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Biomedical subjects

I Parker

Publications and source records attributed to I Parker.

At least 109 records · Page 6Linked to original sources
PubMed1984

Properties of human brain glycine receptors expressed in Xenopus oocytes.

Glycine and gamma-aminobutyric acid (GABA) receptors from the foetal human brain were 'transplanted' into the Xenopus oocyte membrane by injecting the oocytes with poly(A)+-mRNA extracted from the cerebral cortex. Activation of both glycine and GABA receptors induced membrane currents carried largely by chloride ions. However, unlike the GABA-activated current, the glycine current was blocked by strychnine, and was not potentiated by barbiturate. At low doses, the glycine current increased with concentration following a 2.7th power relation, suggesting that binding of three molecules of glycine may be required to open a single membrane channel. The current induced by steady application of glycine decreased with hyperpolarization beyond about -60 mV.

Animals↗
PubMed1984

Separate fractions of mRNA from Torpedo electric organ induce chloride channels and acetylcholine receptors in Xenopus oocytes.

Poly(A)+ mRNA extracted from the electric organ of Torpedo was fractionated by sucrose density gradient centrifugation. After injection into Xenopus oocytes one mRNA fraction induced the appearance of chloride channels in the oocyte membrane. Many of these channels were normally open, and the ensuing chloride current kept the resting potential of injected oocytes close to the chloride equilibrium potential. When the membrane was hyperpolarized, the chloride current was reduced. A separate fraction of mRNA induced the incorporation of acetylcholine receptors into the oocyte membrane. When translated in a cell-free system this fraction directed the synthesis of the alpha, beta, gamma, and delta subunits of the acetylcholine receptor. In contrast, the mRNA fraction that induced the chloride channels caused the synthesis of the delta subunit, a very small amount of alpha, and no detectable beta or gamma subunits. This suggests that the size of the mRNA coding for the chloride channel is similar to the preponderant species of mRNA coding for the delta subunit of the acetylcholine receptor.

Animals↗
PubMed1984

Partial purification and functional expression of brain mRNAs coding for neurotransmitter receptors and voltage-operated channels.

Poly(A)+ mRNAs extracted from embryonic chicken brain and from adult rat brain were fractionated on sucrose density gradients. The fractions were subsequently injected into Xenopus oocytes where the mRNA was translated. The products were processed and incorporated into the oocyte membrane where they formed functional neurotransmitter receptors and voltage-operated channels. Different mRNA fractions induced the incorporation of different transmitter receptors and voltage-operated channels into the oocyte membrane. These experiments provide a useful step towards the understanding of the structure and function of neurotransmitter receptors and channels.

Animals↗
PubMed1984

Extracellular ions and excitation-contraction coupling in frog twitch muscle fibres.

Intracellular calcium transients were recorded from voltage-clamped frog twitch muscle fibres using Arsenazo III. The possible role of extracellular ions in excitation-contraction (e.-c.) coupling was examined using ion substitutions and blocking drugs in the bathing medium. Parameters measured included the Arsenazo response size to a standard depolarizing pulse (5 ms, 0 mV) and the strength-duration curve for threshold Arsenazo signal. Addition of tetrodotoxin (TTX) decreased the response size to small (-30 mV, 5 ms), but not large (+30 mV, 10 ms) depolarizations, probably because of poor voltage clamp of the tubular membrane in the absence of TTX. Clamping TTX-treated fibres with the wave form of a recorded action potential gave an Arsenazo response similar to that elicited by the normal action potential (at 10 degrees C). Complete substitution of sodium (by choline, lithium or Tris) or chloride (by methyl sulphate or maleate) in the bathing solution gave no appreciable changes in the size of the Arsenazo response. Reduction of extracellular free [Ca2+] to low levels using EGTA caused a slight reduction in the calcium signal elicited by the standard depolarization (to 74% after a few hours, and to 62% after 2 days; temperature 5-10 degrees C). The strength-duration curve was unchanged. Arsenazo responses about 75% of the control size could be elicited in high potassium solution (42 mM-K2SO4) by strong (+80 mV, 20 ms) depolarizations, after re-polarizing the fibres to -90 mV for a few minutes. The voltage dependence of activation was shifted to more positive potentials in this solution. Tetraethylammonium (TEA) bromide at a concentration of 20 mM did not alter the Arsenazo signal, whilst 120 mM-TEA reduced the response by 25%. 3,4-diaminopyridine (DAP) reduced the size of the Arsenazo signal at a concentration of 5 mM, and caused spontaneous release of calcium from the sarcoplasmic reticulum (s.r.) in the absence of membrane potential changes. The Arsenazo signal elicited by an action potential was enhanced by 1 mM-DAP, because of prolongation of the action potential, but was depressed by higher concentrations. We conclude that e.-c. coupling does not involve the influx of any external ions into the muscle fibre. If a current flow between the T-tubules and the s.r. is involved in e.-c. coupling, then this is probably carried by an efflux of potassium ions.

