bluuebunny/crossref_metadata_2025 huggingface dataset:
Cl‐ and Na+ homeostasis during anoxia in rat hypoglossal neurons: intracellular and extracellular in vitro studies.
Authors: Jiang C , Agulian S , Haddad G G
Published: December 2014

1. To understand the mechanisms which lead to acute neuronal swelling during anoxia, we studied the ionic movements of Cl‐ and Na+ during O2 deprivation in the hypoglossal (XII) neurons of rat brain slices using double‐barrelled ion‐selective microelectrodes. 2. Baseline extracellular Cl‐ and Na+ activities ([Cl‐]o, [Na+]o) were 128.3 +/‐ 7.4 and 150.0 +/‐ 3.4 mM respectively (n = 12) in the adult. Similar baseline values were obtained from neonatal brain slices. 3. During a period of anoxia (4 min), [Na+]o decreased by about 40 mM in adult slices while [Na+]o did not show any significant change in the neonate (n = 12). Although anoxia induced a significant decrease of [Cl‐]o in both adult and neonate, [Cl‐]o dropped 7 times more in the adult than in the neonate (n = 12). 4. Intracellular Cl‐ activity ([Cl‐]i) was studied in twenty‐seven adult hypoglossal cells. All showed an increase in [Cl‐]i) was studied in twenty‐seven adult hypoglossal cells. All showed an increase in [Cl‐]i with O2 deprivation. Detailed analysis carried out on ten hypoglossal neurons showed a baseline [Cl‐]i of 11.4 +/‐ 4.5 mM and an increase in [Cl‐]i by 20.6 +/‐ 7.2 mM during O2 limitation. 5. Baseline [Cl‐]i in neonatal XII neurons was similar to that of the adult. Anoxia, however, produced an increase in [Cl‐]i by only 4.5 +/‐ 2.4 mM (n = 7). This increase in [Cl‐]i was significantly less than that in the adult (P less than 0.001). Prolonged anoxia (6‐12 min) in the neonate led to a more substantial increase in [Cl‐]i, an observation consistent with the decrease in [Cl‐]o after prolonged O2 deprivation. 7. We conclude that during anoxia: (1) intracellular [Cl‐] increases in the adult and this most likely occurs because of entry of extracellular Cl‐ into the cytosol and (2) there is a major maturational difference in mechanisms regulating Cl‐ and Na+ homeostasis between newborn and adult brain tissue. We speculate that these mechanisms may account, at least partially, for the relative tolerance to anoxia in the newly born.
Role of an inwardly rectifying potassium current in rabbit ventricular action potential.
Authors: Shimoni Y , Clark R B , Giles W R
Published: December 2014

1. Whole‐cell voltage‐clamp measurements were made of the time‐ and voltage‐dependent properties of the inwardly rectifying background potassium current IK1, in single myocytes from rabbit ventricle. The main goal of these experiments was to define the role of IK1 in the plateau and repolarization phases of the action potential (AP). 2. Action potentials from single ventricular myocytes were used as the command signals for voltage‐clamp measurements. In these ‘action potential voltage‐clamp’ experiments, IK1 was isolated from other membrane currents by taking the difference between control currents and currents in K(+)‐free bathing solution. The results show that IK1 is small during the plateau, but then rapidly increases during repolarization and declines in early diastole. 3. Evidence of an important functional role for IK1 in AP repolarization was obtained by comparing the magnitude of IK1 and the rate of change of membrane potential (dVm/dt) in the same cell during the AP. The time courses of IK1 and dVm/dt during the AP were closely correlated, indicating that IK1 was the principal current responsible for final repolarization. 4. Rectangular voltage‐clamp steps were used to study time‐ and voltage‐dependent changes in IK1 at membrane potentials corresponding to the repolarization phase of the AP. ‘Slow’ relaxations or tail currents, lasting 100‐300 ms, were consistently recorded when the cell was repolarized to potentials in the range ‐30 to ‐70 mV, following depolarizations between +10 and ‐10 mV. 5. The close correlation between the magnitude of the steady‐state IK1 (in an external K+ concentration of 5.4 mM), which was outward for membrane potentials in the range ‐30 to ‐70 mV, and the magnitude of the tail currents, suggests that they resulted from a slow increase, or reactivation, of IK1. 6. The component of the slow tails due to reactivation of IK1 can be separated from a previously described component due to Na(+)‐Ca2+ exchange since the IK1 component: (i) does not depend on the presence of the calcium current, ICa; (ii) can be recorded when internal EGTA (5 mM) suppresses large changes in [Ca2+]i; (iii) does not depend on the Na+ electrochemical gradient; (iv) is abolished in K(+)‐free external solution; and (v) is not present in rabbit atrial myocytes, in which IK1 is very small. 7. The time‐ and voltage‐dependent properties of IK1 revealed by these tail current experiments suggest that the measured magnitude of IK1 will be dependent on the voltage‐clamp protocol.(ABSTRACT TRUNCATED AT 400 WORDS)
Volume‐sensitive chloride conductance in bovine chromaffin cell membrane.
Authors: Doroshenko P , Neher E
Published: December 2014

