bluuebunny/crossref_metadata_2025 huggingface dataset:
Contractile properties of skinned muscle fibres from young and adult normal and dystrophic (mdx) mice.
Authors: Williams D A , Head S I , Lynch G S , Stephenson D G
Published: December 2014

1. Single muscle fibres were enzymatically isolated from the soleus and extensor digitorum longus (EDL) muscles of genetically dystrophic mdx and normal (C57BL/10) mice aged 3‐6 or 17‐23 weeks. 2. Fibres of both muscles were chemically skinned with the non‐ionic detergent Triton X‐100 (2% v/v). Ca(2+)‐ and Sr(2+)‐activated contractile responses were recorded and comparisons were made between several contractile parameters of various fibre types of normal and dystrophic mice of similar age. 3. There were no significant differences in the following contractile parameters of skinned fibres of normal and mdx mice of the same age: sensitivity to activating Ca2+ (pCa50) or Sr2+ (pSr50) and differential sensitivity to the activating ions (pCa50‐pSr50). However the maximum isometric tension (Po) and the frequency of myofibrillar force oscillations in EDL fast‐twitch fibres of young mdx mice were significantly lower than those of soleus fast‐twitch fibres of the same animals, or fast‐twitch fibres (EDL or soleus) of normal mice. 4. Age‐related differences were apparent in some contractile parameters of both normal and mdx mice. In particular the steepness of force‐pCa and force‐pSr curves increased with age in normal mice, yet decreased with age in fibres of mdx mice. 5. A fluorescent probe, ethidium bromide, which interchelates with DNA, was used with laser‐scanning confocal microscopy to determine the distribution of myonuclei in fibres. Fibres isolated from either muscle type of normal animals displayed a characteristic peripheral spiral of myonuclei. Fibres from muscles of mdx mice displayed three major patterns of nuclear distribution; the normal peripheral spiral, long central strands of nuclei, and a mixture of these two patterns. 6. The contractile characteristics of mdx fibres were not markedly influenced by the nuclear distribution pattern in that there were no discernible differences in the major contractile parameters (the Hill coefficients nCa and nSr, which are associated with the steepness of the Ca2+ and Sr2+ activation curves, pCa50, pSr50, pCa50‐pSr50) of skinned fibres possessing peripheral or central nuclei. However, except for nSr, these values were all lower in individual fibres which displayed similar proportions of central and peripheral nuclei. The presence of mixed nucleation and absence of fibres with embryonic contractile characteristics in mdx mice suggest that the dystrophin‐negative fibres can repair locally occurring muscle damage.
The central role of corticotrophin‐releasing factor (CRF‐41) in psychological stress in rats.
Authors: Morimoto A , Nakamori T , Morimoto K , Tan N , Murakami N
Published: December 2014

1. We investigated the central role of corticotrophin‐releasing factor (CRF‐41) in psychological stress‐induced responses, including cardiovascular, thermoregulatory and locomotive activity in free‐moving rats. 2. Psychological stress was induced by cage‐switch stress. After rats were placed in the novel environment, blood pressure, heart rate, body temperature and locomotive activity significantly increased. The intracerebroventricular (I.C.V.) injection of alpha‐helical CRF(9‐41), a CRF‐41 receptor antagonist, significantly attenuated the stress‐induced hypertension, tachycardia, hyperthermia and increase in locomotive activity. However, in unstressed rats, the I.C.V. injection of alpha‐helical CRF(9‐41) had no effect on physiological parameters measured in this study. 3. In unstressed rats, the I.C.V. injection of CRF‐41 (1 microgram and 10 micrograms) increased blood pressure, heart rate, body temperature and locomotive activity in a dose‐dependent manner. The changes in these responses were quite similar to those observed during cage‐switch stress. 4. The results suggest that central CRF‐41 plays an important role in psychological stress‐induced hypertension, hyperthermia, tachycardia and increase in locomotive activity. However, it is likely that central CRF‐41 does not contribute to normal cardiovascular and body temperature regulation when rats are free from stress.
Effects of inorganic phosphate analogues on stiffness and unloaded shortening of skinned muscle fibres from rabbit.
Authors: Chase P B , Martyn D A , Kushmerick M J , Gordon A M
Published: December 2014

1. We examined the effects of aluminofluoride (AlFx) and orthovanadate (Vi), tightly binding analogues of orthophosphate (Pi), on the mechanical properties of glycerinated fibres from rabbit psoas muscle. Maximum Ca(2+)‐activated force, stiffness, and unloaded shortening velocity (Vus) were measured under conditions of steady‐state inhibition (up to 1 mM of inhibitor) and during the recovery from inhibition. 2. Stiffness was measured using either step or sinusoidal (1 kHz) changes in fibre length. Sarcomere length was monitored continuously by helium‐neon laser diffraction during maximum Ca2+ activation. Stiffness was determined from the changes in sarcomere length and the corresponding changes in force. Vus was measured using the slack test method. 3. AlF chi and Vi each reversibly inhibited force, stiffness and Vus. Actively cycling cross‐bridges were required for reversal of these inhibitory effects. Recovery from inhibition by AlF chi was 3‐ to 4‐fold slower than that following removal of V1. 4. At various degrees of inhibition, AlF chi and Vi both inhibited steady‐state isometric force more than either Vus or stiffness. For both AlF chi and Vi, the relatively greater inhibition of force over stiffness persisted during recovery from steady‐state inhibition. We interpret these results to indicate that the cross‐bridges with AlF chi or Vi bound are analogous to those which occur early in the cross‐bridge cycle.
The effects of MgADP on cross‐bridge kinetics: a laser flash photolysis study of guinea‐pig smooth muscle.
Authors: Nishiye E , Somlyo A V , Török K , Somlyo A P
Published: December 2014