4-Aminopyridine↗
PubMed1984

Slowly inactivating potassium channels induced in Xenopus oocytes by messenger ribonucleic acid from Torpedo brain.

Poly(A+) messenger RNA was extracted from the electric lobe and medulla of Torpedo and injected into oocytes of Xenopus laevis. The synthesis and processing of proteins coded by the injected messenger RNA led to the incorporation of voltage-activated channels in the oocyte membrane. A large, well maintained outward current was recorded from injected oocytes in response to depolarization. Non-injected oocytes did not show this current. The reversal potential of the current varied according to the Nernst equation with external potassium concentration, indicating that it was largely carried by potassium ions. The maintained potassium current was not reduced by manganese (5 mM) or lanthanum ions (0.1 mM). Tetraethylammonium and aminopyridines blocked the potassium current. The block produced by 3,4-diaminopyridine was enhanced by previous activation, but diminished by strong depolarization. The amplitude of the potassium current increased over the approximate voltage range -30 to +50 mV, but reduced at more positive potentials. The decline of the current during maintained depolarization became slower as the membrane potential was made more positive, and the rate of onset of the current became faster. Estimates from noise analysis indicated that the slow potassium current passes through channels with a mean lifetime of about 14 ms and conductance of 14 pS (at -10 mV and room temperature). Injection of the messenger RNA also induced the formation of fast sodium and potassium channels activated by voltage, and channels activated by kainate.

4-Aminopyridine↗
PubMed1984

Chloride current induced by injection of calcium into Xenopus oocytes.

Membrane currents of Xenopus oocytes were studied with the membrane under voltage clamp. Intracellular injection of the calcium-chelating agent EGTA reduced, or abolished, the transient outward chloride current normally activated by membrane depolarization. Intracellular injection of calcium ions evoked large membrane currents, which inverted direction close to the chloride equilibrium potential. Injections of strontium, or barium, were less effective than calcium, while magnesium was ineffective. Large chloride currents could be evoked by calcium injections in oocytes which showed only small or no transient outward currents. The current activated by calcium injection increased with increasing depolarization up to high (ca. +60 mV) positive potentials, even though the transient outward current was suppressed by strong depolarization. The results indicate that the transient outward current depends upon entry of calcium through voltage-gated calcium ion channels and show that the oocyte membrane contains numerous chloride channels which are activated by intracellular calcium. Only a few of these chloride channels are activated by depolarization.

Animals↗
PubMed1983

Voltage-operated channels induced by foreign messenger RNA in Xenopus oocytes.

Poly(A)+ messenger RNA (mRNA) extracted from rat brains or from cat muscles was injected into Xenopus laevis oocytes. This led to the incorporation of voltage-operated Na+ and K+ channels into the oocyte membrane. These channels are not normally present in the oocyte and presumably result from the synthesis and processing of proteins coded by the injected mRNA. Tetrodotoxin blocked the Na+ channels induced by mRNA derived from either innervated or denervated muscle.

