Prolonged CL Research Articles

Calcineurin Inhibition Impairs the Function of Neuronal Potassium‐Chloride Cotransporter 2

Calcineurin inhibitors (CnI) such as Cyclosporin A (CsA) are instrumental for immunosuppression after organ transplantation but may cause serious neurologic side effects, including seizures.Neuronal excitability depends on intact Cl− homeostasis. Generation of hyperpolarizing synaptic responses to GABA and glycine requires a Cl− gradient across the cell membrane with a low [Cl−]i, mainly established by K+‐Cl− cotransporter 2 (KCC2) and to a lesser extent Na+‐K+‐Clcotransporter 1 (NKCC1); both are cation‐coupled chloride cotransporters (CCCs). Calcineurin has been implicated in the regulation of CCCs, whereas impaired CCC function is a well‐known condition in human pharmacoresistant epilepsy. Therefore, we hypothesized that CsA affects KCC2 or NKCC1 functions, thus causing neuronal hyperexcitability.In ex vivo intracellular recordings with sharp microelectrodes, Wistar rat pyramidal neurons in neocortical slices responded to CsA (5 μM for 1h) with a less negative GABAA reversal potential (+7.2 mV) and prolonged Cl− extrusion time after iontophoretic Cl− loading (+3.9 s). 2‐photon fluorescence lifetime imaging in presence of Cl− sensitive dye MQAE showed an increased [Cl−]i in layer V neurons (+6.9 mM), suggesting reduced KCC2 activity. Co‐immunoprecipitation studies in rodent brain tissue suggested interactions of calcineurin Aβ (CnAβ) with KCC2 and NKCC1.In vivo, CsA administration to rats (5–25 mg/kg i.p.) enhanced levels of inhibitory tyrosine KCC2 phosphorylation at short term (4h; +172%) and reduced levels of activating S940‐KCC2 phosphorylation in the long term (14d; −61%). Reduced phospho‐S940‐KCC2 levels were observed in further models of calcineurin inhibition such as genetic CnAβ‐deficiency in mice (−78%) or CsA treatment of zebrafish larvae (10 μM in water: −54% after 24h). In contrast, NKCC1 and its activating kinase SPAK were stimulated upon calcineurin inhibition in rodents. Similar data were obtained in Drosophila melanogaster.Our results provide evidence that CnI attenuate KCC2 function but may stimulate NKCC1 leading to elevated [Cl−]i. These effects may increase neuronal excitability and contribute to CnI neurological adverse effects. The benefits of KCC2 activators or SPAK inhibitors for enhancing chloride extrusion in patients experiencing CnI neurotoxicity deserve further characterization.Support or Funding InformationThis study was supported by Deutsche Forschergemeinschaft.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Electrical signalling in Nitellopsis obtusa: potential biomarkers of biologically active compounds.

The Nitellopsis obtusa (N.A.Desvaux) J.Groves cell provides a model system for complex investigation of instantaneous effects of various biologically active compounds (BC) on the generation of plant bioelectrical signals in vivo. Experimental evidence using multiple electrical signals as biomarkers of the effects of BC (acetylcholine, asparagine, glutamate, nicotine, aluminium, nickel and cadmium ions) is provided. The effect of BC on membrane transport systems involved in the cell excitability were tested by current clamp, voltage clamp and patch clamp methods. Membrane potential (MP) alterations and action potential (AP) patterns in response to BC were shown to represent the cell state. High discretisation frequency allows precise, high time resolution analysis of real-time processes measuring changes in excitation threshold, AP amplitude and velocity of repolarisation values after application of BC indicating the effect on ion channels involved in AP generation. Application of voltage clamp revealed that changes in AP peak value were caused not only by increment in averaged maximum amplitude of the Cl- current, but in prolonged Cl- channels' opening time also. The cytoplasmic droplet can serve as a model system in which the effects of BC on single tonoplast ion channel can be studied by patch clamping. Investigation of electrical cell-to-cell communication revealed evidence on the electrical signal transduction through plasmodesmata.

Is it time to retire the use of intrauterine glass balls for estrus suppression in mares?

