Factors affecting the cholinomimetic activity of alkyltrimethylammonium compounds in experiments on isolated neurons of the mollusk Planorbarius corneus

It has been shown that the elementary current is independent whereas the duration of channel opening is slightly dependent on the number of methylene groups (from 1 to 9) in the molecule of alkyltrimethylammonium compounds. However, substances with more than 4 methylene groups exhibit lower cholinomimetic activity (i.e. the ability to increase the membrane current) and higher values of Q10 for the reaction with cholinoreceptor. It is suggested that lower activity of these compounds is due to a low rate of formation of a complex with cholinoreceptor because of the higher potential energy barrier.     

Cholinoreceptor sensitivity of the mollusk neuron and frog skeletal muscle to acetylcholine and tetramethylammonium analogs

Studies have been made on the sensitivity of cholinoreceptors in identified isolated neuron from the pedal ganglion of the snail Planorbarius corneus and cholinoreceptors of m. rectus abdominis of the frog Rana temporaria to drugs which differ from acetylcholine by the structure either in cationic group, methylene chain, or ester group. Snail cholinoreceptors were found to be less sensitive to changes in the structure of cationic group and more sensitive to the increase in methylene chain from 3 to 4 groups, as compared to frog cholinoreceptors. The sensitivity of both preparations to changes in ester group, as well as to tetramethylammonium was found to be practically the same. Therefore, the sensitivity of neuronal cholinoreceptors in the snail to the effect of acetylcholine and tetramethylammonium analogues does not significantly differ from the sensitivity of cholinoreceptors in the abdominal muscle of the frog.     

The reaction of the N-cholinoreceptors of the isolated neuron of the mollusk Planorbarius corneus to polymethylene-bis-trimethylammonium compounds

Experiments have been made on isolated giant neurones of the mollusc Planorbarius corneus using clamp technique at temperatures 10 and 20 degrees C. The effect of polymethylene-bis-trimethylammonium compounds with 7-18 methylene groups in the molecule (C7...C18) on N-cholinoreceptors with chloride ionic channels was investigated. All these drugs were found to be agonists. Their cholinomimetic activity depends on the number of methylene groups (up to a certain extent) in their structure. This finding stands true also for skeletal muscles of frog and chick, as it had been shown in our earlier experiments. Analysis of membrane current fluctuations showed that the elementary current, the channel opened time, temperature coefficient (Q10) of the neuronal response to application of an agonist and the calculated Q10 of the reaction rate of the agonist with cholinoreceptor did not significantly differ for C8...C18 from the reaction rate of the agonist with cholinoreceptor. As compared with C8, C12...C18 exhibited 30 ... 40 times higher cholinomimetic activity, all other parameters in them being similar. Presumably, this difference is explained by concentrating capacity of C12...C18 at the membrane site because of their higher hydrophobic properties.     

Sodium-calcium exchange in transverse tubules isolated from frog skeletal muscle

Transverse tubule vesicles isolated from frog skeletal muscle display sodium-calcium exchange activity, which was characterized measuring 45Ca influx in vesicles incubated with sodium. The initial rates of exchange varied as a function of the membrane diffusion potentials imposed across the membrane vesicles, increasing with positive intravesicular potentials according to an electrogenic exchange with a stoichiometry greater than 2 sodium ions per calcium ion transported. The exchange activity was a saturable function of extravesicular free calcium, with an apparent K0.5 value of 3 microM and maximal rates of exchange ranging from 3 to 5 nmol/mg protein per 5 s. The exchange rate increased when intravesicular sodium concentration was increased; saturation was approached when vesicles were incubated with concentrations of 160 mM sodium. The isolated transverse tubule vesicles, which are sealed with the cytoplasmic side out, had a luminal content of 112 +/- 39 nmol calcium per mg protein. In the absence of sodium, the exchanger carried out electroneutral calcium-calcium exchange, which was stimulated by increasing potassium concentrations in the intravesicular side. Calcium-calcium exchange showed an extravesicular calcium dependence similar to the calcium dependence of the sodium-calcium exchange, with an apparent K0.5 of 6 microM. Sodium-calcium and calcium-calcium exchange were both inhibited by amiloride. The sodium-calcium exchange system operated both in the forward and in the reverse mode; sodium, as well as calcium, induced calcium efflux from 45Ca-loaded vesicles. This system may play an important role in decreasing the intracellular calcium concentration in skeletal muscle following electrical stimulation.     

