bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of reducing agents and oxidants on excitation‐contraction coupling in skeletal muscle fibres of rat and toad.
Authors: Posterino G S , Lamb G D
Published: December 2014

1. The mechanically skinned fibre technique was used to examine the role of oxidation‐reduction in the control of Ca2+ release and contraction in rat and toad skeletal muscle fibres under physiological conditions of myoplasmic [Mg2+] and [ATP] and sarcoplasmic reticulum (SR) Ca2+ load. 2. None of the reducing agents, dithiothreitol (DTT, 10 mM), glutathione (GSH, 10 mM) or cysteine (1 and 5 mM), had any detectable effect on the peak force, duration or the total number of depolarization‐induced responses that could be elicited in skinned fibres from either toad or rat muscle, except for a slight alteration in one case (GSH on the duration of the response in rat fibres) caused by an effect of the agent of the Ca2+ sensitivity of the contractile apparatus. 3. Application of the reactive disulphide, 2,2'‐dithiodipyridine (DTDP, 100 microM), a potent oxidizing agent, never induced any measurable force response or noticeable depletion of SR Ca2+ in any fibre under the conditions used. When all Ca2+ uptake was prevented, DTDP treatment of rat fibres was found to cause a 2‐ to 3‐fold increase in the low rate of Ca2+ "leak' from the SR. DTDP treatment also increased the responsiveness of toad muscle fibres to 1 or 2 mM caffeine. These effects could be largely reversed by treatment with DTT. These results indicate that oxidation of the Ca2+ release channel does not cause substantial channel opening under physiological conditions. 4. Depolarization‐induced force responses in both rat and toad fibres were rapidly abolished in the presence of DTDP (10 or 100 microM), in a manner favoured by inactivation of the voltage sensors. The relatively impermeant oxidant, 5,5'‐dithionitrobenzoic acid (DTNB, 100 microM), had an effect very similar to DTDP if applied intracellularly, but unlike DTDP, had little or no effect if applied extracellularly (at 5 mM) before skinning. Depolarization‐induced responses could be restored by treatment with DTT (10 mM). Intracellular application of the sulfhydryl‐alkylating agent, N‐ethylmaleimide (NEM, 100 microM), had effects very similar to DTDP and DTNB. 5. These results are not consistent with the proposal that excitation‐contraction coupling in skeletal muscle primarily involves the oxidative linkage of the voltage sensors to the Ca2+ release channels, but do show that oxidation of an intracellularly accessible site can interfere with the coupling, in a process made more sensitive by voltage sensor inactivation.
Intracellular inositol 1,3,4,5‐tetrakisphosphate enhances the calcium current in hippocampal CA1 neurones of the gerbil after ischaemia.
Authors: Tsubokawa H , Oguro K , Robinson H P , Masuzawa T , Kawai N
Published: December 2014

1. To examine the role of the phosphoinositide cascade triggered by disturbed Ca2+ homeostasis in ischaemic neurones, inositol 1,3,4,5‐tetrakisphosphate (InsP4) was applied to the cytoplasmic face of membrane patches isolated from CA1 pyramidal neurones in the gerbil hippocampus. 2. In outside‐out recordings, InsP4 induced an inward current which was increased by raising the extracellular [Ca2+]. In contrast, no clear channel openings could be observed in patches from neurones of sham‐operated gerbils. 3. Open probabilities of InsP4‐activated channels were significantly decreased upon application of omega‐conotoxin but were not affected by omega‐agatoxin or nifedipine. 4. In inside‐out patches using high concentrations of Ca2+, Ba2+ or Sr2+ in the pipette solution, InsP4 enhanced inward currents. 5. Application of the isomers of InsP4 slightly enhanced the currents, but inositol 1,4,5‐trisphosphate (InsP3) had no effect. 6. In the absence of InsP4 there was a single main Ba2+ current peak of 4.0 pA in amplitude, whereas upon its application two main peaks of 3.0 and 7.2 pA were present. 7. The open probabilities of these channels were apparently increased by InsP4. 8. These findings support the view that a disturbed phosphoinositide cascade occurs in the hippocampal pyramidal neurones after ischaemia and the InsP4 thus formed plays an important role in promoting the Ca2+ accumulation which results in neuronal death.
Endogenously produced prostanoids stimulate calcium reabsorption in the rabbit cortical collecting system.
Authors: van Baal J , de Jong M D , Zijlstra F J , Willems P H , Bindels R J
Published: December 2014

1. The influence of endogenously produced prostanoids on active transepithelial Ca2+ transport and cAMP formation was investigated in immunodissected rabbit kidney connecting and cortical collecting tubule cells grown to confluency on permeable supports. 2. The cyclo‐oxygenase inhibitor indomethacin dose‐dependently (IC50 = 18 nM) reduced the net apical‐to‐basolateral Ca2+ transport by 57%. Inhibition was reversed in medium obtained from monolayers incubated in the absence of indomethacin. 3. HPLC analysis following incubation with 14C‐labelled arachidonic acid revealed the presence of a wide variety of radiolabelled prostanoids in both the apical and basolateral media. These findings are compatible with the endogenous production and subsequent release of stimulatory prostanoids. 4. The inhibitory action of indomethacin was reversed by the addition of the prostanoids PGE1, PGE2 and PGA2, but not PGD2, PGF2 alpha, the stable PGI2 analogue cicaprost or the thromboxane A2 mimetic U‐46619. PGE2 stimulated transepithelial Ca2+ transport dose dependently (EC50 = 3 nM), irrespective of the compartment of which it was added. The stimulatory effect of PGE2 was paralleled by increased cAMP formation, suggesting the apical and basolateral presence of stimulatory prostanoid receptors EP2 and/or EP4. 5. Sulprostone, an analogue selective for EP1 and EP3 receptors, inhibited transepithelial Ca2+ transport in indomethacin‐treated monolayers only when applied basolaterally, suggesting the exclusive presence of inhibitory EP receptors on the basolateral membrane. 6. The percentage by which parathyroid hormone and arginine vasopressin increased both transepithelial Ca2+ transport and cAMP formation was dramatically increased in indomethacin‐inhibited cells as compared with control cells, demonstrating that indomethacin unmasks the actions of these hormones to their full extent.
Histamine induces oscillations of mitochondrial free Ca2+ concentration in single cultured rat brain astrocytes.
Authors: Jou M J , Peng T I , Sheu S S
Published: December 2014