JAVMA, Vol 247, No. 4, August 15, 2015 T ancient medical maxim primum non nocere (first, do no harm) comes to mind when reflecting on the more than 10 years that veterinarians have been using intrauterine glass balls (ie, marbles) for estrus suppression in mares. Although these devices were initially thought to be innocuous, a spate of recent reports has documented the potential for deleterious reproductive consequences associated with their use. In light of these reports, rethinking the use of these devices seems prudent, particularly because they are intended to address a behavioral issue (ie, the real or perceived adverse effects of estrus on performance activities such as showing and racing) rather than a disease condition. Glass balls are inserted into the uterine lumen in an effort to prolong functioning of the CL and allow for continued secretion of progesterone, which suppresses estrous behavior naturally. In nonpregnant mares, the CL secretes progesterone for approximately two weeks after ovulation (ie, the duration of diestrus) and then stops functioning when the endometrium secretes prostaglandin F 2α, causing luteolysis. As a result, serum progesterone concentration falls, and the mare returns to estrus. In 2003, Nie et al reported that placement of a 25or 35-mm-diameter sterile glass ball in the uterine lumen immediately following ovulation resulted in prolonged CL function in 39% of the mares that retained the glass ball after insertion (50% of the smaller glass balls were expelled soon after placement). In those mares that developed prolonged CL function following placement of the glass ball, CL function was maintained for approximately 90 days, during which time serum progesterone concentrations were > 1.0 ng/ mL and estrous behavior was not observed. Clinical interest in the intrauterine glass ball protocol was driven by the desire for nonpharmacological (ie, nonhormonal) methods of estrus suppression that could serve as an alternative to use of the synthetic progestin altrenogest, which is widely considered the gold standard for estrus suppression in mares. Although altrenogest reliably blocks estrous behavior, it is expensive, must be administered daily, and poses safety concerns for personnel who handle the drug. Because of these limitations as well as heightened concerns over the use of exogenous steroid hormones in performance horses, the intrauterine glass ball protocol provided an Is it time to retire the use of intrauterine glass balls for estrus suppression in mares?

Transient acidosis induces a preconditioning chloride conductance that protects mouse nodose neurons from NMDA‐induced apoptosis (1132.13)

Glutamate is a primary neurotransmitter expressed in nodose ganglia where the soma of baroreceptor and vagal afferent neurons are located. Excessive N‐methyl‐D‐aspartate (NMDA) receptor activation by glutamate initiates excitotoxicity and may cause nodose neuronal damage and apoptosis. In previous studies we showed that brief exposure to low pH induces activation of a novel prolonged Cl‐ conductance in nodose neurons. A similar Cl‐ conductance has been shown in cardiac myocytes to be protective from ischemic reperfusion damage (Diaz et al. 1999) . Here we tested the hypothesis that vagal sensory neurons may be protected from NMDA induced excitotoxicity and apoptosis by low pHo preconditioning.Nodose neurons of 3 months old mice were cultured and exposed to 3 brief pulses of low pH (6.0) solution each lasting 15 seconds with intervals of 10 seconds. After 20 minutes, 50 μM NMDA plus 10 μM Glycine (NMDA receptor co‐activator) were added to the cultures. Apoptotic nodose neurons were fluorescently detected by using Alexa Fluor 488 annexin V. Without preconditioning acidosis, the increases in apoptotic neurons as percentages of viable neurons during exposures to NMDA for 20, 40 and 60 minutes were 16 ± 1 (n=2346), 33 ± 4 (n=2056) and 40 ± 8% (n=2093) (p<0.01). After preconditioning acidosis at pH6.0 the increases in NMDA‐induced neuronal apoptosis became less, with levels of 6 ± 2% (n=1764), 10 ± 3% (n=1438) and 16 ± 3% (n=1678) of the total viable neurons. This pHo preconditioning effect was restored partially by 10 μM Tamoxifen, a non‐specific Cl‐ channel blocker, resulting in apoptosis levels of 12 ± 3% (n=831), 22 ± 2% (n=858) and 27 ± 2% (n=804). Thus a transient preconditioning Cl‐ conductance activated by low pHo reduced significantly the NMDA‐induced neuronal damage ( ANOVA, p<0.01). This work provides a putative molecular ionic basis for a neuronal protection during ischemic damage.Grant Funding Source: Supported by NIH with HL14388