Animals↗
PubMed1983

Serotonin receptors induced by exogenous messenger RNA in Xenopus oocytes.

When poly(A)+-mRNA, extracted from rat brain, was injected into Xenopus laevis oocytes, it induced the appearance of serotonin receptors in the oocyte membrane. Application of serotonin to injected oocytes elicited, after a long delay, oscillations in membrane current. The equilibrium potential of this current corresponded with the chloride equilibrium potential. It appears that rat brain mRNA encodes the translation of serotonin receptors into the oocyte membrane. The combination of serotonin with these receptors leads to the opening of membrane channels.

Animals↗
PubMed1983

Calcium transients in frog skeletal muscle fibres following conditioning stimuli.

1. Intracellular Ca(2+) transients were recorded from frog twitch muscle fibres, using arsenazo III as a Ca(2+) monitor. When fibres were stimulated by two action potentials, the arsenazo signal to the second stimulus was smaller than the first, for stimulus intervals of up to several seconds.2. The recovery of the amplitude of the second response followed two exponential time courses; a fast one with a time constant of about 70 msec giving recovery to about 90% of the control value, followed by a slow recovery to 100%, with a time constant of about 12 sec (at 10 degrees C).3. The time constant of the fast recovery component was strongly temperature-dependent, with a Q(10) of approximately 2.7, whilst the Q(10) of the slow component was about 1.4.4. Removal of Ca(2+) in the bathing medium lengthened the time constant of the slow recovery component by a factor of three, but had little effect on the fast recovery component. The lengthening of the slow component was not reversed by addition of Mg(2+), but Sr(2+) ions could substitute for Ca(2+).5. The influence of membrane potential on the recovery time-course was investigated after blocking action potentials with tetrodotoxin, using a voltage clamp to control membrane potential. Paired depolarizing stimuli were used, with the potential held to either low (-60 or -80 mV) or high (-110 or -140 mV) potentials between stimuli. No differences were apparent in either the fast or slow recovery components at these holding potentials.6. The arsenazo response elicited by an action potential following a conditioning tetanus was reduced in size even more strongly than following a single action potential. The time course of recovery of the response following a tetanus again comprised two exponential components. After a 20 Hz tetanus for 0.5 sec, the fast component had a time constant of about 400 msec, and gave a recovery to about 60% of the control value. Subsequent recovery to 100% occurred with a time constant of about 12 sec.7. The time constant of the fast recovery component increased markedly with increasing frequency or duration of the conditioning tetanus. The time constant of the slow component was not appreciably altered by conditioning tetani varying between one impulse and sixty impulses. However, the reduction in response size due to the slow component, extrapolated to zero stimulus interval, increased with increasing number of impulses in the tetanus.8. The time constant of the fast recovery component corresponded closely with the decay time constant of the arsenazo response to the conditioning stimulus. This correspondence held over a nearly fifty-fold range of time constants, and for two different conditions which affected the decay time constant (temperature, and frequency of tetanic stimulation).9. The decay time constant of the arsenazo response elicited by an action potential was slowed by a preceding impulse or tetanus. Following a 20 Hz tetanus for 0.5 sec, recovery of the half decay time appeared to follow an exponential time course, with a time constant of about 12 sec.10. These results suggest that the fast recovery component reflects the re-filling of release stores in the sarcoplasmic reticulum by Ca(2+) ions taken up from the cytoplasm. The origin of the slow component is less clear, but it may arise from inactivation of the excitation-contraction (e-c) coupling process between T-tubule depolarization and Ca(2+) release from the sarcoplasmic reticulum.

Action Potentials↗
PubMed1983

Calcium transients studied under voltage-clamp control in frog twitch muscle fibres.