1. The effects of MgADP on cross‐bridge kinetics were investigated using laser flash photolysis of caged ATP (P3‐1(2‐nitrophenyl) ethyladenosine 5'‐triphosphate), in guinea‐pig portal vein smooth muscle permeabilized with Staphylococcus aureus alpha‐toxin. Isometric tension and in‐phase stiffness transitions from rigor state were monitored upon photolysis of caged ATP. The estimated concentration of ATP released from caged ATP by high‐pressure liquid chromatography (HPLC) was 1.3 mM. 2. The time course of relaxation initiated by photolysis of caged ATP in the absence of Ca2+ was well fitted during the initial 200 ms by two exponential functions with time constants of, respectively, tau 1 = 34 ms and tau 2 = 1.2 s and relative amplitudes of 0.14 and 0.86. Multiple exponential functions were needed to fit longer intervals; the half‐time of the overall relaxation was 0.8 s. The second order rate constant for cross‐bridge detachment by ATP, estimated from the rate of initial relaxation, was 0.4‐2.3 x 10(4) M‐1 s‐1. 3. MgADP dose dependently reduced both the relative amplitude of the first component and the rate constant of the second component of relaxation. Conversely, treatment of muscles with apyrase, to deplete endogenous ADP, increased the relative amplitude of the first component. In the presence of MgADP, in‐phase stiffness decreased during force maintenance, suggesting that the force per cross‐bridge increased. The apparent dissociation constant (Kd) of MgADP for the cross‐bridge binding site, estimated from its concentration‐dependent effect on the relative amplitude of the first component, was 1.3 microM. This affinity is much higher than the previously reported values (50‐300 microM for smooth muscle; 18‐400 microM for skeletal muscle; 7‐10 microM for cardiac muscle). It is possible that the high affinity reflects the properties of a state generated during the co‐operative reattachment cycle, rather than that of the rigor bridge. 4. The rate constant of MgADP release from cross‐bridges, estimated from its concentration‐dependent effect on the rate constant of the second (tau 2) component, was 0.35‐7.7 s‐1. To the extent that reattachment of cross‐bridges could slow relaxation even during the initial 200 ms, this rate constant may be an underestimate. 5. Inorganic phosphate (Pi, 30 mM) did not affect the rate of relaxation during the initial approximately 50 ms, but accelerated the slower phase of relaxation, consistent with a cyclic cross‐bridge model in which Pi increases the proportion of cross‐bridges in detached ('weakly bound') states.(ABSTRACT TRUNCATED AT 400 WORDS)
31P nuclear magnetic resonance studies on the glycogenolysis regulation in resting and contracting frog skeletal muscle.
Authors: Yamada T , Kikuchi K , Sugi H
Published: December 2014

1. Regulation of glycogenolysis in frog skeletal muscle at rest and following contraction was studied by measuring the concentration of phosphate‐containing metabolites and the intracellular pH (pHi) in CN‐treated muscles, in which oxidative phosphorylation was inhibited by NaCN, using the 31P nuclear magnetic resonance (NMR) technique. 2. When CN‐treated muscles were kept at rest, the phosphocreatine (PCr) concentration very slowly decreased with time with a corresponding increase of the inorganic phosphate (Pi) concentration, while the ATP concentration remained unchanged. The pHi changed in the alkaline direction for the first 3 h, and then started to change in the acidic direction. 3. When CN‐treated muscles were tetanized for 10 s, the PCr concentration decreased with a corresponding increase of the Pi concentration and acidification of pHi, while the ATP concentration remained unchanged. 4. When CN‐treated muscles were tetanized repeatedly (each for 2 s) at constant intervals, the pHi changed in the alkaline direction following the first and the second tetani, and then changed in the acidic direction following the subsequent tetani, indicating that the consumed ATP is first replenished by the Lohmann reaction, while glycogenolysis starts only when the total amount of contractile activity exceeds a critical value. 5. Irrespective of whether CN‐treated muscles were kept at rest or tetanized repeatedly, the Pi concentration increased to about 8 mM (mmol/kg wet muscle) when glycogenolysis started, suggesting that the onset of glycogenolysis in CN‐treated muscles is regulated by the Pi concentration. 6. The ‘internal’ buffering power of muscle cytosol was estimated to be 35 mM H+/pH unit in anaerobic muscles and 25 mM H+/pH unit in CN‐ and iodoacetic acid (IAA)‐treated muscles. The ‘internal’ buffering power contains a contribution due to flux of carbon dioxide and lactic acid across the cell membrane. Evidence indicated that lactic acid flux is small.
Within‐breath modulation of left ventricular function during normal breathing and positive‐pressure ventilation in man.
Authors: Innes J A , De Cort S C , Kox W , Guz A
Published: December 2014