1. Bovine chromaffin cells were inflated by pressure applied through a pipette or swollen during intracellular perfusion with hypertonic solutions. Effects of such procedures on electrical properties of the membrane were studied by a combination of the tight‐seal whole‐cell patch‐clamp technique and Fura‐2 fluorescence measurements of free intracellular calcium concentration ([Ca2+]i). 2. Application of air pressure (about +5 cmH2O or 490 Pa) through the patch pipette caused an increase in the cell volume and concomitant development of an inwardly directed transient current at the holding potential of ‐60 mV. The current gradually increased to a peak value and subsequently decayed almost to its initial level within 5‐10 min. A short pulse of pressure (5‐10 s) was sufficient to elicit the whole sequence of events. 3. Intracellular free Ca2+ ion concentration, [Ca2+]i, steeply increased at the beginning of the pressure pulse to about 0.2 microM and either stayed at this level or decayed back to the more usual value of approximately 0.1 microM. 4. Similar changes in the transmembrane current and [Ca2+]i were observed during intracellular perfusion of cells with hypertonic solutions (30‐50 mosM difference relative to the bath solution) or during extracellular application of hypotonic solution. 5. Swelling of non‐perfused cells by extracellular application of hyposmotic solution caused the appearance of inward currents in cell‐attached membrane patches held at a fixed potential ‐30 mV relative to the cell's resting potential. The kinetics of the current resembled those of the whole‐cell current. 6. Intracellular introduction of guanosine triphosphate (GTP, 300 microM) significantly prolonged the duration (from 62 +/‐ 10 s, n = 5, to 98 +/‐ 8 s, n = 4, when measured at the level of half‐amplitude), while introduction of the non‐hydrolysable analogue of guanosine diphosphate (GDP), guanosine 5'‐O‐(2‐thiodiphosphate) (GDP beta S, 300 microM), decreased the maximal rate of increase (from 11.4 +/‐ 2.6 pA/s, n = 6, to 3.2 +/‐ 2.1 pA/s, n = 10) of the current activated by pressure. 7. Lowering of the intracellular free Ca2+ ion concentration by introduction of 10 mM‐EGTA did not significantly affect the current amplitude or time course. However, a rapid increase in the [Ca2+]i to micromolar levels (by activation of the voltage‐operated calcium channels during membrane depolarization) could terminate development of the current activated by pressure and cause its fast decay to zero‐current level.(ABSTRACT TRUNCATED AT 400 WORDS)
The perceptions of force and of movement in a man without large myelinated sensory afferents below the neck.
Authors: Cole J D , Sedgwick E M
Published: December 2014

1. Motor memory and the sense of effort have been investigated in a man with a complete large fibre sensory neuropathy for over 16 years. The perceptions of pain, heat, cold and muscular fatigue remained but he was without perceptions of light touch and proprioception below the neck. 2. The subject was able to discriminate weights held in the hand with an accuracy only slightly worse than control subjects (20 g in 200 g) when forearm movement and visual inspection were allowed. With eyes shut however he could only distinguish a weight of 200 g from 400 g. It is concluded that a crude sense of effort remains which may have a peripheral origin. 3. A limited motor memory was also present, which allowed him to maintain a posture or continue a simple repetitive movement. No novel movement was possible without visual feedback. 4. Differences in movement ability between this subject and others with similar if less pure sensory neuropathies are ascribed to rehabilitation.
The location of the receptors involved in the human diuretic response to drinking an isotonic electrolyte solution.
Authors: Crotty T B , Gebruers E M , Hall W J
Published: December 2014

1. This study aimed to shed light on the receptors involved in the diuretic response to drinking isotonic fluids in man by employing a polyethylene glycol‐based bowel lavage solution (Golytely) which is reported to cause no net movement of fluid across the gut. 2. Drinking Golytely resulted in a transient hypotonic diuresis. Mean urine flow rose from control values of 0.9 ml min‐1 to 10.1 ml min‐1 70 min after the start of drinking. The increase in urine output was accompanied by a fall in urine osmolality from control values of 879 mosM kg‐1 to 105 mosM kg‐1. The diuresis is similar to that produced by ingestion of an equal volume of an absorbable electrolyte‐based solution (Tyrode). 3. Neither solution produced changes in plasma osmolality or electrolytes, but Golytely provoked a 6.8% contraction of plasma volume, whereas drinking Tyrode resulted in plasma expansion. Copious diarrhoea was experienced by all subjects who drank Golytely solution and by none on drinking Tyrode solution. 4. The infusion of Golytely into the stomach resulted in a hypotonic diuresis similar in magnitude to that elicited by drinking. Drinking with simultaneous aspiration of gastric contents ('sham‐drinking') did not produce a significant diuresis. 5. Plasma arginine vasopressin (AVP) levels did not fall following the drinking of Golytely. The assay used was sufficiently sensitive to measure changes of 0.6 pg AVP (ml plasma)‐1. 6. The findings show that signals from the oropharynx do not mediate the diuretic response to drinking Golytely in man and that a mechanism other than inhibition of AVP release appears to be involved. The receptors mediating this response may lie in the stomach and/or small bowel since Golytely is not absorbed, as evidenced by the contraction of plasma volume and diarrhoea.
Synaptic interaction between medullary respiratory neurones during apneusis induced by NMDA‐receptor blockade in cat.
Authors: Feldman J L , Windhorst U , Anders K , Richter D W
Published: December 2014

