The Influence of Non Polar and Polar Molecules in Mouse Motile Cells Membranes and Pure Lipid Bilayers

We report an experimental study of mouse sperm motility that shows chief aspects characteristic of neurons: the anesthetic (produced by tetracaine) and excitatory (produced by either caffeine or calcium) effects and their antagonic action. While tetracaine inhibits sperm motility and caffeine has an excitatory action, the combination of these two substances balance the effects, producing a motility quite similar to that of control cells. We also study the effects of these agents (anesthetic and excitatory) on the melting points of pure lipid liposomes constituted by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and dipalmitoyl phosphatidic acid (DPPA). Tetracaine induces a large fluidization of the membrane, shifting the liposomes melting transition temperature to much lower values. The effect of caffeine is null, but its addition to tetracaine-doped liposomes greatly screen the fluidization effect. A high calcium concentration stiffens pure lipid membranes and strongly reduces the effect of tetracaine. Molecular Dynamics Simulations are performed to further understand our experimental findings at the molecular level. We find a strong correlation between the effect of antagonic molecules that could explain how the mechanical properties suitable for normal cell functioning are affected and recovered.     

Electrophysiological study of effects of chronic injection of caffeine on defensive reflex conditioning in grape snail

It was found that chronic injection of caffeine to grape snail increases a velocity of elaboration of conditioned defensive reflex. It was shown that after daily injection of caffeine immediately after procedure of learning the conditioned defensive reflex elaborated faster than daily injection before procedure of learning. It has been shown, that chronic injection of caffeine both in naive as well as learned snails led to depolarizing shift of membrane potential and to decrease of threshold potential of command neurons of the defensive behaviour of grape snails. It was also found that addition of caffeine in bath solution led to decrease of threshold of generation of action potential of command neurons both in intact and learned snails. The resting membrane potential of command neurons was not changed.     

A sodium leak current regulates pacemaker activity of adult central pattern generator neurons in Lymnaea stagnalis

The resting membrane potential of the pacemaker neurons is one of the essential mechanisms underlying rhythm generation. In this study, we described the biophysical properties of an uncharacterized channel (U-type channel) and investigated the role of the channel in the rhythmic activity of a respiratory pacemaker neuron and the respiratory behaviour in adult freshwater snail Lymnaea stagnalis. Our results show that the channel conducts an inward leak current carried by Na(+) (I(Leak-Na)). The I(Leak-Na) contributed to the resting membrane potential and was required for maintaining rhythmic action potential bursting activity of the identified pacemaker RPeD1 neurons. Partial knockdown of the U-type channel suppressed the aerial respiratory behaviour of the adult snail in vivo. These findings identified the Na(+) leak conductance via the U-type channel, likely a NALCN-like channel, as one of the fundamental mechanisms regulating rhythm activity of pacemaker neurons and respiratory behaviour in adult animals.     

The role of cAMP in generating excitatory serotonin responses of neurons in the edible snail

The neurons of the dorsal surface of snail Helix subesophageal ganglia respond similarly to the application of serotonin and the intracellular cAMP injection. These responses represent membrane depolarization. They increase in amplitude with membrane hyperpolarization and have a reverse potential between +10 and -30 mV. Presumably, these responses are associated with increased conductance for several ions. The values of the reverse potentials of serotonin and cAMP responses coincide in 7 out of 17 cells. Phosphodiesterase inhibitor theophylline caused a reversible increase in the amplitude and duration of both serotonin and cAMP responses and, used at a concentration of 1 mM, simulated them. The results obtained meet 2 out of 4 criteria demonstrating that cyclic nucleotides mediate a neurotransmitter response. It is suggested that cAMP may act as a second messenger in excitatory serotonin responses of snail Helix neurons.     

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.     

Comparative study of the effects of antibodies to S-100 protein and ouabain on neurons of Helix pomatia

Ouabain, used in 5.10(-4) M concentration, elicits 12 +/- 5 mV (15 experiments) depolarization of membrane of snail Helix neurons. In 80% of experiments depolarization is not accompanied by changes in membrane conductance, in 20% of experiments the decrease of the membrane conductance is observed. Application of the antibodies to S-100 protein (their concentration in the micropipette being 0.05 mg/ml) induces similar effects. The effects of ouabain and antibodies to S-100 protein are not additive and the main difference in their action lies in the ability of the cell to recover the resting potential of the membrane in the solution containing ouabain.     

