PubMed HealthSearch

Biomedical subjects

S J Kleene

Publications and source records attributed to S J Kleene.

At least 19 recordsLinked to original sources

Persistence of the olfactory receptor current in a wide variety of extracellular environments.

We measured the current activated by cytoplasmic adenosine 3':5'-cyclic monophosphate (cAMP) in olfactory cilia from the frog Rana pipiens. The odorant-induced current in frog olfactory receptor neurons was also measured for comparison. In both cases, recordings were performed near the neuronal resting potential in a variety of extracellular bath solutions. 2. In Ca(2+)-free baths, cAMP activated an inward current in excised olfactory cilia that was carried entirely by cations. As extracellular Ca2+ was increased, the cationic current decreased while a second current, carried by C1-, increased. Total cAMP-activated current decreased with increasing extracellular CA2+. When external Na+ but not Ca2+ was eliminated, only the C1- component of the current persisted. When external Na+ and Ca2+ were both removed, there was no cAMP-activated current. 3. In receptor neurons, the total odorant-induced receptor current varied in a similar way with the extracellular ionic environment. Under conditions favoring the anionic receptor current, the response amplitude decreased and the latency increased. 4. It is known that olfactory receptor currents persist in a wide variety of extracellular environments. This persistence can be sufficiently explained by the balance between cationic and anionic currents demonstrated here.

Animals

Block by external calcium and magnesium of the cyclic-nucleotide-activated current in olfactory cilia.

Olfactory transduction occurs on the cilia of olfactory receptor neurons, which are in close proximity to the external environment. Transduction is mediated by cyclic AMP, which directly gates channels in the ciliary membrane. Previous evidence indicates that one environmental influence, the level of divalent cations in the mucus, may strongly influence olfactory transduction by blocking the cyclic-AMP-gated channels. In this report the effects of external calcium and magnesium on the ciliary macroscopic current activated by cytoplasmic cyclic AMP were measured. External calcium and magnesium each reduced the cyclic-AMP-activated current at both negative and positive potentials. At the neuronal resting potential (-50 mV), half-maximal inhibition of the current was produced by 250 microM calcium or 1.3 mM magnesium. Reduction in current by external calcium was strongly voltage-dependent, with larger effects at negative potentials. Reduction by magnesium was weaker and less voltage-dependent. Block of the cyclic-AMP-activated current by divalent cations in the mucus may be one element of a system that increases the signal-to-noise ratio for detection of odorants.

Animals

Solving buffering problems with Mathematica software.

Determining ionic concentrations in buffered solutions usually reduces to solving a set of simultaneous polynomial equations. Mathematica software offers a convenient method for doing this. Using buffering of Ca2+ by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) as an example, we provide a Mathematica script to estimate the apparent association constant. A second example shows how to calculate free ion concentrations when two ligands (Ca2+ and Mg2+) compete for one chelator (EGTA). Finally, the concentrations of all species are determined in a complex mixture containing Ca2+, EGTA, and calmodulin, a protein with four Ca(2+)-binding sites. Modifying the examples presented should allow analysis of most practical buffering problems.

Algorithms

Guanine nucleotides modulate steady-state inactivation of voltage-gated sodium channels in frog olfactory receptor neurons.

The voltage for half-inactivation (V1/2) of Na+ currents in frog olfactory receptor neurons (ORNs) under whole-cell voltage clamp showed a shift to more negative potentials with time. Inclusion of guanosine triphosphate (GTP) or its nonhydrolyzable analogue, guanosine-5'-O-3-thiotriphosphate (GTP-gamma-S), which activates G proteins, in the recording pipette, not only gave a more positive V1/2, but also reduced and delayed the negative shift observed in the absence of nucleotides. Guanosine-5'-O-2-thiodiphosphate (GDP-beta-S), a nonhydrolyzable analogue that prevents the binding of GTP to G proteins, did not affect the V1/2 significantly by itself but blocked the positive shift induced by GTP. Since the steady-state activation was not affected, our results indicate that a G protein or a G-protein-dependent process may be important in regulating the steady-state inactivation of Na+ channels in ORNs of the frog.