1. Termination of inspiration is an essential component of respiratory rhythm generation and its perturbation can result in apneusis, i.e. significant prolongation of mechanisms, we studied the postsynaptic events in respiratory neurones during apneustic respiratory periods, and compared them to normal respiratory cycles. 2. Experiments were performed in pentobarbitone‐anaesthetized, paralysed, thoracotomized cats ventilated with a constant volume or a cycle‐triggered constant pressure pump. Apneusis, separated by normal cycles, was induced as follows: the animal was ventilated by a cycle‐triggered pump that normally inflated the lungs during the inspiratory burst of phrenic nerve discharge. The NMDA‐receptor blocker MK‐801 [(+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5, 10‐iminemaleate] (0.3‐0.7 mg/kg) was administered intravenously, and, for designated breaths, inflation of the lungs was withheld during neural inspiration. 3. Membrane potential trajectories of forty‐one late expiratory (E‐2) and eight postinspiratory (PI) neurones of the caudal ventral respiratory group were analysed before and/or after MK‐801 administration, during normal and apneustic periods. 4. Before MK‐801 administration, withholding lung inflation caused modest (10‐20%) lengthening of the inspiratory period; after MK‐801 administration, withholding inflation caused apneusis. Provided that the lungs were inflated during the inspiratory phase, the temporal pattern of phrenic nerve, recurrent laryngeal nerve and membrane potential trajectories of E‐2 and PI neurones were not significantly altered by MK‐801. Apneusis following NMDA‐receptor blockade produced consistent changes in the synaptic activation patterns of E‐2 neurones. In particular, the slow late inspiratory‐related depolarization pattern of E‐2 neurones was consistently retarded during apneustic inspiratory phases when compared to normal inspiratory phases. This was due to continuation of Cl(‐)‐mediated synaptic inhibition of E‐2 neurones. Superior laryngeal nerve stimulation stopped apneusis and sustained membrane hyperpolarization of E‐2 neurones similar to lung inflation. 5. During the plateau phase of apneusis, correlated 10‐20 Hz oscillations could be observed in the integrated phrenic and recurrent laryngeal nerve activities as well as in the membrane potential of E‐2 neurones. 6. We conclude that: (i) the prolonged inhibition of E‐2 neurones during apneusis is indicative of the process responsible for the prolongation of the inspiratory phase.(ABSTRACT TRUNCATED AT 400 WORDS)
Intracoronary veratrine attenuates carotid baroreceptor reflex regulation of blood pressure in conscious dogs.
Authors: Denison A L , Stephenson R B , Hull S S , Cornish K G , Zucker I H
Published: December 2014

1. The effect of activation of left ventricular cardiac receptors on carotid baroreflex control of blood pressure, heart rate, cardiac output, and total peripheral resistance was determined in conscious dogs. Previous studies in conscious subjects assessed only the effect on baroreflex control of heart rate. 2. Dogs with denervated aortic baroreceptors were equipped with aortic flow probes, cardiac pacing electrodes, and catheters in the aorta, vena cava, and left circumflex coronary artery. Both carotid sinus regions were prepared for reversible vascular isolation. 3. Left ventricular receptors were stimulated by an infusion of veratrine (0.1‐1.0 micrograms kg‐1 min‐1) into the left circumflex coronary artery. 4. Veratrine infusion decreased control blood pressure only 10 +/‐ 2 mmHg, but it decreased the range of baroreflex control of blood pressure by 50% and decreased maximum baroreflex gain by 42%. Both the cardiac output and total peripheral resistance components of the baroreflex were attenuated. 5. Baroreflex control of blood pressure was unaffected by intravenous veratrine or by intracoronary infusion of vehicle. 6. Intracoronary veratrine had no effect after autonomic ganglionic blockade. 7. When cardiac output was kept nearly constant (by beta‐adrenergic and cholinergic receptor blockade or by beta‐blockade and cardiac pacing), intracoronary veratrine still attenuated baroreflex control of blood pressure and total peripheral resistance. Veratrine impaired the ability of the baroreflex to utilize alpha‐adrenergic mechanisms to control total peripheral resistance. 8. We conclude that activation of ventricular receptors attenuates baroreflex regulation of blood pressure in conscious dogs through an attenuation of baroreflex control of both cardiac output and total peripheral resistance.
The electro‐mechanical response of canine inspiratory intercostal muscles to increased resistance: the cranial rib‐cage.
Authors: De Troyer A
Published: December 2014

1. The effect of graded increases in inspiratory airflow resistance on the electrical activity and the mechanical behaviour of the three groups of inspiratory intercostal muscles (parasternal intercostal, external intercostal, levator costae) situated in the cranial portion of the rib‐cage has been studied in ten anaesthetized, spontaneously breathing dogs. The mechanical behaviour of the muscles was determined by measuring the respiratory changes in muscle length and the displacements of the rib. 2. During unloaded inspiration, the three muscles were active, the rib moved in the cranial direction, and the parasternal intercostal and levator costae muscles shortened; in most animals, the external intercostals shortened as well. 3. Graded increases in inspiratory airflow resistance elicited a progressive inhibition of parasternal intercostal activity and a gradual facilitation of external intercostal and levator costae activities. Concomitantly, the parasternal intercostals continued to shorten during inspiration. However, both the external intercostals and the levator costae progressively lengthened, and the rib was gradually displaced in the caudal direction. This pattern persisted after increases in chemical respiratory drive had developed. 4. Sectioning the phrenic nerve roots did not alter the electrical or the mechanical response of the parasternal intercostal muscles to loading, but it markedly affected the response of the external intercostals and levator costae. After phrenicotomy, the external intercostals and levator costae continued to shorten during loaded breaths, the rib continued to be displaced in the cranial direction, and although the rate of inspiratory muscle shortening and of rib motion decreased, the facilitation of external intercostal and levator costae activities was markedly reduced or abolished. 5. Lengthening of the external intercostals and caudal displacement of the rib was reproduced by isolated stimulation of the phrenic nerves. 6. The reflex facilitation of external intercostal and levator costae activities that takes place during inspiratory resistive loading thus results primarily from the collapsing action of the diaphragm on the cranial portion of the rib‐cage and the consequent lengthening of these muscles. The mechanical effectiveness of this reflex facilitation, however, appears to be relatively small.
Mechanical properties of skinned rabbit psoas and soleus muscle fibres during lengthening: effects of phosphate and Ca2+.
Authors: Stienen G J , Versteeg P G , Papp Z , Elzinga G
Published: December 2014

