bluuebunny/crossref_metadata_2025 huggingface dataset:
The relationship between light, dopamine release and horizontal cell coupling in the mudpuppy retina.
Authors: Dong C J , McReynolds J S
Published: December 2014

1. The effect of different experimental conditions on electrical coupling between horizontal cells in the mudpuppy retina was studied by comparing the changes in responses to illumination of the central and peripheral portions of the receptive field, using centred spot and annulus stimuli. An increase in the amplitude of the response to a centred spot stimulus and a decrease in the amplitude of the response to a concentric annulus indicated a decrease in coupling, and vice versa. 2. Dopamine (10‐250 microM) caused a decrease in coupling between horizontal cells. The uncoupling effect of dopamine was much greater in dark‐adapted than in light‐adapted retinas. The effect of the D1‐receptor agonist SKF38393 was similar to that of dopamine. The effect of the D2‐receptor agonist LY171555 on coupling was opposite to that of dopamine; this was attributed to a reduction in endogenous dopamine release. 3. The D1 antagonist SCH23390 (15 microM) caused an increase in coupling between horizontal cells. This effect was much greater in light‐adapted than in dark‐adapted retinas. 4. The glutamate analogue 2‐amino‐4‐phosphonobutyrate (APB), which hyperpolarizes on‐centre bipolar cells and blocks their responses to light, caused an increase in coupling between horizontal cells. This effect of APB was greater in light‐adapted retinas than in dark‐adapted retinas. The effect of APB on coupling could be reversed by the addition of dopamine, but the effect of dopamine on coupling could not be reversed by the addition of APB. These results suggest that APB increases horizontal cell coupling by causing a decrease in dopamine release. 5. In dark‐adapted retinas, 2.5 min exposure to an adapting light caused a decrease in coupling between horizontal cells; the uncoupling effect of the adapting light was blocked in the presence of either SCH23390 or APB. 6. The results suggest that coupling between horizontal cells in the mudpuppy retina is decreased by dopamine acting at D1 receptors, that the release of dopamine affecting horizontal cells is greater under light‐adapted conditions, and that the pathway by which exposure to light increases this dopamine release is mainly via on‐centre bipolar cells.
Serotonin facilitates GABAergic transmission in the CA1 region of rat hippocampus in vitro.
Authors: Ropert N , Guy N
Published: December 2014

1. The effect of serotonin on inhibitory synaptic transmission was examined in forty‐one CA1 pyramidal neurones using intracellular voltage recordings in vitro. 2. Serotonin (20‐50 microM) increased the synaptic noise of most (85%) neurones loaded with chloride (n = 33). The duration of this effect was enhanced with increasing concentrations of serotonin and was fully reversible within 5 min. When serotonin was applied at short intervals (less than 10 min), fading of the response was observed. 3. The effect of serotonin on synaptic noise persisted in the presence of the glutamate NMDA and non‐NMDA antagonists, APV (100 microM) and CNQX (10 microM), but it was blocked (n = 5) by a GABAA antagonist, bicuculline (10 microM). 4. The increase in inhibitory synaptic events resulted from an enhanced frequency of unitary IPSPs from 4.6 +/‐ 3.8 Hz in control to 17.2 +/‐ 12.5 Hz (n = 5) in serotonin, especially of large events. Serotonin caused no change in the amplitude and frequency of miniature synaptic events recorded in the presence of TTX (n = 5). The mean amplitude of unitary inhibitory postsynaptic potentials (IPSPs) increased from 1.37 +/‐ 0.35 mV in control to 3.67 +/‐ 1.38 mV in serotonin. The coefficient of variation of unitary IPSPs increased from 0.40 +/‐ 0.11 in control to 0.74 +/‐ 0.23 in serotonin when quantal size appeared unchanged. 5. The 5‐HT3 agonist 2‐methyl‐serotonin (52 microM, n = 4) partially mimicked the effect of serotonin, increasing the inhibitory noise without affecting the pyramidal neurone conductance. The serotonin‐induced facilitation of unitary IPSPs was blocked by the 5‐HT3 antagonists ICS 205‐930 (1‐90 nM, n = 3) and metoclopramide (30 microM, n = 1). 6. These results suggest that serotonin directly excites GABAergic interneurones acting on a 5‐HT3 receptor and consequently increasing the frequency of inhibitory synaptic events recorded in CA1 pyramidal cells.
Synaptic integration at a sensory‐motor reflex in the leech.
Authors: Gu X N , Muller K J , Young S R
Published: December 2014

1. In the medicinal leech the distribution of synapses from the pressure sensory (P) neurone to the annulus erector (AE) motoneurone and the site of impulse initiation in the AE cell were determined to understand better the integration of sensory inputs by the motoneurone. 2. The axon of the AE cell bifurcates before leaving the ganglion. Laser photoablation experiments indicated that the axon proximal to the bifurcation is inexcitable. Two techniques, laser photoablation and measurement of impulse timing, each located the site of impulse initiation at the bifurcation. 3. The medial P cell makes a monosynaptic connection with the AE cell, eliciting an excitatory postsynaptic potential (EPSP) of 1‐3 mV amplitude recorded in the AE cell soma. 4. Intracellular injection of dyes into separate cells showed that P cell branches appear to contact AE cell branches both ipsilaterally and contralaterally. Laser photoablation of selected portions of the P and AE cells' axons revealed functional contacts on both sides. 5. The primary axon bifurcation of the AE cell is the site of integration of synaptic potentials that spread passively from both sides of the ganglion. These summed synaptic potentials account for the concerted activity of the two AE cells in each ganglion.
Limitedly selective action of a delta‐agonistic leu‐enkephalin on the transmission in spinal motor reflex pathways in cats.
Authors: Schmidt P F , Schomburg E D , Steffens H
Published: December 2014

