bluuebunny/crossref_metadata_2025 huggingface dataset:
Dietary restriction sensitizes the rat distal colon to aldosterone.
Authors: Nzegwu H C , Levin R J
Published: December 2014

1. The effects of starvation and undernutrition were assessed on rat colonic electrogenic Na+ absorption in fed controls, 72 h starved and acute undernourished (fed one‐third of the control group's daily food intake for up to 9 days). The basal short‐circuit currents (Isc) of three segments of rat colon (proximal, mid‐ and distal), stripped of their external muscle layers were monitored before and during addition of 0.1 mM‐mucosal amiloride. The decrease in Isc was used as the measure of the electrogenic Na+ absorption. 2. Acute undernutrition and to a lesser extent 72 h starvation elevated the basal Isc only in the distal colon. The increase was inhibited by amiloride (0.1 mM, mucosal) indicating that it was due to electrogenic Na+ transport. 3. Allowing the 9 days acute undernourished rats to drink 0.9% NaCl failed to prevent the increase in the basal Isc in the distal colon but it was reduced by administration of spironolactone. 4. Adrenalectomy completely abolished the increased basal Isc in the distal colon induced by the 9 day undernutrition. However, the plasma aldosterone levels in the fed and 9 day undernutrition groups were not significantly different. 5. Injection of aldosterone into adrenalectomized rats drinking 0.9% NaCl and which were undernourished for 9 days induced a large increase in their distal colonic Isc which was inhibited by mucosal amiloride. Similar treatment of sham‐operated rats on 0.9% NaCl or adrenalectomized control fed rats on 0.9% NaCl had no effect on the distal colonic Isc. 6. The results indicate that acute undernutrition for 9 days makes the distal colonic epithelium more sensitive to the prevailing plasma aldosterone level allowing an enhanced electrogenic Na+ absorptive capacity to be induced.
The inositol 1,4,5‐trisphosphate pathway mediates cholinergic potentiation of rat hippocampal neuronal responses to NMDA.
Authors: Markram H , Segal M
Published: December 2014

1. The cellular mechanism by which acetylcholine (ACh) potentiates neuronal responses to N‐methyl‐D‐aspartate (NMDA) was investigated in CA1 neurones of hippocampal slices using current‐ and voltage‐clamp techniques. 2. Loading cells with 5'‐guanylylimidodiphosphate (GppNHp) caused a gradual increase in response to NMDA. Pulses of ACh accelerated this increase. Guanosine 5'‐O‐(2‐thiodiphosphate) (GDP beta S) blocked the potentiating effect of ACh on responses to NMDA. 3. Acute LiCl caused a gradual decrease in the potentiating effect of ACh, while the potentiation was completely prevented by 3 day chronic 6 mequiv/kg (I.P.) LiCl treatment and restored by acute treatment with 10 mM‐inositol. 4. Loading cells with a general protein kinase inhibitor, H‐7, enhanced the potentiating effect of ACh on responses to NMDA and blocked the effect of ACh on the after‐hyperpolarization (AHP). 5. Ultraviolet irradiation of cells loaded with a photolabile inositol 1,4,5‐trisphosphate (InsP3) caused a transient increase in responses to NMDA, while penetrating cells with active InsP3‐containing pipettes caused a gradual BAPTA‐sensitive increase in responses to NMDA. 6. Reducing the rate of InsP3 metabolism, with 2,3‐diphosphoglyceric acid (DPG), caused an increase and prolongation of the potentiating effect of ACh, while blocking the InsP3 receptor with heparin prevented the cholinergic potentiation. 7. NMDA, by itself, potentiated subsequent responses to NMDA, an effect that was blocked when [Ca2+]i was chelated with BAPTA. NMDA and ACh were also found to compete in potentiating responses to NMDA. Finally, the cholinergic potentiation was blocked when cells were loaded with BAPTA. 8. We propose that activation of the InsP3 branch of the phosphoinositide pathway potentiated responses to NMDA and that InsP3 exerted this effect by elevating [Ca2+]i.
Actions of perchlorate ions on rat soleus muscle fibres.
Authors: Dulhunty A F , Zhu P H , Patterson M F , Ahern G
Published: December 2014

1. The effects of perchlorate (ClO4‐) on contraction have been studied in rat soleus muscle fibres using (i) potassium (K+) contracture and (ii) two‐microelectrode‐point voltage clamp techniques. 2. Membrane potentials (Vm) at all external [K+] were 3‐5 mV more negative in ClO4‐. The hyperpolarization could not be attributed to a change in Na+, K+, or Cl‐ permeability, or to an effect on the Na(+)‐K+ pump. 3. ClO4‐ shifts the voltage dependence of tension activation, and contraction threshold, to more negative membrane potentials without altering maximum tension. Consequently, twitches and submaximal K+ contractures were potentiated, whereas tetanic contractions and 200 mM‐K+ contractures were unaltered. 4. The decay of K+ contractures during steady depolarization with ClO4‐ developed a slow exponential phase with an average time constant of 6.05 +/‐ 0.76 min at ‐38 mV, and 1.68 +/‐ 0.15 min at ‐19 mV. This slow component was (a) under the rapid control of the surface Vm and (b) did not depend on external Ca2+. 5. Inactivation of E‐C coupling was measured with a test 200 mM‐K+ depolarization following 3‐10 min depolarizations in conditioning solutions containing 20‐120 mM‐K+. ClO4‐ induced a negative shift in the curve‐relating test K+ contracture amplitude to conditioning Vm but did not alter the rate of repriming of tension upon repolarization. 6. The results suggest that ClO4‐ increases the amount of activator produced during depolarization and thus allows the slow inactivation step in excitation‐contraction (E‐C) coupling to be reflected in the decay of K+ contracture tension. 7. A ‘perchlorate contracture’, which did not depend on the activation of E‐C coupling, was observed. The contracture depended on external Ca2+, but not on voltage‐dependent Ca2+ channels or Na(+)‐Ca2+ exchange.
Mechanisms of intrinsic tone in ferret vascular smooth muscle.
Authors: Pawlowski J , Morgan K G
Published: December 2014