Different localization of inositol 1,4,5-trisphosphate and ryanodine binding sites in rat liver.

The distribution of inositol 1,4,5-trisphosphate and ryanodine binding sites between plasma membrane, microsomal, and mitochondrial fractions of rat liver were compared. IP3 bound mostly to the plasma membrane fraction (Kd = 6 nM; Bmax = 802 fmol/mg protein). Some IP3 binding sites were also present in the microsomal and mitochondrial fractions (Kd = 2.5 and 2.9 nM; Bmax = 35 and 23 fmol/mg protein respectively). The possibility that these binding sites are due to contamination of the fractions with plasma membrane cannot be excluded. Binding of IP3 to the plasma membrane was inhibited by heparin but not by either caffeine or tetracaine. High-affinity ryanodine binding sites were present mostly in the microsomal fraction (Kd = 13 nM; Bmax = 301 fmol/mg protein). Lower affinity binding sites were also found to be present in the mitochondrial and plasma membrane fractions. Binding of ryanodine to the microsomal fraction was inhibited by both caffeine and tetracaine but not by heparin. These data demonstrate that IP3 and ryanodine binding sites are present in different cellular compartments in the liver. These differences in the localization of the binding sites might be indicative of their functional differences.     

Calcium-activated potassium conductance noise in snail neurons

Current fluctuations were measured in small, 3-6 micrometers-diameter patches of soma membrane in bursting neurons of the snail, Helix pomatia. The fluctuations dramatically increased in magnitude with depolarization of the membrane potential under voltage clamp conditions. Two components of conductance noise were identified in the power spectra calculated from the membrane currents. One component had a corner frequency which increased with depolarization. This component was blocked by intracellular injection of TEA and was relatively insensitive to extracellular calcium levels (as long as the total number of effective divalent cations remained constant). It was identified as fluctuations of the voltage-dependent component of delayed outward current. The second component of conductance noise had a corner frequency which decreased with depolarization. It was relatively unaffected by TEA injection and was reversibly blocked by substitution of extracellular calcium with magnesium, cobalt, or nickel. This second component of noise was identified as fluctuations of the calcium-dependent potassium current. The results suggest that the two components of delayed outward current are conducted through physically distinct channels.     

Effects of local anesthetics on single channel behavior of skeletal muscle calcium release channel

The effects of the two local anesthetics tetracaine and procaine and a quaternary amine derivative of lidocaine, QX314, on sarcoplasmic reticulum (SR) Ca2+ release have been examined by incorporating the purified rabbit skeletal muscle Ca2+ release channel complex into planar lipid bilayers. Recordings of potassium ion currents through single channels showed that Ca(2+)- and ATP-gated channel activity was reduced by the addition of the tertiary amines tetracaine and procaine to the cis (cytoplasmic side of SR membrane) or trans (SR lumenal) side of the bilayer. Channel open probability was lowered twofold at tetracaine and procaine concentrations of approximately 150 microM and 4 mM, respectively. Hill coefficients of 2.0 and greater indicated that the two drugs inhibited channel activity by binding to two or more cooperatively interacting sites. Unitary conductance of the K(+)- conducting channel was not changed by 1 mM tetracaine in the cis and trans chambers. In contrast, cis millimolar concentrations of the quaternary amine QX314 induced a fast blocking effect at positive holding potentials without an apparent change in channel open probability. A voltage-dependent block was observed at high concentrations (millimolar) of tetracaine, procaine, and QX314 in the presence of 2 microM ryanodine which induced the formation of a long open subconductance. Vesicle-45Ca2+ ion flux measurements also indicated an inhibition of the SR Ca2+ release channel by tetracaine and procaine. These results indicate that local anesthetics bind to two or more cooperatively interacting high-affinity regulatory sites of the Ca2+ release channel in or close to the SR membrane. Voltage-dependent blockade of the channel by QX314 in the absence of ryanodine, and by QX314, procaine and tetracaine in the presence of ryanodine, indicated one low-affinity site within the conduction pathway of the channel. Our results further suggest that tetracaine and procaine may primarily inhibit excitation-contraction coupling in skeletal muscle by binding to the high-affinity, regulatory sites of the SR Ca2+ release channel.     