1. The influence of the delta‐opioid receptor agonist (D‐Ser2)‐leu‐enkephalin (Thr6) (DSLET) on different spinal reflex pathways was investigated in anaemically decapitated, high spinal cats. Monosynaptic reflexes were tested to analyse excitatory and inhibitory flexor reflex afferent (FRA) pathways from nociceptive (from the skin of the central pad) and non‐nociceptive (from skin, joint or group II muscle) afferents, as well as an excitatory nociceptive non‐FRA pathway from the central pad to plantaris and intrinsic foot extensors and the inhibitory pathway from Ib muscle afferents. 2. DSLET suffused over the spinal cord (concentration 10(‐3)‐10(‐6) M) caused a concentration‐dependent depression of transmission in nociceptive and non‐nociceptive FRA pathways. The excitatory FRA pathways including those from group II muscle afferents were more sensitive than the inhibitory ones. The nociceptive non‐FRA pathway from the central pad to plantaris and intrinsic foot extensors was less affected than the FRA pathways. The inhibitory pathway from Ib muscle afferents remained almost unaffected. 3. Intravenous injection of DSLET (0.5‐3.6 mg/kg) induced dose‐dependent effects similar to those from local spinal application. The main difference was that I.V. injection more readily caused depression of the inhibitory FRA pathways to extensors. 4. The effects of local spinal application and of I.V. injection of DSLET were antagonized by I.V. injection of naloxone (0.1‐1 mg/kg). 5. The effects of DSLET on spinal reflex pathways in many respects resemble that of monoamines. Possibly there is an interaction and a co‐operation of enkephalins and monoamines in motor control.
Voltage‐activated calcium and potassium currents in human pancreatic beta‐cells.
Authors: Kelly R P , Sutton R , Ashcroft F M
Published: December 2014

1. The whole‐cell configuration of the patch clamp technique was used to study inward and delayed outward currents in beta‐cells isolated from human pancreatic islets. 2. The delayed outward current activated at about ‐20 mV and increased linearly with further depolarization. The instantaneous current‐voltage (I‐V) relation, measured by tail current analysis, reversed at ‐70 mV. This is close to the K+ equilibrium potential and suggests the outward current is carried primarily by potassium ions. In support of this idea, outward currents were abolished when internal K+ was replaced by the impermeant cation N‐methyl‐D‐glucamine (NMG). 3. The voltage dependence of K+ current activation could be fitted by a sigmoidal function with a mid‐point at +1 mV. K+ currents showed voltage‐dependent inactivation which was half‐maximal at ‐25 mV. 4. Inward currents were studied after outward currents were suppressed by replacing internal potassium with NMG. In 5 mM [Ca2+]o, the inward current activated between ‐50 and ‐40 mV, had a peak amplitude at ‐10 mV and reversed at potentials positive to +60 mV. The voltage dependence of inward current activation was sigmoidal with half‐maximal activation at ‐10 mV in 5 mM [Ca2+]o and at ‐22 mV in 5 mM [Ba2+]o. 5. Inward currents were unaffected by tetrodotoxin (TTX), but could be blocked by cadmium ions. Barium was also capable of carrying inward current. This pharmacology is consistent with inward currents flowing through Ca2+ channels. 6. The inactivation of the inward current was dependent on calcium entry. In two‐pulse experiments, the voltage dependence of inactivation was U‐shaped, and resembled that of the calcium current. Barium currents showed little inactivation. 7. In two‐pulse experiments the degree of inward current inactivation during the test pulse was related to the amount of calcium entry during the first pulse. Calcium entering at more positive potentials was less effective at producing inactivation. 8. Calcium and barium currents also showed a slow, voltage‐dependent inactivation when the holding potential was changed between ‐100 and ‐40 mV. This inactivation developed with a time course of seconds. 9. The Ca2+ and K+ currents described here are similar to those reported for rodent beta‐cells and indicate the rodent beta‐cell provides a good model for that of man.
Mechanisms of excitation‐contraction coupling failure during metabolic inhibition in guinea‐pig ventricular myocytes.
Authors: Goldhaber J I , Parker J M , Weiss J N
Published: December 2014