1. Mechanical properties of permeabilized single fibres from rabbit psoas and soleus muscle were determined by measuring the length responses due to abrupt changes in load and the force responses due to isovelocity length changes at different phosphate and Ca2+ concentrations. 2. The length responses due to abrupt increases in load from psoas fibres showed a rapid lengthening during the change in load followed by a phase of lengthening during which the velocity gradually decreased. In soleus fibres an abrupt lengthening during the change in load was followed by a phase of lengthening during which the velocity remained constant or decreased slightly for increases in load to less than 1.45 of the isometric force (F0). For larger increases in load the velocity during this later phase first increased and thereafter decreased. 3. The initial force‐velocity curve, derived from the early part of the isotonic responses after the change in load, as well as the late force‐velocity curve derived from the force level attained during isovelocity length changes, were sensitive to phosphate. Phosphate caused a shift of the absolute force‐velocity curves of both psoas and soleus fibres towards lower values of force. In psoas fibres, the relative force‐velocity curves derived by normalization of the force level to the force developed isometrically was shifted by phosphate to smaller velocities. In soleus fibres, the initial velocity at low and intermediate relative loads (less than 1.75 F0) was increased by phosphate but at higher loads it decreased, while the late force‐velocity curve showed an overall decrease in velocity. 4. The force responses during isovelocity lengthening of psoas fibres showed an early rapid increase in force followed by a slow rise in force. The position of this break point in force was sensitive to the phosphate concentration. In soleus fibres, the force responses without phosphate showed an overshoot followed by a slow rise in force. The overshoot diminished with increasing phosphate concentration. 5. Phosphate and Ca2+ affected the force responses in psoas and soleus fibres in different ways. When the isometric starting levels were the same, force during and after the length change at submaximal activation was always less than at maximal activation in the presence of 15 mM‐phosphate. 6. The changes in the mechanical performance during lengthening caused by phosphate in psoas as well as in soleus fibres, are in agreement with a decrease in the average force per attached crossbridge.(ABSTRACT TRUNCATED AT 400 WORDS)
Effect of an inhaled thromboxane mimetic (U46619) on in vivo pulmonary resistance and airway hyperresponsiveness in dogs.
Authors: Jones G L , Lane C , O'Byrne P M
Published: December 2014

1. We investigated the role of thromboxane A2 in the airway hyperresponsiveness that follows the inhalation of ozone in dogs by examining the responses to an inhaled thromboxane analogue (U46619). 2. Measurements of pulmonary resistance were made in anaesthetized dogs; the concentration of inhaled agonist causing an increase of 5 cmH2O l‐1 s was calculated (provocative concentration). The effect of inhaled U46619 was studied on in vivo canine airway resistance, on airway responsiveness and on airways made hyperresponsive following the inhalation of ozone. 3. Inhaled thromboxane is a potent constrictor of the canine airway. The mean provocative concentration was 2.13 x 10(‐4) M, compared to acetylcholine which was 3.23 x 10(‐2) M. 4. Inhaled thromboxane did not result in the development of airway hyperresponsiveness to acetylcholine. Following U46619 inhalation the mean provocative concentration to acetylcholine was 3.92 x 10(‐2) M. 5. Canine airway was not hyperresponsive to inhaled thromboxane following the inhalation of ozone. This was not due to an inhibition of acetylcholinesterase as the dogs were hyperresponsive to carbachol (a muscarinic agonist not degraded by endplate cholinesterase). 6. These experiments do not support a role for thromboxane in the development of airway hyperresponsiveness following the inhalation of ozone in dogs.
Effects of muscarine on single rat adrenal chromaffin cells.
Authors: Neely A , Lingle C J
Published: December 2014

1. The action of muscarine on membrane currents and cytosolic calcium (Ca2+) of dissociated rat adrenal chromaffin cells was investigated using standard whole‐cell voltage‐clamp techniques and microfluorimetry of unclamped single cells. 2. In cells held at a constant holding potential negative to ‐40 mV, brief (5‐10 s) applications of muscarine produced a transient activation of outward current. The activation of this current by muscarine also occurs in the presence of 5 mM‐Co2+. 3. The outward current activated by muscarine at holding potentials negative to about ‐40 mV is blocked over 90% by either 200 microM‐curare or 200 nM‐apamin. One millimolar TEA produces variable blocking effects at such potentials. 4. The outward current activated by muscarine is transient even in the continuing presence of muscarine. Complete recovery between pairs of muscarine applications occurs over a 1‐2 min period. If sufficient time was allowed for recovery between muscarine applications, the muscarine‐activated outward current could be reliably elicited in dialysed cells for periods of 20‐30 min. 5. Voltage ramps were used to examine effects of muscarine on currents over a range of membrane potentials. Over all potentials, muscarine activates a relatively voltage‐independent component which is blocked almost completely by 200 nM‐apamin and by 200 microM‐curare. At potentials negative to about ‐40 mV, the apamin‐ and curare‐sensitive current accounts for virtually all muscarine‐activated current. This current appears to correspond to a Ca(2+)‐activated, voltage‐independent current found in these cells. Effects of muscarine on currents activated at potentials positive to 0 mV are complex. At potentials above 0 mV, muscarine can produce either an activation or an inhibition of outward current. The outward current activated at positive potentials was primarily voltage dependent and blocked by 1 mM‐TEA. However, in some cells activation of voltage‐dependent current was not observed and, in such cases, muscarine produced an inactivation of the voltage‐dependent component of current. The inactivation of outward current could also be observed in the presence of 5 mM‐Co2+ indicating that the inactivation does not occur secondarily to an effect of muscarine on Ca2+ current. The possibility is discussed that the inactivation of outward current occurs as a result of intrinsic inactivation properties of the voltage‐dependent Ca(2+)‐dependent K+ current. According to this hypothesis, the extent to which inactivation of voltage‐dependent outward current is observed depends on the magnitude of the muscarine‐induced cytosolic Ca2+ elevation and the level of depolarization of the cell.(ABSTRACT TRUNCATED AT 400 WORDS)
Exponential activation of the cardiac Na+ current in single guinea‐pig ventricular cells.
Authors: Mitsuiye T , Noma A
Published: December 2014

