bluuebunny/crossref_metadata_2025 huggingface dataset:
A23187 increases calcium permeability of store sites more than of surface membranes in the rabbit mesenteric artery.
Authors: Itoh T , Kanmura Y , Kuriyama H
Published: December 2014

The effects of a Ca ionophore, A23187, were investigated on intact and skinned smooth muscle tissues of the rabbit mesenteric artery. A23187 (over 10(‐9) M) inhibited, dose dependently, contractions induced by 10(‐5) M‐noradrenaline (NA) or 10 mM‐caffeine in Ca2+‐free solution containing 2 mM‐EGTA. Procaine (3 mM) led to cessation of the caffeine‐ or NA‐induced contractions in the presence or absence of Ca2+. When A23187 (10(‐7) M) was applied, the contractions in the presence of procaine were to some extent restored in Krebs solution. A23187 at a concentration of 10(‐7) M did not modify the resting muscle tone, but this concentration did increase the amplitude of the contraction evoked by 20.2 or 128 mM‐K+ and markedly inhibited the 10(‐7) M‐NA or 10 mM‐caffeine‐induced contraction in Krebs solution. A23187 (10(‐7) M) delayed the onset and rising phase of the 10(‐5) M‐NA‐induced contraction with inhibition of the oscillatory contractions. High concentrations of A23187 (over 10(‐6) M) produced a large contraction in the presence and a small contraction in the absence of 2.6 mM‐Ca2+. These A23187‐induced contractions were not inhibited by 10(‐7) M‐nisoldipine, a Ca2+ antagonist. A23187 (over 10(‐6) M) applied for a long period functionally skinned the muscle tissues. However, the Ca2+ sensitivity of the A23187‐treated skinned muscles was lower than that of saponin‐treated muscles. In saponin‐treated skinned muscles, A23187 (below 10(‐6) M) had no effect on the pCa‐tension relation. After filling the store, A23187 (over 10(‐7) M) generated a larger contraction than did caffeine in Ca2+‐free solution, in the presence or absence of 5 mM‐NaN3. When 10(‐7) M‐A23187 was applied once for 5 min, subsequently applied caffeine (20 mM), following application of Ca2+, no longer produced contraction of skinned muscle tissues. The present results indicate that low concentrations of A23187 show a selective Ca2+‐releasing action on Ca2+ store sites in muscle cells and that high concentrations increase the Ca2+ leakage (influx) and the cell membrane is skinned.
The slow repolarization phase of the action potential in rat heart.
Authors: Schouten V J , ter Keurs H E
Published: December 2014

Intracellular action potentials and isometric force were measured from thin trabeculae of the right ventricle of rat heart. Characteristic for the action potential of rat myocardium is a short plateau and a slow final repolarization phase. We have studied the influence of ionic composition of the medium and of stimulation frequency on the slow phase of repolarization and its relation to peak force. The results confirmed a positive correlation between peak force and the duration of the slow phase of repolarization, as has been reported for other species. An increase of [Ca2+]o caused a shortening of the slow phase of repolarization when peak force was kept constant. In low [Na+]o peak force was increased and the slow phase of repolarization was shortened. Reperfusion with normal medium after a period in low [Na+]o induced a transient prolongation of the slow phase of repolarization and reduction of peak force. The transient lasted about 20 min. In the presence of the Ca2+ entry blocker nifedipine the action potential duration and peak force were reduced. Low [Na+]o caused less shortening of the slow phase of repolarization and a greater increase of peak force. The slow phase of repolarization was prolonged transiently following reperfusion at normal [Na+]o, but only during a few beats. These results are in agreement with the hypothesis that the slow phase of repolarization is due to an inward current generated by Na+‐Ca2+ exchange, as latter mechanism is known to be sensitive to the intracellular and extracellular concentrations of both Na+ and Ca2+.
Sodium‐potassium pump in guinea‐pig parotid gland: secretagogue stimulation of ouabain binding to dispersed acini.
Authors: Hootman S R , Williams J A
Published: December 2014

Dispersed acini were prepared from guinea‐pig parotid glands and incubated at 37 degrees C in HEPES‐buffered Ringer (HR) containing [3H]ouabain. Acini bound the Na+‐K+‐pump inhibitor with an estimated equilibrium dissociation constant (Kd) of 2.05 microM and a capacity of 2.9 X 10(6) ouabain‐binding sites/cell. Carbachol and adrenaline each increased the equilibrium level of binding attained at a medium [3H]ouabain concentration of 0.1 microM by up to 250%, but had no effect on binding at a medium ouabain concentration of 10 microM, a near‐saturating level of the glycoside. These results indicate that the two secretagogues elicit increases in Na+‐K+‐pump activity without increasing the number of pump sites available. The observed stimulation by carbachol was readily reversed by atropine and that evoked by adrenaline was reversed by phentolamine. Incubation of acini in Ca2+‐free HR with 0.2 mM‐EGTA did not alter [3H]ouabain binding in the absence of agonists, but decreased by over 80% the response to both carbachol and adrenaline. The full response to either could be restored by adding 1.5 mM‐Ca2+ to the Ca2+‐free medium after challenge with agonists. The response to a maximally effective dose of carbachol could not be augmented by adrenaline or vice versa in either the presence or absence of extracellular Ca2+. Carbachol and adrenaline also stimulated the ouabain‐sensitive component of acinar oxygen uptake by 230‐260%, but had no significant effect on ouabain‐insensitive respiration. In the absence of extracellular Ca2+, stimulation of oxygen uptake by either agonist was reduced by over 80% and the stimulatory effects disappeared within 5 min. We conclude that alterations in equilibrium level of ouabain bound at 0.1 microM‐[3H]ouabain and in ouabain‐sensitive oxygen uptake by guinea‐pig parotid acini both reflect changes in acinar Na+‐K+‐pump activity. Muscarinic cholinergic and alpha‐adrenergic receptor occupancy strongly stimulates pump activity in a Ca2+‐dependent manner. Na+‐K+ pumps are the primary energy‐requiring ion‐transport component that is activated by these secretagogues.
Comparison of the action of baclofen with gamma‐aminobutyric acid on rat hippocampal pyramidal cells in vitro.
Authors: Newberry N R , Nicoll R A
Published: December 2014

