, marked intracellularly with Lucifer Yellow and post-fixation, immunocytochemically examined. Forty-six cells, typically multipolar, were GABAergic, had resting potentials around -50 mV, and exhibited spontaneous action potentials which were generated by spontaneous depolarizing GABAergic (GABAA) synaptic activity. OP neurons depolarized in response to GABA by increasing Cl- conductance. The biophysical properties of OP-derived GABAergic neurons were distinct from those reported for olfactory receptor neurons but similar to embryonic LHRH neurons. However, unlike LHRH neurons, GABAergic neurons did not migrate large distances in olfactory explants or appear to leave the olfactory pit in vivo.     

Rapid activation of protein C by factor Xa and thrombin in the presence of polyanionic compounds

A recent study indicated that negatively charged substances such as heparin and dextran sulfate accelerate thrombin activation of coagulation factor XI by a template mechanism. Because the serine proteinase of the natural anticoagulant pathway, activated protein C, can bind heparin, it was reasonable to think that these compounds may also bind protein C (PC) and accelerate its activation by thrombin or other heparin binding plasma serine proteinases by a similar mechanism. To test this, PC activation by thrombin and factor Xa (fXa) was studied in the presence of these polysaccharides. With thrombin in the absence of thrombomodulin (TM), these polysaccharides markedly reduced the Km for PC and Gla-domainless PC (GDPC) activation in the presence of Ca2+. With TM containing chondroitin sulfate, heparin did not influence PC activation by thrombin, but with TM lacking chondroitin sulfate, the characteristic high-affinity PC interaction at low Ca2+ (approximately 50 to 100 micromol/L) was largely eliminated by heparin. In EDTA, heparin enhanced thrombin activation of GDPC by reducing the Km, but it inhibited PC activation by increasing the Km. PC activation in EDTA was insensitive to the presence of heparin if the exosite 2 mutant, R93,97,101A thrombin, was used for activation. These results suggest that, when the Gla-domain of PC is not fully stabilized by Ca2+, it interacts with the anion binding exosite 2 of thrombin and that heparin binding to this site prevents this interaction. Additional studies indicated that, in the presence of phospholipid vesicles, heparin and dextran sulfate dramatically accelerate PC activation by fXa by also reducing the Km. Interestingly, on phospholipids containing 40% phosphatidylethanolamine, the activation rate of near physiological PC concentrations ( approximately 80 nmol/L) by fXa in the presence of dextran sulfate was nearly comparable to that observed by the thrombin-TM complex. The biochemical and potential therapeutical ramifications of these findings are discussed.     

Chemosignal transduction in the vomeronasal organ of garter snakes: Ca(2+)-dependent regulation of adenylate cyclase

Earthworm shock secretion contains a 20-kDa vomeronasally mediated chemoattractive protein for garter snakes. Both the ligand-receptor binding and the chemoattractivity of ES20 are Ca(2+)-dependent. When ES20 binds to its G-protein-coupled receptors in the vomeronasal epithelium, the inositol 1,4,5-trisphosphate (IP3) level is increased, but the level of cAMP is reduced. Furthermore, forskolin-stimulated levels of cAMP are completely blocked by ES20-receptor binding or by Ca2+ alone and the effect of calcium ions can be nullified by EGTA. Previously, we hypothesized that the decrease in cAMP was due to activation of a Ca(2+)-dependent phosphodiesterase. In the present study, we provide evidence that the decrease in cAMP is due mainly to the regulation of adenylate cyclase (AC) activity by Ca2+ or is indirectly mediated by ES20. Results obtained with intact vomeronasal sensory epithelium suggest that the binding of ES20 to its receptors facilitates generation of IP3 which mobilizes intracellularly sequestered Ca2+, resulting in an increase of cystosolic Ca2+. A further increase in cytosolic Ca2+ occurs through Ca2+ influx from extracellular sources. Garter snake vomeronasal AC does not require calmodulin for its activity and shows a biphasic response to increasing concentrations of Ca2+; its activity is modulated both positively and negatively by this bivalent cation.     

Influence of male rats on the luteinizing hormone-releasing hormone neuronal system in female rats: role of the vomeronasal organ

Olfactory information processed by the vomeronasal system is reported to influence reproductive functions in a variety of mammals. The present studies were designed to determine if male-associated cues affect the luteinizing hormone-releasing hormone (LHRH) neuronal system, and, if so, to determine the extent to which these cues are processed by the vomeronasal organ (VNO). Ovariectomized rats underwent VNO removal (VNX) or sham surgery (VN-Sham). Forty-eight hours after estrogen priming (5 micrograms), they were subjected to one of the following treatments: repeated mating, repeated exposure to male-soiled bedding or repeated exposure to clean bedding. In animals treated for 180 min, coronal brain sections were double labelled for Fos protein and LHRH. An intense Fos immunoreactivity was induced following mating in the majority of LHRH neurons in the VN-Sham females, whereas removal of the VNO significantly suppressed the mating-induced Fos staining. Exposure of female rats to male-soiled bedding or clean bedding did not induce appreciable Fos immunoreactivity in LHRH neurons. Following 90 min of mating or exposure to bedding, blood samples were assayed for luteinizing hormone (LH). Mating stimulated the release of LH in VN-Sham females, while the removal of the VNO significantly suppressed the mating-induced LH release. Exposure of the females to male-soiled bedding or clean bedding did not induce an LH surge. The present results demonstrate that male-originating sensory cues (i.e. repeated mating) can influence the LHRH neuronal system, as evidenced by the presence of Fos immunoreactivity in LHRH cell bodies, and indicate that this effect is mediated through the VNO to a certain extent.(ABSTRACT TRUNCATED AT 250 WORDS)