Effects of previous activity on the energetics of activation in frog skeletal muscle

Effects of previous activity on the ability of frog skeletal muscle at 0 degrees C to liberate energy associated with contractile activation, i.e., activation heat (AH), have been examined. Earlier work suggests that activation heat amplitude (as measured from muscles stretched to lengths where active force development is nearly abolished) is related to the amount of Ca2+ released upon stimulation. After a twitch, greater than 2 s is required before a second stimulus (AHt) can liberate the same activation heat as a first stimulus (AH infinity), i.e., (AHt)/(AH infinity) = 1 -0.83 e-1.40t, where t is time in seconds. Caffeine introduces a time delay in the recovery of the ability to generate activation heat after a twitch. After a tetanus, the activation heat is depressed to a greater extent at any time than after a twitch. The activation heat elicited by a stimulus 1 s after a tetanus is depressed progressively with respect to tetanus duration up to 3 s. For tetani of 3, 40, and 80 s duration the postetanus activation heat is comparably depressed. The time-course of the recovery of the ability of the muscle to produce activation heat after a tetanus can be described as (AHt)/(AH infinity) = 1 -0.80 e-0.95t - 0.20 e-0.02t. Greater than 90 s is required before the posttetanus activation heat is equal to the pretetanus value. The faster phase of recovery is similar to recovery after the twitch and the slower phase may be associated with the return of calcium to the terminal cisternae from uptake sites in the longitudinal sarcoplasmic reticulum.     

Differential effects of temperature on contractile behavior in isolated frog skeletal muscle.

1. The thermal dependence of contractile behavior at different stimulation frequencies was investigated in isolated frog sartorius muscles. 2. Increasing incubation temperature (10-30 degrees C) produced decreases in Pt (43.7%) and P15 (70.3%), and an increase in Po (26.0%). 3. Thermal ratios (R10) calculated for Pt, P15 and Po indicated high thermal dependence at lower temperatures (10-20 degrees C; 0.60, 0.44 and 1.38, respectively) but relative thermal independence at higher temperatures (20-30 degrees C; 0.95, 0.75 and 0.95, respectively). 4. Contractile ratios (Pt/Po and P15/Po) decreased with increased temperature (10-30 degrees C; 56.3% and 76.0%, respectively). 5. Thermal ratios (R20) calculated for peak tension at different stimulation frequencies demonstrated high thermal dependence at the lower frequencies (10-30 pps, 0.46-0.48) and decreasing dependence at higher frequencies (40-50 pps, 0.69-0.82).     

Inositol 1,4,5-trisphosphate 3-kinase activity in frog skeletal muscle

Frog skeletal muscle contains a kinase activity that phosphorylates inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate. The inositol 1,4,5-trisphosphate 3-kinase activity was mainly recovered in the soluble fraction, where it presented a marked dependency on free calcium concentration in the physiological range in the presence of endogenous calmodulin. At pCa 5, where the activity was highest, the soluble 3-kinase activity displayed a K_m for inositol 1,4,5-trisphosphate of 1.6 μM and a V_max value of 25.1 pmol mg⁻¹ min⁻¹. The removal rates of inositol 1,4,5-trisphosphate by 3-kinase and 5-phosphatase activities of the total homogenate under physiological ionic conditions were very similar, suggesting that both routes are equally important in metabolizing inositol 1,4,5-trisphosphate in frog skeletal muscle.     

Effect of ouabain on electrical activity of the isolated frog muscle spindle

Changes in electrical activity of the isolated frog muscle spindle were studied in Ringer's solution containing ouabain. The presence of ouabain in the solution increased the spontaneous firing rate of the receptors up to a maximum and then reduced it quickly to zero. The amplitude of the action potentials was reduced on the average to 40% of normal. Ouabain causes initial disappearance of the hyperpolarization phase of the receptor potential and a subsequent decrease in amplitude of its dynamic phase to zero. The decrease in amplitude of the receptor potential and action potential and also the changes in firing rate in the solution with ouabain depend on the frequency of their spontaneous activity. The changes observed can be explained by depolarization of the membrane of the nerve endings and the first node of Ranvier, developing as a result of blocking of the sodium pump by ouabain.Translated from Neirofiziologiya, Vol. 5, No. 6, pp. 576–582, November–December, 1973.     

Enzymes with phosphoglycolate phosphatase activity in chicken skeletal muscle and liver

1. Four enzymes with phosphoglycolate phosphatase (EC 3.1.3.18) activity have been detected in extracts of chicken skeletal muscle and liver analyzed by gel-filtration and ion-exchange chromatography. 2. Two enzymes have been found in muscle extracts. One of them acts on glycerate 2,3-P2, in addition to glycolate 2-P. 3. Liver extracts contain two additional enzymes with broad specificity.     

