bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of alcohols on responses evoked by inositol trisphosphate in Xenopus oocytes.
Authors: Ilyin V , Parker I
Published: December 2014

1. The effects of ethanol and other alcohols on inositol 1,4,5‐trisphosphate (InsP3) signalling were studied in Xenopus oocytes by the use of flash photolysis of caged InsP3. Calcium liberation induced by InsP3 was monitored by voltage‐clamp recording of Ca(2+)‐activated membrane currents, and by fluorescence of the Ca2+ indicator Fluo‐3. 2. Membrane current and fluorescence Ca2+ signals evoked by light flashes giving small responses were initially potentiated by bath application of ethanol (80‐400 mM). However, the responses subsequently declined while ethanol was present and were strongly reduced or suppressed when it was removed. 3. These effects did not arise artifactually from changes in photolysis of caged InsP3, as similar results were seen with responses evoked by intracellular injections of InsP3. Also, the effects on the membrane current did not arise primarily through actions on the Ca(2+)‐dependent Cl‐ channels, since currents evoked by intracellular injections of Ca2+ were little changed by ethanol. 4. Ethanol reduced the threshold level of InsP3 required to cause Ca2+ liberation. Thus, potentiation was most prominent with small responses evoked by brief light flashes, whereas the predominant effect on larger responses was inhibitory. 5. The facilitatory and inhibitory actions of ethanol persisted after removing extracellular Ca2+. 6. Intracellular injections of ethanol produced an initial inhibition of InsP3 responses, followed, in some oocytes, by a potentiation. 7. Methanol had little effect on InsP3 responses, whereas butanol and other long‐chain alcohols produced strong inhibition, but little or no potentiation. 8. We conclude that extracellular application of ethanol produces a rapid potentiation of InsP3‐mediated Ca2+ liberation, and a more slowly developing inhibition. The potentiation may arise through stimulation of InsP3 formation at the plasma membrane, whereas the inhibition occurs more deeply in the cell. Both actions were evident at relatively low concentrations (a few tens of millimoles per litre), and might thus be important in the behavioural effects of ethanol intoxication.
Effects of proteins on the permeability of monolayers of cultured bovine arterial endothelium.
Authors: Turner M R
Published: December 2014

1. Monolayers of arterial endothelium on porous membranes were exposed to a pressure of 15 cmH2O at 37 degrees C, or of 30 cmH2O at 0 degree C. At constant pressure, the rate of liquid flow per unit area (Jv/A) through each monolayer decreased with time, in the way previously described for cultured endothelium. This phenomenon has been called sealing. After Jv/A stabilized, the pressure was reduced and the hydraulic permeability (Lp) of the endothelium was calculated from the relationship between Jv/A and pressure. Endothelium was seen to be damaged after some experiments at 37 degrees C, but appeared undamaged after experiments at 0 degree C. 2. Bovine serum albumin (BSA) did not influence the Lp of cultured endothelium. At 37 degrees C, the mean (+/‐ S.E.M.) endothelial Lp was 47.2 +/‐ 7.3 x 10(‐7) cm s‐1 cmH2O‐1 (n = 10) in the presence of BSA (5 g (100 ml)‐1). This is not significantly different from the mean (+/‐ S.E.M.) Lp of 53.4 +/‐ 9.0 x 10(‐7) cm s‐1 cmH2O‐1 (n = 9) in the absence of added protein (P greater than 0.10). At 0 degree C also, there was no significant difference between mean Lps in the presence of BSA (0.1 g (100 ml)‐1) and in the absence of added protein. 3. Solutions of BSA (5 g (100 ml)‐1 or of the neutral polymer Ficoll 70 (4 g (100 ml)‐1) did not exert any effective osmotic pressure across endothelium at 37 or 0 degrees C, respectively. 4. BSA (0.1 g (100 ml)‐1) did not enable solutions of Ficoll 70 (4 g (100 ml)‐1) to exert an effective osmotic pressure across endothelium at 0 degree C. 5. The mean Lp of endothelium at 0 degree C was significantly lower in the presence of cationized ferritin (CF; 0.1 g (100 ml)‐1) than in the absence of added protein (P less than 0.001). Native ferritin (NF; 0.1 g (100 ml)‐1) had no effect on Lp. 6. In the presence of CF (0.1 g (100 ml)‐1), solutions of Ficoll 70 (4 g (100 ml)‐1) exerted a mean effective osmotic pressure of 27.7 cmH2O (n = 5) across endothelium at 0 degree C. The mean effective osmotic pressure exerted across endothelium by solutions of Ficoll 70 (4 g (100 ml)‐1) plus NF (0.1 g (100 ml)‐1) was 1.2 cmH2O (n = 4).
Acetylcholine‐ and caffeine‐evoked repetitive transient Ca(2+)‐activated K+ and C1‐ currents in mouse submandibular cells.
Authors: Smith P M , Gallacher D V
Published: December 2014

1. Resting and acetylcholine‐induced membrane currents were measured in single mouse submandibular acinar cells using the patch‐clamp whole‐cell current recording technique. 2. Micromolar ACh activated a large, sustained outward, Ca(2+)‐dependent K+ current and a single transient inward Ca(2+)‐dependent C1‐current. 3. Nanomolar ACh induced a series of transients in both the K+ and C1‐ currents; C1‐ current activation was now observed throughout the period of agonist application. We consider this repetitive transient current activation better able to support sustained fluid and electrolyte secretion than the response elicited by a high dose of agonist. 4. Repetitive K+ and C1‐ current transients were also induced by 1 mM‐caffeine, consistent with caffeine‐induced Ca2+ release from the Ca(2+)‐sensitive Ca2+ stores which are thought to comprise part of the pathway for activation of secretion. 5. The ACh‐induced current transients were inhibited by 10 mM‐caffeine, 100 microM‐IBMX and 10 microM membrane‐permeable cyclic AMP. Therefore, it seems likely that caffeine is able to inhibit agonist‐induced calcium mobilization via a cyclic AMP‐dependent pathway.
Muscarinic responses of rat basolateral amygdaloid neurons recorded in vitro.
Authors: Washburn M S , Moises H C
Published: December 2014