1. To investigate how intrathoracic pressure affects left ventricular function during normal breathing and positive‐pressure ventilation, beat‐by‐beat measurements of left ventricular stroke volume (LVSV; pulsed Doppler ultrasound), heart rate and blood pressure were made in five normal subjects breathing actively and then undergoing passive positive‐pressure ventilation. 2. To assess the within‐breath effects of positive‐pressure ventilation on left ventricular function in the clinical setting, further measurements were made in six patients sedated, paralysed and ventilated because of brain injuries, who had no disease of the heart or lungs. 3. In the normal subjects, there were minimal phasic variation in LVSV during positive‐pressure ventilation with the subject passive. Heart rate and blood pressure also stayed relatively constant throughout the ventilator cycle. During active breathing at the same depth and rate, there were large phasic respiratory variations in LVSV, with the lowest values occurring during inspiration. 4. In the paralysed and unconscious patients, an increase in LVSV was associated with the increase in airway pressure which occurred during lung inflation; however, the phase lag between the rise in airway pressure and the rise in LVSV varied widely between patients. These changes occurred whether or not sinus arrhythmia was present. 5. Addition of 4.6 +/‐ 1 cmH2O of positive end‐expiratory pressure (PEEP) did not increase the within‐breath LVSV variations, but resulted in a mean depression of LVSV of 5.4% (paired t test, P = 0.035). 6. The smaller variations in LVSV during positive‐pressure ventilation compared to normal breathing in the conscious subjects may reflect (a) the smaller magnitude of positive, compared to negative, pleural pressure excursions which accompany a given tidal volume, and (b) an asymmetry between the effects of positive and negative pleural pressure on the heart. 7. The prominent effects of positive‐pressure ventilation on LVSV in unconscious patients, compared to the minimal effects seen in ventilated normal subjects, may result from reduced lung compliance and a degree of pulmonary vascular congestion in the patients which was undetectable clinically or radiologically.
Properties of GABA‐mediated synaptic potentials induced by zinc in adult rat hippocampal pyramidal neurones.
Authors: Xie X , Smart T G
Published: December 2014

1. Intracellular recording techniques were used to study the actions of the transition ion, zinc, on CA1 and CA3 pyramidal neurones in adult rat hippocampal slices. 2. Zinc (300 microM) hyperpolarized pyramidal neurones, increased the membrane excitability and also induced periodic, spontaneous giant depolarizing potentials associated with a conductance increase mechanism. 3. The occurrence of spontaneous giant depolarizations was dependent on the zinc concentration (10 microM‐1 mM) with an apparent dissociation constant of 98 microM. The frequency of zinc‐induced depolarizations was unaffected by the membrane potential from ‐50 to ‐100 mV. 4. Stimulation of the Schaffer collaterals or mossy fibre pathways evoked an excitatory and inhibitory synaptic potential complex. In the presence of zinc, nerve fibre stimulation evoked, in an all‐or‐none fashion, a giant depolarizing potential with an increased membrane conductance. Both spontaneous and evoked depolarizations were inhibited by 1 microM tetrodotoxin. 5. Evoked giant depolarizations were labile with too frequent stimulation resulting in a failure of generation. A minimum time of 140 s was required between stimuli to ensure successive giant depolarizations. 6. Spontaneous and evoked zinc‐induced depolarizing potentials were inhibited by bicuculline (10 microM) or picrotoxin (40 microM) and enhanced by pentobarbitone (100 microM) or flurazepam (10 microM), suggesting that these potentials are mediated by activation of gamma‐aminobutyric acidA (GABAA) receptors. 7. Ionophoretic application of GABA produced biphasic responses at ‐60 mV membrane potential. The reversal potentials for the depolarizing and hyperpolarizing GABA responses were ‐56 +/‐ 5 and ‐66 +/‐ 8 mV respectively. The giant depolarizations induced by zinc reversed at ‐57 +/‐ 4 mV. This suggests a dendritic location for the generation of these potentials. 8. Excitatory amino acid antagonists, 2‐amino‐5‐phosphonovalerate (APV, 40 microM) or 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 10 microM) did not affect the amplitude but slightly reduced the frequency of the giant depolarizations. 9. It is concluded that zinc induces a synchronized release of GABA, quite independent of intact excitatory synaptic transmission, which acts on GABAA receptors producing large depolarizing synaptic potentials. This increased level of GABA release may be of physiological and pathological importance since zinc is a naturally occurring metal ion endogenous to the central nervous system.
Evidence from fluorescence microscopy and comparative studies that rat, ovine and bovine colonic crypts are absorptive.
Authors: Pedley K C , Naftalin R J
Published: December 2014

1. To test whether colonic crypts are secretory or absorptive interstitial [Na+] in rat descending colonic mucosa is determined using video‐enhanced imaging of the impermeant acid form of the fluorescent Na+ probe SBFI (Molecular Probes) and intracellular [Na+] is monitored with SBFI (AM form). In rat descending colonic mucosa perifused with isotonic Tyrode solution interstitial [Na+] = 500‐650 mM. Following exposure to Tyrode solution containing theophylline (10 mM) interstitial [Na+] falls by 300‐450 mM within 1 min. Exposure to amiloride (0.2 mM) reduces the intracellular [Na+] from ca 25 to 12 mM within 15 min and concurrently decreases [Na+] in the interstitial fluid surrounding the crypts at the mucosal surface by approximately 200 mM. 2. The route of fluid inflow across the rat colonic mucosa is directly traced by perifusing with Tyrode solution containing the impermeant fluorescent dye, fluorescein disulphonate (FS). FS accumulates rapidly within crypt lumens of control tissues to a 2‐fold higher concentration than in the external bathing solution, but FS does not accumulate in crypts of tissues treated with azide (2 mM). The increment in FS accumulation within the crypt lumen above the bulk solution decreases by 80% within 1 min following exposure to theophylline (10 mM), indicating that fluid absorption into crypts is reduced. Estimates of the total fluid influx from the rate and extent of FS concentration polarization within crypts indicate that it is sufficient to account for the entire transcolonic fluid absorption. 3. Comparative studies of isolated bovine and ovine colon were also undertaken to investigate the failure of bovine colon to generate a hypertonic absorbate and hence its incapacity to produce hard faeces. The interstitial fluid surrounding ovine colonic crypts is hypertonic to the bulk solution, whereas the interstitial fluid surrounding bovine colonic crypts is nearly isotonic with the bathing solution. Additionally, fluorescein disulphonate accumulates within ovine colonic crypt lumens by concentration polarization, whereas no concentration of FS occurs within bovine colonic crypt lumens. This corroborates the view that a hypertonic interstitial fluid is absent from bovine colon mainly because of a high rate of transepithelial leakage of low molecular weight solutes via paracellular routes.
State‐dependent radial elasticity of attached cross‐bridges in single skinned fibres of rabbit psoas muscle
Authors: Xu Sengen , Brenner Bernhard , Yu Leepo C.
Published: December 2014