1. The free Ca2+ concentration of mitochondria ([Ca2+]m) in cultured rat brain astrocytes was measured with a fluorescent Ca2+ indicator, rhod‐2, and laser confocal microscopy. 2. Confocal images revealed a rhod‐2 distribution that matched mitochondrial localization. 3. Using a Ca2+ ionophore, ionomycin, to clamp the [Ca2+]m from 0 to 100 microM in order to obtain the minimal and maximal fluorescence of rhod‐2 in situ, a 3.5 +/‐ 0.4‐fold increase in fluorescence intensity was observed, suggesting that the fluorescence of intramitochondrial rhod‐2 was responding in a Ca(2+)‐sensitive manner, thereby allowing measurements of [Ca2+]m in single astrocytes. 4. Exposure of fura‐2‐loaded astrocytes to 100 microM histamine produced a rapid and transient increase in cytosolic Ca2+ concentration ([Ca2+]c) that lasted for several tens of seconds. The spike in [Ca2+]c was frequently followed by variable numbers of repetitive oscillations of Ca2+, which appeared to dampen in amplitude with time. 5. This pattern of histamine‐induced [Ca2+]c oscillations was also observed in rhod‐2‐loaded cells suggesting that [Ca2+]m fluctuated with a similar frequency. 6. The oscillations of [Ca2+]m, but not of [Ca2+]c, were abolished by a proton ionophore, carbonyl cyanide m‐chlorophenyl‐hydrazone (CCCP), and by Ruthenium Red, a mitochondrial Ca(2+)‐uniporter inhibitor. 7. These results suggest that the mitochondrial Ca2+ transport systems in cultured rat brain astrocytes are able to relay receptor‐mediated [Ca2+]m oscillations into mitochondria.
Mitochondria accumulate Ca2+ following intense glutamate stimulation of cultured rat forebrain neurones.
Authors: White R J , Reynolds I J
Published: December 2014

1. In cultures of rat forebrain neurones, mitochondria buffer glutamate‐induced, NMDA receptor‐mediated Ca2+ influx. Here, we have used the fluorescent calcium indicator, indo‐1 AM to record [Ca2+]i from single cells. We varied either the glutamate concentration or the duration of exposure to investigate the cellular mechanisms recruited to buffer [Ca2+]i within different stimulation protocols. 2. For a 15 s stimulus, the recovery time doubled as the glutamate concentration was raised from 3 to 300 microM. Changing the duration of exposure from 15 s to 5 min increased the recovery time tenfold even when the glutamate concentration was held at 3 microM. 3. We used a selective inhibitor of the mitochondrial Na(+)‐Ca2+ exchange, CGP‐37157. When applied immediately after a 15 s, 100 microM glutamate challenge, CGP‐37157 consistently caused a rapid fall in [Ca2+]i followed by a slow rise after the drug was washed out. A similar pattern was seen with the 5 min, 3 microM glutamate stimulus. The effects of CGP‐37157 are consistent with the release of substantial mitochondrial Ca2+ stores during recovery from an intense glutamate stimulus. 4. These studies suggest that mitochondria become progressively more important for buffering glutamate‐induced Ca2+ loads as the stimulus intensity increases. The recovery of [Ca2+]i to baseline following glutamate removal is critically regulated by the release of Ca2+ from mitochondrial stores via mitochondrial Na(+)‐Ca2+ exchange. The data highlight a previously under‐appreciated role for [Na+]i in the regulation of [Ca2+]i in central neurones.
Intracellular pH and calcium in frog early distal tubule: effects of transport inhibitors.
Authors: Cooper G J , Hunter M
Published: December 2014

1. The K+ channels of the apical membrane of the diluting segment (early distal tubule, EDT) of the frog are involved in the regulation of transepithelial NaCl transport. These channels are sensitive to pHi and intracellular Ca2+ (Ca2+i). Inhibition of transport by furosemide (frusemide) results in a compensatory increase in K+ channel activity. The aims of the present study were to determine whether pHi or Ca2+i were altered by furosemide, and to identify the means by which such changes were brought about. 2. Experiments were performed using single, microperfused EDT segments. Measurements of pHi and Ca2+i were made using the intracellular fluorescent probes, 2',7'‐bis(carboxyethyl)‐5,6‐carboxyfluorescein (BCECF) and fura‐2, respectively. 3. Furosemide increased pHi and Ca2+i. The intracellular alkalinization was the result of an alkaline shift in the set‐point of the basolateral Na(+)‐H+ exchanger. This response was dependent upon the increase in Ca2+i. 4. The increase in Ca2+i produced by furosemide was due to the release of Ca2+ from intracellular stores. Depletion of these stores, by 2,5‐di‐t‐butylhydroquinone (TBQ) and caffeine, prevented the furosemide‐induced changes in Ca2+ and pH. 5. Furosemide‐induced activation of Na(+)‐H+ exchange was prevented by the calmodulin antagonist, W‐7. 6. Thus furosemide elicits a rise in Ca2+i which, via calmodulin, results in activation of Na(+)‐H+ exchange. The resulting intracellular alkalinization would be expected to increase channel activity.
Developmental loss of hypoxic chemosensitivity in rat adrenomedullary chromaffin cells.
Authors: Thompson R J , Jackson A , Nurse C A
Published: December 2014