1. The effects of complete metabolic inhibition on excitation‐contraction coupling in heart were studied by exposing patch‐clamped guinea‐pig ventricular myocytes, loaded via the patch pipette with the Ca2+ indicator Fura‐2 (0.1 mM), to carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP, 1 microM) and 2‐deoxyglucose (2‐DG, 10 mM) while simultaneously recording membrane current, Fura‐2 fluorescence, and cell motion. The patch pipette solution contained Cs+ and TEA (tetraethylammonium) to partially block K+ currents. 2. During voltage clamps from a holding potential of ‐40 mV to a test potential of 0 mV, complete metabolic inhibition decreased the Ca2+ current (ICa), activated the ATP‐sensitive K+ current, modestly elevated diastolic [Ca2+]i and markedly reduced the [Ca2+]i transient without altering its voltage dependence. Active shortening was impaired and diastolic cell length decreased prior to large increases in diastolic [Ca2+]i, consistent with rigor induced by ATP depletion. Return of the [Ca2+]i transient to baseline and relaxation upon repolarization were also delayed. 3. Despite the depression of the peak [Ca2+]i transient induced by membrane depolarization during metabolic inhibition, the [Ca2+]i transient induced by a rapid exposure to 5 mM‐caffeine was greater than control. The Na(+)‐Ca2+ exchange current during the caffeine‐induced [Ca2+]i transient was not affected by metabolic inhibition. 4. [Ca2+]i transients depressed by metabolic inhibition could be enhanced by augmenting ICa with elevated [Ca2+]o (10 mM) and Bay K 8644 (5 microM). 5. To study the relationship between the magnitude of ICa and the amplitude of the [Ca2+]i transient, ICa was modulated either by (a) voltage clamping the cell to different membrane potentials at constant [Ca2+]o or by (b) rapidly altering [Ca2+]o immediately prior to a voltage clamp to a fixed membrane potential. Under control conditions, the relationship between the size of ICa and the magnitude of the [Ca2+]i transient was the same whether ICa was modulated by altering membrane potential or [Ca2+]o, suggesting that membrane potential does not significantly modulate the Ca(2+)‐induced Ca2+ release mechanism of cardiac excitation‐contraction coupling. 6. After metabolic inhibition, however, the same ICa released less Ca2+ than under control conditions, consistent with some impairment of the Ca2+ release mechanism. 7. These results suggest that under conditions in which excitability is maintained by controlling membrane voltage and minimizing metabolically sensitive K+ currents, the decreased [Ca2+]i transient observed during metabolic inhibition severe enough to induce rigor is caused primarily by depression of ICa and not by depletion of intracellular Ca2+ stores. Additional factors also modestly hinder Ca2+ release from intracellular stores during metabolic inhibition.
Influence of temperature on the response time of mitochondrial oxygen consumption in isolated rabbit heart.
Authors: Hak J B , van Beek J H , van Wijhe M H , Westerhof N
Published: December 2014

1. In this study we determined the temperature dependence of the mean response time of cardiac mitochondrial oxygen consumption following steps in metabolic demand. Metabolic demand was altered by stepwise changes in heart rate or in left ventricular volume at 20 and 28 degrees C. 2. Ten isolated rabbit hearts were perfused with Tyrode solution at constant oxygen tension and constant arterial flow. A balloon was inserted in the left ventricle and developed pressure was measured. Coronary venous oxygen tension was measured continuously with a Clark‐type oxygen electrode. 3. The mean response time of mitochondrial oxygen consumption is defined as the first statistical moment of the impulse response function. This mean response time of mitochondrial oxygen consumption, following the change in metabolic demand, is calculated from the measured mean response time for the change in coronary venous oxygen tension by subtracting the transport time resulting from diffusion and convective transport in the blood vessels. The transport time is obtained from a model for oxygen transport developed previously. Experimental data, necessary for the model calculation, were obtained from measurement of the coronary venous oxygen tension transients following stepwise changes either in arterial oxygen tension or perfusion flow. 4. The calculated mean response times of mitochondrial oxygen consumption were 26.9 +/‐ 3.0 s (mean +/‐ S.E.M.) at 20 degrees C and 14.9 +/‐ 1.0 s at 28 degrees C. The mean response times of mitochondrial oxygen consumption did not differ significantly for steps in heart rate and in left ventricular volume and between upward and downward steps. 5. We suggest that intracellular calcium concentration is not the sole regulator of mitochondrial oxygen consumption in the isolated rabbit heart, since steps in heart rate and in left ventricular volume showed the same time course of oxygen uptake. 6. The mean response time of mitochondrial oxygen consumption obtained in the isolated rabbit heart at 20 degrees C did not differ significantly from the mean response time of mitochondrial oxygen consumption of isolated rabbit papillary muscle. After combining our data with previously published data on empty beating hearts at 37 degrees C, a Q10, which is the factor by which the mean response time of mitochondrial oxygen consumption increases per 10 degrees C decrease in temperature, of 2.1 was calculated.
On the mechanism of action of picrotoxin on GABA receptor channels in dissociated sympathetic neurones of the rat.
Authors: Newland C F , Cull‐Candy S G
Published: December 2014

1. The mechanism of action of picrotoxin on GABA receptor channels in rat sympathetic neurones has been investigated with whole‐cell clamp. In addition, the action of picrotoxin on single GABA channels has been examined in outside‐out membrane patches from these cells. 2. Picrotoxin, at concentrations which dramatically reduced the amplitude of whole‐cell GABA currents, did not alter the spectral time constants or single‐channel conductance estimated by analysis of GABA‐activated current noise. This was observed at potentials both negative and positive to the GABA reversal potential (i.e. for both inward and outward GABA currents). In control conditions, the slow and fast time constants from GABA noise were 40 +/‐ 14 ms and 2 +/‐ 0.4 ms, while the estimated single‐channel conductance was 14 +/‐ 2 pS. In the presence of picrotoxin, the time constants and estimated single‐channel conductance were 41 +/‐ 5 ms, 2.7 +/‐ 0.6 ms and 15 +/‐ 2.3 pS. 3. Picrotoxin did not alter the shape of the whole‐cell GABA current‐voltage relationship, indicating that the steady‐state block was not voltage dependent. The lack of effect of picrotoxin on the GABA noise spectra and the lack of outward rectification makes it unlikely that picrotoxin acts by a simple voltage‐dependent (or voltage‐independent) channel blocking mechanism. In the presence of picrotoxin the reversal potential for GABA remained at approximately 0 mV in symmetrical chloride. 4. Distributions of total burst durations, obtained from single‐channel records with low concentrations of GABA, were fitted with three or four exponential components. Picrotoxin had no consistent effect on the time constants of the total burst length distributions. It also did not alter the amplitude of the main conductance state. However, picrotoxin did reduce the frequency of channel openings. 5. The application of brief ionophoretic pulses of GABA, to cells under whole‐cell voltage clamp, revealed that the rate of onset of block by picrotoxin was accelerated in the presence of GABA. In the absence of agonist, picrotoxin produced a more slowly equilibrating block. 6. Our data are consistent with a mechanism whereby picrotoxin binds preferentially to an agonist bound form of the receptor and stabilizes an agonist‐bound shut state. This could, for example, mean that picrotoxin enhances the occurrence of a desensitized state or an allosterically blocked state.
Proton‐induced sodium current in freshly dissociated hypothalamic neurones of the rat.
Authors: Ueno S , Nakaye T , Akaike N
Published: December 2014

