In vitro modulation using a synthetic antioxidant of the stimulus-induced formation of cyclic AMP

The in vitro treatment of membranes isolated from different rat organs with a water-soluble synthetic antioxidant has resulted in the change of basal and stimulus-induced adenylate cyclase activity. It is believed that the antioxidant effect is realized rather at the level of signal transfer from activated receptor to adenylate cyclase than at the level of agonist-receptor interaction.     

Demonstration of a large-conduction ion channel in subcellular fractions of the adrenal medulla

The "tip-dip" technique (formation of a lipid bilayer at the tip of a microelectrode) allows the electrophysiological study of organelle membranes. An ionic channel of large conductance has been found by this technique in membrane preparations from adrenal medulla. This channel is voltage-sensitive and it has 4 levels of conductance. Its subcellular origin is to be determined; it is borne by a structure which sediments between 1,500 and 25,000 X g.     

Association of the glucocorticoid receptor binding subunit with the 90K nonsteroid-binding component is stabilized by both steroidal and nonsteroidal antiglucocorticoids in intact cells

The interaction of various antiglucocorticoids with the glucocorticoid receptor from intact rat thymocytes was investigated. Reversible antiglucocorticoids (RU 486, cortexolone, progesterone) underwent more limited nuclear transfer than potent glucocorticoids (dexamethasone, triamcinolone acetonide, progesterone). This behavior was correlated with an impeded dissociation of cytosolic antiglucocorticoid receptor complexes preformed in intact cells, as assayed by high-performance size exclusion chromatography in physiological conditions (i.e., isotonic molybdate-free buffer). Antagonist-receptor complexes remained in a 7-8-nm form whatever the antiglucocorticoid tested (including dexamethasone mesylate and trifluoroperazine, a nonsteroidal antiglucocorticoid) and the incubation time at 37 degrees C, whereas agonist-receptor complexes were rapidly converted into 5-nm species. This stabilization was not detectable by conventional sucrose gradient centrifugation because of artifactual dissociation of untransformed complexes, a pitfall overcome by resorting to vertical tube centrifugation. Moreover, the low amount of nuclear antiglucocorticoid receptor complexes was also in the undissociated form, in contrast with nuclear agonist-receptor complexes. Immunological probes demonstrated that the 90-kDa non-steroid-binding component was associated with the antiglucocorticoid-stabilized receptor. Thus, whatever their chemical structure and their affinity for the receptor, antiglucocorticoids stabilize the oligomeric form of the glucocorticoid receptor in intact cells. Our data, demonstrating for the first time that all antiglucocorticoids probably act via a common mechanism, suggest a key role for subunit dissociation during in vivo receptor activation.     

Mechanism of action of an antiprogesterone, RU486, in the rabbit endometrium. Effects of RU486 on the progesterone receptor and on the expression of the uteroglobin gene

RU486 is a recently described antiprogesterone. In order to be able to understand its mechanism of action it is necessary to analyze its effect on a discrete gene product. We show here that the induction of uteroglobin mRNA by progesterone in the rabbit endometrium may be a suitable model for such studies since RU486 totally inhibits this effect without itself exerting any agonistic activity. Moreover, RU486, which does not bind to the estrogen receptor and is devoid of general antiestrogenic activity, partially inhibits the induction by estradiol of uteroglobin mRNA. Studies of the interaction between [3H]RU486 and the progesterone receptor have been undertaken with the aim of understanding the antagonistic effect of this compound. The binding to DNA-cellulose of heat-activated [3H]RU486-receptor complexes was slightly decreased (37%) when compared with that of the agonist [3H]R5020-receptor complexes (47%). Detailed analysis of this difference showed that it was due to both a decreased activation of complexes and to a diminished affinity of activated complexes towards DNA. The change in activation was shown by the fact that at high concentrations of DNA, where all activated complexes are bound, agonist-receptor complexes were bound to DNA in higher proportion than antagonist-receptor complexes. Moreover a difference was also observed when studying the binding of agonist-receptor and antagonist-receptor complexes to charged resins (phosphocellulose, DEAE-cellulose) which are known to discriminate between activated and non-activated complexes. Decreased affinity to DNA of antagonist-receptor complexes was shown by studying their binding at various concentrations of DNA, either in crude cytosol or after isolating a homogenous population of activated-receptor complexes by DNA-cellulose chromatography and by comparing the salt extraction from DNA-cellulose of agonist-receptor and antagonist-receptor complexes. Both effects (decreased activation and diminished affinity towards DNA) were relatively moderate and could account only for a small decrease in the agonistic activity of RU486. Thus, the fact that this compound is a complete antagonist without any agonistic activity can only be explained by a defect in some further step of hormone action as, for instance in the specific interaction with the regulatory regions of the uteroglobin gene. No immunological difference could be detected between [3H]R5020-receptor and [3H]RU486-receptor complexes, both interacted with the five monoclonal antibodies raised against purified R5020-receptor complexes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of pH in murine tumor and muscle