1. We investigated whether adrenomedullary chromaffin cells (AMCs) derived from neonatal (postnatal day (P) 1‐P2) and juvenile (P13‐P20) rats, and maintained in short‐term culture (1‐3 days), express O2‐chemoreceptive properties. 2. In whole‐cell recordings, the majority (approximately 70%; n = 47) of neonatal AMCs were sensitive to hypoxia. Under voltage clamp, acute hypoxia (PO2 approximately 40 mmHg) suppressed voltage‐dependent K+ current by 25.1 +/‐ 3.4% (mean +/‐ S.E.M.; n = 22); under current clamp, acute hypoxia caused a membrane depolarization of 14.1 +/‐ 1.3 mV (n = 13) from a resting potential of ‐54.8 +/‐ 2.8 mV (n = 13), and this was often sufficient to trigger action potentials. 3. Exposure of neonatal AMC cultures to a moderate (PO2 approximately 75 mmHg) or severe (PO2 approximately 35 mmHg) hypoxia for 1 h caused a dose‐dependent stimulation (approximately 3 or 6 times normoxia, respectively) of catecholamine (CA) release, mainly adrenaline, determined by HPLC. This induced CA release was abolished by the L‐type calcium channel blocker, nifedipine (10 microM). 4. In contrast to the above results in neonates, hypoxia had no significant effects on voltage‐dependent K+ current, membrane potential, or CA release in juvenile AMCs. 5. We conclude that rat adrenal chromaffin cells possess a developmentally regulated O2‐sensing mechanism, similar to carotid body type I cells.
The newborn rabbit sino‐atrial node expresses a neuronal type I‐like Na+ channel.
Authors: Baruscotti M , Westenbroek R , Catterall W A , DiFrancesco D , Robinson R B
Published: December 2014

1. Newborn rabbit sino‐atrial node (SAN) myocytes were recently found to express a tetrodotoxin (TTX)‐sensitive Na+ current. We now report that the dose‐response relation indicates that this SAN Na+ channel has unusually high TTX sensitivity, with half‐maximal inhibition (26 +/‐ 5 nM) which is more typical of neuronal than cardiac tissue. 2. Additional characterization used mu‐conotoxin GIIIA and Cd2+ as relatively selective blockers of the skeletal and cardiac isoforms, respectively. mu‐Conotoxin GIIIA had no effect on the current recorded from SAN myocytes, but the Cd2+ sensitivity was unexpectedly high for a neuronal isoform (half‐maximal inhibition = 185 +/‐ 8 microM). 3. Analysis of the time constant of inactivation did not reveal evidence of multiple inactivation processes, with the data well fitted by a single, relatively rapid exponential (inactivation time constant = 0.58 +/‐ 0.03 ms at 0 mV). 4. In situ hybridization with anti‐sense cDNA probes was used to test for expression of neuronal type I, II and III Na+ channel isoforms. Myocardial cells in newborn SAN tissue exhibited clear hybridization to the type I, but not the type II or III probes. No hybridization was observed in adult SAN tissue with any of the three probes. 5. It is concluded that the newborn SAN expresses a neuronal type I‐like Na+ channel isoform, and that this probably accounts for the unusual characteristic of high sensitivity to both TTX and Cd2+.
Electrophysiological properties of neurones in the internal and external submucous plexuses of newborn pig small intestine.
Authors: Thomsen L , Pearson G T , Larsen E H , Skadhauge E
Published: December 2014

1. Intracellular microelectrodes were used to identify three major electrophysiological categories of neurone in both the internal and external submucous plexuses of the porcine small intestine. 2. Two classes of neurone with a long‐lasting after‐hyperpolarization following their action potential were differentiated by the presence or absence of fast excitatory synaptic inputs (EPSPs) and were termed AH neurones. S neurones received fast EPSPs but did not display after‐hyperpolarizations. 3. The mean resting membrane potentials of the three groups of neurones showed a similar trend in both plexuses, with significantly higher values for the two populations of AH neurone than for S neurones. No significant variation of input resistance with cell type was detected. Neuronal input resistance was significantly greater in the internal submucous plexus than in the external submucous plexus. 4. Over 80% of AH neurones in the internal submucous plexus displayed fast EPSPs but a similar percentage of AH neurones in the external submucous plexus did not show fast EPSPs. S neurones constituted 60% of cells studied in the internal submucous plexus but less than 30% of the cell population in the external submucous plexus. 5. This study of porcine submucous neurones has revealed both similarities and differences to previous work in the guinea‐pig small intestine. The most contrasting features are the relative abundance and subclassification of AH neurones in the pig in addition to the apparent paucity of slow synaptic potentials. The differences in the neuronal profiles of the internal and external submucous plexuses may reflect a differentiation of function between the two enteric nerve networks.
Diabetes and insulin‐induced stimulation of L‐arginine transport and nitric oxide synthesis in rabbit isolated gastric glands.
Authors: Contreras R , Fuentes O , Mann G E , Sobrevia L
Published: December 2014