1. In a single skinned fibre of rabbit psoas muscle, upon attachment of cross‐bridges to actin in the presence of ADP or pyrophosphate (PPi), the separation between the contractile filaments, as determined by equatorial X‐ray diffraction, is found to decrease, suggesting that force is generated in the radial direction.

2. The single muscle fibres were subjected to compression by 0‐8% of dextran T500. The changes in lattice spacings by dextran compression were compared with changes induced by cross‐bridge attachment to actin. Based on this comparison, the magnitude and the direction of the radial force generated by the attached cross‐bridges were estimated. The radial cross‐bridge force varied with filament separation, and the magnitude of the radial cross‐bridge force reached as high as the maximal axial force produced during isometric contraction.

3. One key parameter of the radial elasticity, i.e. the equilibrium spacing where the radial force is zero, was found to depend on the ligand bound to the myosin head. In the presence of ADP, the equilibrium spacing was 36 nm. In the presence of MgPPi the equilibrium spacing shifted to 35 nm and Ca2+ had little effect on the equilibrium spacing.

4. The equilibrium spacing was independent of the fraction of cross‐bridges attached to actin. The fraction of cross‐bridges attached in rigor was modulated from 100% to close to 0% by adding up to 10 mM of ATPγS in the rigor solution. The lattice spacing remained at 38 nm, the equilibrium spacing for nucleotide‐free cross‐bridges at μ = 170 mM.

5. Radial force generated by cross‐bridges in rigor at large lattice spacings (38 nm ≤ d10 ≤ 46 nm) appeared to vary linearly with lattice spacing.

6. The titration of ATPγS to fibres in rigor provided a correlation between the radial stiffness of the nucleotide‐free cross‐bridges and the equatorial intensities. The relation between the equatorial intensity ratio I11/I10 and radial stiffness appeared to be approximately linear.

7. The fibres under different conditions showed a wide range of radial stiffness, which was not proportional to the apparent axial stiffness of the fibre. If the apparent axial stiffness is a measure of the fraction of cross‐bridges bound to actin, it follows that the radial elastic constant is state dependent; or vice versa.

8. Differences in equilibrium lattice spacing and in radial elastic constant, most probably reflect differences in the molecular structure of the acto‐myosin complex and there is more than one single conformation of the various strongly bound cross‐bridge states.

9. Determining equilibrium spacings of the radial elasticity appears to be an effective new approach in detecting structural differences among the attached cross‐bridges, since this approach is independent of the fraction of cross‐bridges attached, a factor that frequently encumbers the interpretation of structural studies of attached cross‐bridge states.
Noradrenaline as a presynaptic inhibitory neurotransmitter in ganglia of the guinea‐pig gall‐bladder.
Authors: Mawe G M
Published: December 2014

1. The effects of noradrenaline on guinea‐pig gall‐bladder ganglia were investigated with intracellular recording techniques. 2. Noradrenaline (0.01‐100 microM) decreased the amplitude of the fast excitatory postsynaptic potential (EPSP) that was evoked by stimulation of interganglionic fibre tracts. High concentrations of noradrenaline (10‐100 microM) caused an inhibition ranging from 93‐100%. The noradrenaline concentration that resulted in half‐maximal inhibition (EC50) of the EPSP was 280 nM. 3. Experiments with selective agonists and antagonists indicated that the alpha 2‐adrenoreceptor was involved in the inhibition of the EPSP. Clonidine (0.001‐100 microM) reduced the EPSP in a concentration‐dependent manner with an EC50 of 30 nM. Yohimbine (100‐300 nM) caused a rightward shift of the noradrenaline concentration‐effect relationship, with a dissociation equilibrium constant of 1.4 nM. 4. Release of endogenous catecholamines by tyramine (100 microM) in the presence of desipramine (1.0 microM), caused a yohimbine‐sensitive decrease in the amplitude of the EPSP. Treatment with tyramine did not affect the amplitude of the EPSP in tissue that had undergone prior chemical sympathectomy with 6‐hydroxydopamine. 5. Electrical stimulation of the vascular plexus (1‐3 s; 10‐20 Hz; 10 mA) decreased the amplitude of the EPSP. In some cases suprathreshold responses were reduced to subthreshold EPSPs following stimulation of the vascular plexus. Yohimbine (300 nM) reversibly inhibited the effects of vascular plexus stimulation. 6. Noradrenaline did not modify the responses of gall‐bladder neurones to exogenously applied acetylcholine. Also, application of noradrenaline, by superfusion (0.001‐100 microM) or by pressure microejection (1.0 mM), had no effect on the resting membrane potential, membrane conductance, or action potential characteristics of gall‐bladder neurones. 7. Immunoreactivity for type A monoamine oxidase (MAO‐A) was found in the vascular plexus and the ganglionated plexus of the gall‐bladder. 8. These results show that noradrenaline has an alpha 2‐adrenoreceptor‐mediated presynaptic inhibitory effect on fast synaptic transmission in the ganglia of the guinea‐pig gall‐bladder. It is proposed that vagal terminals may be an important target of this adrenergic inhibitory input to the gall‐bladder.
N‐ethylmaleimide‐inhibited electrogenic K+ secretion in the ampulla of the frog semicircular canal.
Authors: Ferrary E , Bernard C , Oudar O , Loiseau A , Sterkers O , Amiel C
Published: December 2014