The relationship between intracellular and extracellular pH was investigated in a murine tumor and normal tissue, prior to and following glucose injection. Isotransplants of the murine tumor FSa-II in the dorsum of the hind foot and leg muscle, were investigated in nonanesthetized mice. Extracellular pH was measured with a glass microelectrode, with a tip diameter of approximately 80 microns. Intracellular pH was evaluated by 31P-NMR spectroscopy, using a wide-bore NT-150 spectrometer operating at 60.75 MHz. Five grams per kilogram intraperitoneal glucose led to small changes in extracellular pH of muscle (-0.13 unit) measured with a microelectrode, and no change in intracellular pH measured by NMR. In contrast, tumor intracellular pH decreased by 0.34 units and tumor extracellular pH by 1.13 units. The differential effect of glucose on tumor vs normal tissue, and pronounced pH gradient which develops in tumor cells should markedly affect the intracellular:extracellular distribution of drugs which are weak acids or bases.     

Local changes of resistance in the retinal horizontal cell evoked by changes in membrane potential

A steady current (10·10^–10–6·10^–9 A) was passed by means of a bridge circuit through a recording microelectrode inserted into a horizontal cell of the turtle retina. Illumination of the retina caused an increase in the resistance of the microelectrode circuit (by 10–80 M), causing a change in the shape of the recorded response of the horizontal cell to light. The change in resistance was shown to take place, not on the cell membrane itself, but inside the cell close to the microelectrode tip. The effect described can be reproduced by passing a current through one barrel of a double-barreled microelectrode alongside the recording barrel, but the strength required for this current was greater than that passed through the recording barrel. If the membrane potential of the horizontal cell was made equal to the equilibrium potential (by means of a steady current passed through extracellular electrodes) the hyperpolarization response to light and the effect of the increase in resistance of the microelectrode circuit disappeared simultaneously. On the other hand, artificial hyperpolarization of the cell membrane caused an increase, but depolarization caused a decrease in the resistance of the microelectrode circuit. It is postulated that the observed effect is due to blocking of the microelectrode tip by an intracellular structure whose resistance varies with a change in membrane potential.Institute of Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol.5, No.4, pp.432–441, July–August, 1973.     

Structure-activity relationship in the series of muscarinic acetylcholine receptor agonists: three types of agonist-receptor complexes

A new procedure has been applied to analyze the data available in literature on the structure-activity relationships for agonists of the muscarinic acetylcholine receptor. The agonists were subdivided into three groups, each containing the compounds with the specific structural element. The binding constant logarithms, characterizing the agonist association with the receptor, increase linearly with the increase in the partition coefficient logarithms describing the distribution in the water-octanol system. The performed analysis suggests the existence of three different types of agonist-receptor complexes.     

Spatial approximation between the amino terminus of a peptide agonist and the top of the sixth transmembrane segment of the secretin receptor