1. Circular strips from ferret aorta were used to investigate the mechanism of the intrinsic basal tone. 2. Determinations of stiffness using small sinusoidal length changes showed an abolition of both stiffness and force with cooling, but the temperature dependence of the change in active stiffness did not parallel that of force. At temperatures below 22 degrees C there appeared to be a relatively large population of attached, non‐force‐generating cross‐bridges, indicating that separate mechanisms are involved in regulating cross‐bridge attachment and the force per cross‐bridge. 3. Active intrinsic tone was not affected by removal of extracellular Ca2+ or removal of endothelium. 4. Intracellular ionized Ca2+ concentrations ([Ca2+]i) as measured with the photoprotein aequorin, did not significantly change when intrinsic tone was abolished by cooling. 5. Myosin light chain phosphorylation, as measured by 2‐dimensional polyacrylamide gel electrophoresis, significantly decreased on cooling, but the temperature dependence of phosphorylation did not parallel that of force. The change in phosphorylation in the absence of a change in [Ca2+]i suggests the presence of a constitutively active Ca(2+)‐independent form of myosin light chain kinase. 6. Maximal concentrations of staurosporine inhibited but did not eliminate intrinsic tone. 7. Changes in myosin light chain kinase and protein kinase C activities may explain part but not all of the intrinsic tone.
Arachidonic acid and prostaglandin E2 activate small‐conductance Cl‐ channels in the basolateral membrane of rabbit parietal cells.
Authors: Sakai H , Okada Y , Morii M , Takeguchi N
Published: December 2014

1. Cl‐ channels in the basolateral membrane of non‐stimulated parietal cells in isolated rabbit gastric glands were studied by patch‐clamp and noise analysis techniques. 2. Voltage‐independent whole‐cell currents were recorded from parietal cells equilibrated with Cl(‐)‐containing solutions. Upon reducing the Cl‐ concentration of the basolateral bathing solution, the current‐voltage curve was rapidly shifted to the right. The reversal potential changed according to changes in the equilibrium potential for Cl‐. 3. A Cl‐ channel blocker, 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate (NPPB) inhibited whole‐cell Cl‐ currents. The half‐maximal inhibitory concentration of NPPB was 300 microM, and the inhibition was reversible (at less than or equal to 200 microM). Another Cl‐ channel blocker, 4‐acetamido‐4'‐isothiocyanatostilbene‐2,2'‐dilsulphonic acid or diphenylamine‐2‐carboxylate was much less effective. 4. The power spectra of whole‐cell Cl‐ current fluctuations contained one Lorentzian component. The single Cl‐ channel conductance was estimated to be 0.35 pS by the variance noise analysis, which is in agreement with the value obtained by a method of power spectrum analysis (0.29 pS). 5. The addition of arachidonic acid (10 microM) to the basolateral medium markedly increased the whole‐cell Cl‐ current. The combined application of a cyclo‐oxygenase inhibitor, indomethacin (50 microM), and a lipoxygenase inhibitor, esculetin (100 microM), increased the Cl‐ current, whereas the administration of a phospholipase A2 inhibitor, mepacrine (100 microM), significantly decreased the whole‐cell Cl‐ current. 6. A sizeable increase in the whole‐cell Cl‐ current was also induced by a metabolite of arachidonic acid, prostaglandin E2 (10 microM), but not by leukotriene B4 (5 microM) or D4 (10 microM). 7. The present study has shown that small‐conductance Cl‐ channels are present in the basolateral membrane of rabbit parietal cells, and that the channel was functionally regulated by arachidonic acid and prostaglandin E2.
Cardiovascular responses induced in free‐moving rats by immune cytokines.
Authors: Morimoto K , Morimoto A , Nakamori T , Tan N , Minagawa T , Murakami N
Published: December 2014

1. We investigated the effect of intraperitoneal (I.P.) injections of the immune cytokines, interleukin‐1 beta (IL‐1 beta) and tumour necrosis factor (TNF) on cardiovascular responses in free‐moving rats, using a biotelemetry system. 2. The I.P. injection of a small dose of IL‐1 beta (1 microgram/kg) induced a monophasic increase in the heart rate, and that of a large dose (10 micrograms/kg) induced biphasic increases in the blood pressure and heart rate. However, the I.P. injection of any of several doses of TNF (1, 10 and 50 micrograms/kg) had no effect on cardiovascular responses in rats. 3. Pre‐treatment with I.P. injection of indomethacin (10 mg/kg), an inhibitor of cyclo‐oxygenase, significantly suppressed the cardiovascular responses and the increase in the plasma noradrenaline (NA) concentration induced by I.P. injection of IL‐1 beta. 4. Microinjection of IL‐1 beta (1 and 10 ng) into the preoptic and anterior hypothalamic (PO‐AH) region induced dose‐dependent increases in the blood pressure and heart rate in rats. These responses were also suppressed by pretreatment with I.P. indomethacin (10 mg/kg). In addition, microinjection of prostaglandin E2 (20 and 100 ng) into the PO‐AH region increased blood pressure and heart rate, but that of prostaglandin D2 (100 ng) had no effect. 5. The present results suggest that IL‐1 beta stimulates the release of prostaglandins, presumably E series, near regions of the hypothalamus, which act on the hypothalamus to induce activation of the sympathetic nervous system. Subsequently, the blood pressure, heart rate and the plasma level of NA increase.
Role of cyclic nucleotides and calcium in the nutrient‐induced release of cholecystokinin‐like immunoreactivity in rats.
Authors: Cuber J C , Aucouturier S , Bernard C , Chayvialle J A
Published: December 2014

