Richard A. Gleeson

Olfactory receptors of the spiny lobster: ATP-sensitive cells with similarities to P2-type purinoceptors of vertebrates

Summary1.A distinct population of ATP-sensitive cells, with response characteristics indicative of P2-type purinoceptors found in internal tissues of vertebrates, was identified among the antennular olfactory cells of the spiny lobster,Panulirus argus.2.Extracellular recordings from single cells showed that the ATP-sensitive cells had the following properties in common with P2 purinoceptors: a) potency sequence of ATP>ADP>AMP and adenosine; b) broad sensitivity to nucleotide triphosphates including those with modifications in both the purine and ribose moieties; c) stimulated by slowly degradable analogs of ATP, namely,β, γ-imido ATP (AMPPNP),β, γ-methylene ATP (AMPPCP), and α,β-methylene ATP (AMPCPP).3.The activity sequence of the ATP-sensitive cells for nucleotides and related substances was ATP≥2′-deoxyATP>GTP>CTP≥XTP≥ITP> 8-bromo-ATP=ADP, with pyrophosphate, AMP, and tripolyphosphate being virtually inactive.4.The potency sequence for the slowly degradable analogs was AMPPNP>ATP = AMPPCP> AMPCPP.5.Differences in structure-activity relationships, response duration, and response magnitude clearly distinguished the ATP-sensitive cells from another type of olfactory purinoceptor, the AMP-sensitive cells, also occurring in the antennules of the lobster.6.Comparisons between the ATP-sensitive chemoreceptors of the lobster and of certain insects revealed similarities in the activity sequence of ATP, ADP, AMP, and certain other nucleotides. However differences existed in the relative potencies of ATP, AMPPNP, and AMPPCP, and in the relative inactivity of inorganic pyrophosphate in the lobster.7.The findings of this study lend additional credence to the earlier hypothesis that receptors for transmitters and modulators existing in internal tissues may have evolved from external chemoreceptors of primitive unicellular organisms.

Structure of the aesthetasc (olfactory) sensilla of the blue crab, Callinectes sapidus: transformations as a function of salinity

Abstract.The aesthetasc sensilla of the euryhaline blue crab, Callinectes sapidus, are innervated by the dendrites of from 40 to 160 bipolar chemosensory neurons. Each dendrite forms two cilia within the basal portion of the sensillum, and these subsequently branch yielding approximately 10 outer dendritic segments per neuron. Auxiliary cells surround the inner dendritic segments and also ensheathe the outer dendritic segments up to the terminus of the ”constricted region” (a zone in which there is a slight narrowing of the aesthetasc). Crystal violet staining suggesting access of odor stimuli is limited to that portion of the sensillum distal to the constricted region. In freshwater-acclimated blue crabs the length and level of branching in the dendrites extending beyond the constricted region is significantly reduced relative to that of seawater-acclimated animals (mean lengths: 150 µm versus 517 µm, respectively). After transfer of freshwater-acclimated crabs to seawater there is a rapid increase in length of the outer dendritic segments, reaching 60% of that for seawater-acclimated crabs by 48 h. A similar time course for regrowth is seen for seawater-acclimated crabs in which the outer dendritic segments have been osmotically ablated. Conversely, with rapid transfer of seawater-acclimated animals to lower salinities, there is a correspondingly rapid reduction in length of the outer dendritic segments. The reduced length of the outer dendritic segments in freshwater-acclimated animals may reflect the effective distance over which an appropriate osmotic/ionic microenvironment for neural function can be maintained within the aesthetasc.

Characterization of a Phosphoinositide-mediated Odor Transduction Pathway Reveals Plasma Membrane Localization of an Inositol 1,4,5-Trisphosphate Receptor in Lobster Olfactory Receptor Neurons

The role of phosphoinositide signaling in olfactory transduction is still being resolved. Compelling functional evidence for the transduction of odor signals via phosphoinositide pathways in olfactory transduction comes from invertebrate olfactory systems, in particular lobster olfactory receptor neurons. We now provide molecular evidence for two components of the phosphoinositide signaling pathway in lobster olfactory receptor neurons, a G protein α subunit of the Gq family and an inositol 1,4,5-trisphosphate-gated channel or an inositol 1,4,5-trisphosphate (IP3) receptor. Both proteins localize to the site of olfactory transduction, the outer dendrite of the olfactory receptor neurons. Furthermore, the IP3 receptor localizes to membranes in the ciliary transduction compartment of these cells at both the light microscopic and electron microscopic levels. Given the absence of intracellular organelles in the sub-micron diameter olfactory cilia, this finding indicates that the IP3receptor is associated with the plasma membrane and provides the first definitive evidence for plasma membrane localization of an IP3R in neurons. The association of the IP3receptor with the plasma membrane may be a novel mechanism for regulating intracellular cations in restricted cellular compartments of neurons.