1. Intracellular recordings were obtained from pyramidal‐type neurons in the basolateral amygdaloid nucleus (BLA) in slices of rat ventral forebrain and used to compare the actions of exogenously applied cholinomimetics to the effects produced by electrical stimulation of amygdalopetal cholinergic afferents from basal forebrain. 2. Bath application of carbachol depolarized pyramidal cells with an associated increase in input resistance (Ri), reduced the slow after‐hyperpolarization (AHP) that followed a series of current‐evoked action potentials and blocked spike frequency accommodation. All of these effects were reversed by the muscarinic antagonist atropine but not by the nicotinic antagonist hexamethonium. 3. Electrical stimulation of amygdaloid afferents within the external capsule evoked a series of synaptic potentials consisting of a non‐cholinergic fast excitatory postsynaptic potential (EPSP), followed by early and late inhibitory postsynaptic potentials (IPSPs). Each of these synaptic potentials was reduced by carbachol in an atropine‐sensitive manner. 4. Local application of carbachol to pyramidal cells produced a short‐latency hyperpolarization followed by a prolonged depolarization. The hyperpolarization and depolarization to carbachol were blocked by atropine but not hexamethonium. 5. The carbachol‐induced hyperpolarization was associated with a decrease in Ri and had a reversal potential nearly identical to that of the early IPSP. The inhibitory response was blocked by perfusion of medium containing tetrodotoxin (TTX), bicuculline or picrotoxin, while the subsequent depolarization was unaffected. On the basis of these data, it is concluded that the muscarinic hyperpolarization is mediated through the rapid excitation of presynaptic GABAergic interneurons in the slice. 6. The findings that the carbachol‐induced depolarization was associated with an increase in Ri, often had a reversal potential below ‐80 mV, was sensitive to changes in extracellular potassium concentration and was blocked by intracellular ionophoresis of the potassium channel blocker caesium suggest that it resulted from a muscarinic blockade of one or more potassium conductances. 7. Repetitive stimulation of sites within the slice containing cholinergic afferents evoked a series of fast EPSPs followed by IPSPs. These non‐cholinergic potentials were followed by a slow EPSP that lasted from 10 s‐4 min. The slow EPSP was enhanced by eserine and blocked by atropine. It was also blocked by TTX or cadmium, indicating that it was dependent on spike propagation and calcium‐dependent release of acetylcholine (ACh). 8. Stimulation of cholinergic afferents in the slice mimicked other effects produced by carbachol including blockade of the slow AHP and accommodation of action potential discharge and these actions were potentiated by eserine and blocked by atropine.(ABSTRACT TRUNCATED AT 400 WORDS)
Factors affecting movement of excitatory substances from pulmonary capillaries to type J receptors of anaesthetized cats.
Authors: Paintal A S , Anand A
Published: December 2014

1. Using phenyl diguanide (PDG) as an excitatory substance, the role of certain factors that could influence the movement of such substances across the pulmonary capillaries to the J receptors was studied in cats anaesthetized with sodium pentobarbitone. This was aided by using a new method for estimating continuously in vivo the concentration (C) of PDG in the blood of the pulmonary artery. 2. Reduction of pulmonary blood flow by partial occlusion of the inferior vena cava enhanced the responses of the J receptors to PDG significantly in twelve out of thirteen trials. These effects, which occurred at a time when pulmonary capillary pressure (PCP) had fallen, could be related to the increase in the estimated mean C of PDG over the first 3 s or to the C t (concentration x time) area to 50% of peak C. The responses bore no relation to peak C or rate of rise of C. 3. The responses of the receptors to PDG increased significantly after three out of eight injections of PDG while the PCP was raised by partial occlusion of the mitral orifice; reduced responses were recorded after two injections. These results, showing relatively much weaker stimulation by PDG in spite of the enhanced level of J receptor excitability produced by the raised PCP itself, suggest that movement of PDG out of the capillaries to the J receptors must be influenced primarily by forces governing diffusion, not filtration. 4. In addition to C of PDG there appear to be other factors that influence the responses of the receptors to PDG.
Effect of temperature on relaxation rate and Ca2+, Mg2+ dissociation rates from parvalbumin of frog muscle fibres.
Authors: Hou T T , Johnson J D , Rall J A
Published: December 2014

1. Influence of temperature on relaxation rate as a function of isometric tetanus duration and on Ca2+ and Mg2+ dissociation rates from purified parvalbumin (PA) was examined to test the hypothesis that PA promotes relaxation in frog skeletal muscle. Single fibres and PA IVB from Rana temporaria skeletal muscle were utilized. 2. The magnitude of slowing of relaxation rate with increasing tetanus duration, relative to the final, steady value of relaxation rate, is 3‐fold greater at O than at 10 degrees C. 3. In the 0‐10 degrees C range, the Q10 for relaxation rate increases from 2.3 to 3.7 with increasing tetanus duration. 4. Dissociation of Ca2+ and Mg2+ from PA exhibited: (i) rate constants of 1.03 +/‐ 0.03 s‐1 (mean +/‐ S.D., n = 5) and 3.42 +/‐ 0.14 s‐1 (n = 5) at 20 degrees C and (ii) Q10 values of 2.3 and 1.9 in the 0‐20 degrees C range, respectively. 5. Time courses of slowing of relaxation rate with increasing tetanus duration and recovery of relaxation rate with rest after a prolonged tetanus at 10 degrees C are similar to rates of dissociation of Mg2+ and Ca2+ from PA, respectively, as previously reported at 0 degree C. 6. Both the temperature dependence of the relative magnitude of slowing of relaxation rate and the increased Q10 of relaxation rate with increased tetanus duration can be explained if the Q10 for Ca2+ uptake by the sarcoplasmic reticulum is greater than the Q10 for Ca2+ sequestration by PA during relaxation. When Ca2+ and Mg2+ dissociation rates from PA at various temperatures are compared to other proposed indicators of PA function, it is concluded that PA facilitates relaxation of frog skeletal muscle in the 0‐20 degrees C range.
Changes in the electrical properties of chick ciliary ganglion neurones during embryonic development.
Authors: Dourado M M , Dryer S E
Published: December 2014