Intracellular recordings from CA1 pyramidal cells in the hippocampal slice preparation were used to compare the action of baclofen, a gamma‐aminobutyric acid (GABA) analogue, with GABA. Ionophoretic application of GABA or baclofen into stratum (s.) pyramidale evoked hyperpolarizations associated with reductions in the input resistance of the cell. Baclofen responses were easier to elicit in the dendrites than in the cell body layer. Blockade of synaptic transmission, with tetrodotoxin or cadmium, did not reduce baclofen responses, indicating a direct post‐synaptic action. (+)‐Bicuculline (10 microM) and bicuculline methiodide (100 microM) had little effect on baclofen responses but strongly antagonized somatic GABA responses of equal amplitude. The bicuculline resistance of the baclofen response was not absolute, as higher concentrations of these compounds did reduce it. Pentobarbitone (100 microM) enhanced somatic GABA responses without affecting baclofen responses. (‐)‐Baclofen was approximately 200 times more potent than (+)‐baclofen. The reversal potentials for the somatic GABA and baclofen responses were ‐70 mV and ‐85 mV respectively. When the membrane was depolarized, the baclofen response was reduced. This apparent voltage sensitivity was not seen with somatic GABA responses. Altering the chloride gradient across the cell membrane altered the reversal potential of the somatic GABA response but not that of the baclofen response. It was extrapolated that a tenfold shift in the extracellular potassium concentration would cause a 48 mV shift in the reversal potential of the baclofen response. Barium ions reduced the baclofen response, but not the GABA response. Orthodromic stimulation produced a fast inhibitory post‐synaptic potential (i.p.s.p.) and a slow i.p.s.p. The properties of the fast and slow i.p.s.p.s were remarkably similar to those of the somatic GABA and baclofen responses, respectively. Application of GABA to the pyramidal cell dendrites evoked, in addition to a depolarization, two types of hyperpolarization. One type of hyperpolarization was bicuculline sensitive, had a reversal potential of about ‐65 mV and appeared to be chloride dependent. The other hyperpolarization was more easily observed in bicuculline methiodide (100 microM). This response was similar to that evoked by baclofen since it had a high reversal potential (about ‐90 mV), was relatively insensitive to changes in the chloride gradient across the cell membrane and was reduced by barium. The bicuculline‐sensitive hyperpolarization could be evoked by the dendritic or somatic ionophoresis of muscimol and THIP (4,5,6,7‐tetrahydroisoxazolo‐[5,4‐c]pyridin‐3(2H)‐one.(ABSTRACT TRUNCATED AT 400 WORDS)
Effects of calcium and substrate on force‐velocity relation and energy turnover in skinned smooth muscle of the guinea‐pig.
Authors: Arner A , Hellstrand P
Published: December 2014

Mechanical properties and rate of ATP breakdown (JATP) have been determined in the chemically skinned guinea‐pig taenia coli at 22 degrees C. The influence of varied [Ca2+], [Mg ATP] and muscle length were investigated. The shortening response after a step decrease in force (isotonic quick release) was highly curvilinear in the first 100‐200 ms. This effect was shown to be a time‐dependent response to the force step and not primarily caused by the shift along the length‐force relation associated with shortening. Maximal shortening velocity (Vmax) decreased gradually following the release. At pCa (= ‐log [Ca2+]) 4.5, Vmax at 20 and 1000 ms after release was 0.49 +/‐ 0.07 and 0.041 +/‐ 0.004 (mean +/‐ S.E. of mean, n = 5) lengths s‐1 respectively. Unloaded shortening velocity obtained from length steps of different magnitude (slack test) also showed a gradual decrease after the release, consistent with the isotonic release results. Increasing [Ca2+] from the relaxed state at pCa 9 (1 microM‐calmodulin present) gave increased isometric force to a maximum at pCa 4.5. Half‐maximal response was obtained at pCa 6.1. JATP at maximal force at pCa 4.5 was about 3 times the basal rate at pCa 9. The relation between JATP and force was highly non‐linear, with a marked increase in JATP with little alteration in force at the highest [Ca2+]. When force was reduced to zero at pCa 4.5 by shortening the muscle to 0.3 L0 (L0 being the length giving maximal active force), JATP decreased by about 30%. At two levels of [Ca2+] giving similar force (pCa 5.75 and 4.5) the energetic tension cost obtained by length variations was lower at the low [Ca2+]. At pCa 6.0, Vmax and force were decreased to the same extent relative to their values at pCa 4.5. At pCa 5.75, where there was no reduction in force but a 25% decrease in isometric JATP, Vmax was unchanged relative to pCa 4.5. Force, Vmax and JATP were all dependent on [Mg ATP]. Half‐maximal response was obtained at 0.1 mM for force and Vmax, and at 0.5 mM for JATP. The results are discussed in relation to a possible influence of both Ca2+ and Mg ATP on kinetic properties of the cross‐bridge cycle.
gamma‐Aminobutyric‐acid‐ and pentobarbitone‐gated chloride currents in internally perfused frog sensory neurones.
Authors: Akaike N , Hattori K , Inomata N , Oomura Y
Published: December 2014