1. This study was undertaken with an isolated vascularly perfused rat duodenojejunal preparation to investigate the mechanisms of the release of cholecystokinin measured by immunoassay (cholecystokinin‐like immunoreactivity, CCK‐LI). 2. Intra‐arterial infusion of forskolin (2‐20 microM) evoked a prompt and well‐sustained secretion of CCK‐LI which was increased to a mean value of 600% of basal with the highest dose tested. 3‐Isobutyl‐1‐methylxanthine (IBMX) (10(‐6)‐10(‐4) M) stimulated the secretion of CCK‐LI (maximal increase of 400% of basal at 10(‐4) M). 3. Intra‐arterial infusion of beta‐phorbol 12‐myristate 13‐acetate (5 x 10(‐7)‐5 x 10(‐6) M) and calcium ionophore A23187 (10(‐6)‐10(‐5) M) alone or in combination provoked only a transient increase in the release of CCK‐LI. 4. Luminal infusion of a 5% ovalbumin hydrolysate solution produced an immediate release of CCK‐LI followed by a well‐sustained secretion at 580% of basal. Intra‐arterial infusion of IBMX (10(‐5) or 10(‐4) M) in combination with luminal peptone induced a release of CCK‐LI which was equal to the sum of the CCK responses evoked by IBMX and peptone given separately. 5. Intra‐arterial infusion of EGTA (2 mM) abolished the forskolin‐ and peptone‐induced CCK secretion while luminal EGTA (2 mM) had no inhibitory effect. Verapamil (5 x 10(‐5)‐10(‐4) M) or nifedipine (10(‐5)‐5 x 10(‐5) M) inhibited the peptone‐evoked CCK secretion. A high concentration of trifluoperazine (10(‐4) M) strongly reduced the release of CCK‐LI induced by intraluminal peptone while 10(‐5) M was ineffective. 6. It is concluded that the peptone‐induced secretion of CCK‐LI involves a cyclic AMP‐dependent mechanism and the activation of calcium channels possibly located at the basolateral side of the CCK cell.
Transmucosal electrical resistance in rabbit isolated gastric mucosa during exposure to acid.
Authors: Spencer G E , Spraggs C F , Stables R , Hirst B H
Published: December 2014

1. Transmucosal electrical resistance (Rt) and short‐circuit current (Isc) were determined in rabbit isolated fundic mucosa. Under basal conditions, with a HCO(3‐)‐free HEPES‐buffered solution (pH 7.4) bathing both sides of the mucosae, Rt was 161.5 + 5.0 omega cm2 and Isc 41.8 +/‐ 1.8 microA cm‐2, and these values were not significantly different to values observed in HCO(3‐)‐buffered Krebs‐Hensleit solution. 2. The basal Isc was inhibited by the Cl‐ channel blocker diphenylamine‐2‐carboxylate, and ouabain, but unaffected by the Na+ channel blocker amiloride (10(‐5) M), consistent with electrogenic chloride secretion dependent upon a sodium gradient. Prostaglandin E2 (10(‐7) M) stimulated an increase in Isc which was susceptible to inhibition by diphenylamine‐2‐carboxylate, but not amiloride, again consistent with Cl‐ secretion. 3. Stepwise acidification of the mucosal solution to pH 2.8 resulted in an increase in Rt of 43%, as compared with that measured with mucosal pH 7.4. Isc did not change during the acidification to pH 2.8, indicating retention of tissue viability. Increased Rt while Isc remained constant is consistent with an acid‐induced decrease in the shunt (paracellular) conductance in this Cl(‐)‐secreting tissue. At pH less than 2.8, Rt declined rapidly and Isc declined and reversed, consistent with H+ back‐diffusion. Scanning electron microscopic investigation of tissue exposed to mucosal pH 2.8 revealed little difference from control (pH 7.4) tissue, but there was considerable evidence of cellular damage and membrane disruption in tissue exposed to pH 1.8. 4. Acidification of the serosal solution did not increase Rt, which was maintained until pH 3.7, and then rapidly declined at pH less than 3.7. Bilateral acidification produced a mixed response; Rt increased, as for mucosal acidification, down to pH 2.8, after which there was a rapid decline in Rt following the pattern observed for serosal acidification. 5. Compared at a mucosal pH approximately 2.8, DIDS (4 x 10(‐4) M) and amiloride (10(‐3) M) inhibited the acid‐induced increase in Rt, suggesting a role for both Cl(‐)‐HCO3‐ and Na(+)‐H+ exchange in the response. In contrast, the acid‐induced increase in Rt was unaffected by a lower concentration of amiloride (10(‐5) M), acetazolamide and ouabain. Therefore, neither Na+ channels nor a Na+ gradient appear to be involved in the acid‐induced increase in Rt.(ABSTRACT TRUNCATED AT 400 WORDS)
Cholinergic activation of a non‐selective cation current in canine gastric smooth muscle is associated with contraction.
Authors: Sims S M
Published: December 2014