1. Whole‐cell recording techniques were used to examine the expression of ionic currents in chick ciliary ganglion neurones dissociated acutely at various stages of embryonic development. Currents were also examined in dissociated cells that had been maintained in vitro for several days. 2. Voltage‐activated, tetrodotoxin (TTX)‐sensitive Na+ currents (INa) could be detected in all cells tested between stage 25 and stage 40 (embryonic days 4.5‐14). INa increased in both amplitude and density throughout development, but no obvious changes in kinetics or sensitivity to TTX were observed. 3. High‐threshold Ca2+ currents (ICa) were also detectable between stage 25 and stage 40. ICa increased in both amplitude and density throughout this time. No obvious changes in kinetics or voltage dependence were observed. 4. Delayed rectifier K+ currents (IDR) and A‐currents (IA) could be detected in Ca(2+)‐free salines, and distinguished on the basis of differences in kinetics, voltage dependence, and sensitivity to tetraethylammonium (TEA). IA was either absent, or present at very low densities at stages 26‐30, but showed a sharp increase in density thereafter. In contrast, IDR was detectable as early as stage 25, and did not display a significant increase in density during development. 5. Ca(2+)‐activated K+ currents (IK(Ca)) were either undetectable or present at very low density between stage 26 and stage 30 (embryonic days 5‐9) but showed a large increase in amplitude and density thereafter. 6. Ionic currents were examined in age‐matched cells dissociated acutely on embryonic day 13, or isolated on embryonic day 9 and maintained in vitro for an additional 4 days. Most of the cells maintained in culture for 4 days did not express detectable IK(Ca), and had significantly reduced IA compared to acutely isolated controls. The cultured cells expressed normal densities of IDR, ICa and INa. 7. All ionic currents increased in amplitude during normal embryonic development, and all but IDR increased in density. The largest change in density generally occurred between stages 30 and 40, during which time ciliary ganglion neurones form synapses with target tissues. 8. Isolation of ciliary neurones from the in ovo environment prevented the normal development of IA and IK(Ca), suggesting that the expression of these channels is controlled by one or more extrinsic environmental factors. In contrast, the normal expression of INa, ICa and IDR is not dependent upon extrinsic factors.
Airway epithelial cells regulate membrane potential, neurotransmission and muscle tone of the dog airway smooth muscle.
Authors: Xie Z , Hakoda H , Ito Y
Published: December 2014

1. The effects of epithelial cells were investigated on resting membrane potential and neuro‐effector transmission in smooth muscle cells of the dog tracheal and bronchiolar tissues. 2. The mean value of the resting membrane potential of the epithelium‐intact bronchiolar smooth muscle cells of the dog was‐‐70.0 +/‐ 1.1 mV (+/‐ S.D., n = 40) and mechanical denudation of the epithelial layer depolarized the membrane to ‐57.0 +/‐ 2.5 mV (+/‐ S.D., n = 40). Application of isolated and dispersed epithelial cells (greater than 2 x 10(5) cells/ml) to the perfusing solution repolarized the membrane of epithelium‐denuded bronchiolar smooth muscle cells to ‐67.0 +/‐ 2.7 mV (+/‐ S.D., n = 20). The mean resting membrane potential of the mucosa‐free tracheal smooth muscle cells was ‐59.1 +/‐ 1.4 mV (+/‐ S.D., n = 50), and application of isolated and dispersed cells (greater than 2 x 10(5) cells/ml) hyperpolarized the membrane to ‐67.2 +/‐ 1.8 mV (+/‐ S.D., n = 50). These repolarizing actions were not modified by indomethacin (10(‐5) M). 3. In the epithelium‐denuded bronchioles, ACh (greater than 10(‐9) M) dose‐dependently depolarized the smooth muscle cells, while in the epithelium‐intact bronchioles, ACh (10(‐11) ‐ 10(‐8) M) did not affect the resting membrane potential. At a concentration of 10(‐7) M, ACh significantly depolarized the membrane. 4. Electrical field stimulation (EFS; 50 microseconds in duration and about 10‐20 V in strength) applied to ring preparations of the bronchioles evoked twitch‐like contractions (hereafter referred as twitch contraction), and size of the twitch contractions gradually and continuously decreased in the presence or absence of indomethacin (10(‐5) M) and guanethidine (10(‐6) M). When similar experiments were performed using epithelium‐denuded bronchiolar ring preparations, in no case was there a prominent reduction in the amplitude of the twitch contractions in the presence of indomethacin and guanethidine. 5. The decremental response of the twitch contraction observed in the epithelium‐intact bronchioles was overcome by application of the leukotriene synthesis inhibitor AA861 (10(‐6) M) and the leukotriene antagonist ONO1078 (10(‐5) M). 6. Leukotrienes C4 and D4 (LTC4 and LTD4, greater than 10(‐8) M) evoked muscle contraction with a steady increase in muscle tone, up to a certain level. However, at 10(‐9) M, LTC4 increased and LTD4 decreased the amplitude of the twitch contractions evoked by EFS in the epithelium‐intact bronchioles.(ABSTRACT TRUNCATED AT 400 WORDS)
Changes in presynaptic inhibition of afferents to propriospinal‐like neurones in man during voluntary contractions.
Authors: Burke D , Gracies J M , Meunier S , Pierrot‐Deseilligny E
Published: December 2014