1. The proton‐gated current was investigated in freshly dissociated ventromedial hypothalamic (VMH) neurones from 4‐week‐old Wistar rats, under whole‐cell configuration by the use of the ‘concentration‐clamp’ technique which combines intracellular perfusion with the rapid exchange of external solution within 1‐2 ms under a single‐electrode voltage‐clamp condition. 2. The proton‐gated current increased in a sigmoidal fashion as extracellular pH (pHo) decreased. In external solution containing 2 mM‐Ca2+, the threshold of current activation was at pHo 6.5, and the maximum response appeared at pHo 4.1‐3.9. The dissociation constant (Kd) and Hill coefficient were 10(‐4.9) M (pHo = 4.9) and 1.5 respectively. 3. Decreasing extracellular Na+ concentration reduced the proton‐gated current. The current reversed direction at the Na+ equilibrium potential (ENa), indicating that it was carried by Na+. 4. The activation phase kinetics of proton‐induced current was single exponential. The time constant of activation (tau a) did not have a potential dependence but decreased slightly by decreasing pHo. The inactivation phase kinetics was two‐exponential. The time constant of inactivation (tau i) consisted of fast and slow components (tau if and tau is, respectively). Like tau a, both tau if and tau is did not have any potential dependence, but they slightly increased with decreasing pHo. 5. The steady‐state inactivation curve, constructed by decreasing pHo from various conditioning pHos to 4.1, revealed that the proton‐induced current had a half‐maximum inactivation at pHo 6.2. 6. The proton‐induced current was suppressed as the extracellular Ca2+ concentration ([Ca2+]o) increased from almost free (0.01 mM) to 80 mM. Increasing [Ca2+]o increased tau a, but slightly decreased both tau if and tau is. 7. Recovery of proton‐induced current from complete inactivation of proton‐induced current depended on the degree of pHo change. A bigger change in pHo induced faster recovery than a smaller change. 8. External divalent cations inhibited the proton‐induced current, and the inhibitory potency was in the order of Mn2+ greater than Co2+ greater than Ca2+ greater than Sr2+ greater than Ba2+ greater than Mg2+. 9. Tetrodotoxin (TTX) at relatively low concentration (less than 10(‐7) M) did not inhibit the peak amplitude of the proton‐induced current, but at a higher concentration (10(‐6) M) it slightly inhibited the peak amplitude of the current and accelerated the inactivation process. Scorpion toxin markedly increased the peak amplitude of the proton‐induced current and prolonged the inactivation phase. The tau is was also increased by scorpion toxin in a concentration‐dependent manner. Veratridine had no effect on the proton‐induced current. 10. The membrane properties of the proton‐operated channel were similar to those of the voltage‐gated Na+ channel rather than the Ca2+ channel.
Effects of NG‐nitro‐L‐arginine and L‐arginine on regional cerebral blood flow in the cat.
Authors: Kovách A G , Szabó C , Benyó Z , Csáki C , Greenberg J H , Reivich M
Published: December 2014

1. We studied the effects of NG‐nitro‐L‐arginine (NOLA), a potent inhibitor of the L‐arginine‐nitric oxide pathway, and L‐arginine, the precursor of nitric oxide, on regional cerebral blood flow, electrocortical activity and ex vivo cerebrovascular reactivity in the cat. Flow was measured via radiolabelled microspheres, and vascular responses were studied by measuring isometric tension of isolated middle cerebral arterial rings. 2. NOLA (30 mg kg‐1 bolus followed by 1 mg kg‐1 min‐1 infusion) caused an approximately 40 mmHg elevation in the mean arterial blood pressure, a regionally heterogenous increase of the regional cerebrovascular resistance and a decrease in the regional cerebral blood flow 15 and 40 min after the start of its administration. In contrast L‐arginine (30 mg kg‐1 bolus followed by 10 mg kg‐1 min‐1 infusion) did not alter blood pressure, cerebrovascular resistance nor regional cerebral blood flow 15 min after the start of its administration. The NOLA‐induced changes in tissue flow were the most pronounced in the cerebellum, pituitary and medulla oblongata, whereas there was no decrease in the flow of the cortex and white matter. 3. NOLA caused characteristic changes in total fronto‐occipital EEG power and in power spectra which were unlikely to have been due to cerebral ischaemia. In addition, the ex vivo reactivity of the middle cerebral arteries showed signs of impaired endothelial nitric oxide synthesis: there were enhanced noradrenaline‐induced contractions and N‐ethoxycarbonyl‐3‐morpholino‐sydnonimine (SIN‐1)‐induced relaxations and markedly attenuated acetylcholine‐ and ATP‐induced relaxations after NOLA treatment. 4. The present data indicate that resting cerebral blood flow and cerebrovascular resistance are regulated by nitric oxide derived from L‐arginine in a regionally heterogenous way and that exogenous L‐arginine availability is not a limiting factor in this nitric oxide generation. Possibly, both the vascular endothelium and the neurons contribute to this basal nitric oxide release.
Isoprenaline, Ca2+ and the Na(+)‐K+ pump in guinea‐pig ventricular myocytes.
Authors: Gao J , Mathias R T , Cohen I S , Baldo G J
Published: December 2014