1. The effects of acetylcholine on membrane electrical properties of single smooth muscle cells dissociated from the circular layer of the canine gastric corpus were investigated using the nystatin perforated patch technique. Cells retained their ability to contract during recording, making it possible to correlate changes in membrane potential and membrane currents with contractions. 2. Acetylcholine caused depolarization from ‐59 +/‐ 8 mV to ‐18 +/‐ 7 mV (means +/‐ S.D., n = 12) with no generation of action potentials. The depolarization was associated with a membrane conductance increase, consistent with acetylcholine activating an inward current. In addition, acetylcholine caused contraction of cells to 58% of initial length. Both depolarization and contraction were reversible and were antagonized by atropine. 3. Under voltage clamp, acetylcholine activated inward current associated with increased current noise. The current‐voltage relationship of the acetylcholine‐induced current was studied at steady‐state voltages and with voltage ramp commands. The inward current was largest between ‐40 and ‐20 mV and reversed direction to outward close to 0 mV (reversal potential, Erev = +3 +/‐ 9 mV). Reduction of external Na+ concentration to 21 mM shifted Erev to ‐42 +/‐ 5 mV, as predicted for a non‐selective cation current. The conductance activated by acetylcholine (gACh) increased sigmoidally with depolarization, with about 2.5 nS activated at 0 mV. 4. Cells consistently contracted upon stimulation with acetylcholine, even when studied under voltage clamp at potentials as negative as ‐100 mV. This was consistent with muscarinic receptor activation causing release of Ca2+ from internal stores. When cells were bathed in Ca(2+)‐free solutions, the first application of acetylcholine elicited normal inward current and contraction. Thereafter, both inward current and contractions were greatly diminished or absent, suggesting that the stores of Ca2+ had been depleted. 5. Caffeine caused reversible contraction and activation of inward current similar to that elicited by acetylcholine. 6. It is concluded that muscarinic stimulation of canine gastric smooth muscle cells involves activation of a non‐selective cation conductance and is consistently accompanied by contraction. The release of Ca2+ from internal stores may be a common trigger for both events.
Two types of glutamate receptors differentially excite amacrine cells in the tiger salamander retina.
Authors: Dixon D B , Copenhagen D R
Published: December 2014

1. Excitatory inputs to amacrine cells in the salamander retinal slice preparation were examined using whole‐cell patch pipette voltage‐clamp techniques. In strychnine (500 nM) and bicuculline (100 microM), two types of amacrine cell were easily distinguished by their light‐evoked excitatory responses: transient and sustained. 2. In transient amacrine cells the current‐voltage (I‐V) relation for the peak light‐evoked current was non‐linear with a negative slope region between ‐50 and ‐70 mV. Responses reversed near +10 mV and were prolonged at more positive holding potentials. 3. In DL‐2‐amino‐phosphonoheptanoate (AP7, 30 microM), a selective N‐methyl‐D‐aspartate (NMDA) receptor antagonist, both the negatively sloped region of the light I‐V relation and the prolongation of the response at positive potentials were eliminated. In 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 2 microM), a selective non‐NMDA receptor antagonist, light‐evoked currents at the most hyperpolarized holding potentials were eliminated. At potentials positive to ‐85 mV the light‐evoked currents lacked a fast onset. The light I‐V relation in CNQX had a negative slope region between ‐35 and ‐80 mV. 4. With synaptic transmission blocked, kainate evoked responses in transient cells with a resultant I‐V relation that was nearly linear, whereas glutamate and NMDA elicited responses with non‐linear I‐V relations. 5. Light‐evoked currents in sustained amacrine cells had a nearly linear I‐V relation and reversed near +10 mV. AP7 at a concentration of 30 microM did not affect the light‐evoked currents in sustained cells, but 2 microM‐CNQX eliminated all light‐evoked currents in these cells. 6. With synaptic transmission blocked, sustained amacrine cells responded only to glutamate and kainate, not NMDA. The resultant I‐V relations were linear. 7. We conclude that the light‐evoked responses of transient amacrine cells are mediated by concomitant activation of both non‐NMDA and NMDA receptors whereas the responses of sustained amacrine cells are mediated only by non‐NMDA receptors. Furthermore, these data provide supportive evidence that the primary light‐evoked excitatory neurotransmitter activating amacrine cells is glutamate.
Transient chloride binding as a contributory factor to corneal stromal swelling in the ox.
Authors: Hodson S , Kaila D , Hammond S , Rebello G , al‐Omari Y
Published: December 2014

1. Investigations were made of the cation exchange capacity of fresh isolated ox corneal stroma (Q, units: mequiv fixed stromal charge/kg stromal fluid) at pH 7.4 over a variety of stomal hydrations (H, units: kg stromal fluid/kg dry tissue) both above and below the physiological hydration of 3.2, whilst the stromas were immersed in a variety of sodium chloride solutions (range 5‐1000 mM). 2. At any particular salt concentration, the product QH (dry tissue exchange capacity, units: mequiv/kg dry tissue) appeared constant, over all the hydrations investigated. 3. Dry tissue exchange capacity (QH) varied, however, when the bathing salt concentration was altered. It varied between 55 mequiv/kg dry tissue (e.g. Q = 17 mequiv at H = 3.2) in 5 mM‐NaCl to 240 mequiv/kg dry tissue (e.g. Q = 75 mequiv/l at H = 3.2) in 1000 mM‐NaCl. 4. The variation of stromal exchange capacity in NaCl solutions of different concentrations was similar when detected by three independent procedures: stromal gel pressure measurements, intrastromal sodium ion distributions, and intrastromal electrical potentials. 5. Intrastromal chloride ion distributions were anomalous. Total chloride (measured by radio‐isotopes) was consistently higher than that predicted by Donnan theory. 6. The data were consistent with Elliott's hypothesis that a fraction of intrastromal chloride ions bind to the corneal stromal matrix and in so doing contribute to the fixed negative charge of the stroma. 7. Our observations may be explained by a model of the cation exchange capacity of ox cornea which has two types of components. On is (at constant pH) invariant, and has a dry tissue exchange capacity of about 50 mequiv/kg dry tissue, and is probably generated by the sulphonic and carboxylic acid groups of the glycosaminoglycans. The other is explained by supposing it to consist of a chloride binding ligand which exhibits first order binding, is half occupied at ambient chloride concentrations of 300 mM, and has a total capacity of 240 mequiv/kg dry tissue. 8. Partial stromal extraction with 4 M‐guanidine HCl indicated that the chloride binding ligand is not associated with the collagen molecules in the corneal stromal fibrils. 9. It is suggested that such a stromal chloride ion binding ligand would help to stabilize the hydration and transparency of the living cornea when it is exposed to environments of varying tonicity (such as in river or sea bathing).
A caffeine‐ and ryanodine‐sensitive Ca2+ store in bullfrog sympathetic neurones modulates effects of Ca2+ entry on [Ca2+]i.
Authors: Friel D D , Tsien R W
Published: December 2014