Uptake and metabolism of purine nucleosides and nucleotides in isolated frog skeletal muscle.

When isolated frog skeletal muscles were incubated with ¹⁴C-labeled adenosine, the nucleoside was rapidly taken up by the cells and was either immediately incorporated into adenine nucleotides or deaminated to inosine. Incorporation was predominant at low (micromolar) concentrations whereas, deamination was the major route of metabolism at high (millimolar) concentrations. When muscles were incubated with ¹⁴C-labeled inosine the nucleoside, after entry into the cells, was metabolized to a lesser extent than adenosine. ATP and hypoxanthine were the major products of its metabolism. Intracellular concentrations were calculated using ³H-labeled sorbitol to measure the extracellular space.Because of its lower rate of intracellular metabolism inosine was used to investigate the characteristics of the nucleoside transport system. The uptake of inosine was saturable at high concentrations and was specifically inhibited by the presence of adenosine or uridine in the incubation media. Persantin, a well known specific inhibitor of nucleoside transport, also competitively inhibited inosine uptake, as did theophylline [1, Woo et al. Can J. Physiol. Pharmacol. $\text{52}$, 1063, 1974]. These data, along with the knowledge that in a well-oxygenated muscle, inosine entry follows a downhill chemical potential gradient, strongly support the view that the transport mechanism is facilitated diffusion.The muscle cell membrane does not appear to be permeable to ¹⁴C-labeled ATP under the conditions studied. Investigations of the permeability to the major extracellular degradation products of ATP suggest that AMP was the compound most likely to cross the cell membrane.     

Sarcomere lengthening and tension drop in the latent period of isolated frog skeletal muscle fibers

A laser diffraction technique has been developed for registering small changes in sarcomere length. The technique is capable of resolving changes as small as 0.2 A in isolated frog skeletal muscle fibers. The small sarcomere lengthening that accompanies the drop in tension in the latent period of contraction was investigated. We suggest this lengthening be named latency elongation (LE). The LE is present in a completely slack fiber and must, therefore, be caused by a forcible lengthening process. Furthermore, the LE is dependent on the existence of an overlap between thin and tick filaments. The rate of elongation and the time interval between stimulation and maximum elongation may vary along the fiber. The maximum elongation was 3-5 A per sarcomere. At any instant the drop in tension is a product of the sum of sarcomere lengthenings along the fiber and the slope stiffness of the series elasticity. The latency relaxation (LR) could be registered in the sarcomere length range from 2.2 mum to 3.6-3.7 mum. The amplitude went through a sharp maximum at 3.0-3.1 mum. In the sarcomere length range from 2.2 to 2.8 mum the delay from onset to maximum LR was nearly proportional to the distance from the Z-line to the overlap zone. A working hypothesis is presented. It is suggested that the LE is caused by a lengthening of the thin filaments.     

Respiration and lactate production in isolated frog skeletal muscle: effects of passive stretch

The effects of varying degrees of passive stretch on in vitro oxygen consumption and intracellular lactate efflux have been investigated in paired recti abdomini muscles from small male frogs. Oxygen consumption [mm3 (STP)/mg (dry wt)/hr] was found to be linearly related to load (r = 0.98), increasing from 1.57 +/- 0.11 (SE) at 2 g to 2.30 +/- 0.18 at 10 g, 2.89 +/- 0.16 at 20 g and 3.26 +/- 0.21 at 30 g. Lactate released into the medium [microM/g (dry wt)/hr] was inversely related to load (r = -0.52), increasing initially from 36.84 +/- 3.28 (SE) at 2 g to 108.55 +/- 12.9 at 10 g, then abruptly decreasing with additional loading (18.10 +/- 2.60 at 20 g and 11.71 +/- 2.80 at 30 g). Results suggest that as stretch-related oxidative energy metabolism increases, there is a lessening dependence on anaerobic energy-yielding processes.     

Effect of tetraethylammonium ions on electrical activity of the isolated frog muscle spindle

Changes in electrical activity of the isolated frog muscle spindle in Ringer's solution containing tetraethylammonium (TEA) ions were studied. An increase in the frequency of spontaneour activity was observed, but with continued perfusion with TEA solution both spontaneous afferent impulses and action potentials generated during stretching of the muscle receptor were blocked. The dynamic component of the depolarization phase of the receptor potential was reduced in amplitude and increased in duration. Rinsing the receptor in normal physiological saline did not restore its responses completely.Institute of Physiology, Leningrad State University. Translated from Neirofiziologiya, Vol. 4, No. 2, pp. 208–215, March–April, 1972.