1. The possibility was investigated that the facilitation of the transmission in the propriospinal‐like system during voluntary contraction, documented in the companion paper (Burke, Gracies, Mazevet, Meunier & Pierrot‐Deseilligny, 1992), is due to a decrease in presynaptic inhibition of afferents projecting to propriospinal‐like neurones. 2. The radial nerve was stimulated to evoke presynaptic inhibition of the monosynaptic Ia projections to forearm flexor motoneurones (Berardelli, Day, Marsden & Rothwell, 1987) and, hopefully, of the afferent input to propriospinal‐like neurones projecting to these motoneurones. 3. The propriospinal‐like excitation of forearm motoneurones evoked from mixed afferent inputs was depressed by radial nerve stimulation, and this depression was long‐lasting (200 ms). Despite the convergence of mixed nerve and cutaneous afferents onto common propriospinal‐like neurones, the radial stimulation did not depress the cutaneous‐induced excitation. This differential effect and the time course of the depression suggest that it results from presynaptic inhibition of mixed nerve afferents (presumably large muscle afferents) projecting to propriospinal‐like neurones. 4. With voluntary contractions, phasic or tonic, the radial‐induced depression of the propriospinal‐like excitation evoked by mixed nerve afferents was much greater than at rest, but the cutaneous‐evoked excitation was unchanged. Thus, with voluntary contractions, there was no evidence of decreased gating of the afferent input to propriospinal‐like neurones whether the input was of muscle or cutaneous origin and it is concluded that changes in presynaptic inhibition cannot account for the facilitation of the transmission in the propriospinal‐like system during voluntary contraction. 5. By contrast, presynaptic inhibition of the monosynaptic Ia projections to motoneurones was consistently reduced at the onset of contraction, and to a much lesser extent during a weak tonic contraction.
Cation interactions within the cyclic GMP‐activated channel of retinal rods from the tiger salamander.
Authors: Zimmerman A L , Baylor D A
Published: December 2014

1. The ionic dependence of current through the 3',5'‐cyclic guanosine monophosphate (cyclic GMP)‐activated channels of salamander rods was studied in excised inside‐out membrane patches from isolated outer segments. Voltage‐clamp experiments on transducing rods were performed so that the channels in intact cells could be compared with those in excised patches. 2. The reversal potential of the cyclic GMP‐induced patch current was close to the Na+ equilibrium potential when the concentration of NaCl on the cytoplasmic surface of a patch was varied at constant external NaCl concentration. Fitting the Goldman‐Hodgkin‐Katz equation indicated that the apparent ratio of permeabilities for Na+ and Cl‐ was at least 50. This confirms a previous report that the channel's Na+ permeability is much larger than its Cl‐ permeability. 3. Na+ currents through the channel did not obey the independence principle. The outward patch current at large positive potential began to saturate with increasing concentrations of internal Na+, as if permeation required Na+ to bind to a site with an apparent dissociation constant around 180 mM. 4. In symmetrical NaCl solutions containing very low concentrations of divalent cations the current‐voltage relation measured from excised patches 50 microseconds after switching the voltage showed mild outward rectification. By 1 ms the rectification was more pronounced. The rectification at 50 microseconds is attributed to voltage dependence of Na+ permeation. The additional rectification at later times is attributed to voltage dependence of the channel's probability of being open, depolarization favouring the open state. 5. In symmetrical Mg2+ solutions the cyclic GMP‐induced patch currents were smaller and the outward rectification was more pronounced. 6. Addition of Mg2+ or Ca2+ to an internal Na+ solution blocked the cyclic GMP‐induced Na+ current through the channels, as if by occupying a single binding site with an affinity in the 0.1‐2 mM range. Block by Mg2+ was voltage dependent, suggesting that the binding site was within the channel's transmembrane electric field. Raising the Mg2+ concentration on the external surface of the patch increased the apparent dissociation constant of block by internal Mg2+, as expected if external and internal Mg2+ compete for the same binding site. 7. Block by internal Ca2+ had an opposite and weaker voltage dependence than block by internal Mg2+. 8. In symmetrical solutions containing both Na+ and Mg2+ the outward rectification was more pronounced than in solutions containing Na+ alone. In solutions thought to be close to physiological the outward patch current increased e‐fold for a depolarization of 24‐30 mV.(ABSTRACT TRUNCATED AT 400 WORDS)
Cholinergic excitation of GABAergic interneurons in the rat hippocampal slice.
Authors: Pitler T A , Alger B E
Published: December 2014