1. The cardiac Na+ current of guinea‐pig was recorded using an improved oil‐gap voltage clamp method. When a single ventricular cell was stretched between the internal and external solution compartments across an oil gap of about 40 microns in width, the sealing resistance in the oil gap was higher than 1 G omega and the time constant of the capacitive current was between 10 and 40 microseconds. Effective series resistance (Rs) was less than 50 k omega after Rs compensation. 2. The activation time course (I'Na) was separated from inactivation by dividing the digitized record of Na+ current with the inactivation variable h(t), which was obtained by fitting exponential functions to the decaying phase of current. I'Na started as a single exponential activation at time 0, which was defined by the decay of the capacitive current to 5% of its peak. 3. The Na+ tail current was recorded on repolarization after a short (1.2 ms) depolarizing pulse to ‐10 mV. Its single exponential decay at potentials negative to ‐50 mV, or its major exponential component of decay between ‐50 and ‐30 mV, was attributed to deactivation. The time constants of deactivation were similar to those of activation which were measured from I'Na on depolarization to comparable potentials. The m1 kinetics gave a better fit for Na+ activation than the m3 kinetics. 4. The time constant of deactivation was a linear function of the membrane potential on a semilogarithmic scale with an e‐fold increase per 21.6 +/‐ 1.3 mV (n = 8) depolarization. The steady‐state activation value (m(infinity)) was obtained from the amplitude of I'Na. Fitting a Boltzmann equation indicated a half‐activation potential of ‐21.9 +/‐ 1.7 mV and a slope factor of 7.9 +/‐ 0.4 mV (n = 9). 5. m1 kinetics are more pertinent to a description of the cardiac Na+ current. Limitations in analysing the activation kinetics of Na+ current are discussed for the improved oil‐gap voltage clamp method.
The peptide CGRP increases a high‐threshold Ca2+ current in rat nodose neurones via a pertussis toxin‐sensitive pathway.
Authors: Wiley J W , Gross R A , MacDonald R L
Published: December 2014

1. The whole‐cell variation of the patch clamp technique was used to study the effect of calcitonin gene‐related peptide (CGRP) on voltage‐gated calcium currents in acutely dissociated rat nodose ganglion neurones and to determine if its effects were mediated via a guanine nucleotide binding (G) protein. 2. Both low‐ and high‐threshold calcium current components were present in nodose ganglion neurones. CGRP had no effect on the isolated low‐threshold current component. However, CGRP (1‐1000 nM, ED50 = 50 nM) caused a concentration‐dependent increase in high‐threshold calcium currents. CGRP (1 microM) increased the peak of these calcium currents 21 +/‐ 4% over controls. 3. CGRP enhanced a transient high‐threshold calcium current evoked from a holding potential of ‐80 mV but did not affect the slowly inactivating high‐threshold current evoked from ‐40 mV. Multiple high‐threshold calcium currents have been reported in sensory neurones. We cannot state unequivocally which high‐threshold calcium current component was enhanced by CGRP. However, based on the observation that CGRP increased a transient but not the slowly inactivating high‐threshold calcium current, we believe the peptide enhanced primarily the N‐type calcium current component. 4. CGRP increased the maximal peak current and caused a modest negative shift of < or = 10 mV in the peak of the current‐voltage (I‐V) relation in three of six neurones. In the remaining three neurones the peptide increased the maximal peak current without a detectable shift in the peak of the I‐V relation. 5. To determine if the CGRP‐induced enhancement in calcium current was associated with an increase in calcium conductance, we studied the effect of the peptide on the instantaneous current‐voltage (I‐V) relation when currents were evoked at a clamp potential (Vc) of +30 mV, positive to the observed maximal current (Vc = 0 to +10 mV). CGRP increased the maximal conductance 23 +/‐ 4%. 6. The enhancement of calcium current by CGRP was not due to a shift in the voltage dependency of steady‐state inactivation of the calcium channels. The stimulatory effect of CGRP on calcium current was evaluated by evoking currents from different holding potentials (Vh) at the same Vc (+10 mV). CGRP‐induced increases in calcium currents were similar over the range of (Vh) from ‐60 to ‐110 mV, suggesting that the peptide did not alter voltage‐dependent steady‐state inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)
Ca2+ efflux mechanisms following depolarization evoked calcium transients in cultured rat sensory neurones.
Authors: Benham C D , Evans M L , McBain C J
Published: December 2014