1. We studied how in changes in cytosolic free Ca2+ concentration ([Ca2+]i) produced by voltage‐dependent Ca2+ entry are influenced by a caffeine‐sensitive Ca2+ store in bullfrog sympathetic neurones. Ca2+ influx was elicited by K+ depolarization and the store was manipulated with either caffeine or ryanodine. 2. For a time after discharging the store with caffeine and switching to a caffeine‐free medium: (a) [Ca2+]i was depressed by up to 40‐50 nM below the resting level, (b) caffeine responsiveness was diminished, and (c) brief K+ applications elicited [Ca2+]i responses with slower onset and faster recovery than controls. These effects were more pronounced as the conditioning caffeine concentration was increased over the range 1‐30 mM. 3. [Ca2+]i, caffeine and K+ responsiveness recovered in parallel with a half‐time of approximately 2 min. Recovery required external Ca2+ and was speeded by increasing the availability of cytosolic Ca2+, suggesting that it reflected replenishment of the store at the expense of cytosolic Ca2+. 4. During recovery, Ca2+ entry stimulated by depolarization had the least effect on [Ca2+]i when the store was filling most rapidly. This suggests that the effect of Ca2+ entry on [Ca2+]i is modified, at least in part, because some of the Ca2+ which enters the cytosol during stimulation is taken up by the store as it refills. 5. Further experiments were carried out to investigate whether the store can also release Ca2+ in response to stimulated Ca2+ entry. In the continued presence of caffeine at a low concentration (1 mM), high K+ elicited a faster and larger [Ca2+]i response compared to controls; at higher concentrations of caffeine (10 and 30 mM) responses were depressed. 6. Ryanodine (1 microM) reduced the rate at which [Ca2+]i increased with Ca2+ entry, but not to the degree observed after discharging the store. At this concentration, ryanodine completely blocked responses to caffeine but had no detectable effect on Ca2+ channel current or the steady [Ca2+]i level achieved during depolarization. 7. We propose that, depending on its Ca2+ content, the caffeine‐sensitive store can either attenuate or potentiate responses to depolarization. When depleted and in the process of refilling, the store reduces the impact of Ca2+ entry as some of the Ca2+ entering the cytosol during stimulation is captured by the store.(ABSTRACT TRUNCATED AT 400 WORDS)
Basal Ca2+ and the oscillation of Ca2+ in caffeine‐treated bullfrog sympathetic neurones.
Authors: Nohmi M , Hua S Y , Kuba K
Published: December 2014

1. Effects of caffeine on the intracellular free Ca2+ concentration ([Ca2+]i) in single bullfrog sympathetic neurones in excised tissue were studied by recording Fura‐2 fluorescence excited at 340, 361 or 380 nm and taking their ratios (R340/380 or R361/380). 2. Caffeine (3‐10 mM) produced oscillation of [Ca2+]i and an ‘apparent’ decrease in the basal level of [Ca2+]i during a period between phasic rises. The mechanism of the latter effect was analysed in relation to the mechanism of the former. 3. Caffeine (3‐10 mM) increased Fura‐2 fluorescence in a range of excitation wavelength from 330 to 390 nm. The ratios of fluorescences, R340/380 and R361/380, however, were not significantly affected by caffeine. These results suggest that the ‘apparent’ reduction in the basal [Ca2+]i seen as a decrease in R340/380 or R361/380 results from a true decrease in [Ca2+]i. 4. Caffeine‐induced decrease in [Ca2+]i persisted for every period between phasic rises of [Ca2+]i during [Ca2+]i oscillation, and after the blockade of [Ca2+]i oscillation by ryanodine. The decrease in the latter condition lasted for more than 20 min. 5. The decrease in the basal [Ca2+]i depended on the external Ca2+ concentration and was not mimicked by the action of cyclic nucleotides. 6. Possible mechanisms underlying the decrease in the basal [Ca2+]i produced by caffeine (effects on Ca2+ transport at the cell or Ca(2+)‐storing organelle membrane) and their significance in relation to the [Ca2+]i oscillation were discussed.
On the inhibitory actions of baclofen and gamma‐aminobutyric acid in rat ventral midbrain culture.
Authors: Jarolimek W , Misgeld U
Published: December 2014