1. The mechanisms of K+ secretion into endolymph were studied on a preparation of isolated semicircular canal with different pharmacological inhibitors. Three periods of 5 or 30 min were performed, the first as control, the second in the presence of the drugs added to the apical or the basolateral bathing solution, and the third as recovery. Apical fluid was sampled at the beginning and the end of each period, transepithelial potential was recorded, Na+, K+, and Cl‐ concentrations, and K+ efflux, with 86Rb+ as a tracer, were measured and K+ fluxes were calculated. 2. When both sides of the epithelium were bathed with perilymph‐like solution, the epithelium absorbed Na+, secreted K+, and generated a lumen positive potential. 3. The ATPases inhibitors, ouabain (10(‐5) and 10(‐3) M) and N‐ethylmaleimide (10(‐4) and 10(‐3) M) inhibited the electrogenic K+ secretion when added to the basolateral fluid. N‐ethylmaleimide (10(‐3) M) applied to the apical fluid during a 5 min period decreased the K+ influx by 43% and the transepithelial potential by 66%. Other ATPase inhibitors, harmaline (10(‐3) M), omeprazole (10(‐4) M), vanadate (10(‐4) M and 10(‐3) M), N,N'‐dicyclohexylcarbodiimide (DCC, 10(‐5) M), 7‐chloro‐4‐nitrobenz‐2‐oxa‐1,3‐diazole (NBD‐Cl, 5 x 10(‐6) M and 5 x 10(‐5) M), and bafilomycin (10(‐7) M) did not affect the K+ transport nor the transepithelial potential when they were added to the apical fluid. 4. The Na(+)‐K(+)‐Cl‐ co‐transporter inhibitor, bumetanide, decreased both the transepithelial potential and the K+ transport when added to the basolateral solution but not to the apical one. At 10(‐6) M, bumetanide maximally decreased the K+ influx by about 60%. 5. K+ channel blockers, quinine (10(‐4) M), TEA (5 x 10(‐3) M), added to the apical solution and barium (2 x 10(‐3) M) added to either the apical or the basolateral solutions, did not affect the K+ transport and the transepithelial potential. 6. The carbonic anhydrase inhibitor acetazolamide (10(‐3) M) added to both apical and basolateral solutions did not affect the K+ transport and the transepithelial potential. 7. It is concluded that, in the ampulla of the semicircular canal, a basolateral Na(+)‐K(+)‐Cl‐ co‐transporter energized by the Na+, K(+)‐ATPase was involved for 60% in the K+ secretion into endolymph. The electrogenic K+ transport would partly depend on a N‐ethylmaleimide‐sensitive protein possibly located at the apical plasma membrane or intracellularly.
Effect of endothelin‐3 on vasopressin release in vitro and water excretion in vivo in Long‐Evans rats.
Authors: Rossi N F
Published: December 2014

1. The endothlins (ETs) are a family of homologous peptides originally isolated and purified from cultured porcine endothelial cells. Although initial studies focused on the ETs as potent vasoconstrictor substances, recent findings support a role for ET in vasopressin (AVP) secretion and action. We used cultured explants of the hypothalamo‐neurohypophysial complex (HNC) to examine the effects of ET‐3 on AVP release. 2. ET‐3 produced a significant and concentration‐dependent rise in AVP release from the explants of Long‐Evans rats at 48 h, independent of medium osmolality. AVP release during two sequential control periods did not differ, and osmotically stimulated AVP release was comparable to that exhibited by HNC explants from Sprague‐Dawley rats. ET‐3 (1 nM) induced a 3‐fold rise in AVP that was completely blocked by rabbit antiET serum, which did not alter basal AVP secretion. 3. Clearance experiments were performed in anaesthetized water‐loaded rats given a non‐pressor dose of ET‐3 (0.40 nmol (kg body weight)‐1 intravenously). The means of body weight, arterial blood pressure, plasma Na+ concentrations, and plasma osmolalities did not differ between the first and second periods nor among the groups. Despite no changes in renal plasma flow and inulin clearance, free water clearance significantly increased in the ET‐treated group. This response could not be attributed to changes in osmolal clearances or Na+ reabsorption. The concurrent administration of antiET serum not only blocked this effect, but was associated with the free water clearance falling significantly below the pretreatment level. 4. Taken together, our in vivo and in vitro findings support the hypothesis that ET‐3 stimulates AVP secretion. Furthermore, our data are consistent with the site of action of ET‐3 residing within the blood‐brain barrier, though an independent effect by a higher concentration of ET at neural loci outside the barrier cannot be totally excluded. Finally, the subpressor dose of ET‐3 amplifies free water excretion independent of systemic and renal haemodynamics, Na+ excretion, osmolal clearance, or circulating AVP levels.
Synaptic and intrinsic conductances shape picrotoxin‐induced synchronized after‐discharges in the guinea‐pig hippocampal slice.
Authors: Traub R D , Miles R , Jefferys J G
Published: December 2014