gamma‐Aminobutyric‐acid‐ (GABA) and pentobarbitone‐induced Cl‐ currents (ICl) were studied in isolated frog sensory neurones after suppression of Na+, K+ and Ca2+ currents using a suction‐pipette technique combining internal perfusion with voltage clamp. All GABA‐sensitive neurones responded to pentobarbitone. Both GABA‐ and pentobarbitone‐induced ICl reversed at the Cl‐ equilibrium potential (ECl). The dose‐response curve for maxima of GABA‐induced ICl was sigmoidal with a mean concentration producing a half‐maximum response, Ka of 2 X 10(‐5) M at a Hill coefficient of 1.8. In the presence of pentobarbitone, the GABA dose‐response curve shifted to the left without affecting the saturating maximum current. At high concentrations, both GABA and pentobarbitone could also potentiate the pentobarbitone‐ and GABA‐induced ICl respectively, while pre‐treatment with one of the two markedly attenuated currents induced by the other, indicating a 'cross‐desensitization'. In the presence of pentobarbitone, the augmented response was voltage dependent and this augmentation was much greater in the inward‐current direction than outward. In producing ICl, pentobarbitone and its stereoisomers were potent in the order of (‐) isomer greater than (+/‐) racemic mixture greater than (+) isomer. A stereospecific facilitatory action of pentobarbitone on GABA responses was also found in the same order. Responses to GABA, homotaurine, taurine, beta‐alanine, 5‐aminovaleric acid, (+)‐ and (‐)‐gamma‐amino‐beta‐hydroxybutyric acid and muscimol were equally enhanced by pentobarbitone, though its action on glycine‐induced ICl was less effective. Picrotoxin inhibited the GABA‐ and pentobarbitone‐induced ICl from either side of membrane, while internal application of GABA and pentobarbitone did not exert any effect. It was concluded that pentobarbitone binds to the 'barbiturate receptors' located close to the GABA receptor‐Cl‐ channel complex, and directly affects the GABA‐GABA receptor interactions rather than the ionic channels.
Sino‐aortic denervation in the monkey.
Authors: Cornish K G , Gilmore J P
Published: December 2014

The aortic arch and carotid sinuses were denervated in eleven monkeys. The monkeys were subjected to four sequential surgeries which involved: (1) implantation of an aortic and left atrial catheter; (2) stripping of the adventitia from the aortic arch; (3) stripping the left carotid sinus and associated vessels; and (4) stripping the right carotid sinus and associated vessels. Blood pressure and pulse rate were recorded 6 days after each surgical procedure. Records were taken over a 6 h period while the monkeys were in their home cages. Baroreceptor denervation was confirmed by: (1) absence of heart rate response to blood pressure changes and (2) an increase in the variability of blood pressure. Veratridine given into the left atrium caused a Bezold‐Jarisch reflex both before and after denervation verifying the integrity of the afferent and efferent vagus. Denervation of the baroreceptors resulted in a significant increase in blood pressure when measured from monkeys who were restrained in chairs in the laboratory; however, blood pressure was not significantly elevated in the baroreceptor denervated monkeys while they were tethered in their home cages. It is concluded that denervation of the sino‐aortic baroreceptor does not result in a significant increase in systemic blood pressure.
The action of N‐methyl‐D‐aspartic acid on mouse spinal neurones in culture.
Authors: Mayer M L , Westbrook G L
Published: December 2014

Neurones from the ventral half of mouse embryo spinal cord were grown in dissociated culture and voltage clamped. The current‐voltage relation of responses evoked by N‐methyl‐D‐aspartic acid (NMDA), L‐glutamic acid and kainic acid was recorded in media of different ionic composition. On removal of Mg2+ from the extracellular solution, responses to NMDA and L‐glutamate became less voltage sensitive, such that NMDA responses were no longer associated with a region of negative slope conductance. The antagonism of NMDA responses produced by application of Mg2+ to neurones bathed in nominally Mg2+‐free solutions shows voltage dependence and uncompetitive kinetics. Voltage‐jump experiments showed that the voltage‐dependent action of Mg2+ occurred rapidly, and with complex kinetics. Ni2+ and Cd2+, two potent blockers of calcium currents in spinal cord neurones, had significantly different potencies as NMDA antagonists, Ni2+ being of greater potency than Mg2+, and Cd2+ considerably weaker. The voltage‐dependent block of NMDA responses produced by physiological concentrations of Mg2+ is sufficient to explain the apparent increase in membrane resistance produced by NMDA in current‐clamp experiments, and the ability of NMDA to support repetitive firing. Substitution of choline for Na+ produced a hyperpolarizing shift in the reversal potential for responses evoked by kainic acid consistent with an increase in permeability to Na+ and K+. In choline‐substituted solutions, the reversal potential of NMDA responses was more positive than that recorded for kainic acid, and in addition NMDA responses showed enhanced desensitization.
The influence of 'diastolic' length on the contractility of isolated cat papillary muscle.
Authors: Nichols C G
Published: December 2014

Isometrically contracting cat papillary muscles were studied. Muscle length was changed during diastole and returned to control just before the next contraction such that developed force was always measured at the same length. When the diastolic length was increased from a control length, systolic force at the control length increased slowly over several minutes. When the muscle was then held at the increased length, there was an immediate increase in systolic force followed by a small secondary slow increase. Conversely, a decrease in diastolic length from a control length resulted in a slow decrease in systolic force at the control length. When the muscle was then held at the decreased length there was an immediate decrease in systolic force followed by a small secondary decrease. No change in the time course of contraction accompanied the slow force changes after a maintained change of length or a change of diastolic length alone. The magnitude of the slow change of force was proportional to the duration of time in each diastole for which the length was altered and independent of the onset time of a given duration of diastolic length change. The contractility changes were not linearly related to the amplitude of the diastolic length changes. The potentiating effect of a given stretch was greater than the depotentiating effect of a similar release. The development of inotropic changes as a result of diastolic length changes occurred whether or not the muscle was stimulated during the period of the length changes.
Intracellular pH regulation in the sensory neurone of the stretch receptor of the crayfish (Astacus fluviatilis).
Authors: Moser H
Published: December 2014