1. Whole‐cell voltage‐clamp recordings were used to study the effects of (‐)‐baclofen and of gamma‐aminobutyric acid (GABA) on neurones cultured from the ventral midbrain of embryonic rats. 2. Baclofen induced an outward current (IBac) at a holding potential of ‐60 mV. The maximal current was 80 pA, and half‐maximal current was evoked by 5 microM‐baclofen. The proportion of cells affected by baclofen was greater in 25‐day‐old cultures than in 14‐day‐old cultures. 3. IBac was blocked by barium (1 mM), and it reversed polarity at a potential that changed according to the Nernst equation when the extracellular potassium concentration was changed. The reversal potential was not different when recording electrodes contained caesium instead of potassium. 4. GABA (10‐20 microM), in the presence of picrotoxin (50 microM) and bicuculline (50 microM), also evoked a small potassium current at ‐60 mV. There was no correlation between the amplitude of the potassium current caused by GABA and that caused by baclofen measured in the same neurones. 5. Spontaneous synaptic currents (up to hundreds of picoamps) were observed that were blocked by picrotoxin (20 microM; IPSCs) or by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 10 microM; EPSCs); the amplitude and frequency were strongly reduced by baclofen and by GABA. 6. Spontaneous synaptic currents of lower amplitudes (up to 60 pA) remained in the presence of tetrodotoxin. IPSCs (blocked by picrotoxin, reversal at ‐50 mV) and EPSCs (blocked by CNQX, reversal at 0 mV) were reduced in frequency by baclofen. GABA, in the presence of bicuculline and picrotoxin, had a similar effect on the EPSCs. This action of baclofen persisted in barium (1 mM), and was observed as readily in cells cultured for 14 days as those cultured for 25 days. 7. Some spontaneous synaptic currents remained in the presence of tetrodotoxin and cadmium (100 microM). Their frequency was reduced by baclofen. The effectiveness of baclofen was greater on cells that had been longer in culture. 8. It is concluded that activation of GABAB receptors has two main effects on neurones cultured from rat ventral midbrain. These are potassium conductance increase, and inhibition of the spontaneous release of GABA and excitatory amino acids; both effects can be observed in tetrodotoxin and cadmium.
The electro‐mechanical response of canine inspiratory intercostal muscles to increased resistance: the caudal rib‐cage.
Authors: De Troyer A
Published: December 2014

1. The effect of graded increases in inspiratory airflow resistance and airway occlusion on the electrical activity and the mechanical behaviour of the levator costae and external intercostal muscles situated in the caudal interspaces (zone of apposition of the diaphragm to the rib‐cage) has been studied in spontaneously breathing dogs. 2. The external intercostal and levator costae muscles in the cranial interspaces were invariably active during unloaded inspiration and showed progressive facilitation of activity with increases in inspiratory resistance. In contrast, whether in the supine or in the prone position, the levator costae muscles of the caudal interspaces did not show any facilitation of activity, and the caudal external intercostal muscles never showed any inspiratory electrical activity, including during airway occlusion. 3. With graded increases in inspiratory airflow resistance, the cranial external intercostals demonstrated a gradual inspiratory lengthening and the cranial ribs were progressively displaced in the caudal direction. The caudal ribs, however, were invariably displaced in the cranial direction. As a result, the caudal external intercostals showed a progressive inspiratory shortening. 4. Shortening of the caudal external intercostals and cranial displacement of the caudal ribs were reproduced by isolated stimulation of the phrenic nerves. Thus, as inspiratory resistance increases, contraction of the diaphragm causes unloading, rather than loading, of the spindles present in the caudal external intercostal muscles. 5. After the phrenic nerves were sectioned, however, the caudal external intercostals invariably lengthened a substantial amount during inspiration, but they still did not show any inspiratory electrical activity. Accentuating the inspiratory lengthening of these muscles by external rib fixation and increasing the chemical respiratory drive did not elicit any inspiratory electrical activity either. The alpha‐motoneurones of the external intercostal muscles in the caudal interspaces thus have very small central respiratory drive potentials with respect to their critical firing threshold.
Respiratory‐related activity of lower thoracic and upper lumbar sympathetic preganglionic neurones in the rat.
Authors: Zhou S Y , Gilbey M P
Published: December 2014

1. The segmental distribution of cell bodies of sympathetic preganglionic neurones projecting into the left lumbar sympathetic chain beyond the L4 ganglion was determined in rats using retrograde transport of horseradish peroxidase. Labelled perikarya were found mainly in segments T13‐L2 in the ipsilateral intermediolateral cell column. 2. Neurophysiological studies were conducted on this cell population in anaesthetized, vagotomized and artificially ventilated rats. 3. One hundred and thirty sympathetic preganglionic neurones were studied in spinal segments T13‐L2, inclusive, after their antidromic identification following electrical stimulation of the left lumbar sympathetic chain between the 4th and 5th lumbar ganglia; sixty‐six had on‐going activity and thirty of the remainder were activated by the ionophoretic application of glutamate. Their axonal conduction velocities were 0.5‐12.5 m/s which is in the B and C fibre range. 4. The discharge patterns of these sympathetic preganglionic neurones were analysed in relation to phrenic nerve discharge. Of forty‐eight neurones analysed in this manner five had peak firing during inspiration (inspiratory related), five during early expiration (post‐inspiratory related), four during expiration (expiratory related) with the remainder having a firing pattern unrelated to phrenic nerve discharge (non‐modulated). 5. The ECG‐related patterns of discharge of the same forty‐eight neurones were analysed. Twenty‐one had an ECG‐related pattern of discharge. Glutamate‐activated quiescent neurones and those with on‐going activity had a similar incidence of such modulation. Sympathetic preganglionic neurones with each type of respiratory modulation were found to have an ECG‐related firing pattern. 6. The patterns of discharge of these neurones have been compared to those recorded, in previous studies, from sympathetic preganglionic neurones located at T2 which had axons projecting into the cervical sympathetic nerve and 'sympathoexcitatory' neurones of the rostral ventrolateral medulla. There appear to be some differences in the respiratory modulations of baroreceptor‐sensitive neurones found within each of the three populations. Consequently, it is suggested that supraspinal pathways, other than those arising in the rostral ventrolateral medulla, may be important in influencing the activity of baroreceptor‐sensitive sympathetic preganglionic neurones.
Cerebro‐cerebellar projections from the ventral bank of the anterior ectosylvian sulcus in the cat.
Authors: Kyuhou S
Published: December 2014