1. The properties of L‐arginine transport have been characterized and correlated with cGMP production (index of nitric oxide (NO)) in whole gastric glands isolated from non‐diabetic and alloxan‐diabetic rabbits. 2. In non‐diabetic and diabetic glands, transport of L‐arginine was stereoselective, Na+ and pH independent and inhibited by other cationic amino acids. L‐Arginine transport was slightly inhibited by L‐leucine and L‐phenylalanine, but unaffected by other neutral amino acids. 3. Diabetes enhanced the Vmax for saturable L‐arginine transport from 10.7 +/‐ 1.0 to 17.7 +/‐ 0.5 pmol (mg protein)‐1 s‐1, with negligible changes in K(m). 4. Accumulation of the membrane potential‐sensitive probe tetra[3H]phenylphosphonium (TPP+) was increased 2‐fold in diabetic compared with non‐diabetic gastric glands, suggesting a membrane hyperpolarization. 5. Basal intracellular cGMP levels were elevated 2‐fold in diabetic gastric glands, and in non‐diabetic glands histamine, vasoactive intestinal peptide, and bradykinin increased cGMP levels. The NO synthase inhibitor NG‐nitro‐L‐arginine methyl ester (100 microM) abolished basal cGMP accumulation. 6. Addition of extracellular L‐arginine induced a concentration‐dependent increase in cGMP levels in gastric glands isolated from non‐diabetic rabbits, but had no effect on elevated cGMP levels in diabetic glands. 7. Insulin induced a rapid (5 min) concentration‐dependent increase in cGMP levels in non‐diabetic gastric glands, but reduced elevated cGMP levels in diabetic gastric glands. 8. The present study has identified a specific transport system for L‐arginine in gastric glands which resembles the classical system y+. Our findings also provide the first direct evidence that diabetes increases the basal activity of system y+ and NO synthase in gastric glands. The differential modulation of L‐arginine transport by insulin and L‐arginine identified in non‐diabetic and diabetic glands, may be of importance in protecting the gastric mucosa from injuries associated with diabetes.
alpha 1‐adrenoceptor‐mediated negative inotropy of adrenaline in rat myocardium.
Authors: Kissling G , Blickle B , Ross C , Pascht U , Gulbins E
Published: December 2014

1. The effect of alpha‐ and beta‐adrenoceptor stimulation on isotonic contraction was investigated on right ventricular papillary muscles of the rat, stimulated at a rate of 0.5 Hz. 2. Adrenaline (0.5 microM) induced a slight but significant negative inotropic effect: shortening decreased from 0.137 +/‐ 0.058 to 0.122 +/‐ 0.059 muscle lengths (mean +/‐ S.D.; ‐11%, P < 0.0001) and maximum shortening velocity from 2.9 +/‐ 1.2 to 2.7 +/‐ 1.3 muscle lengths s‐1 (‐7%, P < 0.025). 3. The negative inotropic effect of adrenaline was enhanced after blocking the beta‐adrenoceptors with 50 microM atenolol. On the other hand, exposure to adrenaline after blocking the alpha‐adrenoceptors with 50 microM phentolamine resulted in an increase in shortening as well as in maximum shortening velocity. 4. Stimulation of the beta‐adrenoceptors with 0.5 microM isoprenaline caused marked positive inotropic effects, whereas stimulation of the alpha 1‐adrenoceptors with 0.5 microM phenylephrine regularly resulted in a long‐lasting decrease in shortening and maximum shortening velocity. 5. 1,2‐Dioctanoyl‐sn‐glycerol (1,2‐DOG) and adrenaline induced an activation of protein kinase C (PKC) with translocation of this enzyme from the cytosol to the sarcolemma. 6. Activation of PKC with 10 microM 1,2‐DOG and 0.5 microM adrenaline was accompanied by a decrease in shortening and maximum shortening velocity. Inhibition of PKC with 0.1 microM staurosporine abolished the negative inotropic effect of adrenaline. 7. From these results we conclude that a low dose of adrenaline stimulates not only beta‐but also alpha‐adrenoceptors and that the observed negative inotropic effect of adrenaline is mediated by alpha 1‐adrenoceptors, linked to the diacylglycerol‐PKC signal transduction pathway.
Three different Cl‐ channels in the bovine ciliary epithelium activated by hypotonic stress.
Authors: Zhang J J , Jacob T J
Published: December 2014

1. Hypotonic solution induced transient Cl‐ channel activity in both pigmented and non‐pigmented ciliary epithelial cells in cell‐attached patches. 2. The activation time constants for these currents were 63 and 97 s for non‐pigmented and pigmented ciliary epithelial (NPCE and PCE) cells, respectively. The currents inactivated during the exposure to hypotonic solution with time constants of 59 and 304 s for NPCE and PCE cells, respectively. 3. Single channel analysis revealed the presence of a low‐conductance Cl‐ channel in both the NPCE (7.3 +/‐ 0.4 pS) and PCE (8.6 +/‐ 0.9 pS) cells. In addition, an intermediate‐conductance (18.8 pS) channel was found alone in the NPCE cells and a maxi Cl‐ channel (105 pS) was found in the PCE cells. The similarities between the 18.8 pS Cl‐ channel and the volume‐activated organic osmolyte‐anion channel (VSOAC) are discussed. 4. The hypotonic activation of all three Cl‐ channels was prevented by the inclusion of 100 microM 5‐nitro‐2‐(3‐phenylpropylamino)benzoic acid (NPPB) in the patch pipette. 5. Inclusion of tamoxifen in the patch pipette inhibited only the intermediate‐conductance channel in the NPCE cells. This is consistent with the identification of the intermediate 18 pS Cl‐ channel, found only in NPCE cells, as VSOAC. 6. Negative pressure also evoked single Cl‐ channel activity but the activation kinetics were quite different, suggesting that, even if the channels are the same, the second messenger pathways involved are different. 7. The activation/inactivation kinetics of these Cl‐ channels correlate well with the time course of regulatory volume decrease (RVD) in these cells, suggesting that these channels may well participate in the process of RVD. 8. The asymmetrical distribution of Cl‐ channels in the ciliary epithelium may be significant for aqueous humour secretion. For instance, the presence of the VSOAC‐like channel in the NPCE cells may allow one‐way traffic of anions and organic osmolytes into the eye and, if these solutes were loaded by the PCE cells, then a vectorial flow from the blood into the eye would occur.
Properties of ionic currents from isolated adult rat carotid body chemoreceptor cells: effect of hypoxia.
Authors: López-López J R , González C , Pérez-García M T
Published: December 2014