Distinct spatial approximations between residues within the secretin pharmacophore and its receptor can provide important constraints for modeling this agonist-receptor complex. We previously used a series of probes incorporating photolabile residues into positions 6, 12, 13, 14, 18, 22, and 26 of the 27-residue peptide and demonstrated that each covalently labeled a site within the receptor amino terminus. Although supporting a critical role of this domain for ligand binding, it does not explain the molecular mechanism of receptor activation. Here, we developed probes having photolabile residues at the amino terminus of secretin to explore possible approximations with a different receptor domain. The first probe incorporated a photolabile p-benzoyl-l-phenylalanine into the position of His(1) of rat secretin ([Bpa(1),Tyr(10)]secretin-27). Because His(1) is critical for function, we also positioned a photolabile Bpa as an amino-terminal extension, in positions -1 (rat [Bpa(-1),Tyr(10)]secretin-27) and -2 (rat [Bpa(-2),Gly(-1),Tyr(10)]secretin-27). Each analog was shown to be a full agonist, stimulating cAMP accumulation in receptor-bearing Chinese hamster ovary-SecR cells in a concentration-dependent manner, with the position -2 probe being most potent. They bound specifically and saturably, although the position 1 analog had lowest affinity, and all were able to label the receptor efficiently. Sequential specific cleavage, purification, and sequencing demonstrated that the sites of covalent attachment for each probe were high within the sixth transmembrane segment. This suggests that secretin binding may exert tension between the receptor amino terminus and the transmembrane domain to elicit a conformational change effecting receptor activation.     

Response of acetylcholine receptors to photoisomerizations of bound agonist molecules.

In these experiments, agonist-induced conductance is measured while a sudden perturbation is produced at the agonist-receptor binding site. A voltage-clamped Electrophorus electroplaque is exposed to trans-Bis-Q, a potent agonist. Some channels are open; these receptors have bound agonist molecules. A light flash isomerizes 3(-35)% of the trans-Bis-Q molecules to their cis form, a far poorer agonist. This causes a rapid decrease of membrane conductance (phase 1), followed by a slower increase (phase 2). Phase 1 has the amplitude and wavelength dependence expected if the channel closes within 100 mus after a single bound trans-Bis-Q is isomerized, and if the photochemistry of bound Bis-Q resembles that in solution. Therefore, the receptor channel responds rapidly, and with a hundred-fold greater closing rate, after this change in the structure of a bound ligand. Phase 2 (the conductance increase) seems to represent the relaxation back toward equilibrium after phase 1, because (a) phase 2 has the same time constant (1(-5) ms) as a voltage- or concentration-jump relaxation under identical conditions; and (b) phase 2 is smaller if the flash has led to a net decrease in (trans-Bis-Q). Still slower signals follow: phase 3, a decrease of conductance (time constant 5(-10 ms); and phase 4, an equal and opposite increase (several seconds). Phase 3 is abolished by curare and does not depend on the history of the membrane voltage. We consider several mechanisms for phases 3 and 4.     

Calcium dependence of acetylcholine induced conductance changes.

The influence of external calcium variation (0.7–28 mM) on acetylcholine (ACh) induced conductance changes was examined under voltage clamp conditions in snail neurons in which ACh elicitis hyperpolarizing responses and in which the main current-carrying ion species is Cl^?. Raising external calcium to 28 mM as well as lowering external calcium to 1.75 mM decreased the ACh-induced condutance change without altering the reversal potential for ACh-induced currents. Lowering external calcium to 0.7 mM increased the ACh-induced conductance change and shifted the reversal potential to less negative values. The results at 28 mM calcium can be best explained on the assumption that excess calcium interferes with the interaction between ACh and the receptor. The results at 1.75 mM calcium can be satisfactorily explained in terms of the concept that fixed membrane charges play a role in regulating Cl^? permeation through transmitter sensitive membranes. Evidence was also obtained that in snail neurons receptor inactivation increases when external calcium is reduced.     

Vessel-diameter distribution in roots of grapevines (Vitis vinifera L. cv. Shiraz)

The distribution frequency patterns of diameter of xylem vessels and percentage of total predicted axial conductances were studied in 190-day and 212-day-old main roots of grapevine (Vitis vinifera L. cv. Shiraz) grown under well-watered and stressed conditions. The protoxylem were the first to mature and were responsible for most of the theoretical conductance in root segments between the tip and 2.5 cm from the tip. Some large xylem vessels retained cross walls and protoplasm up to 22.5 cm from the tip. Statistical tests using the Kolmogorov-Smirnov two sample test showed that the pattern of distribution frequency of xylem vessels classified in different diameter classes varied with distance from the root tip. The distribution frequency of xylem vessels was similar in both well-watered and stressed plants from the tip up to 15 cm from the tip. At distances further from the tip the distribution frequency of xylem vessels of well-watered plants was significantly different from that of stressed plants, with the former having more larger vessels than the latter. The pattern of vessel distribution frequency was different from that of percent total axial conductance (K_h) predicted with fewer large vessels carrying most of the axial flow.     