1. We have used a combination of microfluorimetry and patch‐clamp techniques to investigate cytoplasmic Ca2+ ([Ca2+]i) buffering in response to physiological Ca2+ loads in neurones cultured from the dorsal root ganglia of the rat. 2. In cells loaded with Indo‐1 AM and using high resistance microelectrodes to initiate and record action potentials, single action potentials were associated with a measurable rise in [Ca2+]i. Short trains of action potentials evoked [Ca2+]i transients with monoexponential recovery rates with time constants of around 5 s. 3. Similar Ca2+ buffering properties were seen in cells perfused with patch‐clamp pipettes in the whole‐cell recording mode suggesting that the slow (seconds) Ca2+ buffering properties were not seriously perturbed by the recording technique. 4. In cells held under voltage clamp, reversal of the Na(+)‐Ca2+ exchanger driving force had a small but significant effect on the rate of Ca2+ removal. 5. Increasing extracellular pH or adding vanadate (200 microM) to the internal solution dramatically slowed the rate of recovery. Addition of calmidazolium to the pipette solution also produced a significant but much less dramatic slowing of Ca2+ efflux. 6. The results demonstrate that the activity of a plasmalemmal Ca(2+)‐ATPase is important for the removal of somatic Ca2+ loads of a similar amplitude to those generated by the firing of a few action potentials.
Macroscopic and unitary properties of physiological ion flux through L‐type Ca2+ channels in guinea‐pig heart cells.
Authors: Rose W C , Balke C W , Wier W G , Marban E
Published: December 2014

1. We investigated the currents through L‐type Ca2+ channels when Ca2+ (1‐10 mM) was the charge carrier, as is the case physiologically. 2. Na+ was removed from both the external and internal solutions to eliminate currents through Na+ channels and Na(+)‐Ca2+ exchange. 3. From a holding potential of ‐50 mV only L‐type channels were available to open with depolarization. Macroscopic L‐type currents were maximal during depolarizing pulses to +10 mV (peak current density of 4.7 +/‐ 0.3 nA nF‐1). 4. During depolarizing steps as long as 180 ms, the decay of current through L‐type channels was incomplete, in contrast to that of T‐type current. 5. Unitary currents recorded with comparable ionic conditions and voltage protocols exhibited a single‐channel conductance of 6.9 pS in 10 mM Ca2+. Ensemble average currents reproduced accurately the features of whole‐cell L‐type current, including the maintained component. 6. Convolution analysis was employed to clarify the single‐channel basis of the complex current waveform of L‐type channels. First openings underlie the peak, while the maintained pedestal is generated by multiple re‐openings. As with T‐type channels, single openings are brief and contribute little to the time course of the average current. 7. The prominent maintained component of macroscopic and ensemble average L‐type current cannot be explained by simple Markov models in which current decay reflects the progressive entry of channels into an absorbing inactivated state. 8. We considered the possibility that the maintained component of current arises from the existence of multiple distinct gating patterns, one of which lacks inactivation. Individual sweeps were sorted among three patterns of gating (no openings, active‐early and active‐late). Patterns of activity are not randomly distributed; instead, they tend to cluster over time. 9. Most of the maintained current is attributable to the ‘active‐late’ pattern of gating. Considered separately, this pattern can be well described by a simple Markov chain lacking an inactivated state. The ‘active‐early’ gating pattern accounts entirely for the initial current transient, and for about one‐third of the maintained component; thus, inactivation, even when present, must be reversible rather than absorbing. 10. The unitary current amplitudes and peak open probabilities measured for single L‐type channels, when compared to the average macroscopic L‐type current density, predict 170 functional channels per picofarad, or 28,000 L‐type channels per typical ventricular myocyte.(ABSTRACT TRUNCATED AT 400 WORDS)
Expression and characterization of a canine hippocampal inwardly rectifying K+ current in Xenopus oocytes.
Authors: Cui J , Mandel G , DiFrancesco D , Kline R P , Pennefather P , Datyner N B , Haspel H C , Cohen I S
Published: December 2014

1. An inwardly rectifying potassium current expressed in Xenopus laevis oocytes injected with canine hippocampal poly(A)+ RNA was investigated with the two‐microelectrode voltage clamp technique. 2. Xenopus oocytes injected with canine hippocampal poly(A)+ RNA expressed a current activated by hyperpolarization. This current contained an instantaneous and a time‐dependent component. Both components were inwardly rectifying and could be blocked by extracellular Cs+ or Ba2+. 3. The expressed current was carried mainly by K+. Its reversal potential measured in different [K+]os could be fitted by the Nernst equation with a slope of ‐50.7 per tenfold change in [K+]o. Extracellular Cl‐ and Na+ made minimal contributions to the current. 4. The activation of the expressed current depended on both voltage and [K+]o. Activation started near EK and the activation curve shifted along the voltage axis in parallel with EK when [K+]o was altered. 5. The activation time constants of the expressed current also depended on both voltage and [K+]o. The voltage dependence of the time constants was bell‐shaped and the peak value was at a potential 30‐50 mV more negative than EK. The voltage dependence of the time constants shifted along the voltage axis when EK was changed. 6. The poly(A)+ RNA extracted from canine hippocampus was fractionated in a 10‐31% linear sucrose gradient. The size of the mRNA required to express the inwardly rectifying current was estimated to be around 4 kb. 7. In conclusion, the expressed current is an inwardly rectifying potassium current. The canine hippocampal mRNA should be an excellent source for expression‐cloning of the inward rectifier channel.
Responses of macaque ganglion cells to the relative phase of heterochromatically modulated lights.
Authors: Smith V C , Lee B B , Pokorny J , Martin P R , Valberg A
Published: December 2014

