bluuebunny/crossref_metadata_2025 huggingface dataset:
Block of ATP‐regulated and Ca2(+)‐activated K+ channels in mouse pancreatic beta‐cells by external tetraethylammonium and quinine.
Authors: Bokvist K , Rorsman P , Smith P A
Published: December 2014

1. The whole‐cell and outside‐out patch configurations of the patch‐clamp technique were used to investigate the effects of extracellular tetraethylammonium ions (TEA+) and quinine on both Ca2(+)‐activated and ATP‐regulated K+ channels in mouse pancreatic beta‐cells. 2. The Ca2(+)‐activated K+ channel has a single‐channel K+ permeability of 4.7 x 10(‐13) cm3 s‐1 when recorded with physiological ionic gradients. This value decreased to 2.9 x 10(‐13) cm3 s‐1 after addition of 0.3 mM‐TEA+. 3. Two exponentials with time constants of 0.2 and 4.7 ms were required to describe the distribution of the channel openings suggesting that the Ca2(+)‐activated K+ channel has at least two open states. The fast and slow components comprised 16 and 84% of the total number of openings respectively. 4. TEA+ caused a concentration‐dependent decrease in the single‐channel amplitude and open probability of the Ca2(+)‐activated K+ channel. A Kd for the reduction in the mean current of 0.14 mM was observed. The stoichiometry was approximately 1:1. 5. Quinine blocked the Ca2(+)‐activated K+ channel in a concentration‐dependent manner. Half‐maximal block was observed at 0.10 mM and binding was 1:1. Inhibition by 20 microM‐quinine was not associated with a decrease in channel amplitude but markedly reduced the lifetime of the channel openings. Two exponentials, with time constants of 0.5 and 1.3 ms, were required to describe the channel openings. The rapid component contained 55% of the events. 6. TEA+ reduced the single‐channel amplitude of the ATP‐regulated K+ channel in a concentration‐dependent manner. Kd for the block was 22 mM and the binding approximately 1:1. The block was not associated with changes in the open probability or channel kinetics. Two exponentials were required to describe the distribution of the open times. The time constants for the fast and slow components were approximately 2 and approximately 20 ms respectively. The rapid component accounted for approximately 35% of the events. 7. Quinine (10‐20 microM) almost abolished activity of the ATP‐regulated K+ channels. Inhibition was characterized by slow onset and reversibility but not associated with a change in the appearance of the single‐channel events. Quinine‐induced block could not be reversed by diazoxide. 8. We conclude that TEA+ produces rapid block of both Ca2(+)‐activated and ATP‐regulated K+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)
Measurement of filtration coefficient in single cerebral microvessels of the frog.
Authors: Fraser P A , Dallas A D , Davies S
Published: December 2014

1. This study reports the first results of measurements of filtration coefficient (Lp) and osmotic reflection coefficient to sucrose (sigma suc) in single brain microvessels. 2. Microvessels on the surface of frog brain were cannulated with a micropipette and perfused with an artificial cerebrospinal fluid (CSF) containing the low molecular weight impermeant dye carboxyfluorescein (MW 376). The superfusing solution was a similar CSF which could be made hypertonic by the addition of 40‐125 mmol l‐1 sucrose. 3. Vessels were assessed for dye retention using video‐intensified microscopy after occlusion with a glass microneedle. Only six vessels out of a total of ninety‐five were tight under the experimental conditions used. Those vessels which were tight were occluded while an osmotic load was applied across them. When this load was 50 mosmol l‐1 and less, the steady‐state dye concentration within the vessel lumen was similar to that predicted assuming the endothelium behaves as a perfect semipermeable membrane, with concentration polarization of solute. 4. The product Lp sigma was estimated in two ways: (i) from the fitted monoexponential function that described the rising dye concentration within the occluded segment, and (ii) from the initial rate of increase in dye concentration. The two values obtained were similar and it was concluded that sigma NaCl = sigma suc = 1, and the best estimate for filtration coefficient Lp = 2.0 x 10(‐9) cm (cmH2O s)‐1. 5. At the osmotic loads of 100 mosmol l‐1 and more, the initial rate of increase estimate of Lp sigma was less than half of the whole curve estimate, the axial dye distributions were dissimilar from those predicted by a mathematical model based on the perfect semipermeable membrane, and the steady‐state concentration was less than 70% of that expected. These findings are consistent with a diffusive pathway having opened. The model was modified to include patches of vessel wall which had developed leaks and a good fit to the data was obtained with a sucrose permeability and an Lp similar to skeletal muscle endothelium. 6. The possibility that water passes through a paracellular pathway across the intact blood‐brain barrier is discussed. It is concluded that this pathway could not be detected by the methods used and can carry no more than 50% of the water driven by a hydrostatic pressure gradient.
Renin and angiotensin converting enzyme concentrations in the fetal and neonatal guinea‐pig.
Authors: Raimbach S J , Thomas A L
Published: December 2014