The ionic mechanisms of intracellular pH (pHi) regulation were studied in the slowly adapting sensory cell of the crayfish stretch receptor by using pH‐, Na+‐ and Cl(‐)‐sensitive liquid ion exchanger electrodes. Under control conditions a mean pHi of 7.23 +/‐ 0.12 (S.D.) at a mean membrane potential of 68.3 +/‐ 4.1 mV S.D. was found in sixteen cells. Thus pHi is about 1 pH unit more alkaline than predicted from passive distribution, implying the presence of an acid extrusion mechanism. In order to acidify the cytoplasm, the cell was either acid‐loaded by NH4Cl or exposed to CO2 and CO2/HCO3‐ solutions. During CO2 exposures pHi was regulated only if calculated amounts of HCO3‐ were added to keep external pH (pHo) constant. The pHo per se was found to be an important determinant of pHi and its regulation. Substitution of external Na+ by choline inhibited pHi recovery almost completely. As soon as Na+ was readmitted H+ extrusion occurred immediately at a rate similar to that of the control. The internal Na+ activity (aiNa) ranged between 6 and 13 mM with a mean of approximately 9.1 +/‐ 2.5 mM (S.D.; n = 8). The effects of various solutions on aiNa and the temporal relationship between aiNa and pHi in NH4Cl acid‐loaded cells were investigated. The amount of aiNa increased during cell internal acidification and recovered in parallel with pHi recovery in NH4Cl acid‐loaded cells. Experiments with 10(‐4) M‐ouabain and K+‐free conditions suggest that neither the Na+‐K+ pump nor external K+ are directly involved in pHi regulation. The internal chloride activity (aiCl), which was lower than predicted from a passive distribution, fell during exposure to HCO3‐/CO2. Regulation of pHi was inhibited if the cell was completely depleted of Cl‐ by prolonged exposures to Cl(‐)‐free solution (isethionate and/or gluconate substituted). The pHi‐regulating system of the sensory cell requires Na+ and Cl‐ which probably operate in a combined mechanism such as Na+ ‐H+‐Cl(‐)‐HCO3‐ or an equivalent.
Three serial neurones in the innervation of the colon by the sacral parasympathetic nerve of the dog.
Authors: Fukai K , Fukuda H
Published: December 2014

Electrophysiological and histological techniques were used to study the nature of the innervation of the colon by the sacral parasympathetic nerve of the dog. Many cells which incorporate horseradish peroxidase (HRP) were observed in ganglia of the pelvic plexus, but not in the sacral cord, after HRP was injected into the wall of the distal colon. Stimulation of the pelvic ganglia by an application of dimethylphenylpiperazinium contracted the distal colon. The contraction was reduced by nicotine applied to the colon. Compound action potentials in responses of rectal strands of the pelvic nerve to stimulation of the sacral ventral roots decreased or disappeared after hexamethonium bromide (C6) was applied to the pelvic plexus. Conduction velocities of the potentials showed that most of the preganglionic fibres were B fibres, and nearly all the post‐ganglionic fibres were C fibres. Many C fibres were observed by electron microscopy in the rectal strands after degeneration of the pelvic and hypogastric nerves. Stimulation of such rectal strands caused a contraction of the colon, which diminished after C6 was applied to the distal colon. These results show that the sacral parasympathetic nerve innervating the colon comprises three serial neurones located in the sacral spinal cord, the pelvic plexus and the myenteric plexus.
Effects of stimulation and diphenylhydantoin on the intracellular sodium activity in Purkinje fibres of sheep heart.
Authors: Ellis D
Published: December 2014

The effects of stimulus frequency on the intracellular Na+ activity (aiNa) of Purkinje fibres from sheep hearts have been investigated using ion‐sensitive micro‐electrodes. aiNa increased by approximately 44% at 3 Hz and approximately 6.5% at 0.2 Hz. The size of the increase was very variable between preparations and did not appear to be related to the initial level of aiNa before stimulation. During stimulation, the rise of aiNa was exponential and gave a new steady‐state level within about 15 min. The time constant of this rise was independent of the frequency of stimulation. The recovery of aiNa following a period of stimulation also appeared to be exponential but the time constant was dependent upon the extent to which aiNa had been raised during stimulation, with shorter time constants following larger rises. The effects of stimulation on aiNa were compared to inhibition of the Na+‐K+ pump using high concentrations of cardioactive steroids. The influence of diphenylhydantoin (DPH) on the Na+ permeability of the cell membrane was assessed by measuring the rate of rise of aiNa following Na+‐K+‐pump inhibition. DPH decreased the rate of rise of aiNa by approximately 37% at concentration of 10(‐4) M. The decrease of Na+ permeability produced by DPH is probably responsible for the observed reduction of aiNa in quiescent preparations. DPH also produced substantial decreases of aiNa in stimulated preparations. These results are discussed in relation to its known anti‐arrhythmic effects.
Changes in sodium activity during light stimulation in photoreceptors, glia and extracellular space in drone retina.
Authors: Coles J A , Orkand R K
Published: December 2014