1. Intracellular calcium transients were recorded from frog twitch muscle fibres in response to voltage-clamped depolarizing pulses, using arsenazo III as an intracellular calcium monitor. The object was to investigate the time- and voltage-dependent characteristics of the coupling process between membrane depolarization and calcium release from the sarcoplasmic reticulum (s.r.)2. To examine the extent to which the T-tubule membrane potential was controlled during clamp pulses, the dye NK 2367 was used as an optical probe of tubular potential. This indicated that the tubular time constant is about 0.6 msec.3. Strength-duration curves were obtained for depolarizing pulses required to give both threshold mechanical contraction and calcium signal. Curves measured in these two ways were closely similar.4. Changes in holding potential altered the strength-duration curve for calcium release so that at more positive holding potentials a shorter pulse was needed to obtain a response for any given pulse amplitude.5. A latency of a few milliseconds was observed between the onset of depolarization and the initial rise of the calcium signal. This became shorter with stronger depolarizations, but approached a minimum at potentials above about +25 mV.6. Subthreshold depolarizations applied before a test pulse increased the size and decreased the latency of the calcium signal. Conditioning hyperpolarizations had opposite effects.7. The rate of build-up of potentiation or depression of response size seen with subthreshold de- and hyperpolarizing conditioning pulses was examined using conditioning pulses of different durations. For both pulses this process showed a time constant of about 3 msec (at 10 degrees C).8. The rate of decay of potentiation or depression was similarly measured, using a gap of variable duration between conditioning and test pulses. For both de- and hyperpolarizing pulses this showed a time constant of about 5 msec (10 degrees C).9. The relationship between conditioning pulse potential, and the size of calcium signal elicited by a following test pulse was non-linear.10. Subthreshold pulses immediately following a brief test pulse affected the size of the calcium signal in a similar way to preceding conditioning pulses.11. The relationship between potential and size of the calcium signal was examined using pulses of 3 and 20 msec duration. With the long pulse the relation was roughly sigmoid, but with the short pulse continued to rise even at strongly positive potentials.12. The results are discussed in terms of a model in which the exponential build-up of a hypothetical coupler in the excitation-contraction (e.-c.) coupling process is presumed to lead to calcium release when a threshold level is exceeded.

Animals↗
PubMed1983

Changes in threshold for calcium transients in frog skeletal muscle fibres owing to calcium depletion in the T-tubules.

Strength-duration curves were measured for voltage-clamp depolarizations required to elicit a just detectable rise in intracellular calcium, as monitored using arsenazo III, in frog twitch muscle fibres. In normal Ringer solution, the threshold for a 5 sec duration depolarization was about 5 mV more negative than for a 200 msec duration pulse. The shift in threshold comparing 200 msec and 5 sec pulses was almost abolished in bathing solutions including magnesium or nickel (4 mM), or where the free calcium concentration was buffered. The shift in threshold was little changed by substitution of barium for calcium. These results can be explained by supposing that the 5 sec depolarization activates an inward calcium flux across the T-tubule membrane, which decreases the calcium concentration in the tubules, and hence alters the threshold for activation of excitation-contraction (e.-c.) coupling because of surface charge effects.

Animals↗
PubMed1982

Synthesis of chick brain GABA receptors by frog oocytes.

Poly(A)-mRNA, extracted from the optic lobe of chick embryos, directs the synthesis of gamma-aminobutyric acid (GABA) receptors in Xenopus laevis oocytes. The receptors are inserted into the oocyte membrane, where they form receptor--channel complexes. When activated by GABA, and related agonists, the chick brain receptors open membrane channels that are permeable to chloride ions. Thus, Xenopus oocytes provide a novel and useful approach to the study of brain receptors.

Animals↗
PubMed1982

Calcium transients evoked by action potentials in frog twitch muscle fibres.