1. The whole‐cell patch clamp technique was employed to study the effects of the beta‐agonist isoprenaline (ISO) on the Na(+)=K+ pump current, Ip, in acutely isolated ventricular myocytes from guinea‐pig hearts. Propranolol, a beta‐adrenergic antagonist, was used to demonstrate that all of the effects of ISO, stimulatory or inhibitory, are mediated by beta‐receptors. 2. Below about 150 nM [Ca2+]i, we find that ISO reduces Ip, while above this [Ca2+]i ISO increases Ip. The stimulatory and inhibitory effects of ISO on Ip are independent of either intracellular sodium ([Na+]i) or extracellular potassium ([K+]o). These results suggest that the end‐effect of ISO is directly on the maximum pump turnover rate (Vmax) rather than indirectly through changes in [Na+]i or [K+]o or modulatory effects on Na+ or K+ affinity. 3. The maximum effect of ISO increases Ip by 25% when [Ca2+] is buffered at 1.4 microM. A half‐maximal effect is reached at roughly 10 nM‐ISO and a near‐maximal effect by 0.5 microM. 4. The permeabilized patch technique, using amphotericin B (Horn & Marty, 1988; Rae, Cooper, Gates & Watsky, 1991), was employed to minimize changes in the normal second messenger systems and calcium buffers. In these experiments, we used a high intracellular sodium solution (pipette sodium was 50 mM), thus sodium‐calcium exchange was depressed and we expected [Ca2+]i to be above 150 nM. ISO increases Ip in these conditions as in the dialysed cells. 5. Our results suggest that beta‐stimulation can increase Ip, but only if [Ca2+]i is above about 150 nM. In the beating heart [Ca2+]i rises well above this value during systole and the average [Ca2+]i, which depends on heart rate, is expected to normally be above this level. During beta‐stimulation, the increase in Ip along with a concomitant increase in IK (Giles, Nakajima, Ono & Shibata, 1989; Duchatelle‐Gourdon, Hartzell & Lagrutta, 1989) helps prevent action potential lengthening and allows an increase in heart rate even in the presence of increased calcium current. Further, beta‐stimulation will compensate for the effects on Ip of either hypokalaemia or digitalis toxicity, and so reduce the expected rise in both [Na+]i and [Ca2+]i.
Tetrodotoxin‐sensitive calcium‐conducting channels in the rat hippocampal CA1 region.
Authors: Akaike N , Takahashi K
Published: December 2014

1. Tetrodotoxin (TTX)‐sensitive Ca2+ conducting channels which produce a transient inward current were investigated in pyramidal neurones freshly dissociated from the dorsal part of rat hippocampal CA1 region by the use of the suction‐pipette technique, which allows for intracellular perfusion under a single‐electrode voltage clamp. 2. In all cells superfused with Na(+)‐ and K(+)‐free external solution containing 10 mM‐Ca2+ and 10(‐5) M‐La3+, a transient inward Ca2+ current was evoked by a step depolarization to potentials more positive than about ‐50 mV from a holding potential (VH) of ‐100 mV. This current was inhibited by either removing the extracellular Ca2+ or adding TTX (termed as ‘TTX‐ICa’). 3. Activation and inactivation processes of the TTX‐ICa were highly potential dependent at 20‐22 degrees C, and the latter was fitted by a double exponential function. The time to peak of the current decreased from 5.0 to 2.3 ms at a test potential change from ‐50 to 0 mV. The time constants of the current decay decreased from 2.8 to 2.2 ms for fast component (tau if) and from 16.0 to 8.2 ms for slow component (tau is) at a potential change from ‐35 to ‐10 mV. 4. The TTX‐ICa was activated at threshold potential of about ‐55 mV and reached full activation at ‐30 mV. The steady‐state inactivation of TTX‐ICa could be fitted by a Boltzmann equation with a slope factor of 6.0 mV and a half‐inactivation voltage of ‐72.5 mV. 5. Biphasic recovery (reactivation) from the complete inactivation of TTX‐ICa was observed. The time constant of the major component (78.8 to 91.6% of total) of the reactivation was 13.1 ms, and that of the minor one was 120 to 240 ms. Therefore, TTX‐ICa remained fairly constant at a train of stimulation up to 3 Hz. However, the inhibition of current amplitude occurred as the repetitive stimulation increased more than 10 Hz, and considerable tonic inhibition occurred with increasing stimulation frequency. 6. When the peak amplitudes in the individual current‐voltage (I‐V) relationships of TTX‐ICa at various extracellular Ca2+ concentrations ([Ca2+]o) were plotted as a function of [Ca2+]o, the current amplitude increased linearly without showing any saturation. 7. The ratio of peak amplitude in the individual I‐V relationships of Ca2+, Sr2+ and Ba2+ currents passing through the TTX‐sensitive Ca2+ conducting channel was 1:0.33:0.05, although the current kinetics were much the same.(ABSTRACT TRUNCATED AT 400 WORDS)
Activation and desensitization of embryonic‐like receptor channels in mouse muscle by acetylcholine concentration steps.
Authors: Franke C , Költgen D , Hatt H , Dudel J
Published: December 2014