1. Plasma renin concentration (PRC) and plasma and pulmonary angiotensin converting enzyme (ACE) concentration were measured in fetal and neonatal guinea‐pigs from 45 days gestational age (GA) until 21 days post‐partum. 2. Fetal PRC increased towards term to reach values greater than those measured in normal adult males. Pentobarbitone anaesthesia of the mother resulted in significant elevation of fetal PRC after 66 days GA but not before this time. 3. PRC were very high in the newborn guinea‐pig, decreased rapidly during the first 24 h after birth and then more gradually, to reach approximately adult values by day 21. 4. Fetal plasma ACE concentration increased towards term to reach values greater than those measured in adult males and decreased subsequently. 5. Pulmonary ACE concentrations were very low throughout gestation but increased considerably between days 3 and 14 post‐partum. Low concentrations of ACE were measured in other fetal tissues but placental concentrations were relatively high. 6. Propranolol (0.1 mg I.P.) or saline was administered (under halothane‐nitrous oxide anaesthesia) to fetuses of litters of various GA from 55 days to term. Fetal PRC were measured 3 h later. Propranolol treatment resulted in significantly lower fetal PRC than saline treatment in litters aged 63 days to term but not in younger litters. 7. These data indicate that the renin‐angiotensin system is functional in the fetal guinea‐pig during the last third of gestation. Fetal plasma renin concentrations near term are greater than those measured in normal adult males. This may, in part, reflect an increased influence of the fetal sympathetic nervous system.
The renal and vascular effects of central angiotensin II and atrial natriuretic factor in the anaesthetized rat.
Authors: Al‐Barazanji K A , Balment R J
Published: December 2014

1. The interaction between atrial natriuretic factor (ANF) and angiotensin II (Ang II) within the brain to influence renal function and blood pressure was studied in Inactin‐anaesthetized male Sprague‐Dawley rats. 2. Central infusion of ANF produced a diuresis which was associated with a significant decrease in plasma arginine vasopressin (AVP) level. There was no change in sodium excretion rate over the 80 min of intracerebroventricular ANF infusion and ANF produced no detectable change in mean arterial blood pressure. 3. Central Ang II administration produced a significant decrease in urine flow, which was associated with elevated plasma AVP, an increase in sodium excretion and a rise in mean arterial blood pressure. 4. Combined ANF and Ang II infusion produced an antidiuresis, which was associated with increased plasma AVP concentration. Both the natriuretic and vasopressor actions of central Ang II were abolished when ANF was co‐administered. 5. It is concluded that ANF and Ang II interact centrally; ANF antagonizes the pressor and natriuretic effects but not the antidiuretic effects of central Ang II. These data suggest the possibility of distinct and separate sites within the brain through which Ang II influences vasopressin release and renal sodium handling and elevates blood pressure.
Calcium release in skinned muscle fibres of the toad by transverse tubule depolarization or by direct stimulation.
Authors: Lamb G D , Stephenson D G
Published: December 2014

1. Skeletal muscle fibres from the toad were mechanically skinned under paraffin oil and then bathed in a potassium HDTA solution (HDTA: hexamethylenediamine‐tetraacetate) which mimicked the ionic composition of the myoplasm. 2. Rapid transient contractions could be triggered by substitution of K+ with Na+ (with no change of anion), which should have virtually no direct effect on the electrical polarization of the sarcoplasmic reticulum (SR) membrane. Up to thirty or more contractions could be evoked by repeated substitutions if there was sufficient ‘repriming’ time (about 30 s) between them; these rapid contractions were analagous to potassium contractures in intact fibres. 3. When the SR was not heavily loaded, substitution of potassium HDTA with choline chloride also produced a rapid, brief contraction. 4. All treatments designed to ‘inactivate’ the voltage sensor in the T‐system invariably abolished the rapid contractions. Thus, rapid contractions were absent if (i) the T‐system was permanently depolarized by pre‐soaking the muscle in a high potassium solution with ouabain before skinning, (ii) a fibre was split rather than skinned, (iii) the T‐system was temporarily depolarized by Na+ substitution immediately before choline chloride substitution, or vice versa, (iv) a skinned fibre was briefly exposed to saponin (50 micrograms/ml) to selectively disrupt the T‐system membrane or (v) the muscle was pre‐soaked in a solution with 1 mM‐EGTA and no Ca2+ or Mg2+ before skinning. In contrast to (v), if 10 mM‐Mg2+ was present in the EGTA solution before skinning, rapid contractions could be elicited, presumably because the presence of Mg2+ prevented the inactivation of the T‐system voltage sensor in low [Ca2+]. 5. These results unequivocally demonstrate that (a) the T‐system reseals and repolarizes after mechanical skinning under oil and (b) the fast contractions are produced by activation of the voltage sensor in the T‐system. 6. When the SR had been heavily loaded, choline chloride substitution (but not Na+ substitution) could also induce an unphysiological, slow contraction ('second component’). In total contrast to the fast contraction, this slow component was unaffected by any of the treatments (i‐v) above, indicating that it did not depend on activation of the voltage sensor in the T‐system but resulted from a direct action of choline chloride on the SR.(ABSTRACT TRUNCATED AT 400 WORDS)
Heart rate and arterial blood pressure at the onset of static exercise in man with complete neural blockade.
Authors: Friedman D B , Jensen F B , Mitchell J H , Secher N H
Published: December 2014