1. Intracellular Ca(2+) transients were recorded from frog twitch muscle fibres in response to action potentials and repetitive stimulation, using ionophoretically injected arsenazo III as a Ca(2+) monitor. A dual wave-length optical system was used to measure absorbance changes of the injected dye from small areas of single fibres within the cutaneous pectoris muscle.2. The absorbance spectrum of the injected arsenazo III in a resting fibre was consistent with an intracellular free Mg(2+) level of a few hundred micromolar, assuming an intracellular pH of 7.1. The resting free Ca(2+) concentration was below the limit of resolution.3. The wave-length dependence of the arsenazo light absorbance signal during twitches followed that expected for Ca(2+) binding to the dye. Recordings made at wave-lengths where the dye is maximally sensitive to pH or Mg(2+) concentration changes indicated that interference from these sources is minimal at the usual wave-length pair (650-700 nm) used for Ca(2+) recordings.4. Over a wide range of intracellular dye concentrations, the size of the arsenazo response to an action potential increased linearly with dye concentration (100-1000 muM), although there were deviations from this relationship at low and high concentrations.5. An approximate estimate of 8 muM was obtained for the peak free Ca(2+) concentration change following a single action potential. Changes in temperature (6-25 degrees C) did not significantly affect the size of the free Ca(2+) transient. During maximal tetanic stimulation the signal rose to about three times higher than the twitch response. An approximate minimum estimate of the increase in total cytoplasmic Ca(2+) concentration during a twitch gave a value of 220 muM.6. A latency of about 1.5 ms (at 10 degrees C) was observed between the foot of an action potential and the onset of the arsenazo response. Recordings made using a narrow measuring light slit, placed either at the edge or the centre of a fibre, suggested that only a small part of this latency could be due to inward conduction of the action potential along the T-tubules.7. The decay phase of the arsenazo response to an action potential followed an exponential time course, with a time constant of 71 ms at 10 degrees C. This time constant was strongly temperature-dependent, with a Q(10) of about 2.4. An Arrhenius plot of the decay time constant gave a straight line.8. During repetitive stimulation, the arsenazo responses evoked by successive impulses showed two changes: a progressive decrease in amplitude and a slowing of the decay. The extent to which successive responses summated during a tetanus depended upon the balance between these two effects.

Action Potentials↗
PubMed1981

Effects of membrane polarization on sarcoplasmic calcium release in skeletal muscle.

Calcium release from the sarcoplasmic reticulum was investigated in voltage-clamped, tetrodotoxin-treated frog skeletal muscle fibres injected with arsenazo III. Short (5 ms) depolarizing pulses (test pulses) produced a transient change in arsenazo III absorption, signalling an increase in intracellular calcium in concentration (calcium transient). Conditioning subthreshold depolarizations, which preceded the test pulse, potentiated the calcium transient triggered by the test pulse. Conditioning hyperpolarizations, applied either before or after the test pulse, inhibited the calcium transient. These effects of conditioning polarizations on the calcium transient may explain similar effects of subthreshold polarizations on muscle contraction that have previously been reported. The potentiating effect of subthreshold depolarizations was observed only when the test pulse was short (5 ms). The potentiating effect develops at -48 mV with a time constant of about 7 ms at 6.5 degrees C; this seems to be slower than that predicted by the potential spread from the surface along the tubular system. Thus, part of the effect could arise from the coupling process between tubular depolarization and calcium release.

Animals↗
PubMed1981

Calcium transients recorded with arsenazo III in the presynaptic terminal of the squid giant synapse.

Transient changes in free intracellular Ca2+ concentration were monitored in the presynaptic terminal of the giant synapse of the squid, by means of the Ca2+-sensitive dye arsenazo III. Calibration experiments showed a linear relation between the amount of Ca2+ injected by iontophoresis into the terminal, and the peak size of the arsenazo light absorbance record. A light signal could be detected on tetanic stimulation of the presynaptic axon bathed in sea water containing 45 mM Ca2+. During a 1 s tetanus the light signal rose approximately linearly, even though transmitter release declined rapidly and the light signal subsequently declined with a half-time of 2-6 s. The Ca2+ transient elicited by single nerve impulses was recorded by signal averaging, and showed a time course very much slower than the duration of transmitter release.

Animals↗