1. Stimulation of the ventral bank of the anterior ectosylvian sulcus (AESv) induced marked mossy fibre (MF) and climbing fibre (CF) responses in the cerebellar posterior vermis (lobules VI‐VII) and moderate sized ones in the paraflocculus, paramedian lobules and crus I and II of the cat. The relay stations for these responses to the posterior vermis were investigated morphologically and electrophysiologically. 2. It can be considered that the MF responses were relayed at least in part via the dorsolateral, peduncular and paramedian pontine nuclei, since in these nuclei there were units orthodromically responsive to AESv stimulation and antidromically responsive to stimulation of the posterior vermis. The MF responses are thought to be relayed monosynaptically, since the distribution of axon terminals labelled after injection of wheatgerm agglutinin‐conjugated peroxidase (WGA‐HRP) into the AESv overlapped in these pontine nuclei with that of neurons labelled after injection of WGA‐HRP into the posterior vermis. 3. It is thought that the CF responses are relayed in the caudomedial part of the medial accessory olive (MAOcm), because neurons in the MAOcm were orthodromically responsive to AESv stimulation and antidromically responsive to stimulation of the posterior vermis. 4. It is suggested that the cerebro‐olivary projection which transmits the orthodromic responses in the MAOcm is indirect, via the superior colliculus (SC), because injection of WGA‐HRP into the AESv labelled axon terminals not in the MAOcm but in the SC, and injection of WGA‐HRP into the MAOcm gave rise to retrograde labelling of cells in the SC. Synaptic connections between the axon terminals of the cerebrotectal projection and the tecto‐olivary neurons were demonstrated by extracellular unit studies in the SC. 5. The hypothesis that the CF responses were transmitted via the SC was supported by the finding that the CF responses disappeared transiently after muscimol or lidocaine was injected into the SC. 6. These findings provide evidence that the MF responses are transmitted at least in part via the cerebro‐ponto‐cerebellar projection, while the CF responses are relayed via the cerebro‐tecto‐olivo‐cerebellar projection. These cerebro‐cerebellar pathways from the AESv are suggested to participate in conducting visual information to the posterior vermis.
The inhibitory action of cyclic AMP on responses to carbachol dependent on calcium stores in rat gastric smooth muscle.
Authors: Ohta T , Ito S , Noto T , Tachibana R , Nakazato Y , Ohga A
Published: December 2014

1. The effects of cyclic AMP on contraction and Ca(2+)‐activated K+ currents induced by carbachol (CCh), caffeine and inositol 1,4,5‐trisphosphate (InsP3) were examined in intact and skinned smooth muscle fibres and in whole‐cell voltage‐clamped smooth muscle cells of the rat stomach. Intracellular Ca2+ level, [Ca2+]i, was monitored in intact muscle fibres loaded with Fura‐2. 2. In intact muscle fibres, dibutyryl cyclic AMP, 8‐bromo‐cyclic AMP and forskolin inhibited a phasic contraction induced by CCh (100 microM) much more extensively than that induced by caffeine (30 mM) in Ca(2+)‐free solution containing 2 mM‐EGTA. A rise in [Ca2+]i evoked by CCh was also reduced by dibutyryl cyclic AMP. 3. In skinned muscle fibres, InsP3 (40 microM) produced a contraction of amplitude similar to that evoked by caffeine (30 mM) in Ca(2+)‐free solution containing 0.05 mM‐EGTA. Cyclic AMP suppressed the InsP3‐induced contraction to a much greater extent than that induced by caffeine. 4. In cells voltage‐clamped at 0 mV, CCh (100 microM) evoked a transient Ca(2+)‐activated outward K+ current in 61% of cells tested. After wash‐out of CCh, caffeine (10 mM) evoked a similar K+ current in all cells. In cells loaded with cyclic AMP (100 microM), the percentage of cells responding to CCh was reduced to 26% and the magnitude of current response tended to decrease. Cyclic AMP caused a small increase in the caffeine‐induced K+ current. 5. An outward current was elicited immediately after the patch membrane was ruptured at a holding potential of 0 mV, using a patch pipette containing InsP3 (40 microM), in 76% of cells tested. In cells treated with dibutyryl cyclic AMP, the percentage of cells responding to InsP3 was reduced to 50% and the magnitude of current response tended to decrease. 6. In intact muscle fibres loaded with Fura‐2, the relationship between [Ca2+]i and tension development shifted to the right in the presence of dibutyryl cyclic AMP. In skinned muscle fibres, cyclic AMP shifted the pCa‐tension relation to the right, suggesting that cyclic AMP inhibits the contractile machinery directly. 7. These results suggest that the inhibitory effect of cyclic AMP on muscarinic receptors mediating both the contraction and the Ca(2+)‐activated K+ current, is partly due to the inhibition of InsP3‐induced Ca2+ release from intracellular stores in rat gastric smooth muscle cells.
The effect of a calmodulin inhibitor on intracellular [Ca2+] and contraction in isolated rat ventricular myocytes.
Authors: Frampton J E , Orchard C H
Published: December 2014

1. The effect of the calmodulin inhibitor N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulphonamide (W7; 10 microM) on intracellular [Ca2+] ([Ca2+]i) and [H+], and on contraction, has been studied in myocytes isolated from the ventricles of rat hearts. [Ca2+]i and [H+] were monitored using the fluorescent dyes Fura‐2 and 2',7'‐bis(carboxyethyl)‐5,6‐carboxyfluorescein (BCECF) respectively. 2. W7 decreased the size of both the Fura‐2 fluorescence (a function of [Ca2+]i) transient and twitch, but had no effect on their time course. 3. The decrease in the size of the Fura‐2 fluorescence transient in the presence of W7 was accompanied by a decrease in the increase of Fura‐2 fluorescence that could be elicited by releasing Ca2+ from the sarcoplasmic reticulum using 10 mM‐caffeine. 4. There was a decrease in the apparent sensitivity of the contractile proteins to Ca2+ in the presence of W7 which may account, in part, for the decrease in the twitch observed in the presence of W7. 5. Test beats were interpolated at different test intervals after a train of steady‐state contractions. Mechanical restitution curves were constructed by plotting the size of the test beat against the test interval. Both the size and the duration of the twitch increased as the test interval was prolonged. W7 slowed this mechanical restitution but had no effect on the changes in the duration of the twitch. 6. Intracellular pH was not altered by W7. 7. These results are discussed in terms of the known actions of calmodulin and W7.
Second‐messenger regulation of sodium transport in mammalian airway epithelia.
Authors: Graham A , Steel D M , Alton E W , Geddes D M
Published: December 2014