1. Pulses of acetylcholine (ACh) in concentrations between 0.1 and 1000 microM were applied repetitively to outside‐out patches of enzymatically denervated (14 days) mouse muscle with the liquid filament switch. Solutions superfusing the patch could be changed rapidly (within 0.2 ms). 2. Single‐channel activity was studied under steady‐state conditions in the outside‐out and in the cell‐attached mode. The single‐channel conductance was 26 pS in outside‐out patches, characteristic for embryonic‐like channels. Apparent mean open time was about 2.5 ms, a shorter component of closed times was 800 microseconds and burst length was about 5 ms. 3. Channel currents elicited by pulses of ACh were averaged. The time‐to‐peak current was concentration dependent and decreased from a level of about 10 ms below 10 microM to about 400 microseconds at 100 microM‐ACh. 4. For a typical experiment, the average peak current, imax, increased from ‐0.4 pA with 0.1 microM to ‐82 pA with 1000 microM‐ACh, close to the value at saturation. The half‐maximal response was at 60 microM‐ACh. The dose‐response curves for imax had double‐logarithmic slopes of 1.1‐1.3, consistent with two binding sites at the embryonic nicotinic acetylcholine receptor (nAChR). 5. The current elicited by ACh pulses decreased rapidly after the peak. The time constant of desensitization increased from 20‐50 ms with 1000 microM‐ACh to up to more than a second with 1 microM‐ACh. 6. The current in steady state (fully desensitized) increased up to 10 microM‐ACh, but decreased slightly to values of imax/100 to imax/500 when higher concentrations were applied. 7. In addition to the well‐known differences between adult and embryonic nAChR concerning the apparent mean open time and burst length, we found differences in the slope of the dose‐response curve for imax, in the ratio of peak to steady‐state response, and in the rise time of the response.
Calcium current modulation in frog sympathetic neurones: multiple neurotransmitters and G proteins.
Authors: Elmslie K S
Published: December 2014

1. Whole‐cell calcium currents of bullfrog sympathetic neurones were partially inhibited by noradrenaline (NA), chicken‐II‐luteinizing hormone‐releasing hormone (LHRH), muscarine, ATP, substance P, or intracellular dialysis with guanosine 5'‐O‐(3‐thiotriphosphate)(GTP‐gamma‐S) or aluminium fluoride. These agents had similar effects on the activation kinetics of calcium current. 2. The amplitude of the LHRH effect varied from cell to cell. This did not correlate with cell size or the time of whole‐cell dialysis. 3. The response to LHRH desensitized rapidly. Desensitization to LHRH did not affect inhibition by NA, ATP or substance P. 4. The effects of LHRH and NA were partially additive. 5. Cells dialysed with GTP‐gamma‐S still responded to NA or LHRH. However, NA or LHRH inhibited a smaller fraction of the calcium current than usual, and second applications of the same transmitter to GTP‐gamma‐S‐dialysed cells were ineffective. 6. In GTP‐gamma‐S‐dialysed cells, application of LHRH occluded the response to NA, but LHRH was still effective after application of NA. 7. The effect of GTP‐gamma‐S decreased during prolonged dialysis. 8. The effect of NA was selectively reduced by intracellular dialysis with the A‐protomer of pertussis toxin (PTX), or extracellular pretreatment with high concentrations of whole PTX at room temperature. These treatments had little or no effect on the action of LHRH or ATP. 9. It is concluded that multiple G proteins can produce identical changes in calcium channel gating. The adrenergic receptor preferentially couples to a PTX‐sensitive G protein.
Role of potassium in the reflex regulation of blood pressure during static exercise in man.
Authors: Fallentin N , Jensen B R , Byström S , Sjøgaard G
Published: December 2014

1. The relationship between [K+] in venous effluent blood and alterations in mean arterial blood pressure was studied during static handgrip contractions at 15 and 30% of maximal voluntary contraction (MVC). 2. To further elucidate the importance of K+ in the reflex regulation of blood pressure a situation with normal recovery was compared with a situation in which 3 min of post‐exercise occlusion was applied by arresting the circulation to the forearm just prior to the cessation of the contraction. 3. There was a temporal as well as quantitative correlation between venous [K+] and the blood pressure response during and after static exercise. During 30% MVC mean arterial blood pressure (MAP) attained 161.7 mmHg and venous [K+] 5.8 mM, while the corresponding values during 15% MVC were 121.5 mmHg and 5.0 mM. 4. In the occlusion period mean arterial blood pressure remained elevated above resting level and provided a measure of the magnitude of muscle chemoreflexes. In the same period venous [K+] was maintained at 5.3 mM and 4.6 mM following 30% MVC and 15% MVC respectively. This is indicative of interstitial concentrations of above 8‐10 mM. This level is sufficiently high to stimulate type III and IV muscle afferents involved in the reflex regulation of blood pressure, and strengthens the notion that K+ may play an important role in eliciting the pressor reflex. 5. In contrast to [K+] the time course of venous blood concentrations of lactate and ammonia (NH3) exhibited a clear dissociation from the blood pressure recordings.
Control of the hyperpolarization‐activated cation current by external anions in rabbit sino‐atrial node cells.
Authors: Frace A M , Maruoka F , Noma A
Published: December 2014

1. Effects of varying concentrations of anions on the hyperpolarization‐activated current (I(f)) were studied in myocytes isolated from the rabbit sino‐atrial node. Substituting Cs+ for the intracellular K+ clearly separated I(f) from the delayed rectifier K+ current. Control properties, including gating kinetics and ion selectivity, similar to previous studies were obtained. 2. Substitution of extracellular Cl‐ with larger anions including isethionate, glutamate, acetate, and aspartate, reduced the amplitude of I(f) without changing the reversal potential. Substitution with small anions such as iodide or nitrate supported an intact I(f). These effects were reproduced in the excised outside‐out patch conformation. 3. The conductance for I(f) was a saturating function of the extracellular Cl‐ concentration ([Cl‐]o) with an equilibrium binding constant (K1/2) of 11 mM and a slope factor of about 1 when substituted with large anions. Total removal of small anions completely abolished I(f). 4. The voltage‐dependent gating of I(f) was not affected by changing ([Cl‐]o), suggesting that Cl‐ modulates conductance properties of I(f). 5. The results indicate that I(f) conductance is unique in that it is dependent on an extracellular anion (Cl‐), yet it is carried exclusively by cations, K+ and Na+. These effects are independent of any measurable voltage‐dependent gating parameters.
A study of the bovine adrenal chromaffin nicotinic receptor using patch clamp and concentration‐jump techniques.
Authors: Maconochie D J , Knight D E
Published: December 2014