1. A computer model was constructed of the guinea‐pig hippocampal region in vitro, containing 100 pyramidal neurones. This approach has contributed to the understanding of brief (usually less than 100 ms) epileptic events known as ‘interictal spikes’. The present study addresses the cellular mechanisms of more prolonged epileptic events, lasting 200 ms and more, that may represent short‐duration seizures. Each neurone was simulated with a nineteen‐compartment model using six voltage‐dependent ionic conductances. The neurones were randomly interconnected with excitatory synapses, each synapse exerting a fast voltage‐independent alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) component and a slower voltage‐dependent N‐methyl‐D‐aspartate (NMDA) component. Each neurone received input from twenty other neurones. 2. This model was able to generate, in response to synaptic noise or to stimulation of one neurone, a series of synchronized population bursts, the initial (primary) burst being longer than later (secondary) bursts, terminating in a prolonged after‐hyperpolarization. The simulated after‐discharge potentials resemble those recorded experimentally from pyramidal neurones during perfusion of the hippocampal slice with media containing picrotoxin, a blocker of synaptic inhibition mediated by GABAA receptors. 3. Simulated after‐discharges agree with the following experiments: over a certain range of total NMDA conductance, blockade of AMPA receptors will prevent the occurrence of synchronized firing, whereas, blockade of NMDA receptors will, in contrast, abolish the secondary bursts, leaving a shortened and somewhat smaller primary burst. Dendritic potential oscillations occur in phase with somatic oscillations. When interneurones (some generating GABAA‐mediated IPSPs, others generating GABAB IPSPS) are included in the model, the occurrence of synchronized events was suppressed, the most significant suppressant effect coming from GABAA IPSPS. 4. The model predicts that: a dendritic calcium spike occurs during each secondary burst; AMPA receptors serve to maintain the synchrony of secondary bursts, as well as to initiate the primary burst; and that with sufficient total NMDA conductance, synchronized firing can occur even with AMPA receptors blocked. 5. The model suggests, in addition, that the duration of the initial burst is determined in part by the experimentally observed delay between Ca2+ entry and peaking of the after‐hyperpolarization (AHP) conductance, and hence reflects properties of the individual pyramidal neurones. Specifically, a pattern of a long initial burst followed by brief secondary bursts is elicited in single‐cell simulations by injection of a steady depolarizing current into the apical dendrite. The same pattern is produced when the single‐cell model includes only calcium and calcium‐dependent conductances.(ABSTRACT TRUNCATED AT 400 WORDS)
The control of intracellular pH in cultured avian chondrocytes.
Authors: Dascalu A , Nevo Z , Korenstein R
Published: December 2014

1. Mechanical loading of cartilaginous tissue generates an increase in the concentration of cations in the extracellular matrix. This includes a decrease of the extracellular pH (pHo), which is known to affect the intracellular pH (pHi), thereby modifying the intracellular metabolism. Thus, the regulation of pHi is essential for the physiological function of cartilage. The fluorescent pH‐sensitive dye 2',7'‐bis(carboxyethyl)‐5(6)‐carboxyfluorescein acetoxymethyl ester (BCECF AM) was employed in order to assess the mechanisms responsible for control of the pHi in an embryonic avian chondrocyte cell suspension. 2. Steady‐state pHi in the absence of physiological HCO3‐ was 7.15 +/‐ 0.01 pH units as compared to a pHi of 6.94 +/‐ 0.02 pH units in its presence (P < 0.01). The intrinsic buffering power of chondrocytes (beta i) was 38.9 mM/pH unit and the total buffering capacity (beta T) was 65.8 mM/pH unit. 3. Cells maintained in a Hepes‐buffered solution were exposed to an intracellular acid load by the NH4+ prepulse technique (20 mM NH4Cl). The initial rate of pHi recovery was 0.106 pH units/min (n = 18). Amiloride (0.33 mM), an inhibitor of the Na(+)‐H+ exchanger, or replacement of external sodium [Na+]o with choline induced a 60% inhibition of the recovery rate, indicating a predominant involvement of this antiporter in the response to intracellular acidification. 4. H(+)‐ATPase inhibitors (oligomycin 20 micrograms/ml; N,N;‐dicyclohexylcarbodiimide (DCC), 0.5 mM; N‐ethylmaleimide (NEM), 0.25 mM) and iodomycin (2 mM), a metabolic cell suppressor, reduced acid extrusion by 25% as measured by the NH4Cl prepulse in Hepes‐bathed cells. 5. Chondrocytes transferred from a Hepes‐buffered solution to a 5% CO2‐25 mM HCO3‐ medium (HCO3‐ solution) underwent a pHi decrease of approximately 0.20 pH units, followed by a regulatory alkalinizing response of 0.118 pH units/min. The Na(+)‐H+ exchanger was responsible for only 15% of this alkalinization (amiloride, 0.33 mM), in contrast to its primary role in HCO(3‐)‐free solution. 6. The activity of a Na(+)‐dependent Cl(‐)‐HCO3‐ exchanger in physiological HCO3‐ solution was estimated by addition of the inhibitors 4‐acetamido‐4'‐isothiocyanatostilbene‐2,2'‐disulphonic acid (SITS; 0.5 mM) or diisothiocyanatostilbene‐2,2'‐disulphonic acid (DIDS; 100 microM) and by the suspensions of chondrocytes in a Na(+)‐free solution. Acidification performed under these conditions resulted in a 45% inhibition of the recovery rate as compared to control rates.(ABSTRACT TRUNCATED AT 400 WORDS)
Gating of transmission to motoneurones by stimuli applied in the locus coeruleus and raphe nuclei of the cat.
Authors: Jankowska E , Riddell J S , Skoog B , Noga B R
Published: December 2014