1. The electrical properties of chemoreceptor cells from neonatal rat and adult rabbit carotid bodies (CBs) are strikingly different. These differences have been suggested to be developmental and/or species related. To distinguish between the two possibilities, the whole‐cell configuration of the patch‐clamp technique was used to characterize the ionic currents present in isolated chemoreceptor cells from adult rat CBs. Since hypoxia‐induced inhibition of O2‐sensitive K+ currents is considered a crucial step in O2 chemoreception, the effect of hypoxia on the adult rat chemoreceptor cell currents was also studied. 2. Outward currents were carried mainly by K+, and two different components could be distinguished: a Ca(2+)‐dependent K+ current (IK(Ca)) sensitive to Cd2+ and charybdotoxin (CTX), and a Ca(2+)‐insensitive, voltage‐dependent K+ current (IK(V)). IK(V) showed a slow voltage‐dependent activation (time constant (tau) of 87.4 ms at ‐20 mV and 8.8 ms at +60 mV) and a very slow inactivation, described by the sum of two exponentials (tau 1 = 684 +/‐ 150 ms and tau 2 = 4.96 +/‐ 0.76 s at + 30 mV), that was almost voltage insensitive. The kinetic and pharmacological properties of IK(V) are typical of a delayed rectifier K+ channel. 3. Voltage‐dependent Ca2+ currents (ICa) were present in nineteen of twenty‐seven cells. TTX‐sensitive Na+ currents were also observed in about 10% of the cells. 4. Low PO2 (< 10 mmHg) reduced the whole outward current amplitude by 22.17 +/‐ 1.96% (n = 27) at +20 mV. This effect was absent in the presence of Cd2+. Since low PO2 did not affect ICa, we conclude that hypoxia selectively blocks IK(Ca). 5. The properties of the currents recorded in adult rat chemoreceptor cells, including the specific inhibition of IK(Ca) by hypoxia, are similar to those reported in neonatal rat CB cells, implying that the differences between rat and rabbit chemoreceptor cells are species related.
Intracellular alkalinization mobilizes calcium from agonist‐sensitive pools in rat lacrimal acinar cells.
Authors: Yodozawa S , Speake T , Elliott A
Published: December 2014

1. We have investigated interactions between intracellular pH (pHi) and the intracellular free calcium concentration ([Ca2+]i) in collagenase‐isolated rat lacrimal acinar cells. The fluorescent dyes fura‐2 and 2',7'‐bis(carboxyethyl)‐5‐carboxyfluorescein (BCECF) were used to measure [Ca2+]i and pHi, respectively. 2. Application of the weak base NH4Cl alkalinized the cytosol and caused a dose‐dependent increase in [Ca2+]i. Trimethylamine (TMA) also alkalinized the cytosol and increased [Ca2+]i. The increase in [Ca2+]i evoked by NH4Cl or TMA was much smaller than that evoked by the secretory agonist acetylcholine (ACh). 3. Application of NH4Cl also increased [Ca2+]i in cells bathed in Ca(2+)‐free medium, indicating that NH4Cl released Ca2+ from an intracellular pool. 4. Ammonium chloride had no effect on [Ca2+]i in cells bathed in Ca(2+)‐free medium if agonist‐sensitive intracellular Ca2+ pools had been depleted with either ACh or the microsomal Ca(2+)‐ATPase inhibitor 2,5‐di(tert‐butyl)hydroquinone. Treatment of cells with NH4Cl in Ca(2+)‐free medium reduced the amount of Ca2+ released by ACh. These results suggest that NH4Cl released Ca2+ from the same intracellular pool released by ACh. 5. Calcium release from the agonist‐sensitive pool was also triggered when the cytosol was alkalinized by removing the weak acid acetate. 6. Ammonium chloride caused a modest increase in inositol phosphate production, suggesting that NH4Cl may have released stored Ca2+ via an increase in the intracellular inositol 1,4,5‐trisphosphate concentration. 7. The increase in [Ca2+]i evoked by NH4Cl was not sustained even in the presence of extracellular Ca2+. In contrast, when a low dose of ACh was used to evoke intracellular Ca2+ release of similar magnitude, sustained Ca2+ entry was observed. 8. Alkalinizing the cytosol appeared to partially inhibit Ca2+ entry triggered by thapsigargin or by ACh. 9. We suggest that alkalinizing the cytoplasm in unstimulated lacrimal acinar cells can release Ca2+ from the intracellular agonist‐sensitive Ca2+ pool. However, releasing stored Ca2+ via alkalinization does not appear to trigger significant Ca2+ entry, perhaps because intracellular alkalinization inhibits either the Ca2+ entry pathway or the mechanism which couples the entry pathway to store depletion.
Functional evidence for calcium‐induced calcium release in isolated rat vibrissal Merkel cell mechanoreceptors.
Authors: Senok S S , Baumann K I
Published: December 2014