1. Voltage clamp records have been obtained from bovine adrenal chromaffin cells in the outside‐out and whole‐cell configurations, in response to step changes of acetylcholine (ACh) concentration. The concentrations used ranged from 50 nM to 20 mM. 2. At high acetylcholine concentrations, the activation and desensitization kinetics of the nicotinic receptor, as observed in outside‐out patches, may be described by a model incorporating a single, fast agonist binding step, and relatively slow isomerization to the open state. The affinity of the closed receptor for ACh is 310 microM, the channel opening rate constant is 460 s‐1, and the closing rate constant is 29 s‐1. 3. Single channel events, observed when nanomolar ACh concentrations are applied to whole cells, have two distinct channel lifetimes: 0.6 ms and 11‐15 ms. The variation of the frequencies of the events with ACh concentration, suggests that the short lifetimes are openings of a singly liganded receptor and the longer lifetimes are openings of a doubly liganded receptor. 4. Only a single exponential associated with receptor desensitization is seen with outside‐out patches, but two are seen with whole cells. It is postulated that there are two nicotinic receptor types present on adrenal chromaffin cells. 5. The rate of desensitization (9 s‐1 and 26 s‐1, whole cells; 24 s‐1, patches), is fast enough to be significant in determining the open channel lifetime. 6. A sudden increase in current (rebound) is observed when a high concentration of ACh is abruptly removed from outside‐out patches. This is evidence for a blocked state. The affinity of the blocking site for ACh is 1400 microM (outside‐out patches). 7. The total number of activatable nicotinic channels per whole cell is estimated to be 2600.
Inhibition of cytokine‐induced nitric oxide production by transforming growth factor‐beta 1 in human smooth muscle cells.
Authors: Junquero D C , Scott‐Burden T , Schini V B , Vanhoutte P M
Published: December 2014

1. Experiments were performed to investigate the effects of human recombinant interleukin‐1 beta on the production of vasoactive substances by human aortic smooth muscle cells in culture. Smooth muscle cells were cultured either on microcarrier beads for bioassay experiments, or in multiwell plates for the determination of nitrite levels. 2. Cells were grown on microcarrier beads, treated with interleukin‐1 beta or vehicle (control) for 24 h, and packed in a column which was perfused with oxygenated Krebs‐Ringer solution in the presence of indomethacin. The activity of the perfusates was bioassayed by measuring the changes in tension of a contracted ring of Wistar rat aorta without endothelium, and by evaluating the modulation of thrombin‐induced platelet aggregation. 3. Perfusates from interleukin‐1 beta treated cells evoked relaxations of the contracted detector tissues, and microcarrier beads covered with treated cells inhibited thrombin‐induced platelet aggregation. Superoxide dismutase enhanced these effects whereas Methylene Blue abolished them. Control cells evoke neither relaxation nor inhibition of platelet aggregation. Interleukin‐1 beta induced a time‐ and concentration‐dependent production of nitrite. Cycloheximide and nitro‐L‐arginine inhibited the relaxations and the production of nitrite evoked by interleukin‐1 beta‐treated cells. L‐Arginine but not D‐arginine overcame the blockade elicited by nitro‐L‐arginine. Transforming growth factor‐beta 1 reduced the interleukin‐1 beta‐dependent generation of nitrite by cultured smooth muscle cells and relaxation of contracted bioassay tissues. 4. Interleukin‐1 beta, transforming growth factor‐beta 1, Methylene Blue and L‐arginine‐related compounds did not induce significant variations of tension of the detector rings. 5. These data demonstrate that the inflammatory and immunological mediator interleukin‐1 can stimulate the production of a nitric oxide‐like substance(s) in cultured human smooth muscle cells leading to the activation of soluble guanylate cyclase. Liberation of transforming growth factor‐beta by activated platelets may inhibit these reactions.
A patch clamp study of the nicotinic acetylcholine receptor of bovine adrenomedullary chromaffin cells in culture.
Authors: Nooney J M , Peters J A , Lambert J J
Published: December 2014