1. Intracellular recordings were made from CA1 pyramidal cells in the rat hippocampal slice to study the cholinergic modulation of GABAergic inhibition. The cholinergic receptor agonist, carbamylcholine (carbachol), depressed evoked excitatory postsynaptic potentials (EPSPs) and evoked inhibitory postsynaptic potentials (IPSPs), but enhanced small spontaneously occurring membrane potential fluctuations that resembled IPSPs. Both atropine (1 microM) and picrotoxin (25‐60 microM) abolished the small fluctuations. 2. Recording from cells with potassium or caesium chloride (KCl or CsCl)‐filled microelectrodes enhanced and inverted spontaneous Cl(‐)‐dependent GABAA‐mediated IPSPs. These events appeared to result from the spontaneous firing of GABAergic interneurons since they could be inhibited by picrotoxin or bicuculline and nearly eliminated by tetrodotoxin. 3. Muscarinic acetylcholine (ACh) receptor activation significantly increased the frequency of spontaneous‐activity‐dependent IPSPs from 1.7 +/‐ 0.4 s (mean +/‐ S.E.M.) in control saline to 7.0 +/‐ 1.1 s in carbachol (10‐50 microM)‐containing saline, although evoked IPSPs were inhibited. All effects of carbachol were completely reversed by atropine. 4. The increase in frequency of spontaneous IPSPs observed in carbachol was not secondary to changes in the postsynaptic cell and was not blocked by high doses of 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 5‐10 microM) and 2‐amino‐5‐phosphonovaleric acid (APV, 10‐20 microM), which abolished evoked excitatory transmission. Amplitude histograms showed an increase in mean size as well as of frequency of spontaneous IPSCs in carbachol. 5. Stimulation of cholinergic afferents in stratum oriens in the presence of the acetylcholinesterase inhibitor eserine (1 microM) also increased spontaneous IPSP frequency, and the time course of this response was similar to that of the muscarinic slow EPSP. Postsynaptic factors or the activation of glutamatergic excitatory pathways could not account for this effect. 6. Evoked monosynaptic IPSCs in CNQX and APV were diminished by carbachol. 7. We conclude that GABAergic inhibitory interneurons possess muscarinic receptors, that activation of these receptors increases the excitability of the interneurons and that synaptically released ACh increases interneuronal activity. Cholinergic reduction of the monosynaptic IPSC may point to additional complexity in cholinergic regulation of the GABA system.
Somatosensory stimulation causes autonomic vasodilatation in cat lip.
Authors: Izumi H , Karita K
Published: December 2014

1. Electrical stimulation of the infra‐orbital nerve and the maxillary buccal gingiva caused an increase in ipsilateral lip blood flow in a stimulus intensity‐dependent manner in anaesthetized cats. 2. The reflex vasodilator response was resistant to blockade by antimuscarinic (atropine), antiadrenergic (phentolamine and propranolol) and antihistaminergic (tripelennamine) but sensitive to ganglionic blocking agent (hexamethonium). 3. The reflex vasodilator response was unaffected by section of the ipsilateral cervical sympathetic trunk and facial nerve root, but was completely abolished by ipsilateral section of the glossopharyngeal nerve root. 4. The vasodilator response elicited by stimulation of the facial and glosso‐pharyngeal nerves was never affected by lesion of the ipsilateral pterygopalatine ganglion. 5. Local anaesthesia or section of the inferior alveolar nerve abolished the vasodilator effects of stimulation of the facial and the glossopharyngeal nerves. 6. These results suggest that there is a somato‐autonomic reflex vasodilator system mediated via final neurons that are not cholinergic, and that the parasympathetic vasodilator fibres emerge from the brain stem with the glossopharyngeal nerve and reach the blood vessels via the otic ganglion and the inferior alveolar nerve in the cat mandibular lip.
Calcium requirements for secretion in bovine chromaffin cells.
Authors: Augustine G J , Neher E
Published: December 2014

1. Measurements of membrane capacitance and intracellular Ca2+ concentration, [Ca2+]i, were used to examine the Ca2+ dependence of secretion in single adrenal chromaffin cells. 2. Intracellular dialysis of Ca2+, through a patch pipette, promoted secretion; the rate of secretion increased monotonically as [Ca2+]i was elevated, while the total amount of secretion reached a maximum at 1.5 microM‐Ca2+ and declined at high [Ca2+]i. 3. Release of Ca2+ from internal stores, using bradykinin or ionomycin, transiently elevated [Ca2+]i and the rate of secretion. 4. Considering responses to both Ca2+ dialysis and release from internal stores, it appears that the rate of secretion increases over a range of [Ca2+]i levels above 0.2 microM and saturates at concentrations greater than 10 microM, if at all. Secretion appears to have a Hill coefficient for Ca2+ of about 2. At [Ca2+]i greater than 1‐2 microM, prolonged elevation of [Ca2+]i, via dialysis, produced lower rates of secretion than transient elevation of [Ca2+]i caused by release from internal stores. This may have been caused by a depletion of readily releasable chromaffin granules during prolonged elevation of [Ca2+]i. 5. Brief depolarizing pulses produced transient rises in both [Ca2+]i and the rate of secretion. The ability of these pulses to evoke secretion ‘washed out’ during prolonged intracellular dialysis, due to both reduced Ca2+ influx and a diminished ability of the cell to secrete in response to a given Ca2+ load. 6. The kinetics of the secretory response depended upon the size of the depolarization‐induced Ca2+ load; small rises in [Ca2+]i increased membrane capacitance only during the depolarization, while larger rises in [Ca2+]i produced increases both during and following the depolarization. The secretory responses that outlasted the depolarization appeared to be due to persistent elevation of [Ca2+]i. Secretory responses were sometimes followed by a slower decline in membrane capacitance, probably due to endocytosis of membrane. 7. Comparison of the rates of secretion measured during depolarization to those produced by Ca2+ dialysis or release from internal stores suggests that [Ca2+]i at secretory sites can exceed 10 microM during depolarization. The spatially averaged measurements of [Ca2+]i indicate much smaller levels of [Ca2+]i; thus, there must be pronounced spatial gradients of [Ca2+]i during depolarization.
Calcium gradients and buffers in bovine chromaffin cells.
Authors: Neher E , Augustine G J
Published: December 2014