Ion‐selective micro‐electrodes were used to measure Na+ activity, aNa, in the two types of cell, photoreceptors and glial cells, and in the extracellular space, in superfused slices of the retina of the honey‐bee drone, Apis mellifera male. Movements of Na+ were induced by light stimulation, or by increasing [K+] in the superfusate. In the dark, aNa in the photoreceptors was 10 mM (S.E. of the mean = 1 mM); in the glial cells it was higher: 37 +/‐ 2 mM. We estimate that in this preparation about 2/3 of the free Na+ in the tissue is in the glial cells. Stimulation with a train of light flashes, 1 s‐1 for 90 s caused aNa in the photoreceptors to increase by 16 +/‐ 2 mM. K+ activity, aK, decreased by 21 +/‐ 3 mM. During the standard train of light flashes, aNa in glial cells decreased by only 1.5 +/‐ 0.3 mM, much less than the increase in aK (7 +/‐ 2 mM). One possible interpretation of this result is that most of the increase in aK is due to K+ uptake by a mechanism other than Na+‐K+ exchange. In extracellular fluid, stimulation caused aNa to fall to a relatively steady value in about 10 s. Unlike aK, there was no tendency for aNa to return to the base line during the remainder of the 90 s stimulation. The fall in aNa was 14 +/‐ 1 mM: a greater fall is prevented by extracellular electric currents and a decrease in extracellular volume. When [K+] in the superfusate was increased from 7.5 to 18 mM, aNa decreased in the glial cells but not in the photoreceptors. In this tissue, stimulation causes changes in aNa in the neurones that might be large enough to modify the biochemistry of the cells. But in the glia, the fractional changes are small.
Regulation of cytosolic calcium concentration in presynaptic nerve endings isolated from rat brain.
Authors: Nachshen D A
Published: December 2014

The regulation of cytosolic Ca concentration ([Ca]i) was studied with the fluorescent Ca indicator, quin2, in pinched‐off presynaptic nerve endings (synaptosomes) isolated from rat brain. The resting [Ca]i is 0.1‐0.2 microM, in solutions containing 1‐2 mM‐Ca. [Ca]i increases by only 100‐150 nM when the external Ca concentration is increased from 0.02 to 2 mM. The mitochondrial inhibitors valinomycin and fluoro‐carbonyl cyanide phenylhydrazone (FCCP) increase [Ca]i by 100‐200 nM. This increase is not correlated with the resting level of [Ca]i prior to the addition of inhibitors, but it is dependent on the presence of external Ca. It seems likely that the effect of these inhibitors on [Ca]i is a secondary consequence of metabolic inhibition. [Ca]i increases by about 2‐fold when the external Na concentration is lowered from 145 to 5 mM, and returns to its initial level when external Na is restored. This recovery occurs also in the presence of FCCP. These results suggest that Na/Ca exchange, but not mitochondrial Ca uptake, plays a role in regulating [Ca]i and in allowing the nerve terminals to recover from Ca loading.
Calcium mobilization in enzymically isolated single intact and skinned muscle cells of the porcine coronary artery.
Authors: Ueno H
Published: December 2014

The mobilization of 45Ca2+ was investigated in collagenase‐treated single smooth muscle cells of the porcine coronary artery. After removal of extracellular 45Ca2+ by 10 mM‐EGTA at 0 degree C, the content of exchangeable Ca2+ was estimated to be 0.42 +/‐ 0.02 nmol/2 X 10(5) cells at rest and 0.62 +/‐ 0.03 nmol/2 X 10(5) cells in 102.5 mM‐external K solution. The efflux of 45Ca2+ into Ca2+‐free solution, estimated from the 45Ca2+ remaining in the cells, increased temperature dependently and was reduced by oligomycin. The muscle cells at rest had a substantial amount of stored Ca2+ which was releasable by caffeine or acetylcholine. Saponin‐treated (skinned) muscle cells accumulated 45Ca2+ in the presence of Mg ATP. Two mechanisms of ATP‐dependent Ca2+ sequestration were observed: one exhibited a low affinity for Ca2+ but a high‐capacity uptake which was sensitive to sodium azide; this was thought to be located in the mitochondria. The other had a high‐affinity (1.5/microM) and low‐capacity uptake (0.92 nmol/2 X 10(5) cells), which was insensitive to sodium azide, potentiated by oxalate and was thought to be mainly mediated via the sarcoplasmic reticulum (s.r.). The minimum concentration of free Ca2+ required for the ATP‐dependent Ca2+ uptake in the saponin‐treated cells was about 20 nM by the s.r. and 1 microM by the mitochondria. Thus, the mitochondria seem to play a minor role in regulating cytoplasmic Ca2+ during the contraction‐relaxation cycle. These results indicate that enzymically isolated muscle cells are functionally intact, and may facilitate direct measurement of Ca2+ movements when attempting to estimate the physiological role of Ca2+ in vascular smooth muscles.
A rabbit jejunal isolated enterocyte preparation suitable for transport studies.
Authors: Brown P D , Sepúlveda F V
Published: December 2014

A method is described for isolating viable enterocytes from rabbit jejunum. Estimates of sucrase and gamma‐glutamyl transferase activities in cells isolated by this method suggest that they originate from the upper villus only. Isolated cells accumulate both alpha‐methyl‐D‐glucoside and alanine, maintaining high intracellular concentrations for at least 60 and 40 min respectively. Accumulation of alpha‐methyl‐D‐glucoside is inhibited by the presence of phloridzin. The cells accumulate 42K and 86Rb in an identical manner. This uptake, which is maintained for at least 60 min, is inhibited in the presence of ouabain. Passive efflux of 42K and 86Rb occurs with rate constants which are virtually identical. The efflux follows a single exponential suggesting that it originates from only one intracellular compartment. It is suggested that the preparation can be used to study the effect of sugars and amino acids on K efflux. The advantages of using such a preparation are discussed.
Pyramidal effects in dorsal neck motoneurones of the cat.
Authors: Alstermark B , Pinter M J , Sasaki S
Published: December 2014