1. Single unit recordings were made from Merkel cell (sinus hair type I; St I) and sinus hair type II (St II) mechanoreceptors in isolated rat vibrissae. Responses were determined as the number of spikes evoked by controlled mechanical displacement of the hair shaft for 5 s every 30 s. 2. Superfusion of caffeine (10 mM) increased the responses of Merkel cell receptors by 50‐180% of control (mean +/‐ S.E.M., 64 +/‐ 12.6%, n = 6, P < 0.001). Similar concentrations of caffeine inhibited St II receptor responses by 20‐60% (mean +/‐ S.E.M., 35 +/‐ 8%, n = 5, P < 0.01). In both receptor types, caffeine induced a low‐frequency increase in spontaneous firing. 3. When Merkel cell receptor responses were completely blocked by superfusion of high Mg2+‐containing solution (to competitively block Ca2+ influx) caffeine had no effect when added after complete inhibition, but when added during partial inhibition of responses, the Mg2+‐induced inhibition was transiently reversed or halted. This suggests that Ca2+ influx was a prerequisite for the action of caffeine. 4. Ryanodine (1 microM) increased the responses of Merkel cell receptors to mechanical stimulation by 7‐60% (mean +/‐ S.E.M., 32 +/‐ 10.9 %, n = 5, P < 0.05) but had no effect on St II receptor responses. 5. The Ca2+‐induced Ca2+ release (CICR) inhibitor procaine inhibited St I receptor responses in a concentration‐dependent manner. Near‐maximal inhibition was attained with 100 microM procaine. In four St I units, mean responses were depressed to 25% of control values. When both procaine (100 microM) and caffeine (10 mM) were introduced together, no net effect was seen. The responses of St II receptors were little affected by up to 100 microM procaine superfusion. 6. It is concluded that the mechano‐electrical transduction process in St I receptors (but not St II) includes a CICR pathway. Taken with previous findings on the role of Merkel cells, it is likely that CICR is occurring in the Merkel cells.
G‐protein‐mediated desensitization of metabotropic glutamatergic and muscarinic responses in CA3 cells in rat hippocampus.
Authors: Guérineau N C , Bossu J L , Gähwiler B H , Gerber U
Published: December 2014

1. Desensitization of a metabotropic response was investigated in CA3 pyramidal neurons in hippocampal slice cultures using the patch‐clamp technique. 2. 1S,3R‐1‐aminocyclopentane‐1,3‐dicarboxylate (1S,3R‐ACPD), an agonist at metabotropic glutamate receptors (mGluRs), and metacholine (MCh), an agonist at muscarinic receptors, induced a cationic current that appears to be activated through a G‐protein‐independent transduction process, as previously shown. Prolonged or repetitive bath application of agonists led to rapid desensitization of the cationic current with a time constant of approximately 20 s. 3. Complete recovery from desensitization was observed within 6 min. 4. These responses mediated by mGluRs and muscarinic receptors cross‐desensitized. 5. Preventing the activation of G‐proteins by loading cells with GDP beta S strongly reduced or suppressed desensitization, and resulted in a sustained inward cationic current. When cells were filled with GTP gamma S to irreversibly activate G‐proteins, the desensitization process was enhanced such that a first application of agonist caused a markedly reduced response. 6. These results show that a cationic current induced by metabotropic agonists in hippocampal pyramidal cells undergoes apparent desensitization and suggests that this process occurs through a G‐protein‐mediated inhibition of the underlying membrane conductance.
Intracellular ATP activates inwardly rectifying K+ channels in human and monkey retinal Müller (glial) cells.
Authors: Kusaka S , Puro D G
Published: December 2014

1. In the vertebrate retina, the inwardly rectifying K+ (KIR) channels of the Müller (glial) cells are pathways for the redistribution of excess extracellular K+. Due to this role in K+ homeostasis, the activity of Müller cell KIR channels is likely to have significant functional consequences for the retina. In this study we asked whether intracellular ATP regulates the function of KIR channels expressed by Müller cells, the principal glia of the retina. 2. Freshly dissociated Müller cells from the human and monkey (Macaca fascicularis) retina were studied with various configurations of the patch‐clamp technique. 3. Whole‐cell recordings from Müller cells revealed that a run‐down of the inwardly rectifying K+ current (IK(IR)) was prevented if the pipette solution contained Mg‐ATP. Chemical ischaemia induced by inhibitors of glycolysis and oxidative phosphorylation caused a nearly 10‐fold reduction in the IK(IR)) that was fully restored when metabolically inhibited Müller cells were internally perfused with ATP. 4. In recordings from membrane patches of fresh primate Müller cells, we found that inward‐rectifying channels with a conductance of 20 pS in 100 mM Ko+ were the predominant type of KIR channel. In excised patches these 20 pS KIR channels were activated when Mg‐ATP was at the cytoplasmic surface. Experiments with inside‐out patches indicated that the activity of the 20 pS KIR channels can be maintained by ATP synthesized at sites located close to the channel. 5. The inability of the non‐hydrolysable ATP analogue 5'‐adenylylimidodiphosphate (AMP‐PNP) to prevent the run‐down of IK(IR))and the Mg2+ dependence of the ATP effect on KIR channels are consistent with a mechanism of activation requiring the hydrolysis of ATP. 6. These observations suggest that the metabolic state of a Müller cell regulates the activity of its 20 pS KIR channels and thus influences the function of the glial cell in maintaining K+ homeostasis in the retina.
Measurement and Analysis of Laminar Heat Transfer Coefficients in Micro and Mini-Scale Ducts and Channels
Authors: Muzychka Y. S. , Ghobadi M.
Published: December 2014