1. Digital imaging and photometry were used in conjunction with the fluorescent Ca2+ indicator, Fura‐2, to examine intracellular Ca2+ signals produced by depolarization of single adrenal chromaffin cells. 2. Depolarization with a patch pipette produced radial gradients of Ca2+ within the cell, with Ca2+ concentration highest in the vicinity of the plasma membrane. These gradients dissipated within a few hundred milliseconds when the voltage‐gated Ca2+ channels were closed. 3. Dialysis of Fura‐2 into the chromaffin cell caused concentration‐dependent changes in the depolarization‐induced Ca2+ signal, decreasing its magnitude and slowing its recovery time course. These changes were used to estimate the properties of the endogenous cytoplasmic Ca2+ buffer with which Fura‐2 competes for Ca2+. 4. The spatially averaged Fura‐2 signal was well described by a model assuming fast competition between Fura‐2 and an endogenous buffer on a millisecond time scale. Retrieval of calcium by pumps and slow buffers occurs on a seconds‐long time scale. No temporal changes indicative of buffers with intermediate kinetics could be detected. 5. Two independent estimates of the capacity of the fast endogenous Ca2+ buffer suggest that 98‐99% of the Ca2+ entering the cell normally is taken up by this buffer. This buffer appears to be immobile, because it does not wash out of the cell during dialysis. It has a low affinity for Ca2+ ions, because it does not saturate with 1 microM‐Ca2+ inside the cell. 6. The low capacity, affinity and mobility of the endogenous Ca2+ buffer makes it possible for relatively small amounts of exogenous Ca2+ buffers, such as Fura‐2, to exert a significant influence on the characteristics of the Ca2+ concentration signal as measured by fluorescence ratios. On the other hand, even at moderate Fura‐2 concentrations (0.4 mM) Fura‐2 will dominate over the endogenous buffers. Under these conditions radiometric Ca2+ concentration signals are largely attenuated, but absolute fluorescence changes (at 390 nm) accurately reflect calcium fluxes.
Permeability and Mg2+ blockade of histamine‐operated cation channel in endothelial cells of rat intrapulmonary artery.
Authors: Yamamoto Y , Chen G , Miwa K , Suzuki H
Published: December 2014

1. In the cell‐attached and inside‐out patch‐clamp experiments using undispersed endothelial cells of the rat intrapulmonary artery, the majority of channels were cation selective. 2. Under physiological ionic conditions, the I‐V relationship for the inward currents fell to ‐80 mV and the slope conductance was 22.5 pS. There was an inward rectification and the outward currents were smaller than the inward currents. 3. Under symmetric high‐K+ conditions, the slope conductance for the inward currents was 26.4 pS and the inward rectification was observed when the high‐K+ solution contained 1 mM‐Mg2+. The channel activity was weakly voltage dependent at negative membrane potentials, while it was much enhanced at positive potentials. 4. The channel activity did not depend on intracellular Ca2+ concentrations. 5. Mg2+ was not only impermeant, it also blocked this channel in a voltage‐dependent manner and rectifications appeared in the I‐V relationship. Mg2+ blocked the channel from both sides of the membrane. 6. Ca2+ permeated this channel and the permeability ratios calculated from the reversal potentials using the constant‐field theory were; PK:PNa:PCa = 1:1:15.7. 7. Histamine but not acetylcholine applied to the pipette activated this channel. Guanosine 5'‐O‐(3‐thiotriphosphate) (GTP gamma S) applied to the intracellular surface of the patch did not mimic the effect of histamine. 8. Thus, in the endothelial cell membrane of the rat intrapulmonary artery, there exists a cation channel which is selective to Ca2+ but also permeable to Na+ and K+. This channel has inward rectifying properties, possibly due to intracellular Mg2+. Histamine, but not acetylcholine, activates this cation channel to elevate endothelial [Ca2+]i.
Time‐resolved changes in intracellular calcium following depolarization of rat brain synaptosomes.
Authors: Lentzner A , Bykov V , Bartschat D K
Published: December 2014

1. Changes of cytoplasmic free calcium levels ([Ca2+]i) in isolated rat brain nerve terminals (synaptosomes), previously loaded with the fluorescent intracellular calcium indicator Fura‐2, were measured 1‐2 ms after depolarization with elevated K+ by stopped‐flow fluorescence spectroscopy. 2. In physiological saline (PSS) containing 4 mM‐K+, intraterminal Ca2+ was estimated to be in the range 150‐250 nM. Depolarization of the nerve terminals with elevated external K+ in the presence of Ca2+ induced a prompt rise in [Ca2+]i, which occurred in two phases. No change in [Ca2+]i was seen when the terminals were depolarized in nominally Ca(2+)‐free solutions, and only a small change was seen when the terminals were acutely exposed to Ca2+ in 4 mM‐K+. 3. Predepolarization of the nerve terminals with K+ in nominally Ca(2+)‐free solutions several seconds before the introduction of Ca2+ greatly decreased the magnitude of the fast phase, whilst leaving the slow phase largely intact. 4. In Na(+)‐depleted nerve terminals, the fast phase of K(+)‐stimulated Ca2+ uptake was essentially unaltered, but the slow phase of Ca2+ uptake was dramatically reduced. 5. The rapid phase of K(+)‐stimulated uptake displayed voltage‐dependent inactivation (tau approximately 50 ms at ‐10 mV), and the rate of inactivation was accelerated with increasing depolarization. In contrast, at constant [K+]o, increasing [Ca2+]o had little or no effect on the rate of inactivation, but did increase the initial rate of Ca2+ uptake. 6. The dihydropyridine calcium channel blockers nifedipine and nitrendipine had little effect on either component of Ca2+ uptake. However, the inorganic Ca2+ channel blockers La3+, Cd2+, and Co2+ were potent blockers of the fast phase of Ca2+ uptake, but blocked the slow phase only at higher concentrations. No consistent effect of the peptide neurotoxin omega‐conotoxin was observed on either component of the Ca2+ rise. 7. These studies demonstrate that the dynamics of depolarization‐activated intraterminal Ca2+ changes can be studied on a millisecond time scale in isolated nerve terminals. Moreover, our results indicate that two pathways contribute to depolarization‐induced [Ca2+]i changes, namely a voltage‐activated, inactivating Ca2+ channel, possibly of the N‐type, and Na(+)‐Ca2+ exchange operating in the ‘reverse’ mode.
Activation and desensitization of N‐methyl‐D‐aspartate receptors in nucleated outside‐out patches from mouse neurones.
Authors: Sather W , Dieudonné S , MacDonald J F , Ascher P
Published: December 2014