1. Acetylcholine‐induced currents recorded from bovine adrenal medullary chromaffin cells maintained in culture were studied during pressure or ionophoretic applications of ACh, using the ‘whole‐cell’ and ‘outside‐out’ configurations of the patch clamp technique. In standard salines, ACh evoked whole‐cell currents of ‐38 pA to ‐1 nA at ‐60 mV, which had a reversal potential (EACh) of ‐7.1 +/‐ 0.6 mV. The ACh current‐voltage relationship was characteristically linear at negative holding potentials and biphasic at positive holding potentials, displaying a region of almost zero slope conductance between 0 and +40 mV followed by a region of positive slope conductance at more positive potentials. 2. Relative permeation to cations was examined. Substitution of external Na+ by sucrose resulted in a ‐42 mV shift of EACh for a 10‐fold reduction in [Na+]o. Using isotonic substitutions, the permeability ratios (relative to Na+) for monovalent cations were determined to be 1.32 +/‐ 0.02 for Cs+ (n = 11), 1.03 +/‐ 0.02 for Li+ (n = 8) and 0.18 +/‐ 0.02 for Tris+ (n = 7). Elevated external Ca2+ salines were found to shift EACh to more positive potentials, especially in the presence of low external Na+. 3. The nicotinic agonists nicotine, tetramethylammonium and lobeline evoked inward currents in bovine chromaffin cells. In contrast, decamethonium and the muscarinic agonist, methacholine, had no effect. 4. The nicotinic antagonists mecamylamine, trimetaphan, (+)‐tubocurarine and hexamethonium caused dose‐dependent reductions in the amplitude of ACh‐evoked inward currents. The estimated IC50's were 0.25, 0.33, 0.63 and 2.2 microM respectively, for cells voltage clamped at ‐60 mV. High concentrations (> 2 microM) of the muscarinic antagonist, atropine, also produced a dose‐dependent reduction in the amplitude of ACh‐induced currents. 5. Inhibition by trimetaphan was voltage independent. With the other drugs the antagonism was voltage sensitive, increasing with membrane hyperpolarization. The voltage sensitivity was most marked for hexamethonium. Neither hexamethonium nor mecamylamine were found to depress ACh‐evoked outward currents at concentrations which severely depressed inward currents. In addition to its antagonist actions, (+)‐tubocurarine activated unitary currents in these cells and on isolated membrane patches. 6. The results indicate that nicotinic ion channels of bovine chromaffin cells have a similar ionic selectivity to monovalent cations, but that Ca2+ ions permeate the channels to a greater degree than at the motor endplate. The ACh current‐voltage relationship resembles that described for other types of ‘neuronal’ nicotinic receptors.(ABSTRACT TRUNCATED AT 400 WORDS)
The ventilatory response to CO2 of the peripheral and central chemoreflex loop before and after sustained hypoxia in man.
Authors: Berkenbosch A , Dahan A , DeGoede J , Olievier I C
Published: December 2014

1. The ventilatory response to sustained hypoxia is characterized by a fast increase due to the peripheral chemoreceptors followed by a slow decline. The mechanism of this decline is unknown. 2. To investigate the characteristics of the ventilatory response to sustained hypoxia ten healthy subjects were exposed to two consecutive periods of isocapnic hypoxia (arterial saturation 78%) separated by a 5 min exposure to isocapnic normoxia. 3. The acute hypoxic response to the second exposure to hypoxia (mean increase in ventilation +/‐ S.E.M., 7.2 +/‐ 0.8 l min‐1) was significantly depressed (P = 0.04) compared to the first one (9.5 +/‐ 1.3 l min‐1). 4. To investigate whether this depression was due to central or peripheral effects or both we measured, in the same ten subjects, the normoxic ventilatory response to CO2 before and after a period of 25 min of hypoxia using the technique of dynamic end‐tidal forcing. 5. Each response was separated into a fast peripheral and slow central component characterized by a CO2 sensitivity, time constant, time delay and an off‐set. 6. A total of thirty‐six prehypoxic and thirty posthypoxic responses were analysed. The ventilatory CO2 sensitivities of the peripheral and central chemoreflex loops and the overall off‐set (apnoeic threshold) after 25 min of hypoxia were somewhat larger than their prehypoxic values, but this effect was not significant. 7. We argue that the hypoxic ventilatory decline in man is due to a change in the off‐set of the peripheral chemoreflex loop.
Effects of left atrial receptor stimulation on carotid chemoreceptor‐induced renal responses in dogs.
Authors: Karim F , al‐Obaidi M
Published: December 2014

1. Dogs were anaesthetized with thiopentone sodium and alpha‐chloralose and artificially ventilated. The carotid sinus regions were vascularly isolated and perfused with arterial or venous blood to stimulate the chemoreceptors. Left atrial receptors were stimulated by distending four balloons, three in the left pulmonary vein‐atrial junctions and one in the left atrial appendage. Mean aortic pressure was held constant by means of a pressure control device. Atenolol and atropine (2.0 and 0.5 mg kg‐1, respectively), and gallamine triethiodide (3.0 mg kg‐1 h‐1) were given I.V. Renal blood flow was measured by an electromagnetic flowmeter, glomerular filtration rate by creatinine clearance, urinary sodium by flame photometry and solute excretion by osmometry. 2. In fifteen tests in eight dogs (in one dog responses of both left and right kidneys were determined), at a constant aortic pressure (AoP) of 92.0 +/‐ 3.2 mmHg, and carotid sinus pressure (CSP) of 95.0 +/‐ 2.0 mmHg, stimulation of left atrial receptors with balloon inflation resulted in significant increases in renal blood flow (RBF) by 8.3 +/‐ 0.9 from 255.0 +/‐ 14.6 ml min‐1 (100 g kidney weight)‐1 (n = 9), in glomerular filtration rate (GFR) by 4.1 +/‐ 0.6 from 21.2 +/‐ 1.9 ml min‐1 (100 g)‐1, in filtration fraction (FF) by 0.04 +/‐ 0.003 from 0.20 +/‐ 0.01, in urine flow rate (V) by 0.08 +/‐ 0.02 from 0.33 +/‐ 0.05 ml min‐1 (100 g)‐1, in sodium excretion (UNaV) by 4.4 +/‐ 0.9 from 27.7 +/‐ 4.2 mumol min‐1 (100 g)‐1, in osmolar excretion (UosmV) by 62.0 +/‐ 5.6 from 303.0 +/‐ 28.3 mu osmol min‐1 (100 g)‐1, and in a decrease in free water clearance (CH2O) by 0.13 +/‐ 0.03 from ‐0.63 +/‐ 0.04 ml min‐1 (100 g)‐1. Left atrial pressure (LAP) and heart rate (HR) did not change significantly from 6.9 +/‐ 0.3 cmH2O, and 133.0 +/‐ 3.4 beats min‐1 respectively.(ABSTRACT TRUNCATED AT 400 WORDS)