1. Neuronal systems activated by stimulation in the region of the locus coeruleus/subcoeruleus (LC/SC) and raphe nuclei have previously been shown to depress transmission from group II muscle afferents in regions of the midlumbar spinal segments in which premotor interneurones are located. The aim of the present investigation was to determine the extent to which such depression is paralleled by depression of the reflex actions of group II afferents on motoneurones. 2. The effects of short trains of conditioning electrical stimuli applied within the LC/SC and raphe nuclei were examined on postsynaptic potentials (PSPs) evoked by group I and group II muscle afferents in hindlimb motoneurones. The effects were examined over a wide range of conditioning‐test intervals but particular emphasis was placed on the effects produced at long intervals (> 100 ms) since such effects are more likely to be mediated by the descending noradrenergic and serotonergic neurones of the LC/SC and raphe nuclei which are of slow conduction velocity. In addition, conditioning stimuli alone evoked PSPs in motoneurones (with latencies of 7‐15 ms and a duration of 50‐80 ms) and effects evoked at short conditioning‐test intervals might therefore have been secondary to changes in motoneurone membrane properties. 3. At conditioning‐test intervals between 100 and 350 ms synaptic actions of group II origin were strongly and consistently depressed. Both EPSPs and IPSPs were affected, two‐thirds of those tested being reduced in amplitude by 50% or more. A similar depression was exerted on PSPs evoked from the quadriceps and deep peroneal nerves mediated predominantly by interneurones located in the midlumbar segments and on PSPs evoked from the hamstring and triceps surae nerves mediated by interneurones located in more caudal segments. It is thus concluded that neuronal systems activated by stimuli applied in the LC/SC and raphe nuclei are capable of gating transmission in all those interneuronal pathways which mediate the reflex actions of group II afferents on motoneurones in anaesthetized animals.
Co‐regulation of cAMP‐activated Na+ current by Ca2+ in neurones of the mollusc Pleurobranchaea.
Authors: Huang R C , Gillette R
Published: December 2014

1. The cAMP‐gated Na+ current (INa, cAMP) was studied in axotomized neurons of the pedal ganglion of the sea slug Pleurobranchaea. INa, cAMP responses were elicited by iontophoretic injection of cAMP and recorded in voltage clamp. 2. The current‐voltage relation for INa, cAMP was flat between ‐90 and ‐50 mV, but declined steeply with depolarization from ‐50 to ‐30 mV. Depolarizing pulses also suppressed the INa, cAMP response, which recovered slowly over tens of seconds. 3. The inactivating effects of depolarization on the current were abolished both by blockade of Ca2+ current and intracellular injection of Ca2+ chelator. Thus, Ca2+ influx through voltage‐dependent Ca2+ channels probably mediates inactivation of INa, cAMP within its normal physiological range of action. 4. Increasing intracellular cAMP levels antagonized the effects of Ca2+ influx on INa, cAMP. The mutual antagonism of the ions suggests that cAMP and Ca2+ act competitively in regulation of the INa, cAMP channel. 5. Measures of fractional inactivation of INa, cAMP provided evidence for the existence of an appreciable basal level of current, and hence cAMP, in the unstimulated neuron. Since INa, cAMP is a direct function of cAMP activity, measures of fractional inactivation permit quantification of cAMP levels in the living neuron. 6. Calcium inactivation of INa, cAMP completes a negative feedback loop that can contribute to endogenous burst activity. Over the burst cycle, depolarization and action potential activity driven by INa, cAMP would lead to Ca2+ influx, consequent inactivation of the inward current, and hyperpolarization. This mechanism of endogenous bursting resembles other in which the burst cycle has been found to be regulated by kinetics of Ca2+ influx and removal. However, INa, cAMP may vary in its Ca2+ sensitivity in different neurons and these variations may affect the functional expression of endogenous oscillatory activity.
Common ionic mechanisms of excitation by substance P and other transmitters in guinea‐pig submucosal neurones.
Authors: Shen K Z , Surprenant A
Published: December 2014

1. Intracellular recordings were made from submucosal neurones and single‐electrode voltage‐clamp methods were used to record membrane currents. The actions of substance P (SP), 5‐hydroxytryptamine (5‐HT), muscarine, vasoactive intestinal polypeptide (VIP), forskolin and nerve stimulation were studied. 2. Substance P, 5‐HT (in the presence of 5‐HT3 receptor antagonists), muscarine, VIP, forskolin and slow excitatory synaptic transmission all produced identical responses: an inward current associated with a membrane conductance decrease at the resting potential. The actions of any one occluded the actions of any other and all responses were pertussis‐toxin insensitive. 3. These agonists produced a voltage‐independent decrease in a ‘leak’ potassium conductance between ‐40 and ‐120 mV in 14% of neurones. 4. These agonists decreased a voltage‐dependent, calcium‐activated potassium conductance between ‐40 and ‐80 mV in all other (86%) neurones. The agonists still evoked an inward current without apparent conductance change at potentials between ‐90 and ‐130 mV. 5. In a low calcium solution containing cobalt or cadmium, the agonists produced an inward current associated with a conductance increase from ‐40 to ‐120 mV. Ion replacement studies indicated this current was due to an increase in a cation‐selective (mainly sodium) conductance. 6. The agonists also reduced the inwardly rectifying potassium current that is activated by somatostatin and alpha 2‐adrenoceptor agonists in these neurones. The agonists did not alter the inwardly rectifying potassium current that is present in these neurones in the absence of somatostatin or alpha 2‐agonists. 7. Thus, SP, 5‐HT, muscarine, VIP and the release of slow excitatory transmitters all appear to act through a common intracellular transduction pathway, an increase in adenylate cyclase. This results in an activation of a sodium‐selective cation current and an inhibition of three distinct potassium conductances: the background potassium conductance, the calcium‐activated potassium conductance and the inwardly rectifying potassium conductance activated by somatostatin and alpha 2‐adrenoceptor agonists.
Effects of isoprenaline on cytosolic calcium concentrations and on tension in the porcine coronary artery.
Authors: Ushio‐Fukai M , Abe S , Kobayashi S , Nishimura J , Kanaide H
Published: December 2014