Animals

Inhibition of olfactory cyclic nucleotide-activated current by calmodulin antagonists.

1. In amphibian olfactory receptor neurones, much of the depolarizing current in response to odours is carried by cationic channels that are directly gated by cyclic AMP. The effects of four calmodulin antagonists on the cyclic AMP-activated receptor current were studied in single olfactory cilia of the frog. 2. Two antagonists, W-7 and trifluoperazine, were potent and reversible inhibitors of the cyclic AMP-activated current. IC50 values were 5 microM for W-7 and 13 microM for trifluoperazine. A third antagonist, calmidazolium, irreversibly blocked the current. The fourth, mastoparan, had little effect. 3. Calmodulin was unable to reverse the effects of W-7 and trifluoperazine, suggesting that these inhibitors act directly on the cyclic AMP-gated channels. 4. Neither W-7 nor trifluoperazine inhibited a Ca(2+)-activated Cl- current which also contributes to the odorant response. These compounds thus allow the two components of the olfactory receptor current to be discriminated.

Animals

An electrophysiological survey of frog olfactory cilia.

Individual olfactory receptor neurons vary widely in their responses to odorants. Olfactory stimulus reception occurs in the cilia of the receptor neurons. Thus, the variability among individual neurons could in part be due to differences among the olfactory cilia. We have quantified the known conductance properties of each of 117 frog olfactory cilia. From a strictly qualitative viewpoint, the cilia were very homogeneous. All but a few of them had a basal conductance in the absence of odorants and second messengers, conductances stimulated by cytoplasmic cyclic AMP and by Ca2+ and a conductance measured in the presence of ATP and stimulated by GTP gamma S. However, the magnitudes of the conductances varied widely among the cilia. Amplitudes of the cyclic-AMP- and Ca(2+)-activated ciliary currents correlated strongly with one another across the 117 cilia and 24 frogs studied, suggesting that expression of the underlying channels may be co-regulated. None of the conductance properties correlated strongly with ciliary length, a marker of cell maturity. Given cytoplasmic MgATP as substrate, ciliary adenylate cyclase apparently produced cyclic AMP, which in turn gated membrane channels and increased the ciliary conductance. In some cilia, MgATP alone caused a very large increase in conductance. In others, there was little effect unless GTP gamma S, which increases cyclase activity, was also added. No effect of cytoplasmic inositol trisphosphate on ciliary conductance was detectable.

Adenosine Triphosphate

The cyclic nucleotide-activated conductance in olfactory cilia: effects of cytoplasmic Mg2+ and Ca2+.

Olfactory receptor neurons depolarize in response to odorants. This depolarization is mediated by an increase in intracellular cyclic AMP, which directly gates channels in the membranes of the neuronal cilia. Previous evidence suggests that a Ca2+ influx during the odorant response may ultimately play a role in terminating the response. One way Ca2+ inside the cell could terminate the odorant response would be to directly inhibit the cAMP-gated channels. In this report the effects of cytoplasmic Ca2+ and Mg2+ on the cAMP-activated current were measured in single olfactory cilia. Near the neuronal resting potential, cytoplasmic Ca2+ and Mg2+ only slightly reduced the cAMP-activated current. Even at high levels (1.0 mM Ca2+ or 5.0 mM Mg2+), the average inhibition was only around 20%. It is therefore unlikely that an influx of divalent cations terminates the odorant response by a direct effect on the cAMP-gated channels.

Ambystoma

A simple intrapipette salt bridge.

A simple method is described for achieving a salt bridge within a patch pipette. The tip of the pipette is filled with a Cl(-)-free solution that bathes the membrane patch. Above this is a Cl(-)-containing solution that surrounds the recording Ag/AgCl wire electrode. Addition of 0.07% (w/v) agarose to the solution in the tip prevents bulk mixing of the 2 phases within the pipette. This configuration makes it possible to remove Cl- from the membrane surface but maintain it at the electrode surface. The presence of the Cl(-)-free phase in the bottom of the patch pipette does not reduce the electrical stability of the recording electrode.