1. We tested the ‘muscle‐heart reflex’ hypothesis for the immediate increases in heart rate and blood pressure at the onset of static exercise in man by performing complete blockade of afferent nerves from the working muscles. Brief (5 s) maximal static hand‐grip contractions were performed without performing a Valsalva‐like manoeuvre and with no increase in central venous pressure both before and after combined axillary and radial blockade with lidocaine. Muscle strength was reduced to near zero. The effectiveness of the afferent neural blockade was evaluated by recording the heart rate and blood pressure responses and rating the perceived pain during a cold pressor test of the blocked and contralateral unblocked hand. 2. The cold pressor test increased blood pressure but had no effect on heart rate. Afferent neural blockade eliminated the increase in blood pressure and the perceived pain associated with the cold pressor test. Maximal hand‐grip contractions resulted in immediate and similar increases in heart rate and blood pressure before and after afferent neural blockade of the arm. 3. The results of this study suggest that the immediate increases in heart rate and blood pressure at the onset of static exercise in man occur when the ‘muscle‐heart reflex’ is inoperable.
Potassium, chloride and non‐selective cation conductances opened by noradrenaline in rabbit ear artery cells.
Authors: Amédée T , Benham C D , Bolton T B , Byrne N G , Large W A
Published: December 2014

1. The action of noradrenaline on cells isolated from the rabbit ear artery was studied with the whole‐cell configuration of the patch clamp technique. In normal potassium‐containing solutions at a holding potential of ‐50 mV noradrenaline elicited either outward, inward or mixed outward and inward currents. These responses were blocked by the alpha‐adrenoceptor antagonist, phentolamine (10(‐6) M). 2. The outward current occurred as a consequence of an increase in membrane conductance and the reversal potential was close to the potassium equilibrium potential (EK). It was possible to record outward currents without external calcium but not when the concentration of EGTA in the pipette was increased to 10 mM or when potassium was absent from the pipette solution. It is concluded that the outward current evoked by alpha‐adrenoceptor stimulation is produced by a calcium mediated increase in potassium conductance. 3. The ionic basis of the inward current was investigated in potassium‐free external and pipette solutions. When sodium chloride was the major constituent of the external and pipette solutions the reversal potential (Er) of the noradrenaline‐induced current was 0.63 mV, close to ENa and ECl. 4. When most of the external sodium chloride was replaced by sucrose Er was intermediate between ENa and ECl but had shifted significantly towards ENa. Further ion substitution experiments suggest that noradrenaline increased the membrane conductance to both anions and cations. 5. When the current was carried predominantly by anions, depolarizing steps (from ‐50 mV) produced outward current relaxations with a time constant of about 40 ms. Bath‐applied caffeine also produced a membrane current which rectified in the outward direction. 6. When the response to noradrenaline was mediated mainly by cations, the relationship between the membrane current and clamp potential was non‐linear and the amplitude of the currents at potentials positive to ‐50 mV became disproportionately smaller. In addition on repolarization to ‐50 mV the instantaneous current was followed by an inward relaxation. 7. In high external barium solution noradrenaline evoked a membrane current with a reversal potential much more positive than ENa or ECl. This current was recorded in the presence of 10(‐5) M‐nifedipine and diltiazem and in 10(‐4) M‐cadmium which suggests that the voltage‐dependent calcium channel is not implicated in the generation of the non‐selective cation current to noradrenaline.(ABSTRACT TRUNCATED AT 400 WORDS)
Centrifugal motion bias in the cat's lateral suprasylvian visual cortex is independent of early flow field exposure.
Authors: Brenner E , Rauschecker J P
Published: December 2014

1. Neurones in the postero‐medial part of the cat's lateral suprasylvian visual cortex (area PMLS) show an overall preference for centrifugal motion, suggesting that the PMLS may be specialized in the analysis of expanding optic flow fields associated with forward locomotion. 2. We examined whether the visual experience young kittens normally receive during forward locomotion guides the development of the centrifugal preference in the PMLS. 3. Seven kittens were reared in the dark and exposed to either expanding or contracting flow fields for at least 100 h during their 4th‐11th weeks of life. Specific experience was achieved by exposing kittens either to flow field patterns generated on a screen or by actually moving them forward or backward in a carousel. 4. Our results show that although the development of directional selectivity in the PMLS requires visual experience, the centrifugal bias is independent of specific visual exposure. The preference for centrifugal motion among PMLS cells was just as evident in kittens exposed to contracting as in kittens exposed to expanding flow fields. 5. We conclude that the preference for centrifugal motion in the PMLS is not the result of anisotropic stimulation kittens receive during locomotion in early ontogeny, but is probably innately determined as a phylogenetic adaptation.
Effects of hypoxia on high‐energy phosphagen content, energy metabolism and isometric force in guinea‐pig taenia caeci.
Authors: Ishida Y , Paul R J
Published: December 2014

1. Previously, Ishida, Takagi & Urakawa (1984) reported that, in the presence of high K+ (45.4 mM) under hypoxia (95% N2‐5% CO2 bubbling), tension and ATP content of the smooth muscle of the guinea‐pig taenia caeci increased concomitantly when the glucose concentration was raised. Tension and energy metabolism of the taenia seemed to be closely correlated. In the present experiments, we investigated the metabolic changes during the relaxation phase after the taenia was exposed to hypoxia in the presence of high K+ by measuring the content of phosphagen, inorganic phosphate (Pi) and lactate in the tissue. Oxygen consumption and lactate release from the tissue were also determined to estimate the rate of ATP synthesis. 2. Under hypoxic conditions, high‐K(+)‐induced tension decreased to one‐tenth of maximum in aerobic conditions (95% O2‐5% CO2 bubbling); increasing the calcium concentration from 2.5 to 10 mM had no effect. To test receptor‐associated stimuli, carbachol or histamine (both 5 microM) had little effect on tension in hypoxia. The calcium ionophore A23187 (10 microM) also did not produce any significant contraction in the presence of high K+ under hypoxia. 3. Hypoxia in the presence of high K+ elicited a rapid decrease in phosphocreatine, approximately in parallel with the decrease in tension. The ATP content decreased gradually while Pi content increased. Lactate content increased rapidly and then partially decreased. 4. The rate of ATP synthesis estimated from the oxygen consumption and lactate release of the taenia was linearly correlated with tension development under aerobic and hypoxic conditions. 5. These results suggest that the decrease in tension of the taenia observed under hypoxic conditions is due to an inhibition of energy metabolism, and not due to an oxygen‐sensing step in excitation‐contraction coupling.
Effect of membrane potential on acetylcholine‐induced inward current in guinea‐pig ileum.
Authors: Inoue R , Isenberg G
Published: December 2014