1. We measured the response of macaque ganglion cells to sinusoidally modulated red and green lights as the relative phase, theta, of the lights was varied. 2. At low frequencies, red‐green ganglion cells of the parvocellular (PC‐) pathway with opponent inputs from middle‐wavelength sensitive (M‐) and long‐wavelength sensitive (L‐) cones were minimally sensitive to luminance modulation (theta = 0 deg) and maximally sensitive to chromatic modulation (theta = 180 deg). With increasing frequency, the phase, theta, of minimal amplitude gradually changed, in opposite directions for cells with M‐ and L‐cone centres. 3. At high frequencies (at and above 20 Hz), phasic cells of the magnocellular (MC‐) pathway were maximally responsive when theta approximately 0 deg and minimally responsive when theta approximately 180 deg, as expected from an achromatic mechanism. At lower frequencies, the phase of minimal response shifted, for both on‐ and off‐centre cells, to values of theta intermediate between 0 and 180 deg. This phase asymmetry was absent if the centre alone was stimulated with a small field. 4. For PC‐pathway cells, it was possible to provide an account of response phase as a function of theta, using a model involving three parameters; phases of the L‐ and M‐cone mechanisms and a L/M cone weighting term. For red‐green cells, the phase parameters were monotonically related to temporal frequency and revealed a centre‐surround phase difference. The phase difference was linear with a slope of 1‐3 deg Hz‐1. If this represents a latency difference, it would be 3‐8 ms. Otherwise, temporal properties of the M‐ and L‐cones appeared similar if not identical. By addition of a scaling term, the model could be extended to give an adequate account of the amplitude of responses. 5. We were able to activate selectively the surrounds of cells with short‐wavelength (S‐) cone input to their centres, and so were able to assess L/M cone weighting to the surround. M‐ and L‐cone inputs added linearly for most cells. On average, the weighting corresponded to the Judd modification of the luminosity function although there was considerable inter‐cell variability. 6. To account for results from MC‐pathway cells, it was necessary to postulate a cone‐opponent, chromatic input to their surrounds. We developed a receptive field model with linear summation of M‐ and L‐cones to centre and surround, and with an additional M,L‐cone opponent input to the surround.(ABSTRACT TRUNCATED AT 400 WORDS)
Influence of hypertonic‐hyperoncotic solution and furosemide on canine hydrostatic pulmonary oedema resorption.
Authors: Wickerts C J , Berg B , Frostell C , Schmidt J , Blomqvist H , Rösblad P G , Kihlström I , Messmer K , Hedenstierna G
Published: December 2014

1. This study aimed at enhancing the clearance of experimental hydrostatic pulmonary oedema in dogs using hypertonic‐hyperoncotic solution (HHS) and furosemide. 2. Anaesthetized dogs (n = 20) were mechanically ventilated with a positive end‐expiratory pressure of 10 cmH2O (1.0 kPa). 3. Hydrostatic pulmonary oedema was induced by inflating a balloon inserted into the left atrium and simultaneously infusing isotonic saline rapidly. Oedema formation was terminated by deflating the balloon and reducing the infusion rate. 4. Four groups were studied: A, control; B, furosemide; C, HHS and D, HHS+furosemide. HHS, 6 ml kg‐1, was given as a bolus injection and furosemide, 1 mg kg‐1, intravenously as a bolus followed by an infusion of 0.5 mg kg‐1 h‐1. All dogs were studied for 4 h. 5. Serum osmolarity, plasma colloid oncotic pressure and diuresis in groups C and D (HHS groups) substantially increased; haemoglobin concentration decreased and pulmonary arterial wedge pressure remained constant. 6. Despite the combination of these factors favouring fluid flux from the extravascular to the intravascular compartment, extravascular lung water measured with the double indicator dilution technique decreased no faster in the HHS groups than in the two other groups (from over 26 to approximately 19 ml kg‐1 in groups A, C and D and to 14.7 in group B (only furosemide)). 7. This was confirmed by postmortem gravimetric measurements of extravascular lung water; A, 11.0 +/‐ 5.7; B, 9.7 +/‐ 3.3; C, 10.5 +/‐ 3.1 and D, 10.6 +/‐ 1.8 g kg‐1. 8. We speculate that mechanisms other than effective Starling gradients and enhanced diuresis might define a maximal rate of pulmonary oedema clearance.
Inorganic phosphate decreases the Ca2+ content of the sarcoplasmic reticulum in saponin‐treated rat cardiac trabeculae.
Authors: Smith G L , Steele D S
Published: December 2014

1. Measurements of [Ca2+] were made in saponin‐permeabilized rat ventricular trabeculae using the fluorescent indicator Indo‐1. Application of caffeine (20 mM) caused a transient rise in [Ca2+] within the preparation as a result of Ca2+ release from the sarcoplasmic reticulum (SR). The size of the caffeine‐induced Ca2+ transient was related to the amount of Ca2+ accumulated by the SR prior to addition of caffeine. Caffeine‐induced Ca2+ release was abolished by ryanodine (10 microM), an inhibitor of SR Ca2+ release. 2. At a bathing [Ca2+] of 0.2 microM, the amount of Ca2+ released from the SR on addition of caffeine was sufficient to generate a tension transient. Ca2+ and tension responses were stabilized by application of caffeine at regular intervals (2 min). Addition of 10 mM inorganic phosphate (Pi) induced a transient increase in [Ca2+] within the preparation due to a net release of Ca2+ from the SR. The amplitude of subsequent caffeine‐induced Ca2+ transients were reduced to 65 +/‐ 7.5% (mean +/‐ S.D., n = 13) of control. In addition, the accompanying tension transient fell to 45 +/‐ 6.9% of control. Removal of Pi caused a transient decrease in the [Ca2+] within the preparation consistent with a net increase in Ca2+ uptake by the SR. Subsequent caffeine‐induced Ca2+ and tension transients returned to control levels. 3. Inclusion of Pi (2‐30 mM) in the perfusing solution decreased the size of caffeine‐induced Ca2+ and tension transients in a dose‐dependent manner. 4. Addition of 10 mM ADP caused a transient increase in [Ca2+] and depressed subsequent caffeine‐induced Ca2+ transients to a greater extent than 10 mM Pi. Despite the reduction in Ca2+ release from the SR, tension responses were larger in the presence of 10 mM ADP than under control conditions. This is a consequence of an increase in Ca(2+)‐activated force by ADP. 5. A decrease in the amplitude of caffeine‐induced Ca2+ transients also occurred on changing from a solution containing 1 mM ADP and 10 mM Pi to a solution with 10 mM ADP and 1 mM Pi. This confirms the previous observation that ADP is more effective than Pi at reducing caffeine‐induced Ca2+ released from the SR. 6. Spontaneous oscillations of [Ca2+] and tension occurred in the presence of 0.5 microM Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)
Intracellular pathways triggered by galanin to induce contraction of pig ileum smooth muscle cells.
Authors: Botella A , Delvaux M , Bueno L , Frexinos J
Published: December 2014