Biochemistry of an Olfactory Purinergic System: Dephosphorylation of Excitatory Nucleotides and Uptake of Adenosine

Abstract: The olfactory organ of the spiny lobster, Panu‐lirus argus, is composed of chemosensory sensilla containing the dendrites of primary chemosensory neurons. Receptors on these dendrites are activated by the nucleotides AMP, ADP, and ATP but not by the nucleoside adenosine. It is shown here that the lobster chemosensory sensilla contain enzymes that dephosphorylate excitatory nucleotides and an uptake system that internalizes the nonexcitatory dephosphorylated product adenosine. The uptake of [3H]‐adenosine is saturable with increasing concentration, linear with time for up to 3h, sodium dependent, insensitive to moderate pH changes and has a Km of 7.1 μM and a Vmax of 5.2 fmol/sensillum/min (573 fmol/μg of protein/min). Double‐label experiments show that sensilla dephosphorylate nucleotides extracellularly; 3H from adenine‐labeled AMP or ATP is internalized, whereas 32P from phosphate‐labeled nucleotides is not. The dephosphorylation of AMP is very rapid; 3H from AMP is internalized at the same rate as 3H from adenosine. Sensillar 5′‐ectonucleotidase activity is inhibited by ADP and the ADP analog α,β‐methylene ADP. Collectively, these results indicate that the enizymes and the uptake system whereby chemosensory sensilla of the lobster inactivate excitatory nucleotides and clear adenosine from extracellular spaces are very similar to those present in the internal tissues of vertebrates, where nucleotides have many neuroactive effects.

Amino acid suppression of taurine-sensitive chemosensory neurons.

Single unit recordings from chemoreceptors on the antennule of the spiny lobster revealed a population of taurine-sensitive cells whose response is suppressed when taurine is presented in mixture with certain amino acids. A synthetic mixture of 21 amino acids plus betaine, which mimics the composition of a natural food stimulus (crab muscle tissue) and itself contains taurine, totally and reversibly blocked the taurine response of this group of receptor cells. An analysis of the contribution to this suppression by the six major components (based on concentration) in the mixture revealed partial or complete inhibitory activity by five of the compounds. In a sample group of the inhibited cells, mean percent suppression of the taurine response was 99% for glycine and L-arginine, 98% for L-glutamine, 60% for L-alanine and 43% for L-proline. Both glycine and alanine in binary mixture with taurine caused a right-shift in the concentration-response function for taurine, suggesting a competitive mechanism of suppression. pA2 values determined from these data yielded 4.17 for glycine and 3.55 for alanine. These results suggest that the processing of chemical information in quality and/or intensity coding of natural stimulus mixtures can be tempered by interactions of the components at the receptor-cell level, and possibly at the receptor-sites themselves.

Ectonucleotidase Activities Associated with the Olfactory Organ of the Spiny Lobster

Abstract: The olfactory system of the Florida spiny lobster, Panulirus argus, has olfactory receptors that are excited by the purine nucleotides AMP, ADP, and ATP. These receptors reside on chemosensory neurons that are contained within aesthetasc sensilla on the lateral filaments of the antennules. Also associated with the lobsters olfactory system are ectonucleotidase activities that dephosphorylate excitatory nucleotides, resulting in the production of the nonstimulatory nucleoside adenosine. Our studies of the 5′‐ectonucleotidase, ecto‐ADPase, and ecto‐ATPase activities of this olfactory system showed that each activity was characterized by Michaelis–Menten kinetics; Michaelis constants ranged from 6.9 to 33.5 μM, and maximum velocities ranged from 2.5 to 28.8 fmol/sensillum/s. Evidence that AMP dephosphorylation may serve as an inactivation process was shown by the close correlation between the kinetics of 5′‐ectonucleotidase activity and the periodicity of olfactory sampling. Decreased magnesium ion concentration or increased calcium ion concentration resulted in increased ecto‐ATPase activity; this activity was insensitive to vanadate ion. Ectonucleotidase activities may have multiple effects on the detection of exogenous nucleotides by a chemosensory system. These effects can be either direct, such as the conversion of an odorant to an inactive compound, or indirect, such as the conversion of an odorant to another compound that can activate or inhibit either receptors or enzymes associated with the system.