1. The whole‐cell patch clamp technique with caesium aspartate internal solution was used with single isolated cells from the longitudinal muscle layer of guinea‐pig ileum, to investigate the voltage‐dependent gating of ACh‐induced inward current. 2. In voltage clamp experiments, at holding potentials ranging from ‐80 to ‐30 mV, ACh (300 microM) produced a slow sustained inward current in physiological salt bath solution (PSS). The measurements of the reversal potentials on substituting Na+ by other monovalent and divalent cations showed that this current is through non‐selective cation channels (Ins, ACh). 3. During hyperpolarizations, Ins, ACh instantaneously increased in amplitude and then relaxed to a new steady‐state level. The I‐V relationship of the instantaneous peak was linear with a reversal potential of 0 mV, while that of the steady state was bell‐shaped. The time course of relaxation appeared to be monoexponential and its time constants were reduced by stronger hyperpolarizations. 4. These results were not affected by the organic Ca2+ antagonists D600 or nitrendipine (10 microM). Under this condition, maximal chord conductance of Ins, Ach which was observed at 0 mV was about 1.5 nS. The steady‐state activation relationship was well fitted by Boltzmann's equation with a half‐maximal activation (Vh) of ‐50 mV and a slope factor (k) of ‐15 mV at membrane potentials negative to 0 mV, but over 0 mV the degree of activation was again decreased. The time constants for relaxation also appeared to follow a sigmoid curve. 5. In current clamp experiments, superfusion of ACh (300 microM) depolarized the membrane up to ‐10 to 0 mV. Inward current injection resulting in the moderate hyperpolarization of the membrane (‐70 to ‐80 mV) attenuated ACh‐induced depolarization and stronger hyperpolarization (less than ‐80 mV) abolished it. 6. These results show that ACh‐induced depolarization is controlled by the membrane potential, which is explained by the voltage‐dependent gating of Ins, Ach.
Currents carried by monovalent cations through cyclic GMP‐activated channels in excised patches from salamander rods.
Authors: Menini A
Published: December 2014

1. Ionic selectivity and affinity for monovalent cations of channels activated by guanosine 3',5'‐cyclic monophosphate (cyclic GMP) were studied in excised inside‐out patches of plasma membrane from retinal rods of the tiger salamander. Channels were activated by addition of cyclic GMP to the medium bathing the cytoplasmic side of the membrane. The ionic solution at the cytoplasmic side was rapidly changed using the method of Nunn (1987 a). 2. Permeability ratios were calculated with the Goldman‐Hodgkin‐Katz potential equation from reversal potential measurements for alkali monovalent cations in bi‐ionic conditions. The permeability sequence was: Li+:Na+:K+:Rb+:Cs+ = 1.14:1:0.98:0.84:0.58. 3. The selectivity sequence obtained from macroscopic current measurements in bi‐ionic conditions at +100 mV was: Na+:K+:Rb+:Li+:Cs+ = 1:1:0.67:0.36:0.25. 4. The organic cations tetramethylammonium (TMA+), choline and tetraethylammonium (TEA+) were not permeant through the cyclic GMP‐activated channels and caused a reduction of the Na+ inward current. At ‐100 mV the current ratio for inward current was 1:0.75:0.58:0.2 in the presence, at the cytoplasmic side, of 110 mM‐Na+, TMA+, choline or TEA+ respectively. 5. The concentration dependence of the macroscopic current and the reversal potential was studied by changing the internal concentration of Na+ or K+ or Li+ from 5 mM to 500 mM. The permeability ratios were nearly constant regardless of the permeant ion concentration. 6. The current as a function of internal ion activity could be described by a Michaelis‐Menten relation with a half‐saturating activity, Km, at +90 mV equal to 249, 203 and 160 mM for Na+, K+ and Li+ respectively. The ratio of the extrapolated saturating current Imax at +90 mV was 1:0.86:0.26 for Na+, K+ and Li+ respectively. 7. The outward currents and the reversal potentials measured in different mixtures of Na+ and Li+ were monotonic function of the mole fraction. 8. These results can be explained by assuming that, at least in a narrow region, the cyclic GMP‐activated channel is a one‐ion channel, possibly with other poorly voltage‐dependent binding sites in a large inner vestibule.
Whole‐cell calcium current in guinea‐pig ventricular myocytes dialysed with guanine nucleotides.
Authors: Shuba Y M , Hesslinger B , Trautwein W , McDonald T F , Pelzer D
Published: December 2014