Selective effect of the antibody to protein SMP-69 on activity of the defence behavior command neurons in grape snail

Effects of antibody against serotonin-modulated protein SMP-69 on defence behavior command neurons L-RP11 were studied in semi-intact preparation of snail Helix lucorum. An increase in membrane excitability as well as selective facilitation of neural responses evoked with chemical sensory stimulation of the snail head (0.25-0.5% quinine solution) were determined 1-1.5 hours after antibody application to the neurons. The antibody did not change neural responses evoked with tactile stimulation of the snail head. These effects were similar to those found in L-RP11 neurons after serotonin or cAMP applications as well as after nociceptive sensitization of the snail. It was suggested that protein homologically related the SMP-69 in mammalians was involved in mechanisms of excitability as well as long-term specific plasticity regulation of L-RP11 neurons synaptic inputs from the head chemoreceptors in snail Helix lucorum.     

The ionic mechanism associated with the action of putative transmitters on identified neurons of the snail, Helix aspersa

Intracellular recordings were made from identified neurons in the suboesophageal ganglionic mass of the snail, Helix aspersa. The ionic mechanisms associated with acetylcholine excitation and inhibition, dopamine excitation and inhibition, gamma-aminobutyric acid (GABA) excitation and inhibition and serotonin excitation were investigated. Acetylcholine excitation was found to involve an initial increase in sodium conductance while acetylcholine inhibition was a pure chloride event which reversed at membrane potentials more negative than the chloride equilibrium potential. Dopamine excitation appeared to involve only an increase in sodium conductance while serotonin excitation involved an increase in conductance to both sodium and calcium ions. Dopamine inhibition was associated with an increase in potassium conductance but failed to reverse at membrane potentials more negative than the potassium equilibrium potential. GABA excitation involved conductance increases to both sodium and chloride ions while GABA inhibition was a pure chloride event. An attempt was made to estimate the degree of co-operativity of the putative transmitters with their receptors using log-log and Hill plots. The slopes of the line for the log-log plots for acetylcholine excitation and inhibition were 0.88 and 1.1, respectively, suggesting the interaction of one molecule of acetylcholine with the receptor. The slope of the log-log plot for dopamine inhibition was 0.46 while that for serotonin excitation was 0.75. The Hill plots for GABA excitation and inhibition were 1.64 and 1.42, respectively, suggesting that two molecules of GABA are required for receptor activation.     

Blockers of the uncoupling action of caffeine on the Ca2+-transport function of sarcoplasmic reticulum membranes

The influence of caffeine on the efficiency of Ca2+ transport in the presence of oxalate by different fractions of sarcoplasmic reticulum (SR) membranes from rabbit skeletal muscle was studied. It was shown that caffeine (5 mM) decreases 4-fold the value of the Ca/ATP ratio in terminal cisterns of the SR without having any appreciable influence on the efficiency of Ca2+ transport by the light fraction of the SR. The uncoupling effect of caffeine is completely blocked by ruthenium red and by the local anesthetics, tetracaine, procaine, and benzocaine.     

Effect of caffeine on the Ca2+-transport function of sarcoplasmic reticulum vesicles in the rat myocardium

The effects of caffeine on active transport of Ca2 by heavy and light fractions of rat myocardial microsomes were investigated with the use of a Ca2+-selective electrode and nephelometry. It was found that under the effect of caffeine (5 mM) the rate of Ca2 transport in the presence of oxalate decreased by 30 to 40%. The caffeine-induced inhibition was prevented by ruthenium and tetracaine, thus suggesting the inhibitor specificity. Since caffeine is a specific blocker of Ca2 transport to the terminal cisterns of the skeletal muscle sarcoplasmic reticulum, it is assumed that the microsomal fraction of rat myocardium contains terminal cistern fragments.     