Animals

Origin of the chloride current in olfactory transduction.

In the cilia of amphibian olfactory receptor neurons, odorants elicit a receptor current that has two components: a cationic current through cAMP-gated channels and a Cl- current. Here, a cascade of ciliary currents that accounts for the total receptor current is demonstrated. In isolated olfactory cilia, cAMP sequentially activates two currents. The first is a primary cationic current through channels directly gated by cAMP. Part of this current is carried by Ca2+, which in turn activates a Cl- current. This secondary current is eliminated by the presence of Cl- channel inhibitors, replacement of Cl- with methanesulfonate-, removal of external Ca2+, or blockers of the cAMP-gated cationic channels. When cytoplasmic Ca2+ buffering is low, small cationic currents can activate Cl- currents that are 20 times larger.

Animals

Basal conductance of frog olfactory cilia.

The conductance of isolated frog olfactory cilia in the absence of odorants and second messengers has been measured. Current flowing through the pipette-membrane seal rather than the ciliary membrane was subtracted. In normal physiological solutions, each cilium has a conductance averaging 92 pS at the neuronal resting potential. This basal conductance allows current to be carried by K+ or Na+ but not by Cl-. In some cases, single channels with a unit conductance of 153 pS were observed. The conductance of the ciliary membrane implies a length constant for electrotonic conduction of about 160 microns. Since the reversal potential of the basal conductance is near the neuronal resting potential, it should help to stabilize the ciliary potential at some cost to stimulus transduction efficiency.

Animals

Transmembrane currents in frog olfactory cilia.

We have measured transmembrane currents in intact single cilia from frog olfactory receptor neurons. A single cilium on a neuron was sucked into a patch pipette, and a high-resistance seal was formed near the base of the cilium. Action potentials could be induced by applying suction or a voltage ramp to the ciliary membrane. A transient current was seen in some cells on stimulation with odorants. After excision from the cell, most of the cilia showed increased conductance in a bath containing cAMP, indicating that the cytoplasmic face of the ciliary membrane was accessible to the bath. The estimated resistance of a single cilium was surprisingly low.

Action Potentials

Calcium-activated chloride conductance in frog olfactory cilia.

We have measured the effects of cytoplasmic Ca2+ on the conductance of single cilia excised from frog olfactory receptor neurons. When free cytoplasmic Ca2+ is buffered at 0.1 microM, ciliary conductance is low. As Ca2+ is increased, ciliary conductance increases. Maximal conductance averages sevenfold higher than that measured in the absence of Ca2+. We estimate that the K1/2 for Ca2+ activation is 5 microM; the dose-response curve indicates some positive cooperativity of Ca2+ binding. Activation by Ca2+ is rapid and fully reversible. Most of the Ca(2+)-activated current is carried by Cl- and persists in the absence of Na+ and K+. The Cl- channel inhibitor 3',5-dichlorodiphenylamine-2-carboxylate (300 microM) reduces the Ca(2+)-activated current by 90%. Odorants induce a Ca2+ influx in some olfactory receptor neurons, but the consequences of this influx for neuronal function are not well understood. Our findings allow us to predict that a Ca2+ influx would increase the permeability of the olfactory cilia to Cl-. How this would affect the neuronal potential is uncertain, since the equilibrium potential for Cl- in olfactory receptor neurons is unknown.

Animals

Comparative study of immature and mature olfactory receptor cells in adult frogs.