1. Whole‐cell calcium current (ICa) was recorded in guinea‐pig ventricular myocytes superfused with Na+,K(+)‐free solution and dialysed with a substrate‐free solution (minimum intracellular solution, MICS). A dual tight‐seal pipette method was often used to permit pressure‐enhanced dialysis of a test solution after a given pre‐dialysis. 2. In dual‐pipette experiments, test dialysates contained 100 mM‐GTP‐gamma‐S (guanosine 5'‐O‐(3‐thiotriphosphate] or 100 microM‐GMP‐PNP (guanyl‐5'‐imidodiphosphate). These non‐hydrolysable analogues of guanosine triphosphate (GTP) enhanced ICa amplitude (+ 10 mV) by 20‐40%. Dialysates containing 100 microM‐GTP or GDP‐beta‐S (guanosine 5'‐O‐(2‐thiodiphosphate] were ineffective, and pre‐dialysis with GDP‐beta‐S blocked stimulation by GTP‐gamma‐S. 3. Non‐hydrolysable GTP analogues slowed the inactivation of ICa and shifted the voltage eliciting maximum ICa by 5‐10 mV in the negative direction. 4. ICa enhancement by GTP analogues was attributed to the activation of three GTP‐binding regulatory (G) proteins (Gi, Gp and Gs). In single‐pipette experiments, the inactivation of Gi by pre‐treatment with pertussis toxin did not block enhancement, and a Gp‐activating regimen (external acetylcholine‐internal GTP) was without effect. Thus, it is probable that the effects of GTP analogues on ICa were primarily mediated by Gs activation. 5. PI‐MICS dialysates contained phosphorylation‐pathway inhibitors and were used to inhibit Ca2+ channel phosphorylation via the adenyl cyclase pathway. These were deemed effective since forskolin (1‐5 microM) doubled ICa during control dialysis but was without effect after 8 min PI‐MICS dialysis. However, 0.1 microM‐isoprenaline increased ICa by 35% in myocytes totally unresponsive to forskolin, suggesting that beta‐adrenergic receptor occupation can stimulate ICa even when the phosphorylation pathway is blocked. 6. After prolonged dialysis of myocytes with PI‐MICS, ICa was still enhanced by pressure‐assisted dialysis of 100 microM‐GTP‐gamma‐S or GMP‐PNP. We conclude that activated Gs has a direct effect on cardiac Ca2+ channels.
Allosteric potentiation of quisqualate receptors by a nootropic drug aniracetam.
Authors: Ito I , Tanabe S , Kohda A , Sugiyama H
Published: December 2014

1. Allosteric potentiation of the ionotropic quisqualate (iQA) receptor by a nootropic drug aniracetam (1‐p‐anisoyl‐2‐pyrrolidinone) was investigated using Xenopus oocytes injected with rat brain mRNA and rat hippocampal slices. 2. Aniracetam potentiates the iQA responses induced in Xenopus oocytes by rat brain mRNA in a reversible manner. This effect was observed above the concentrations of 0.1 mM. Kainate. N‐methyl‐D‐aspartate and gamma‐aminobutyric acid responses induced in the same oocytes were not affected. 3. The specific potentiation of iQA responses was accompanied by an increase in the conductance change of iQA and alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionic acid (AMPA) responses, but the affinity of receptors for agonist and the ion‐selectivity of the channels (reversal potentials) were not changed. 4. Aniracetam reversibly potentiated the iQA responses recorded intracellularly from the pyramidal cells in the CA1 region of rat hippocampal slices. The excitatory postsynaptic potentials (EPSPs) in Schaffer collateral‐commissural‐CA1 synapses were also potentiated by aniracetam. 5. Population EPSPs recorded in the mossy fibre‐CA3 synapses as well as Schaffer‐commissural synapses were also potentiated by aniracetam. The amplitudes of the potentiation were not changed by the formation of long‐term potentiation.
Effects of modified chromophores on the spectral sensitivity of salamander, squirrel and macaque cones.
Authors: Makino C L , Kraft T W , Mathies R A , Lugtenburg J , Miley M E , van der Steen R , Baylor D A
Published: December 2014

1. Chemically modified retinal chromophores were used to investigate the mechanisms that produce the characteristic spectral absorptions of cone pigments. Spectral sensitivities of single cones from the salamander, squirrel and macaque retina were determined by electrical recording. The chromophore was then replaced by bleaching the pigment and regenerating it with a retinal analogue. 2. Exposing a bleached cone to 9‐cis‐retinal for a brief period (less than 20 min) caused its flash sensitivity to recover to about 0.2 of the pre‐bleach value. Similar exposure to a locked 6‐s‐cis, 9‐cis analogue gave a recovery to about 0.03 of the pre‐bleach value. 3. Unlike the flash sensitivity, the saturating photocurrent amplitude often recovered completely after bleaching and regenerating the pigment. 4. When the 3‐dehydroretinal chromophore in the salamander long‐wavelength‐sensitive (red) cone was replaced with 11‐cis‐retinal, shortening the conjugated chain in the chromophore, the spectral sensitivity underwent a blue shift of 67 nm. 5. Pigments containing the planar‐locked 6‐s‐cis.9‐cis‐retinal analogue absorbed at substantially longer wavelength than those containing unmodified 9‐cis‐retinal. The opsin shift, a measure of the protein's ability to modify the chromophore's absorption was larger for the locked analogue than for 9‐cis‐retinal. This suggests that the native chromophore assumes a twisted 6‐s‐cis conformation in these pigments. 6. The spectral sensitivities of red and green macaque cones containing 9‐cis‐retinal or planar‐locked 6‐s‐cis.9‐cis‐retinal retained the 30 nm separation characteristic of the native pigments. This suggests that the different absorptions of of the 6‐7 carbon bond in the retinal chromophore.
Increased airway reactivity in the guinea‐pig follows exposure to intravenous isoprenaline.
Authors: Sanjar S , Kristersson A , Mazzoni L , Morley J , Schaeublin E
Published: December 2014