Extracellular calcium and microwave enhancement of membrane conductance in snail neurons

Summary Microwave irradiation has been shown to decrease the input resistance of snail neurons. In this study, we examined the role of extracellular calcium in triggering the microwave-induced enhancement of membrane conductance. Two sets of experiments were conducted. In the first set, nerve cells were superfused using Ringer solution with added Cd²⁺ (0.9 mM) which is a known blocker of calcium channels. In the second set, cells were superfused with low Ca²⁺ (0.7 mM) Ringer solution. Microwave irradiation was conducted at 2,450 MHz for 30 min with a specific absorption rate of 13 mW/g. It was found that 7 mM to 0.7 mM lowering of Ca²⁺ in bathing solution as well as blocking of calcium channels in neuronal membrane by means of Cd²⁺ did not influence the fall in membrane resistance induced by microwave radiation. In fact, the observed changed in membrane resistance in these experiments were nearly equal to those observed for neurons superfused by normal Ringer's. Thus, these results rule out the possible contribution of external Ca²⁺ in the observed microwave effect. Experiments with high Ca²⁺ solution also support this conclusion.     

Actions of reactive oxygen species on AH/type 2 myenteric neurons in guinea pig distal colon

With conventional intracellular recording methods, we investigated the mechanism of actions of reactive oxygen species (ROS) derived from hypoxanthine and xanthine oxidase (HX/XO) reactions on AH/type 2 myenteric neurons in the guinea pig distal colon. Of the 54 neurons to which HX/XO was applied, 32 neurons showed a transient membrane hyperpolarization(s) followed by a long-lasting membrane depolarization. Two additional groups of 10 myenteric neurons exhibited only a membrane hyperpolarization(s) or a late-onset membrane depolarization, respectively, and the remaining two neurons did not show any response to HX/XO. Analysis of changes of the input resistance induced by HX/XO indicated that suppression and augmentation of the conductance of Ca(2+)-dependent K(+) channels are the ionic mechanisms underlying the membrane hyperpolarization and depolarization, respectively. The effects of HX/XO on myenteric neurons were mimicked by application of caffeine or H(2)O(2). The results suggest that OH(.), but neither H(2)O(2) nor O(2)(.-), is responsible for HX/XO-induced responses. The intracellular Ca(2+) store may be the acting site of ROS in colonic AH/type 2 neurons.     

He-Ne激光对大鼠离体颈上神经节后神经元膜电导的影响

应用细胞内生物电记录技术 ,观测不同功率、不同照射时间的 He- Ne激光 (脉冲频率 1Hz)对大鼠离体颈上神经节后神经元快兴奋性突触后电位 (f- EPSP)期间膜电导的影响。功率密度为 2 m W/ cm2 的 He- Ne激光在照射初期 (1min～ 2 min)引起快兴奋性突触后电位 (f- EPSP)幅值增大 ,同时膜电导增大 ;而在激光照射后期 (后 3m in～8m in)引起节后神经元膜电导减少。功率密度为 5 m W/ cm2 的 He- Ne激光照射期膜电导无明显变化 .结果表明 :功率密度为 2 m W/ cm2 的 He- Ne激光照射初期引起膜电导 (Gl=34.6± 5 .4 n S)较照射前 (Gf=2 6 .8± 6 .2 n S)有明显增大 (P<0 .0 5 ) ,照射后期膜电导减少。提示 :He- Ne激光照射可能是通过两时相效应改变节后神经元膜电导来影响交感神经节内兴奋传递过程。这可能是低功率激光对神经细胞的一种作用机制。     

Tetracaine stimulates insulin secretion through the mobilization of Ca2+ from thapsigargin- and IP3-insensitive Ca2+ reservoir in pancreatic beta-cells