The Receptor Potential of the Taste Cell of the Rat

The electrical responses of the taste cell of the rat to chemical stimuli were studied by means of microelectrode techniques. Although large positive potential changes in the taste cell were usually elicited by taste stimuli, the response was a small negative potential change with respect to surrounding tissues if the microelectrode was thrust deeply into the taste bud. Both FeCl₃ and cocaine produced a positive change in the steady potential. If this new potential is larger than a certain equilibrium potential, reversal of the polarity of the potential change caused by a taste stimulus is observed. Gamma-aminobutyric acid and acetylcholine had no effect on the receptor steady potential nor on the receptor responses elicited by taste stimuli.     

Morphology and electrophysiology of water receptors on legs of the cricket Gryllus bimaculatus

To identify the sensory organs that are sensitive to water stimuli in the cricket Gryllus bimaculatus, cuticular structures on the legs and the number of sensory neurons innervating them were studied. Some small hair sensilla on the legs were innervated by 2-5 sensory neurons. All such sensilla had a tiny pore at the tip of their hairs. The diameter of the pore was approximately 0.2 mum. These findings suggest that these are chemosensitive hairs (LCS: leg chemosensillum). Of the three pairs of legs, the anterior legs (forelegs) possessed the largest number of LCSs. Of the five leg segments (i.e., coxa, trochanter, femur, tibia and tarsus), the tarsus possessed the largest number of LCSs on each leg. Electrophysiological investigation by tip recording revealed that some of the LCSs contained water-receptor cells. Because the basitarsus possessed a larger number of LCSs than the other tarsomeres, the distribution of water-receptor-containing LCSs in the basitarsus of a foreleg was investigated morphologically and electrophysiologically. LCSs that contained water-receptor cells were mainly distributed on the ventral surface of the basitarsus. There were two types of water receptor that showed different response patterns to a stimulus, that is, phasic- and tonic-type water receptors. From the distribution of LCSs on the legs, the roles of these different types of water receptors in behavioral selection, that is, the initiation of swimming and the inhibition of flying, will be discussed.     

Membrane permeability and receptor function.

This paper presents a model of receptor function for receptors that mediate selective increases in ionic permeabilities (e.g. receptors that are sensitive to acetylcholine and gamma-aminobutyric acid). The model distinguishes itself from previous models by the qualitative and quantitative application of current biophysical approaches to ion permeation. Explicit consideration of receptor-controlled permeation sites and membrane permeability results in a specific and testable theory into which complex receptor phenomena can easily be incorporated.Examination of agonist concentration-conductance data from a well-studied GABAnergic receptor allows invalidation of a standard assumption for determining kinetic constants of agonist-receptor interactions. Specifically, calculations are presented which indicate that the size of an individual receptor (elementary) conductance event can vary with agonist concentration. The following section develops the expressions relating agonist concentration to total membrane conductance for possible receptor models which incorporate variations in the amplitude of elementary conductance events.In the case of desensitization, the “inactivated” receptor is suggested to result from occupation and inactivation, not at the agonist-binding (activation) site of the receptor, but at the ionophore. The ionophore occupation model provides a parsimonious explanation of different types of desensitization. In addition, incorporation of this model into the cyclic reaction model of Katz &; Thesleff (1957) provides increased specification for the cyclic model and results in new experimental predictions.     

MRI-based localization of electrophysiological recording sites within the cerebral cortex at single-voxel accuracy

The localization of microelectrode recording sites in the layers of primate cerebral cortex permits the analysis of relationships between recorded neuronal activities and underlying anatomical connections. We present a magnetic resonance imaging method for precise in vivo localization of cortical recording sites. In this method, the susceptibility-induced effect thickens the appearance of the microelectrode and enhances the detectability of the microelectrode tip, which usually occupies less than a few percent of the volume of an image voxel. In a phantom study, the optimized susceptibility-induced effect allowed tip detection with single-voxel accuracy (in-plane resolution, 50 mum). We applied this method to recording microelectrodes inserted into the brains of macaque monkeys, and localized the microelectrode tip at an in-plane resolution of 150 mum within the cortex of 2-3 mm in thickness. Subsequent histological analyses validated the single-voxel accuracy of the in vivo tip localization. This method opens up a way to investigate information flow during cognitive processes in the brain.     