1. Intravenous infusion of (+/‐) isoprenaline (1‐100 micrograms kg‐1 h‐1) enhanced airway responses (resistance, RL; and compliance, Cdyn) to histamine (1.0‐1.8 micrograms kg‐1) and bombesin (100‐240 ng kg‐1), whereas airway responses to vagal stimulation remained unchanged. 2. Bilateral vagotomy before intravenous infusion of (+/‐)isoprenaline (100 micrograms kg‐1 h‐1) prevented development of airway hyperreactivity to histamine or bombesin, yet vagotomy after infusion of isoprenaline was without effect. 3. Prior treatment with atropine (1 mg kg‐1) did not influence the capacity of (+/‐)isoprenaline (100 micrograms kg‐1 h‐1) to increase airway reactivity to bombesin. 4. Despite a 500‐fold difference in spasmolytic potency in vivo, infusion of (+)isoprenaline (100 micrograms kg‐1 h‐1) or (‐)isoprenaline (100 micrograms kg‐1 h‐1) increased reactivity of the airways to histamine or bombesin to a comparable extent. 5. Neither adrenaline (100 micrograms kg‐1 h‐1) nor forskolin (600 micrograms kg‐1 h‐1) increased reactivity of the airways to histamine or bombesin. 6. Intravenous infusion of dopamine (100 micrograms kg‐1 h‐1) or noradrenaline (100 micrograms kg‐1 h‐1) increased reactivity of the airways to histamine or bombesin. 7. Intravenous infusion of (+/‐) propranolol (100 micrograms kg‐1 h‐1) increased reactivity of the airways to histamine or bombesin which was partially inhibited by bilateral vagal section. 8. Depletion of circulating platelets by lytic anti‐platelet serum or concomitant infusion of an antagonist of platelet‐activating factor (PAF), ginkgolide B (1 mg kg‐1 h‐1) did not diminish the capacity of (+/‐)isoprenaline (100 micrograms kg‐1 h‐1) to induce hyperreactivity of the airways to histamine or bombesin. 9. These observations indicate that (+/‐)isoprenaline can induce airway hyper‐reactivity by a mechanism unrelated to beta‐adrenoceptor activation, but which is dependent upon intact vagus nerves.
Tetraethylammonium blockade of apamin‐sensitive and insensitive Ca2(+)‐activated K+ channels in a pituitary cell line.
Authors: Lang D G , Ritchie A K
Published: December 2014

1. The pharmacological sensitivities and physiological contributions of two types of Ca2(+)‐activated K+ channels (BK and SK) in GH3 cells were examined by the outside‐out, whole‐cell and cell‐attached modes of the patch‐clamp technique. 2. BK channels (250‐300 pS in symmetrical 150 mM‐K+) in outside‐out patches were blocked by external tetraethylammonium (TEA) and by 50 nM‐charybdotoxin (CTX), but were not blocked by apamin. 3. SK channels (9‐14 pS in symmetrical 150 mM‐K+) in outside‐out patches were blocked by external TEA and by apamin, but were not blocked by 50 nM‐CTX. 4. The dissociation constant (Kd) for TEA block of SK channels (3.1 +/‐ 0.37 mM) was 12‐fold greater than the Kd for the BK channels (260 +/‐ 21 microM). The TEA blockade of both channels was not strongly voltage dependent: for both channels the TEA binding site sensed less than 20% of the membrane electric field. 5. Application of blockers of the BK channels (1 mM‐TEA and 50 nM‐CTX) to whole cells under current clamp prolonged action potential duration; whereas application of apamin, a selective blocker of the SK channel, inhibited a slowly decaying after‐hyperpolarization and had little effect on action potential duration. Apamin also increased the firing rate in 30% of the spontaneously pacing cells. 6. It is suggested that BK channels contribute to action potential repolarization: whereas SK channels contribute to the regulation of action potential firing rate.
Fast gating kinetics of the slow Ca2+ current in cut skeletal muscle fibres of the frog.
Authors: Feldmeyer D , Melzer W , Pohl B , Zöllner P
Published: December 2014

1. Calcium currents and intramembrane charge movements were measured in cut twitch muscle fibres of the frog and the time course of activation of the current was studied using various conditioning pulse protocols. 2. When a conditioning activation was produced by a depolarizing pulse which ended before inactivation occurred, a subsequent depolarization led to a faster onset of activation, indicating that the system had not completely returned to the initial state during the interval between the two pulses. 3. The interval between conditioning and test pulse was varied at different subthreshold potentials to study the time course of restoring the steady‐state conditions. Complete restoration required a waiting period of about 1 min at the holding potential of ‐80 mV due to a very slow process but partial recovery was reached within 100 ms. This initial recovery process was strongly voltage dependent and became considerably slower when the interval potential approached the threshold for current activation. 4. Stepping to a roughly 10 mV subthreshold potential without applying a conditioning activation caused no change in the time course of the current produced by a subsequent test depolarization. Depolarizing just to the current threshold caused a slowly progressing acceleration of test current activation. 5. The peak current‐voltage relation in the fast gating regime caused by a conditioning activation coincided with the current‐voltage relation measured under steady‐state conditions, indicating not that a new channel population had become activated but that the same channels showed a different gating behaviour. 6. Intramembrane charge movements measured in 2 mM‐Cd2+ and tested at potentials between ‐40 and +40 mV showed negligible changes when preceded by a strong depolarization. 7. We discuss several possible models which can explain the fact that the current is speeded up by a conditioning activation while the charge movements remain unchanged. It is possible that the fast voltage‐dependent transition which becomes visible after conditioning pulses reflects a rapid conformational change of the Ca2+ channel molecule which also occurs during its normal gating mode but remains undetectable in terms of conductance. In view of the hypothesis that the Ca2+ channel molecule forms a voltage sensor for excitation‐contraction coupling this fast transition could be coupled to the control of Ca2+ release from the sarcoplasmic reticulum.
Characteristics of Na+ channels and Cl‐ conductance in resealed muscle fibre segments from patients with myotonic dystrophy.
Authors: Franke C , Hatt H , Iaizzo P A , Lehmann‐Horn F
Published: December 2014