The effects of contralateral pyramidal stimulation have been investigated with intracellular recording from cat alpha‐motoneurones that innervate the dorsal neck musculature. A short train of stimuli evoked three types of synaptic effects: predominant excitation or inhibition and mixed effects characterized chiefly by early excitation followed by inhibition. Latency measurements indicated a minimal disynaptic linkage for excitation and for inhibition. Splenius motoneurones received primarily excitation whereas biventer cervicis‐complexus motoneurones received a more varied input characterized by mixed effects or inhibition. Following transection of the pyramid just rostral to the decussation (lower pyramidal lesion) pyramidal stimulation above the lesion still produced disynaptic excitation and longer latency (possibly trisynaptic) inhibition. Pyramidal stimulation just caudal to this transection evoked inhibition with a minimal disynaptic latency, as well as longer latency excitation. The incidence of longer latency excitation was found to be reduced in cats with corticospinal tract transections at the level of the second cervical spinal segment. No post‐synaptic potentials were evoked by pyramidal stimulation rostral to a pyramidal transection at the level of the trapezoid body. It is suggested that disynaptic excitation evoked by pyramidal stimulation above the lower pyramidal lesion is mediated by medullary reticulospinal neurones possessing monosynaptic excitatory connexions with neck motoneurones. Longer latency excitation appears to be mediated by neurones that receive corticospinal tract input and are located in the spinal segments containing the neck motoneurones. Disynaptic inhibition is mediated by neurones likely to be situated between the second cervical spinal segment and the level of the lower pyramidal lesion. The results also suggest that the first neurone in the chain mediating longer latency inhibition is located in the brain stem. The differences in pyramidal synaptic input between splenius and biventer cervicis‐complexus motoneurones are considered in relation to the roles these muscles may serve in head position control.
Respiratory activation of the facial nerve and alar muscles in anaesthetized dogs.
Authors: Strohl K P
Published: December 2014

Comparisons were made of the activities of the dorsal buccal branch (d.b.b.) of the facial nerve which innervates the alae nasi and of the alar muscles themselves (alae nasi) with the activity of the phrenic nerve in eight anaesthetized, spontaneously breathing dogs during hypercapnia, end‐expiratory airway occlusion, and chest wall compression, before and after vagotomy. Nerve and muscle activities were recorded from bipolar cuff or wire electrodes respectively and processed by a moving average technique. Peak facial d.b.b., alae nasi, and phrenic activity all increased linearly with hypercapnia. Airway occlusion prolonged the duration of phrenic as well as facial d.b.b. and alae nasi activity. A comparison of electrical activity during unoccluded and occluded inspirations indicated a facilitation of phrenic activity but an inhibition of both facial d.b.b. and alae nasi activities associated with volume feed‐back. These volume‐related feed‐back effects were abolished by vagotomy. After vagotomy, peak facial d.b.b., alae nasi and phrenic activities increased during lower chest wall compression and decreased with upper chest wall compression when compared to the preceding control breath. Alae nasi and facial nerve activities, like that of the phrenic nerve, respond to respiratory chemical and reflex influences.
Post‐natal development of pyramidal tract neurones in kittens.
Authors: Oka H , Samejima A , Yamamoto T
Published: December 2014

The post‐natal development of pyramidal tract neurones (p.t.n.s) was investigated in twenty‐one barbiturate‐anaesthetized kittens from birth to 28 days of age using a combination of electrophysiological and anatomical techniques. P.t.n. responses were recorded intracellularly as well as extracellularly with glass micropipettes filled with horseradish peroxidase (HRP) on stimulation of the medullary pyramid and cerebellar nuclei. Latency histograms of antidromic responses of p.t.n.s were compared at various ages. In the neonate, p.t.n.s were divided into two groups which were presumed to be analogous with fast and slow p.t.n.s in adult animals. During the first post‐natal week, latency shortening was not conspicuous, but by the end of the second post‐natal week, the faster group showed a marked decrease of latencies (up to around 10 ms at 14 days of age), while those of the slower group did not change so much. The slower group increased their conduction velocity during the third post‐natal week (latencies up to around 18 ms). At the end of the fourth post‐natal week, the distribution of antidromic latencies was in a narrower range, but the values were still longer than those reported in adult animals. Intracellular HRP staining revealed that apical dendrites of p.t.n.s spread fully to the pial surface even at birth. The somata of these neurones were characteristically covered with somatic appendages and development of the basal dendritic tree was immature in 0‐1‐day‐old kittens. Basal dendrites developed nearly completely by 7 days, but somata were still covered with appendages. During the fourth post‐natal week, these appendages disappeared almost completely. The sizes of the dendritic field, especially of apical dendrites, became larger in parallel with the development of cortical layers. From the morphological point of view, differentiation of fast and slow p.t.n.s was not clear until 28 days of age except in somatic volumes, which were already different in the first post‐natal week. At the end of the fourth post‐natal week, p.t.n.s with short antidromic latencies had a tendency to bear spines more sparsely over the secondary and tertiary dendritic surface in comparison with p.t.n.s with longer latencies. Intracortical axonal trajectories developed fairly well in the immature cerebral cortex, and the general pattern of ramification changed little during the first month after birth.(ABSTRACT TRUNCATED AT 400 WORDS)
Selective block of Y optic nerve fibres in the cat and the occurrence of inhibition in the lateral geniculate nucleus.
Authors: Burke W , Burne J A , Martin P R
Published: December 2014