1. Activation and desensitization of N‐methyl‐D‐aspartate (NMDA) receptors were studied in large outside‐out patches excised from cultured embryonic neurones dissociated from mouse forebrain. The patches were exposed to rapid changes of NMDA or L‐glutamate concentrations in the presence of glycine at concentrations (10‐20 microM) saturating the modulatory site of the NMDA receptor. 2. Immediately after formation of the patch the responses to NMDA and L‐glutamate showed a slow and small desensitization, even with high concentrations of agonist. During the following hour, the peak response either decreased or remained relatively stable, but in all cases the desensitization increased and accelerated until it stabilized. In this 'stabilized’ state, the desensitization produced by high concentrations of NMDA (1 mM) or L‐glutamate (300 microM) had an exponential time course, with a time constant of about 30 ms. The ratio of the peak over the steady‐state current was in the order of 40 for NMDA and about 30 for L‐glutamate. 3. Concentration‐response curves were built for the peak and the plateau responses, for NMDA and for L‐glutamate. The comparison of these curves indicated that (i) the EC50 of the peak (K(app) was always higher than the EC50 of the plateau (Kss); (ii) the two EC50 values for NMDA (K(app) and Kss) were higher than those for L‐glutamate; (iii) the Hill coefficient was close to 1.4 for each of the four curves. 4. The application of NMDA or L‐glutamate at a low concentration for 3 s periods reduced the response to a subsequent application of the same agonist at a saturating concentration. The IC50 of this ‘predesensitization’, termed Kpre, was lower than the EC50 of the steady‐state response, Kss. 5. The onset rates of desensitization increased with the concentration of agonist. The EC50 of this relation was close to the value of K(app). 6. The decay of the currents at the end of a 3 s application of agonist was usually well described by the sum of two exponentials both of which were faster for NMDA than for L‐glutamate. 7. The recovery from desensitization after a long (3 s) pulse of agonist was approximately exponential, with a time constant of about 0.5 s for NMDA and about 3.5 s for L‐glutamate.(ABSTRACT TRUNCATED AT 400 WORDS)
The effects of a high sodium diet on the metabolism and secretion of vasoactive intestinal peptide in the rabbit.
Authors: Davis R E , Shelley S , MacDonald G J , Duggan K A
Published: December 2014

1. In view of previous observations that the metabolism of vasoactive intestinal peptide (VIP) is significantly increased in sodium‐depleted rabbits, we wished to determine whether a high sodium intake also leads to alterations in VIP metabolism. We performed metabolic clearance studies in rabbits maintained on a high sodium diet and normal control diets. These studies were performed both before and after the administration of 1.5 mmol kg‐1 of sodium intravenously to observe the effects of an acute increase in body sodium. 2. The rabbits maintained on the high sodium diet had a significantly lower basal plasma VIP level (P less than 0.025), a lower metabolic clearance rate (MCR) of the peptide (P less than 0.025) and a lower secretion rate (P less than 0.005), compared with the normal control animals. These differences were maintained following the intravenous sodium infusion. 3. The administration of the intravenous sodium infusion resulted in a further decrease in MCR in the rabbits on the high sodium diet (P less than 0.05). 4. These results confirm that VIP metabolism is affected by high dietary intake of sodium, as well as a low sodium intake, adding further support to the hypothesis that VIP may be involved in sodium homeostasis.
Sarcoplasmic reticulum buffering of myoplasmic calcium in bovine coronary artery smooth muscle.
Authors: Sturek M , Kunda K , Hu Q
Published: December 2014

1. We tested the hypothesis that the sarcoplasmic reticulum (SR) buffers (attenuates) the increase in averaged myoplasmic free [Ca2+] (Ca(im)) resulting from Ca2+ influx. 2. Fura‐2 measurements of Ca(im) were obtained in single smooth muscle cells freshly dispersed from bovine coronary artery. 3. Caffeine (5 x 10(‐3) M) elicited a transient increase in Ca(im) and depleted the SR Ca2+ store. In the continued presence of caffeine or 10(‐5) M‐ryanodine SR buffering of Ca(im) was inhibited. Subsequent exposure to high extracellular [K+] (greater than 30 mM, equimolar Na+ removal) elicited a 2‐fold more rapid and 2‐fold greater peak increase in Ca(im) than high K+ elicited when SR buffering of Ca(im) was normal. The augmented increase in Ca(im) was inhibited 35% by 10(‐5) M‐diltiazem, 65% by 2 x 10(‐4) M‐LaCl3, and 87% in Ca(2+)‐free external solution. 4. When Ca(im) buffering capacity was increased by partially depleting the SR with a transient (1 min) exposure to caffeine, subsequent exposure to 80 nM‐K+ solution increased Ca(im) almost 2‐fold more slowly than 80 mM‐K+ before depletion of Ca2+ from the SR. However, the influxing Ca2+ was sequestered by the SR and refilled it, as evident by the subsequent caffeine‐induced Ca(im) transient being identical to the first. Increasing extracellular [K+] (thus, increasing depolarization and Na+ removal) caused proportional increases in Ca(im) and the subsequent caffeine‐induced Ca(im) transients were proportionally larger, indicating a graded filling of the SR by Ca2+ influx. 5. Diltiazem (10(‐5) M) inhibited the refilling of the SR achieved by 80 mM‐K+, by 26%. Refilling was inhibited 76% by 80 mM‐K+, Ca(2+)‐free solution, indicating the fraction of refilling dependent on influx of Ca2+ through voltage‐gated Ca2+ channels, leak channels, and other influx pathways. Mild depolarization with 35 mM‐K+ (no Na+ removal) often caused no increase in Ca(im), but influx through voltage‐gated Ca2+ channels occurred because the SR Ca2+ store was refilled. Also, 10(‐5) M‐diltiazem or 10(‐6) M‐TA3090 inhibited the refilling to levels attributable only to leak influx of Ca2+. 6. All data support our hypothesis that the SR significantly attenuates the amount of Ca2+ influx that accumulates to increase Ca(im).(ABSTRACT TRUNCATED AT 400 WORDS)
Association of cyclic GMP‐gated channels and Na(+)‐Ca(2+)‐K+ exchangers in bovine retinal rod outer segment plasma membranes.
Authors: Bauer P J , Drechsler M
Published: December 2014