Ecto-ATPase/phosphatase activity in the olfactory sensilla of the spiny lobster, Panulirus argus : localization and characterization

SummaryElectrophysiological studies have shown that the olfactory organ (antennule) of the spiny lobster, Panulirus argus, has chemoreceptors that are selectively excited by adenine nucleotides in seawater. Biochemical studies have revealed that these same nucleotides can be rapidly dephosphorylated by ectoenzymes associated with the olfactory sensilla (aesthetascs). In this study the distribution of ecto-ATPase/phosphatase activity within aesthetascs was determined cytochemically and the nature of the adenine-nucleotide dephosphorylating activity was dissected biochemically. Cytochemically, the distribution of ATP-dephosphorylating activity was similar to that shown previously for AMP and β-glycerol phosphate; i.e., cerium phosphate reaction product was specifically localized to the transitional zone where the sensory dendrites develop cilia and branch to form the outer dendritic segments. Unlike the dephosphorylation of AMP and β-glycerol phosphate, Mg2+ or Ca2+ was required for ecto-ATPase/phosphatase activity. Biochemical measures of both AMP-and ATP-dephosphorylating activity within aesthetascs corroborated the cytochemical evidence that these activities are localized to the transitional zone. A major portion of the AMP dephosphorylation (about 67%) derives from nonspecific alkaline phosphatase activity that is insensitive to levamisole and L-bromotetramisole. In contrast, nonspecific phosphatase activity accounted for a much smaller part of the ATP dephosphorylation (about 15%). Ectoenzymatic activity in the transitional zone may be an important means of removing excitatory/inhibitory nucleotides from this region.

Modulation of behavior by biogenic amines and peptides in the blue crab, Callinectes sapidus.

Using the blue crab Callinectes sapidus as a model system, we have investigated the effects of potential neuromodulators on freely behaving animals. Of interest is the modulatory effect of a number of drugs on three rhythmic behaviors of the blue crab: courtship display (CD) of the male crab, sideways swimming and backward swimming. The drugs tested were proctolin, dopamine, octopamine, serotonin, and norepinephrine. Injection of each drug elicited a unique posture or combination of limb movements. These experiments showed two results pertinent to CD behavior: A posture identical to the CD posture was displayed after dopamine injection; and rhythmic leg waving similar to CD was evoked by proctolin. An unusual combination of flexion and extension of all limbs and movements of some limbs occurred after serotonin injection. Injection of octopamine led to a posture antagonistic to CD posture. The effects of these drugs were concentration- and time-dependent. Injection of dopamine, octopamine, or serotonin produced effects that were seasonally-dependent, and the influence of proctolin proved to be dependent on developmental stage. Quantitative analysis of leg waving movements after proctolin injection allowed for comparison of these movements to naturally-occurring behavior.

ATP-sensitive chemoreceptors: antagonism by other nucleotides and the potential implications of ectonucleotidase activity

As measured by extracellular single-cell recording, the responses to adenosine triphosphate (ATP) by ATP-sensitive chemoreceptors (ATP cells) on the olfactory organ of the spiny lobster are markedly suppressed by adenosine diphosphate (ADP), adenosine monophosphate (AMP) and to a lesser extent, adenosine, when each is presented in binary mixture with ATP. In the presence of ADP, the dose-response function for ATP exhibits an apparent parallel displacement to the right suggesting that this antagonism may occur via competition at the ATP receptor. Structure-activity relationships reveal that the structural requirements for antagonism by diphosphate analogs of ADP bear little relationship to the requirements for the agonistic activity of corresponding triphosphate analogs. Under Mg2+-free conditions, the desensitization of ATP cells tends to be delayed resulting in enhanced responses to ATP. Desensitization does not appear to be related to the generation of the antagonist, ADP, from ATP via ecto-ATPase activity. The results of this study suggest that the responses of ATP cells to the ATP contained in natural stimulus (odor) mixtures can be tempered by the suppressive interactions of other nucleotides in the mixtures. Furthermore, these interactions may be mitigated and/or intensified by the actions of sensillar ectonucleotidases.

Ionic and elemental analyses of olfactory sensillar lymph in the spiny lobster, panulirus argus

Abstract 1. 1. The elemental composition of the olfactory sensillar lymph of the spiny lobster, Panulirus argus , was determined by energy dispersive X-ray analysis. Sodium activity was also measured using a Na-selective microelectrode. 2. 2. Sensillar lymph has an elemental composition of Na, K, Ca, Mg and S similar to that of both seawater and hemolymph. Cl may be present at a reduced level in sensillar lymph. 3. 3. Marine and terrestrial arthropods exhibit major differences between both the absolute and proportional concentrations of Na and K in the milieu bathing their olfactory receptors. 4. 4. Significant levels of bromine occur in the sensillar cuticle and antennular integument of three crustaceans.