We describe a method to produce frog olfactory epithelium with a population of developmentally synchronized receptor cells suitable for electrophysiological studies. The epithelium is ablated with ZnSO4 and allowed to regenerate for 10 days. Generation of new cells is then blocked by continuous treatment with hydroxyurea. Since new receptor cells are generated beginning on the 6th day after ablation, it is reasonable to assume that receptor cells in the preparation originated between the 6th and 10th days following ablation. The age range of these cells would be no more than 5 days. The cells develop normally and in relative synchrony. Olfactory epithelia with developmentally synchronized receptor cells were used for a physiological study of response properties as a function of developmental age. Developmental stages of receptor cells were confirmed by fine-structure analysis. There were no differences in the polarities or shapes of electro-olfactograms (EOGs) recorded from olfactory epithelia composed of immature or mature receptor cells. However, amplitudes of the main negative components in EOGs recorded from epithelia composed of mature receptor cells were generally higher than those from epithelia composed of immature cells. The majority of immature olfactory receptor cells had low spontaneous activity or none at all. Mature olfactory receptor cells had a variety of frequencies of spontaneous activity ranging from less than one to more than 70 spikes/min. Extracellular single unit activity recordings showed that, contrary to what has been observed in rat embryos, olfactory receptor cells in regenerating epithelial of adult frogs do not go through a stage in which they respond to all odorants.

Animals

Suppression of mitotic activity and synchronization of cell development in olfactory epithelium.

Prolonged continuous application of hydroxyurea to frog olfactory epithelium suppresses mitotic activity in this tissue without side effects. After ZnSO4-induced coagulation necrosis of the olfactory epithelium, application of hydroxyurea prevents the usual epithelial regeneration. A method for obtaining frog olfactory epithelium with a developmentally synchronized neuronal population is introduced. The epithelium is first ablated by perfusion of the nasal cavity with ZnSO4. Then the epithelium is allowed to regenerate for 6 days, after which continuous introduction into the nasal cavity of hydroxyurea suppresses mitotic activity and thus the generation of new cells in the tissue. Cells formed during the 6 days prior to hydroxyurea treatment continue to develop. We found that the olfactory receptor cells are generated beginning on the 6th day after ablation, and so the epithelia contain a highly synchronized population of neurons all generated within about 24 h.

Animals

Dissociation of frog olfactory epithelium.

We report a method for producing cell suspensions from frog olfactory epithelium. The tissue is incubated for 45 min at room temperature in a solution which causes dissociation of the epithelium. The solution is an isotonic saline buffered to maintain a pH of 10.3 and a free Ca2+ ion concentration of 10(-6) M. The method uses no degradative enzymes, except for a brief DNase treatment. The resulting cell suspension contains single olfactory receptor neurons, sustentacular cells, glandular cells, and respiratory epithelial cells. The cells are viable as judged by vital staining, ciliary motility, and synthesis of RNA. Some types of cells lose their normal columnar shapes and become rounded in the suspension.

Animals

Dissociation of frog olfactory epithelium with N-ethylmaleimide.

Treatment of frog olfactory epithelium with 8 mM N-ethylmaleimide for 2 min results in extensive dissociation of the epithelium. The resulting cell suspension contains single olfactory receptor neurons, sustentacular cells, respiratory epithelial cells, and cells of Bowman's glands. The cells in suspension exhibit the same morphologies seen in histological sections of intact epithelium.

Animals

Attractants and repellents influence methylation and demethylation of methyl-accepting chemotaxis proteins in an extract of Escherichia coli.

During bacterial chemotaxis, attractants and repellents alter the methylation levels of the methyl-accepting chemotaxis proteins (MCPs). These methylation levels represent a balance between two enzymatic processes: methylation and demethylation. In vivo experiments previously have shown that chemoeffectors influence the demethylation process; effects on the methylation system have not been reported. Here we show that in a cell-free extract of Escherichia coli both methylation and demethylation of the MCPs are affected by attractants and repellents. Attractants enhance methylation and inhibit demethylation. Repellents inhibit methylation and stimulate demethylation. The cell-free system provides an opportunity for further study of the mechanisms by which attractants and repellents influence the levels of methylation of the MCPs.

Bacterial Proteins