1. Using front‐surface fluorometry and fura‐2‐loaded medial strips of the porcine coronary artery, cytosolic Ca2+ concentration ([Ca2+]i) and tension development were simultaneously monitored in an attempt to determine the mechanisms of vasorelaxation induced by l‐isoprenaline (Iso). 2. Iso actively decreased [Ca2+]i of the strips at rest, both in the presence and absence of extracellular Ca2+. 3. In the presence of extracellular Ca2+, depolarization with high‐external K+ solution induced an elevation of [Ca2+]i and tension of the rapid increase and sustained, steady‐state type; both levels depended on external K+ concentration. When Iso was applied at the time of steady state of high‐K(+)‐induced [Ca2+]i elevation, there was an initial transient reduction (the first component) followed by a subsequent sustained reduction (the second component) of [Ca2+]i. For a given [Ca2+]i level during high‐K+ depolarization, the tension developed in the presence of Iso was smaller than that in its absence. Thus, the [Ca2+]i‐tension relationship during the steady state of high‐K(+)‐induced contraction was shifted to the right by Iso. Pretreatment with ryanodine, a compound which depletes Ca2+ stored in the sarcoplasmic reticulum, abolished the first component, but not the second sustained decrease in [Ca2+]i by Iso. 4. In the presence of extracellular Ca2+ (1.25 mM), histamine (Hist) induced an abrupt (the first component) and then sustained (the second component) elevations of [Ca2+]i, while the tension rose rapidly to reach the peak, and then, gradually declined. The second, but not the first, component of [Ca2+]i elevation depended on extracellular Ca2+. Iso inhibited both the first and the second components of [Ca2+]i elevation and the contraction induced by Hist, in a concentration‐dependent manner (IC50, 2 x 10(‐8) M for the first component, and 5 x 10(‐8) M for the second component). Cumulative application of Hist (10(‐7)‐10(‐4) M) increased [Ca2+]i and tension with the [Ca2+]i‐tension relationship shifting to the left from that observed with high K+. The [Ca2+]i‐tension relationship during the Hist‐induced contraction shifted to the right by Iso. In contractions induced by a higher concentration (> or = 6 x 10(‐5) M) of Hist, despite the negligible decrease in [Ca2+]i, Iso could relax the muscle in a concentration‐dependent manner. 5. In the absence of extracellular Ca2+, Hist induced transient elevations of [Ca2+]i and tension, possibly due to a release of Ca2+ from intracellular stores, and with similar time courses to those of the first component observed in the presence of extracellular Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)
Tetraethylammonium‐sensitive apical K+ channels mediating K+ secretion by turtle colon.
Authors: Wilkinson D J , Kushman N L , Dawson D C
Published: December 2014

1. Apical membrane K+ channels in turtle colon were identified and characterized using current fluctuation analysis. 2. Under short‐circuit conditions in NaCl‐Ringer solution, the power density spectrum (PDS) of the short‐circuit current (Isc) sometimes exhibited a clearly discernible Lorentzian component, indicating spontaneous fluctuations produced by a population of apical ion channels. The Lorentzian component had a characteristic corner frequency (fc) which averaged 10.2 +/‐ 0.9 Hz (mean +/‐ S.E.M., n = 20). 3. The power of the spontaneous fluctuations was enhanced (So increased) by manoeuvres that depolarize the apical membrane electrical potential (Va). Discernible fluctuations were enhanced or induced by raising the serosal K+ concentration ([K+]s = 50‐115 mM, Na+ replacement), by clamping the transepithelial potential (Vt) to serosa‐positive values, or by blocking basolateral K+ channels with Ba2+. 4. Mucosal amiloride (100 microM) attenuated the spontaneous fluctuations observed in NaCl‐Ringer solution but had no effect in the presence of serosal high K+, indicating that amiloride did not block the K(+)‐permeable channels but these channels resided in the same cells as the amiloride‐sensitive Na+ channels. 5. Raising the mucosal K+ concentration attenuated spontaneous fluctuations. 6. In the presence of serosal high K+ and mucosal amiloride, the spontaneous fluctuations were often accompanied by a reversed Isc consistent with K+ secretion. These conditions were used to test the effects of putative channel blockers. 7. Mucosal Ba2+ and tetraethylammonium (TEA+) were effective inhibitors of the spontaneous fluctuations and the reversed Isc. At a concentration of 10 mM, TEA+ was maximally effective but the TEA+ analogues tetramethylammonium (TMA+) and tetrapropylammonium (TPrA+) were much less effective. Mucosal Rb+ or Cs+ did not inhibit at a concentration of 10 mM. 8. Mucosal lidocaine (200 microM), quinidine (200 microM), or diphenylamine‐2‐carboxylate (DPC, 1 mM) had little or no effect on the spontaneous fluctuations and reversed Isc. Quinine (100 microM), 4‐aminopyridine (1 mM), and apamin (100 nM) were also without effect. 9. Mucosal TEA+ (10 mM) abolished the active secretory K+ flux measured in the presence of serosa‐positive transepithelial potentials. 10. These experiments identified a population of TEA(+)‐sensitive, apical K+ channels which mediate active K+ secretion in turtle colon. Sensitivity to external TEA+ distinguishes these channels from basolateral K+ channels in turtle colon and demonstrates similarity to apical K+ channels in mammalian colon.