The effect of tetracaine on 45Ca efflux, cytoplasmic Ca2+ concentration [Ca2+]i, and insulin secretion in isolated pancreatic islets and beta-cells was studied. In the absence of external Ca2+, tetracaine (0.1-2.0 mM) increased the 45Ca efflux from isolated islets in a dose-dependentOFF efflux caused by 50 mM K+ or by the association of carbachol (0.2 mM) and 50 mM K+. Tetracaine permanently increased the [Ca2+]i in isolated beta-cells in Ca2+-free medium enriched with 2.8 mM glucose and 25 microM D-600 (methoxiverapamil). This effect was also observed in the presence of 10 mM caffeine or 1 microM thapsigargin. In the presence of 16.7 mM glucose, tetracaine transiently increased the insulin secretion from islets perfused in the absence and presence of external Ca2+. These data indicate that tetracaine mobilises Ca2+ from a thapsigargin-insensitive store and stimulates insulin secretion in the absence of extracellular Ca2+. The increase in 45Ca efflux caused by high concentrations of K+ and by carbachol indicates that tetracaine did not interfere with a cation or inositol triphosphate sensitive Ca2+ pool in beta-cells.     

Mytilus inhibitory peptide (MIP) induces a Na+-activated K+-current in snail neurons

Two microelectrode voltage-clamp and single-channel recordings were performed on D-cluster neurons of snail right parietal ganglion in order to study the properties of MIP-activated potassium current. It was found that the octapeptide member of the MIP-family, ASHIPRFVa elicits an outward current, which possesses all the properties characteristic for the hexapeptide(s) inward membrane response. The main component of the peptide elicited response is highly [K+]o dependent, however the response was attenuated in Na-free extracellular saline. The peptide elicited response was mimicked by raising the [Na+]i by pressure injection of Na+ into the cell. Single channel recordings indicated that MIP-induced outward K-current is Na-dependent. The probability to find a channel in open state increases with increasing intracellular Na+-concentration. Excised inside-out patches obtained from D-neurons contained I(K(Na)) channels could be activated by exposure of the cytoplasmic face of the patch membrane to 40 mM Na+, and 40 mM Li+, as well. The single channel current amplitude at -60 mV is 15 pA and the single channel conductance is 212 pS between -80 and 0 mV. It was concluded that MIP's activate a novel type of K+-current in the snail neurons. This current is the Na-activated K+-current. The single channel properties of the MIP activated channel is in concert with I(K(Na)) data obtained on different vertebrate and invertebrate preparations.     

Activation of the caffeine center of the sarcoplasmic reticulum at a reduced concentration of magnesium ions

The action of caffeine and Mg2+ on the efficacy of Ca2+ transport by terminal cisterns and longitudinal tubules of rabbit skeletal muscle sarcoplasmic reticulum (SR) was studied and compared. Addition of 5 to 10 mM caffeine to the incubation medium or a decrease in Mg2+ concentration from 4 to 0.1 mM led to a 3-fold diminution of the Ca/ATP ratio for the terminal cistern fraction. In longitudinal tubules, that effect was far less pronounced. The effects of caffeine and decreases in Mg2+ concentration were blocked by ruthenium red, tetracaine and dimethylsulfoxide. It is assumed that the decrease in Mg2+ concentration is accompanied by activation of the caffeine site of the SR, induced by the intravesicular caffeine-like factor.     

The action of substance P on neurons of the myenteric plexus of the guinea-pig small intestine.

Extracellular and intracellular recordings were made in vitro from single neurons of the myenteric plexus of the guinea-pig small intestine. Synthetic substance P was applied to the neurons by means of the perfusing solution or by electrophoresis from micropipettes. Extracellular recording showed that substance P (100 pm-30 nm), applied by perfusion, increased the firing rate of myenteric neurons. Intracellular recording indicated that perfusion with substance P caused a dose-dependent membrane depolarization which was unaffected by hexamethonium, hyoscine, naloxone or baclofen. The depolarization was also evoked by electrophoretic application of substance P. It was associated with an increase in membrane resistance, augmented by membrane depolarization and reduced by membrane hyperpolarization. The relation between the substance P reversal potential and the logarithm of the extracellular potassium concentration was linear with a slope of 54 mV/log10[K+], which indicates that substance P inactivates the resting potassium conductance of the myenteric neurons. This effect on ion conductance is the same as that of an unknown substance that mediates slow synaptic excitations with the myenteric plexus.