1. Cyclic GMP‐gated channels and Na(+)‐Ca(2+)‐K+ exchangers from bovine photoreceptors were examined by investigation of the Ca2+ fluxes from vesicles of rod outer segment (ROS) membranes and from proteoliposomes obtained by solubilization of the ROS membrane proteins and reconstitution in soy bean L‐alpha‐phosphatidylcholine (PC). 2. Whereas vesicles obtained by mild sonication of ROS membranes in a Ca(2+)‐containing buffer yielded a maximal cyclic GMP‐induced Ca2+ release of about 2.5% and a maximal Na(+)‐induced Ca2+ release of about 7%, freezing and thawing of ROS membranes prior to sonication elevated these maximal Ca2+ releases to about 17% for cyclic GMP, and to about 34% for Na+. These observations are in agreement with the view that cyclic GMP‐gated channels and Na(+)‐Ca(2+)‐K+ exchangers are localized only in the plasma membrane of the photoreceptors (which in bovine ROS makes up about 6% of the total membrane), whereas freezing and thawing results in fusion of disc and plasma membranes, thus leading to a distribution of these proteins over a much larger membrane area. 3. For fused ROS membranes, the cyclic GMP‐releasable fraction of Ca2+ of 17% is an upper bound; assuming that the cyclic GMP‐gated channels are randomly distributed we estimate that about 37% of the vesicles contain at least one cyclic GMP‐gated channel. The mean diameter of the vesicles prepared by sonication was determined to be 0.12 +/‐ 0.04 micron, and therefore the fused ROS membranes contain about sixteen cyclic GMP‐gated channels/microns 2. If all cyclic GMP‐gated channels originated from the plasma membrane, we estimate that the plasma membrane contains about 270 cyclic GMP‐gated channels/microns 2. 4. In vesicles prepared from fused ROS membranes, Na(+)‐Ca2+ exchange after activation of the cyclic GMP‐gated channels. On the other hand, after an exhaustive Na(+)‐Ca2+ exchange, only little, if any, Ca2+ was released upon addition of cyclic GMP, demonstrating that cyclic GMP‐gated channels and Na(+)‐Ca(2+)‐K+ exchangers occur on the same vesicle fraction. This observation suggests that Na(+)‐Ca(2+)‐K+ exchangers do not distribute independently of the cyclic GMP‐gated channels upon membrane fusion but are apparently associated with the cyclic GMP‐gated channels.(ABSTRACT TRUNCATED AT 400 WORDS)
Impact of antral mechanoreceptor activation on the vago‐vagal reflex in the rat: functional zonation of responses.
Authors: McCann M J , Rogers R C
Published: December 2014

1. Activation of gastric sensory afferents alters gastric motor and secretory function via the gastric vago‐vagal reflex. In this report, we investigated in the rat the impact of gastric mechanoreceptor activation on the brain stem components of the reflex, which are located in the dorsal vagal complex (DVC), i.e. the nucleus of the solitary tract (NTS) and the subjacent dorsal motor nucleus (DMN). 2. In our extracellular recordings of single‐cell activity in the DVC, we observed a relation between the response to antral distention and the location of the cell in the DVC. Specifically, cells that were excited by antral distention (ON cells) were located dorsal to those that were inhibited (OFF cells) by the same stimulus (mean depth = 536 +/‐ 15 and 627 +/‐ 14 microns for ON and OFF cells, respectively). 3. For a subset of DVC cells, the location was marked by ionophoretic ejection of Pontamine Blue from the recording barrel. Histological analysis indicated that ON cells were located in the NTS, and OFF cells were located in the ventral NTS or within the boundaries of the DMN. Together, these data led to the hypothesis that ON and OFF cells are functionally different groups of neurones, i.e. ON cells may be NTS neurones, and OFF cells may be DMN neurones. We tested this directly by employing both an intragastric balloon and a non‐traumatic vagal stimulating electrode to determine whether inflation‐related cells were NTS or DMN cells via orthodromic and antidromic activation, respectively. 4. Almost all ON cells (12/13) were orthodromically activated by vagal stimulation, i.e. they were NTS neurones. One ON cell was antidromically activated, and therefore was a DMN neurone. Of the twenty‐eight OFF cells that were encountered, ten were classified as NTS neurones because they were orthodromically inhibited by vagal stimulation. The remaining eighteen OFF cells were orthodromically inhibited and antidromically activated (i.e. DMN neurones). Thus, our results support the hypothesis that ON and OFF cells can be functionally distinct populations of neurones, in that almost all ON cells are NTS cells and approximately 2/3 of the OFF cells are DMN neurones. 5. The response to mechanoreceptor activation was different for NTS and DMN neurones. NTS cells were activated (55%) or inhibited (45%) by balloon distention of the stomach, whereas DMN cells were almost exclusively inhibited (95%) by this stimulus. This information provides insight into the organization of excitatory and inhibitory connections of the brain stem components that mediate gastric vago‐vagal reflexes.