Heat transfer in micro and mini-scale ducts and channels is considered. In particular, issues of thermal performance are considered in systems with constant wall temperature at low to moderate Reynolds numbers or small dimensional scales which lead to conditions characteristic of thermally fully developed flows or within the transition region leading to thermally fully developed flows. An analysis of two approaches to representing experimental data is given. One using the traditional Nusselt number and another using the dimensionless mean wall flux. Both approaches offer a number of advantages and disadvantages. In particular, it is shown that while good data can be obtained which agree with predicted heat transfer rates, the same data can be problematic if one desires a Nusselt number. Other issues such as boundary conditions pertaining to measuring thermally developing and fully developed flow Nusselt numbers are also discussed in detail.
Multiscale Simulation of Nano-Fluidic Networks
Authors: Stephenson Dave , Lockerby Duncan A. , Borg Matthew K. , Reese Jason M.
Published: December 2014

We present a hybrid molecular-continuum method for the simulation of general nano-fluidic networks, which consist of a multiscale system of channels with high aspect ratios. This develops on the hybrid molecular-continuum internal multiscale method recently devised by Borg et al. [1, 2] with three main additions: a) method generalisation to accurately model any nano-fluidic network, rather than just serial channel systems; b) density correction, enabling the modelling of compressible fluids; and c) utilisation as a design tool, rather than just a simulation tool, by replacing pressure boundary conditions for the network with mass flow rate boundary conditions (inlet/outlet pressures are the output of the simulation).

We compare our multiscale method with a full molecular dynamics (MD) simulation for a bifurcating channel network, and show that it converges quickly, within 3 iterations, and with good agreement. The multiscale method produces errors of < 2%, while providing a computational speed up of 2.1. The speed up demonstrated is far more modest than it would be for larger networks, but our validation case is restricted by the need to perform a full MD simulation.
Modeling Drug Delivery in Gravity-Driven Microfluidic System
Authors: Mäki Antti-Juhana , Kreutzer Joose , Kallio Pasi
Published: December 2014

Human cells cultivated in a microfluidic system provide an interesting alternative for animal experiments in drug screening. For these tests, a pumpless system based on hydrostatic pressure could be used for drug delivery. The objective of this paper is to provide a method to analyze drug distribution in a gravity-driven microfluidic system to reduce the design cycle of these systems. The approach is based on an analytical model combined with a finite element method (FEM). The paper presents simulation of gravity-driven drug delivery in a polydimethylsiloxane (PDMS)-based microfluidic cell culture system. In the study, a simple but commonly used system including two reservoirs, inlet and outlet, connected through a microchannel, is modeled. In the proposed method, time-dependent working pressure based on hydrostatic and capillary pressures is first approximated analytically. Secondly, using the calculated pressure, a velocity profile of single-phase fluid flow is solved across the system using the FEM. Finally, a distribution of a selected drug compound over the system is simulated and analyzed. Based on the results, the initial geometry is improved for better performance. The paper demonstrates how the modified system provides faster and more uniform drug concentration profile on the cells compared to the initial structure.
Heat Transport Through a Solid-Liquid Interface System and its Thermal Rectification Phenomena by Molecular Dynamics Simulations
Authors: Yuan Feng , Xin-Gang Liang
Published: December 2014

Solid-liquid interfaces widely exist in microdevices, it is necessary to study the heat transfer mechanism through solid-liquid interfaces. We compose a sandwich structure of solid-liquid system. The liquid is argon and the solid has FCC structure with different atomic mass. Heat transfer and rectification are investigated by molecular dynamics method. The effect of wettability of liquid argon is found to be essential to heat transport at solid-liquid interfaces. Asymmetric mass distribution affects the heat flux through the system. Thermal rectification is produced when the atomic mass of virtual solid walls differs. The asymmetry of interfacial thermal resistances between liquid and solid is observed, as well as the thermal rectification when the depth of potential well of virtual solid walls change. The larger the asymmetry is, the more obvious the thermal rectification is.
Finite Element Modeling of Micro-Particle Separation Using Ultrasonic Standing Waves
Authors: Büyükkoçak Süleyman , Çetin Barbaros , Özer Mehmet Bülent
Published: December 2014

Acoustophoresis which means separation of particles and cells using acoustic waves is becoming an intensive research subject. The method is based on inducing an ultrasonic compression standing wave inside a microchannel. A finite element approach is used to model the acoustic and electro-mechanical behavior of the piezoelectric material, the micro-channel geometry as well as the fluid inside the channel. The choices of silicon and PDMS materials are investigated as the chip materials for the resonator.