A method of selectively blocking the larger nerve fibres in the optic nerve of the cat is described. The optic nerve is compressed between a small balloon and the inside of a small metal cylinder. The block is monitored by stimulating and recording on opposite sides of the blocked region. The t1 (Y) response can be eliminated usually with some loss of the t2 (X) response. Stimulation of the blocked optic nerve initially excites certain cells (r2 or X) in the dorsal lateral geniculate nucleus (d.l.g.n.) but this is followed by an inhibition of both r1 (Y) and r2 (X) cells. This inhibition is similar in time course to that produced by stimulation of t1 fibres, suggesting that the effect may be mediated via a similar mechanism. The effect is present in a preparation in which all t1 fibres have degenerated, removing the possibility that it might have been due to inadvertent stimulation of t1 fibres. Experiments on a small number of r2 (X) d.l.g.n. cells have confirmed the inhibitory effect of stimulation of the smaller optic nerve fibres on X cells.
Effects of thyroid hormone on calcium handling in cultured chick ventricular cells.
Authors: Kim D , Smith T W
Published: December 2014

Mechanisms underlying thyroid hormone‐induced changes in myocardial contractile state were investigated by studying the effects of triiodothyronine (T3) on Ca2+ fluxes across the sarcolemmal membrane and Ca2+ handling by the sarcoplasmic reticulum, using spontaneously contracting monolayers of cultured chick embryo ventricular cells. Cells were grown in serum‐free medium containing either no T3 or 10(‐8) M‐T3 for 48 h. At [Ca2+]o levels of 0.6 and 1.2 mM, the velocity of cell contraction was significantly greater in cells grown in 10(‐8) M‐T3 than in its absence. At higher [Ca2+]o, no differences in the velocity of contraction were noted. 45Ca2+ exchange kinetic studies showed a biexponential pattern with a rapid and a slow component of uptake in cells grown both with and without 10(‐8) M‐T3. The rate of the rapid phase of uptake and total Ca2+ content were higher in cells grown in T3, with the increment in content ascribable to the rapidly exchangeable Ca2+ pool. Verapamil partially inhibited the T3‐induced increase in the rapidly exchangeable pool. 45Ca2+ uptake in response to a step change to Na+‐free medium in the presence of 1 microM‐verapamil was significantly greater in cells grown in 10(‐8) M‐T3 than in T3‐free medium. Cells grown in T3 showed 20% greater beating rate than cells grown in its absence. A similar increase in beating rate achieved by lowering [K+]o from 4.0 to 3.0 mM or by electrical stimulation failed to affect the rate of 45Ca2+ uptake or the size of the rapidly exchangeable pool; pacing‐induced increases in rate resulted in reduction rather than augmentation of contractile state. Ca2+ efflux rate was greater in cells grown in 10(‐8) M‐T3 than in T3‐free medium, whereas cells loaded with various levels of Ca2+ acutely by incubation at selected [Ca2+]o levels had similar efflux rates. Replacement of Na+ by choline in the efflux medium resulted in elevated Ca2+ efflux rates in cells grown both with and without T3; however, it remained greater in cells grown in 10(‐8) M‐T3 than in its absence. Caffeine (20 mM) in the efflux medium increased Ca2+ efflux to a greater degree in cells grown in T3 than without it. Caffeine also produced a greater tonic contraction in T3‐treated cells than in cells grown in absence of T3 in Na+‐ and Ca2+‐free medium.(ABSTRACT TRUNCATED AT 400 WORDS)
The mechanism of the increase of tonic tension produced by caffeine in sheep cardiac Purkinje fibres.
Authors: Eisner D A , Valdeolmillos M
Published: December 2014

The effects of caffeine were examined on contraction and membrane current in voltage‐clamped sheep cardiac Purkinje fibres. The photoprotein aequorin was injected into several cells in order to measure the intracellular ionized Ca concentration [( Ca2+]i). When the Na‐K pump was inhibited, depolarization produced a twitch followed by a tonic component of tension. Repolarization produced an after‐contraction. These components of tension were accompanied by corresponding increases of aequorin light. Caffeine (10 mM) decreased both the twitch and the after‐contraction while increasing the tonic component. The application of caffeine also produced a transient increase of aequorin light, both during depolarization and at rest, which was followed by a maintained decrease in all three components of the light signal. In particular, although caffeine decreased the rise of aequorin light during prolonged depolarization it increased the tonic tension. The possibility that the effects of caffeine on tonic tension could be due to suppression of spontaneous Ca oscillations was rejected for the following reasons. (i) Ryanodine (which also abolishes Ca oscillations) decreased the magnitude of the tonic tension. (ii) Caffeine still increased tonic tension when it was added to a fibre exposed to ryanodine (1‐10 microM). In the presence of ryanodine it was possible to measure [Ca2+]i and tonic tension without interference from Ca oscillations. The increase of tonic tension produced by caffeine could not be accounted for by a rise of [Ca2+]i. The results showed that, at a given level of Ca, caffeine increased tension. The results show that a large part of the increase of tonic tension produced by caffeine is due to an increase of the Ca sensitivity of the contractile apparatus rather than to changes of [Ca2+]i. The consequence of this observation for the experimental use of caffeine is discussed.
A dual mechanism for intracellular pH regulation by leech neurones.
Authors: Schlue W R , Thomas R C
Published: December 2014