1. Electrical and contractile properties of resealed fibre segments were investigated by a variety of in vitro techniques. The preparations were removed from skeletal muscles of normal subjects and of eight patients with myotonic dystrophy. 2. Several hours after removal, fibre segments from normal subjects and those patients in whom myotonia was the primary symptom had resting membrane potentials of approximately ‐80 mV. In contrast, fibre segments obtained from patients in whom muscle dystrophy was more expressed were depolarized (‐60 to ‐70 mV). 3. Contractions induced in fibre segments of myotonic muscle which had normal potentials were characterized by slowed relaxation which was due to electrical after‐activity. 4. After single stimuli, long‐lasting (3‐100) runs of action potentials were recorded intracellularly from the myotonic muscle. In some of these fibre segments complex repetitive discharges were observed: multiple sites of locally gated currents were identified. 5. The three‐electrode voltage clamp was used to determine the total membrane conductance, gm, and the ion component conductances. All fibres of a particular patient had similar conductances. However, the Cl‐ conductance varied from patient to patient from normal (74% of gm) to low values (30% of gm). The K+ conductance was normal in all fibres of all patients. 6. The patch‐clamp technique was used to record currents through single Na+ channels of the sarcolemma. After treatment of the fibre segments with collagenase gigaohm seals were routinely obtained. The rate of success was greater when using the cell‐attached mode than the inside‐out mode. 7. Sodium channel currents were elicited by depolarizing voltage steps which produced an initial burst of Na+ channel openings. Up to ten channels were activated simultaneously when the patch was depolarized to potentials more positive than ‐30 mV. The Na+ channels re‐opened very rarely in controls. The macroscopic sodium current, INa, was reconstructed by averaging depolarizing pulses. The time constant of rapid decay of INa reflecting macroscopic inactivation, the onset of INa and the amplitude of INa were voltage dependent. The mean amplitude of the current produced by re‐openings was on average only 0.11 +/‐ 0.04% of the amplitude of the peak current. 8. Late openings of the Na+ channels were frequent in patches on the myotonic fibre segments. The amplitude of the current produced by re‐openings was as high as about 0.75 +/‐ 0.11% of the amplitude of the peak current.(ABSTRACT TRUNCATED AT 400 WORDS)
pH dependence of intrinsic H+ buffering power in the sheep cardiac Purkinje fibre.
Authors: Vaughan‐Jones R D , Wu M L
Published: December 2014

1. Intrinsic, intracellular H+ buffering power (beta) was estimated in the isolated sheep cardiac Purkinje fibre at various values of intracellular pH (pHi) in the range 6.2‐7.5 and for various values of extracellular pH (pHo) in the range 6.5‐8.5. Buffering power was calculated from the fall of pHi (recorded with an intracellular pH‐selective microelectrode) induced by addition and removal of extracellular, permeant weak acids and bases (NH4Cl, trimethylamine chloride, sodium propionate). Experiments were performed under conditions nominally free of CO2‐HCO3. 2. beta was estimated firstly following acid loads induced by NH4Cl removal (10‐20 mM) under conditions where Na(+)‐H+ exchange was operational (i.e. in Na(+)‐containing Tyrode solution). At constant pHi, the value of beta appeared to double (from a control level of 39.7 mM) as pHo was increased from 7.5 to 8.5. Notably, raising pHo in this range greatly accelerated pHi recovery from an intracellular acid load, indicating stimulation of acid extrusion. It is likely that this stimulation results in an overestimation of beta because it blunts the intracellular acid load. The apparent elevation of beta at high pHo may therefore be an artifact. 3. Estimates of beta were compared (NH4Cl removal) before and after inhibiting Na(+)‐H+ exchange in Na(+)‐free solution or with amiloride (1 mM). The acid load was larger and in many (but not all) cases the apparent value of beta decreased after inhibition of acid extrusion. This indicates that, if Na(+)‐H+ exchange is operational, it can result in an overestimate of beta. In amiloride, beta was 26.6 +/‐ 1.4 mM (n = 8) at a mean pHi of 6.84 +/‐ 0.03. 4. Small stepwise reductions of external NH4Cl (from 40 to 0 mM), in the presence of Na(+)‐free solution plus 5 mM‐BaCl2 at constant pHo, resulted in small stepwise reductions of pHi (approximately 0.1 units). When these were used to calculate beta, we observed that beta increased roughly linearly as pHi became more acid. For a pHi of 7.2, beta approximately 20 mM. 5. An almost identical relationship between beta and pHi was found when using the method of sodium propionate addition (10‐50 mM): amiloride (1 mM) was present and pHi was manipulated to various test levels by changing pHo. This confirms that beta varies inversely with pHi and also that it is independent of pHo. We conclude that the apparent variation of beta with pHo observed earlier was indeed an artifact.(ABSTRACT TRUNCATED AT 400 WORDS)
Effects of muscarine and adrenaline on neurones from Rana pipiens sympathetic ganglia.
Authors: Selyanko A A , Smith P A , Zidichouski J A
Published: December 2014