A separation channel geometry which is commonly used in the literature is implemented in this study and the fluid flow inside the microchannel geometry is simulated using computational fluid dynamics. The acoustic field inside the fluid channel is also be simulated using the finite element method. For the separation process to be successful micro-particles of different diameter groups should end up in different channels of the micro-separator. In order to simulate real life scenarios, each particle size group have a size distribution within themselves. For realistic simulation results the particles will be released into the micro separator from a different starting locations (starting location distribution). The results of this Monte-Carlo based finite element simulation approach will be compared with the reported experimental results.
Investigation of the Hydrodynamics of Suspended Cells for Reliable Inkjet Cell Printing
Authors: Cheng Eric , Ahmadi Ali , Cheung Karen C.
Published: December 2014

Reliable inkjet drop-on-demand dispensing of cells has numerous applications including cell assays and tissue engineering. Previous work on inkjet cell printing has demonstrated that the cell count per droplet is inhomogeneous and does not follow the expected Poisson distribution. In the present work, the flow-induced cell behaviour is characterised to better understand the hydrodynamic mechanisms behind unreliable cell printing. A glass piezoelectric inkjet nozzle with an 80 μm diameter orifice is mounted on a PDMS cast which acts as a refractive index matching material for cell tracking through an inverted microscope. Droplet formation is achieved by a bipolar waveform. A high-speed camera focused on the centre plane of the nozzle captures images which are then analysed by a cell tracking algorithm to obtain the horizontal and vertical position of the cells over time. High-speed tracking of cells within a transparent inkjet nozzle revealed three possible cell behaviours caused by the formation and break-off of droplets. These behaviours are cell travel, cell ejection and cell reflection, determined as a function of the position of the cell at the onset of droplet formation. The first behaviour, cell travel, is characterised as the displacement of the cell towards the orifice during droplet formation followed by a small backwards motion due to the retracting meniscus after droplet pinch-off. Cell travel results in a net forward displacement of the cell towards the nozzle orifice. The second observed cell behaviour is cell ejection, where a cell is ejected with a droplet and can no longer be observed within the nozzle after the droplet break-off. The third observed cell behaviour is cell reflection. In this case, hydrodynamic forces produced during droplet ejection acts on the cell to move it further away from the nozzle orifice resulting in a net displacement of the cell away from the orifice after droplet ejection. Through the cell tracking information, it is hypothesized that cell reflection is caused by fluid flow reversal during the droplet ejection process.

As a result of cell reflection, certain cells within a region close to the orifice will not be printed; instead they are pushed to a location further away from the orifice. Therefore, mapping of cell positions before droplet formation is performed to identify regions within the nozzle that exhibit a high probability of cell ejection and reflection. Overall, the results from this study will greatly contribute to our understanding of the cell printing process, which will allow us to optimize current inkjet systems for cell printing applications.
Flow of Taylor Bubble in Microchannel Having an Obstacle
Authors: Thippavathini Sudhakar , Moharana Manoj Kumar
Published: December 2014

A novel concept of mixing based on 2-D numerical study is proposed where Taylor bubble flows past an obstacle inside a horizontal microchannel. A square shaped obstacle of size 0.02 × 0.02 mm2 is considered inside a microchannel of diameter 0.2 mm. Water and air enters at the two inlet ends of a T-junction and creates Taylor bubble flow at the junction. The obstacle is placed in the downstream at a sufficient distance from the junction where air and water meet. This ensures stability of the Taylor bubble by the time it touches the obstacle. The position of the obstacle is varied along the perpendicular to the flow direction. First, the obstacle is placed exactly at the centre, thus providing equal space of 0.09 mm each on its either side. When Taylor bubble touches this obstacle, it splits and moves through both sides of the obstacle with perfect symmetric flow. The bubbles again join to form the original bubble as it moves past the obstacle. This is inline with the prior expectation. Next, the obstacle is moved by 0.02 mm away from the centre line towards one side, thus providing gap of 0.11 mm and 0.07 mm respectively on the two sides of the obstacle. Now it is found that when the bubble touches the obstacle it do not split in to two, rather the whole bubble moves through the bigger opening of 0.11 mm and only water flows through the smaller opening of 0.07 mm. Similar phenomena is observed when the bubble is further moved away from the centre line towards one side. The liquid-gas interface is found to be continuously changing its shape due to disturbance created by the presence of an obstacle. This causes turbulence inside the liquid plug between two consecutive bubbles, which is confirmed from velocity vector fields. This raises a hope to enhance heat and mass transfer in microchannels by placing multiple obstacles.
Prediction of Cell Growth Over a Circular Strand of a Scaffold Using the Lattice Boltzmann Method
Authors: Hossain Md Shakhawath , Bergstrom Donald J. , Chen X.-B.
Published: December 2014

The role of a bioreactor during the in vitro tissue culture process is important as the cell growth can be significantly enhanced by providing adequate nutrient supply and favorable mechanical stimuli, i.e. shear stress to the cells. Within a certain range, flow-induced shear stress has a positive impact on the cell growth. In the past, bioreactors and scaffold structures were designed based on empirical evidence, e.g. the flow rate inside the bioreactor was selected based on a trial and error method. More recently, mathematical and computational modeling of such complex process has been able to provide insight into the culture process and predict the overall cell and tissue growth. Although a number of computational studies which provide such cell and tissue growth information can be found in the literature, a comprehensive simulation to predict the overall tissue growth based on the supply of multiple nutrients and shear stress level acting on the cells is not yet available. In this study a simultaneous fluid flow and mass transfer analysis has been performed using the lattice Boltzmann method. A cell growth equation which considers the transport of multiple nutrients as well as the shear stress induced on the cells to predict the cell growth rate has been implemented. The overall model integrates the momentum, convection-diffusion and cell growth equations in a coupled fashion to predict the cell growth on a single strand of a scaffold placed inside a perfusion bioreactor. It is found that the maximum shear stress on the strand surface occurs near the strand shoulder. The nutrient supply and cell growth rate at the front of the cylinder is about ten times higher than at the rear of the cylinder. The cell growth rate obtained in the present study compares well with the results of other models documented in the literature.