1. Sodium absorption is the dominant ion transport process in conducting airways and is a major factor regulating the composition of airway surface liquid. However, little is known about the control of airway sodium transport by intracellular regulatory pathways. 2. In sheep tracheae and human bronchi mounted in Ussing chambers under short circuit conditions, the sodium current can be isolated by pretreating tissues with acetazolamide (100 microM) to inhibit bicarbonate secretion, bumetanide (100 microM) to inhibit chloride secretion and phloridzin (200 microM) to inhibit sodium‐glucose cotransport. This sodium current consists of amiloride‐sensitive (57%) and amiloride‐insensitive (43%) components. 3. The regulation of the isolated sodium current by three second messenger pathways was studied using the calcium ionophore A23187 to elevate intracellular calcium, a combination of forskolin and the phosphodiesterase inhibitor zardaverine to elevate intracellular cyclic AMP, and the phorbol ester 12,13‐phorbol dibutyrate (PDB) to stimulate protein kinase C. 4. In sheep trachea, A23187 produces a dose‐related inhibition of the sodium current with maximal effect (38% of ISC) at 10 microM and IC50 1 microM. This response affects both the amiloride‐sensitive and insensitive components of the sodium current and is not altered by prior stimulation of protein kinase C or elevation of intracellular cyclic AMP. In human bronchi, A23187 (10 microM) produced a significantly greater inhibition of ISC (68%), a response which was unaffected by prior treatment with PDB or forskolin‐zardaverine. 5. In sheep trachea, stimulation of protein kinase C with PDB produced a dose‐related inhibition of ISC maximal (56% of ISC) at 50 nM (IC50 7 nM). This response was abolished by amiloride (100 microM) pretreatment suggesting a selective effect on the amiloride‐sensitive component of the sodium current. The response was not altered by prior elevation of intracellular calcium or cyclic AMP. PDB (10 nM) caused a similar inhibition of ISC in human bronchi (43%). The effect of PKC stimulation following pretreatment with A23187 was diminished in human bronchi. Elevating intracellular cyclic AMP did not alter this response. 6. Addition of forskolin (1 microM) together with the phosphodiesterase inhibitor zardaverine (100 microM) produced a mean 35‐fold increase in intracellular cyclic AMP in sheep trachea. This was associated with a small, but significant, 6% transient increase in ISC followed by a significant 4% fall. Neither effect could be abolished by amiloride pretreatment. In human bronchi, a small decrease in ISC which could not be distinguished from that occurring in controls was observed.(ABSTRACT TRUNCATED AT 400 WORDS)
Properties of the pacemaker current (If) in latent pacemaker cells isolated from cat right atrium.
Authors: Zhou Z , Lipsius S L
Published: December 2014

1. Single latent pacemaker cells were isolated from the Eustachian ridge of cat right atrium using Langendorff perfusion and enzyme dispersion techniques. Whole‐cell patch‐clamp techniques were used to study the hyperpolarization‐activated inward current (I(f)). 2. All cells studied beat rhythmically. Pacemaker activity was recorded in the voltage range ‐68 +/‐ 1 to ‐54 +/‐ 2 mV and its cycle length was 901 +/‐ 67 ms (72 +/‐ 5 beats min‐1) at 34‐36 degrees C. Cells were elongated with tapered ends, and appeared bent or crinkled without obvious striations. Mean cell diameter and length were 7.4 +/‐ 0.5 microns and 93.1 +/‐ 5.9 microns, respectively (n = 15). Input resistance and total membrane capacitance were 2.2 +/‐ 0.2 G omega and 27.8 +/‐ 3.1 pF, respectively. 3. Hyperpolarizing clamp steps more negative than ‐50 mV elicited a time‐dependent increasing inward current that was maximally activated at ‐120 mV. Activation of I(f) was well within the pacemaker voltage range. Half‐maximal activation voltage and slope factor were calculated, using a Boltzmann function, to be ‐80.5 mV and 8.4, respectively. 4. The fully activated current‐voltage (I‐V) relationship was approximately linear at voltages more negative than ‐30 mV and showed outward rectification at more positive voltages. The reversal potential of I(f) was ‐26 mV and the fully activated conductance was 1.75 +/‐ 0.14 nS (n = 21). Caesium (2 mM) blocked I(f) at voltages more negative than the reversal potential. Reducing extracellular Na+ or K+ shifted the reversal potential more negative, and increasing extracellular K+ exerted the opposite effect. Reducing extracellular Na+ decreased I(f) amplitude and the slope of the fully activated I‐V relationship, and elevated extracellular K+ increased I(f) amplitude and the slope of the fully activated I‐V relationship. 5. Some pacemaker cells exhibited a short delay in the onset of I(f) activation whereas other pacemaker cells exhibited little, if any, delay in activation. I(f) currents exhibiting no delay in activation were best fitted by a single exponential function with a mean time constant of 3.20 +/‐ 1.03 s at ‐70 mV (n = 4). 6. A nystatin‐permeabilized patch recording method was used to record spontaneous pacemaker action potentials and I(f) from the same pacemaker cell. Caesium (2 mM) inhibited I(f) by more than 90% (at ‐70 mV), and decreased the slope of diastolic depolarization, resulting in a 48 +/‐ 5% decrease in spontaneous rate.(ABSTRACT TRUNCATED AT 400 WORDS)