1. In order to determine the intracellular mechanisms by which galanin induces contraction of isolated smooth muscle cells from pig ileum, we examined the effects of external Ca2+, relaxing agents, pertussis toxin and forskolin on the galanin‐induced contraction and compared these effects to those observed on the cholecystokinin derivative CCK8‐induced contraction. 2. Galanin induced a concentration‐dependent cell contraction. The maximal contraction (24.5 +/‐ 2.1% of the length of resting cells) was observed at 1 nM of galanin. When cells were incubated in the simultaneous presence of concentrations of galanin (10 fM) and CCK8 (1 pM) which were ineffective alone, or galanin (10 fM) and acetylcholine (100 pM), a synergistic action was observed corresponding to a submaximal contraction. 3. Incubation of cells in Ca(2+)‐free medium caused a significant decrease in galanin‐ but not in CCK‐induced contraction. Nifedipine, a Ca2+ channel blocker, provoked a concentration‐dependent inhibition of galanin‐induced contraction while it had no effect on the contraction induced by CCK8. 4. Vasoactive intestinal polypeptide (VIP) and isoprenaline, known to induce cell relaxation through an increase in intracellular cAMP level, inhibited CCK‐induced cell contraction at concentrations ranging from 1 pM to 1 microM but failed to inhibit cell contraction induced by galanin. 5. When cells were pre‐incubated for 3 h in the presence of 200 ng/ml of pertussis toxin, the contraction induced by galanin was abolished while the CCK‐induced contraction remained unchanged. On the contrary, 10 microM forskolin abolished the contraction induced by 10 nM CCK but had no effect on galanin‐induced contraction. 6. These results indicate that galanin induces a concentration‐dependent contraction of pig ileum smooth muscle by a direct myogenic effect. This effect of galanin involves the activation of a pertussis toxin‐sensitive G protein, which results in an influx of Ca2+ into the cell. This intracellular pathway is insensitive to the relaxing effect of cAMP.
Relaxant innervation of the guinea‐pig trachealis: demonstration of capsaicin‐sensitive and ‐insensitive vagal pathways.
Authors: Canning B J , Undem B J
Published: December 2014

1. The guinea‐pig trachea was isolated with its extrinsic innervation intact and placed in a water‐jacketed dissecting dish containing warmed, oxygenated Krebs solution. The trachea was not separated from the oesophagus. Two adjacent cartilage rings of the rostral portion of the trachea were cut open opposite the trachealis and prepared for isometric tension measurements. 2. Following the addition of atropine and contraction of the trachealis with prostaglandin F2 alpha (PGF2 alpha), stimulation of the cervical sympathetic trunks elicited relaxations that were abolished by propranolol or hexamethonium. Stimulation of the vagus nerves caudal to the nodose ganglia also elicited relaxations. These vagally mediated relaxations were unaffected by propranolol but were abolished by hexamethonium or by cutting the recurrent laryngeal nerves. 3. After cutting the vagi caudal to the nodose ganglia, stimulation of the vagi rostral to the nodose ganglia elicited relaxations of the trachealis that were not significantly affected by either propranolol or hexamethonium but were abolished by cutting the superior laryngeal nerves. Stimulation of right vagi which had undergone supranodose vagotomy 14 days prior to experimentation was without effect on the smooth muscle of the guinea‐pig trachea while the response to stimulation of the left vagus was unchanged. 4. Acute capsaicin desensitization abolished relaxations of the guinea‐pig trachealis elicited by stimulation of the vagal fibres carried by the superior laryngeal nerves. In contrast, capsaicin desensitization only modestly inhibited relaxations elicited by stimulation of the preganglionic parasympathetic fibres carried by the recurrent laryngeal nerves and had no effect on sympathetic nerve‐induced relaxations. 5. Removing the oesophagus selectively abolished relaxations elicited by stimulation of both vagal pathways of non‐adrenergic relaxant innervation. Non‐adrenergic relaxations of the trachealis elicited by electrical field stimulation were unaffected by removing the oesophagus. Oesophagus removal also had no effect on the parasympathetic‐cholinergic contractile innervation or the sympathetic relaxant innervation of the trachealis. 6. The results indicate that the guinea‐pig trachealis receives non‐adrenergic relaxant innervation from both parasympathetic and capsaicin‐sensitive vagal pathways. The results also suggest that the neurones mediating non‐adrenergic relaxations of the trachea are sensitive to oesophagus removal. The observation that oesophagus removal abolishes parasympathetic relaxations of the trachealis while having no effect on parasympathetic contractions supports the hypothesis that the guinea‐pig trachealis receives excitatory and inhibitory innervation from distinct vagal parasympathetic pathways.