1. Neurones dissociated from Rana pipiens paravertebral sympathetic ganglia were studied by means of the whole‐cell patch‐clamp technique. Responses to agonists were best recorded when cyclic AMP was included in the patch pipette. 2. Two populations of cells were identified on the basis of size (input capacitance, Cin) and the presence or absence of a fast, transient outward current (A‐current, IA). This current was usually present in the ‘large’ cells (Cin = 40.5 +/‐ 1.5 pF, n = 66) but absent from 'small’ cells (Cin = 21.0 +/‐ 0.8 pF, n = 70). 3. Both cell types exhibited a slowly activating, non‐inactivating K+ current (M‐current, IM) which was suppressed by luteinizing hormone‐releasing hormone (LHRH, 10‐100 microM). Threshold for activation of IM was about ‐75 mV, half‐maximal activation was at ‐50 mV and the M‐conductance GM increased e‐fold for at 7 mV change in membrane potential. The maximum value for IM studied in large cells by patch‐clamp procedures was less than 0.2 nA. More M‐channels were available per unit membrane area in the small cells (GM = 1495 microS cm‐2) than in the large cells (GM = 1034 microS cm‐2). Time constants for IM deactivation at ‐70 mV were faster in the large cells (37.2 +/‐ 4.6 ms, n = 16) than in the small cells (66.1 +/‐ 5.9 ms, n = 9). 4. Muscarine (10 microM) produced inward current in the large cells as a result of IM suppression. In 40% of the large cells, some of the M‐channels were also sensitive to adrenaline (10‐100 microM). In a few large cells (less than 10%) adrenaline produced outward current by increasing IM. 5. Muscarine failed to effect IM in the small cells and instead produced an inwardly rectifying K+ current which activated within 5 ms at ‐110 mV. The outward current produced in twenty out of thirty‐seven small cells by adrenaline was occluded by that produced by muscarine, suggesting that both agonists affect the same K+ channels. 6. Inclusion of the protein kinase inhibitors, 1‐(5‐isoquinolinyl‐sulphonyl)‐2‐methyl piperazine (H‐7, 50 microM) or gold sodium thiomalate (GST, 50 microM) in the pipette solution failed to antagonize either muscarine‐induced current. Both currents were prolonged when the ‘internal solution’ contained GTP‐gamma‐S (50 microM). 7. Phorbol‐12‐myristate‐13‐acetate (PMA, 2‐5 microM) produced an inward current as a result of IM suppression in both small and large cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Correlation between quantal secretion and vesicle loss at the frog neuromuscular junction.
Authors: Hurlbut W P , Iezzi N , Fesce R , Ceccarelli B
Published: December 2014

1. We measured the rate of occurrence of miniature endplate potentials (MEPPs) at identified endplates in frog cutaneous pectoris muscles treated with crude black widow spider venom (BWSV) or purified alpha‐latrotoxin (alpha‐LTX) in calcium‐free solutions, and we examined the relationship between the length of the nerve terminal and the total number of quanta secreted, and the relationship between the number of quanta secreted and the number of vesicles remaining at different times. 2. The venom, or toxin, was applied in a modified Ringer solution with tetrodotoxin, 1 mM‐EGTA and no divalent cations, and quantal secretion was started by applying Ca2(+)‐free solutions with Mg2+. This was done to synchronize the quantal discharge at the various junctions in a muscle. Ringer solution was applied after the MEPP rate had declined to low levels, and then the muscle fibre was injected with Lucifer Yellow, the endplate stained for acetylcholinesterase and the length of the nerve terminal and the length of a sarcomere were measured on the fluorescent fibre. 3. The total number of quanta secreted by a terminal was measured under a wide variety of experimental conditions: the weights of the frogs ranged from 13 to 68 g, the temperature from 9 to 28 degrees C, and the concentration of Mg2+ from 2 to 10 mM. In one series of experiments the Mg2+ was withdrawn after 3‐4 min and reapplied 35‐40 min later in order to divide the total output of quanta into two approximately equal bouts of secretion that were well separated in time. 4. The total number of MEPPs recorded at a junction was loosely correlated with the length of its nerve terminal, but it was not affected by the temperature, the concentration of Mg2+ or the division of secretion into well‐separated bouts of quantal release. The average total secretion per unit length was about 3700 quanta/sarcomere or about 1200 quanta/microns. 5. The average time course of quantal secretion per micrometre of terminal was determined at single junctions in muscles held at 22‐23 degrees C or at 9‐10 degrees C. Other muscles were fixed at various times during the course of secretion at each temperature and the number of synaptic vesicles remaining in cross‐sections of the terminals were counted on electron micrographs. The number of vesicles remaining per micrometre of terminal was determined from the number per cross‐section and the section thickness.(ABSTRACT TRUNCATED AT 400 WORDS)