Two neuronal nicotinic acetylcholine receptors, alpha4beta4 and alpha7, show differential agonist binding modes

Nicotinic acetylcholine receptors (nAChRs) are pentameric, neurotransmitter-gated ion channels responsible for rapid excitatory neurotransmission in the central and peripheral nervous systems, resulting in skeletal muscle tone and various cognitive effects in the brain. These complex proteins are activated by the endogenous neurotransmitter ACh as well as by nicotine and structurally related agonists. Activation and modulation of nAChRs has been implicated in the pathology of multiple neurological disorders, and as such, these proteins are established therapeutic targets. Here we use unnatural amino acid mutagenesis to examine the ligand binding mechanisms of two homologous neuronal nAChRs: the α4β4 and α7 receptors. Despite sequence identity among the residues that form the core of the agonist-binding site, we find that the α4β4 and α7 nAChRs employ different agonist-receptor binding interactions in this region. The α4β4 receptor utilizes a strong cation-π interaction to a conserved tryptophan (TrpB) of the receptor for both ACh and nicotine, and nicotine participates in a strong hydrogen bond with a backbone carbonyl contributed by TrpB. Interestingly, we find that the α7 receptor also employs a cation-π interaction for ligand recognition, but the site has moved to a different aromatic amino acid of the agonist-binding site depending on the agonist. ACh participates in a cation-π interaction with TyrA, whereas epibatidine participates in a cation-π interaction with TyrC2.     

An improved pCO2 microelectrode

By utilizing the recently developed glass-membrane pH microelectrode an improved pCO₂ microelectrode has been manufactured. The new pCO₂ microelectrode described here has been made with tip diameters ranging from 2 to 200 μm. In addition, the sensitivity (slope) was nearly theoretical, 56 to 60 mV/log pCO₂, the response time was 1–3 min, and the intercept stability (drift) was less than 3 mV/20-min time interval. Finally, the lifetime of this electrode was several days to a week when stored correctly. The most unique quality of this pCO₂ microelectrode was that the eperatienal characteristics, tip diameter, sensitivity, and response time, could be controlled by adhering to predetermined design considerations.     

A glass-membrane pH microelectrode.

By miniaturizing the original MacInnes and Dole glass-membrane pH electrode a new pH microelectrode has been developed. The technique developed utilizes the tip of a high electrical resistance glass pipet that can be sealed with a thin membrane of H⁺-sensitive glass. Single-barreled electrodes have been made with tip diameters ranging from 1.5 to 100 μm and double-barreled electrodes with tip diameters from 2 to 28 μm. The glass-membrane pH microelectrode provided a means for sensing the pH of biological solutions with an electrode having theoretical slope and tip configurational control. The most unique characteristics of the electrode were: the pH sensing surface was quite small, the tip diameter could be controlled, and the problem of electrode insulation was eliminated.     

Calcium and the tonic release of transmitter at a non-impulsive synapse in the crab

Depolarization-transmitter release coupling was studied in the promotor stretch receptor/motoneuron synapse of the crab. Callinectes sapidus, a preparation in which presynaptic action potentials do not occur. Intracellular microelectrode recordings were made from the presynaptic terminal and from the somata of postsynaptic motoneurons while injecting current pulses into the peripheral stretch receptor dendrite with the aid of the sucrose-gap. 1. For short current pulses, the relationship between presynaptic potential and postsynaptic response was found to be similar to that demonstrated in the giant synapse of the squid stellate ganglion, indicating a common reliance on the properties of voltage-dependent calcium channels. 2. The crab synapse was found to be capable of continuous transmission in the range of seconds and minutes without the pronounced depletion of transmitter seen in the squid, and without inactivation of the release process (i.e., the calcium conductance is non-inactivating). 3. A graded, transient response to depolarising current in the presynaptic fibre was found to be calcium-dependent, and probably to reflect the presence of a separate, inactivating calcium conductance. 4. It was concluded that the graded response of the presynaptic membrane could function in helping to compensate for capacitative distortion of receptor potentials decrementally conducted in the sensory dendrite, and was therefore a specialisation for non-impulsive transmission.