Neutral‐carrier pH‐sensitive micro‐electrodes were used to investigate intracellular pH (pHi) in leech neurones. When used in snail neurones such electrodes gave very similar pHi values to those recorded simultaneously by recessed‐tip glass micro‐electrodes. Leech Retzius neurones superfused with a pH 7.4 HCO3‐‐free physiological saline were found to have a pHi of 7.3, too high to be explained by a passive distribution of H+ or OH‐. To investigate pHi regulation the pHi was decreased by one of three methods: by exposure to propionate, by adding and then removing NH4Cl or by exposure to CO2. Acidification by any method was followed by a recovery to normal pHi values within 15‐20 min. In HCO3‐‐free solutions, pHi recovery from acidification was blocked by removing external Na or by amiloride (2 mM). In solutions buffered with 2% CO2 and 11 mM‐HCO3‐, amiloride slowed but did not block pHi recovery. The anion exchange inhibitor SITS (4‐acetamido‐4'‐isothiocyanato‐stilbene‐2,2'‐disulphonic acid) also slowed pHi recovery in the presence of HCO3‐. In CO2/HCO3‐ solution the removal of external Na either slowed or blocked pHi recovery, and blocked it completely in the presence of amiloride. We conclude that in HCO3‐‐free solutions pHi regulation is by a Na‐H exchange system; but in the presence of HCO3‐ there is an additional mechanism which is probably a Na‐dependent Cl‐HCO3 exchanger.
Two calcium‐sensitive spike after‐hyperpolarizations in visceral sensory neurones of the rabbit.
Authors: Fowler J C , Greene R , Weinreich D
Published: December 2014

Intracellular recordings were made from rabbit nodose neurones in vitro. Two temporally distinct spike after‐hyperpolarizations (a.h.p.s) were identified in a subpopulation of C‐type neurones. The fast a.h.p. after a single spike lasted no longer than 500 ms, while the slow a.h.p. persisted for seconds. Both a.h.p.s. were increased in amplitude in low K+ (0.56 mM) solutions and decreased in amplitude in high K+ (11.2 mM) solutions, and both were reversed at hyperpolarized membrane potentials. The slow a.h.p. was reduced in low Ca2+ (0.22 mM), in the presence of Ca2+ antagonists (Ni2+, 1 mM; Cd2+, 100 microM; or Co2+, 1 mM) and was enhanced in tetraethylammonium (5 mM). In approximately half of the cells tested, the fast a.h.p. was reduced in low Ca2+ and in the presence of the Ca2+ antagonists. In the remaining cells the fast a.h.p. was insensitive to these procedures. Prostaglandin (PGE1, 1‐10 micrograms/ml) reduced the slow a.h.p. in all cells tested. Neither the Ca2+‐sensitive nor the Ca2+‐insensitive fast a.h.p. was affected by the prostaglandin. It is concluded that there is a subpopulation of C‐type nodose neurones possessing a slow a.h.p. which is due to a Ca2+‐dependent K+ current. This subpopulation of neurones can further be divided on the basis of the presence of a Ca2+‐sensitive fast a.h.p. Furthermore, PGE1 pharmacologically separates the fast and slow a.h.p.s by selectively blocking the slow one. The blockage by the PGE1 is most probably not due to a reduction in Ca2+ influx.
Pharmacological evidence for L‐aspartate as the neurotransmitter of cerebellar climbing fibres in the guinea‐pig.
Authors: Kimura H , Okamoto K , Sakai Y
Published: December 2014

Climbing fibre responses (c.f.r.s) evoked by white matter stimulation and the depolarizations induced by iontophoretically applied L‐glutamate and L‐aspartate were recorded intracellularly from the proximal dendrites of Purkinje cells in in vitro slice preparations of the guinea‐pig cerebellum. Short pulses of L‐glutamate and L‐aspartate dose‐dependently depolarized the Purkinje cell dendrite. Even small doses of these amino acids reduced the input resistance. The maximum decrease in input resistance induced by L‐glutamate was 36% and that by L‐aspartate was 38%. Intracellular injection of Cs+ allowed Purkinje cell dendrites to be depolarized to a range of ‐15 to +30 mV. The mean reversal potential for the c.f.r. (Ec) was found to be +10.2 mV (n = 4). The mean reversal potentials obtained for L‐glutamate (Eg) and for L‐aspartate (Ea) were +7.3 mV (n = 7) and +5.6 mV (n = 7) respectively. When external Na+ concentration was reduced, Ec, Ea and Eg were linearly and similarly shifted in the negative direction, indicating that all these reversal potentials are determined primarily by a Na+ conductance. The effects of the glutamate antagonists 2‐amino‐5‐phosphonovaleric acid (APV), gamma‐D‐glutamylglycine (gamma‐DGG), N‐methyl‐DL‐aspartic acid (NMDLA) and glutamic acid diethylester (GDEE) were compared as to the responses to L‐glutamate and L‐aspartate and Ca2+‐activated focal climbing fibre responses (c.f.c.f.r.s) in order to investigate the receptor type at the synapses formed by the climbing fibres with Purkinje cell dendrites. The order of antagonistic potency to the c.f.c.f.r. was : APV (mean percentage blockade = 99%) greater than gamma‐DGG (87%) greater than NMDLA (71%) greater than GDEE (28%). The order of antagonistic potency to the response to L‐aspartate was: gamma‐DGG (69%) greater than APV (66%) greater than NMDLA (60%) greater than GDEE (31%), and that to the response to L‐glutamate was: GDEE (63%) greater than NMDLA (22%) greater than gamma‐GDD (15%) greater than APV (14%). APV was found to be the most effective anatagonist of the c.f.c.f.r. Its action was reversible, selective for L‐aspartate‐induced depolarization and had no effect on the responses to L‐glutamate. NMDLA, which has no activity as an agonist, was a greater suppressant of the responses to L‐aspartate than those to L‐glutamate. These electrophysiological and pharmacological findings suggest that the receptor for the transmitter at the synapses formed by climbing fibres with Purkinje cell dendrites is of the L‐aspartate‐preferring type, and are thus consistent with the bio‐and histochemical findings that L‐aspartate may be the endogenous transmitter at this synapse.