Prolonged systemic infection in patients with endocardial leads predicts high mortality despite complete device extraction

be disclosed. The surface ECG during ILVT exhibited QRS complexes with right bundle blanch block and right axis deviation. Both the QRS morphology (#1: RS in V2, #2: QS in V2) and the preceding CL (#1 300ms, #2 280ms) showed alternans (Figure). Verapamil (4mg) prolonged both CL#1 ( 180ms) and CL#2 ( 50ms) with no changes in QRS morphologies. Electroanatomical mapping during VT revealed that the distance between two earliest activation site (QRS#1 and QRS#2) was 1.2cm, both located in the mid-anterior LV. Mid-diastolic dull potential (P1 in Figure) that preceded the onset of QRS by 80 msec could be recorded at the site 1.2 cm basal to the earliest ventricular activation site. A pre-systolic potential (P2 in Figure) could be also recorded at the site with P1. The intervals between P2 and P1 were constant, and the CL was dependent on the P1-P2 intervals. Radiofrequency energy delivered at this site successfully eliminated ILVT within 3 seconds. Interpretation: It is known that P1 reflects slow conduction zone (SCZ). The exit of QRS#1 and QRS#2 may apart 1.2cm each other, but have a common SCZ, since both QRS#1 and QRS2 was eliminated by an RF application at the site with P1. A 2:1 conduction block between the common SCZ to the exit of QRS#2 may cause the CL and QRS alternans. The CL alternans was due to a difference of conduction time from P1 to the two exits. Further, the two pathways from the common SCZ to the two exits may have different sensitivity to verapamil, since verapamil prolonged CL#1 and CL#2 to a different degree.

Chloride ion efflux regulates adherence, spreading, and respiratory burst of neutrophils stimulated by tumor necrosis factor-alpha (TNF) on biologic surfaces.

Chloride ion efflux is an early event occurring after exposure of neutrophilic polymorphonuclear leukocytes (PMN) in suspension to several agonists, including cytokines such as tumor necrosis factor-alpha (TNF) and granulocyte/macrophage-colony stimulating factor (Shimizu, Y., R.H. Daniels, M.A. Elmore, M.J. Finnen, M.E. Hill, and J.M. Lackie. 1993. Biochem. Pharmacol. 9:1743-1751). We have studied TNF-induced Cl- movements in PMN residing on fibronectin (FN) (FN-PMN) and their relationships to adherence, spreading, and activation of the respiratory burst. Occupancy of the TNF-R55 and engagement of beta 2 integrins cosignaled for an early, marked, and prolonged Cl- efflux that was accompanied by a fall in intracellular chloride levels (Cl-i). A possible causal relationship between Cl- efflux, adherence, and respiratory burst was first suggested by kinetic studies, showing that TNF-induced Cl- efflux preceded both the adhesive and metabolic response, and was then confirmed by inhibition of all three responses by pretreating PMN with inhibitors of Cl- efflux, such as ethacrynic acid. Moreover, Cl- efflux induced by means other than TNF treatment, i.e., by using Cl(-)-free media, was followed by increased adherence, spreading, and metabolic activation, thus mimicking TNF effects. These studies provide the first evidence that a drastic decrease of Cl-i in FN-PMN may represent an essential step in the cascade of events leading to activation of proadhesive molecules, reorganization of the cytoskeleton network, and assembly of the O2(-)-forming NADPH oxidase.

Corpus luteal function in nonpregnant mares following intrauterine administration of prostaglandin E2 or estradiol-17β

The objective of this study was to test the hypothesis that intrauterine administration of prostaglandin E2 (PGE2) or estradiol-17β (E-17β) would prolong CL function in nonpregnant mares. Nonpregnant mares were continuously infused with 240 μg/d of PGE2, 6 μg/d of E-17β, or vehicle (sham-treated) on Days 10 to 16 post ovulation (ovulation = Day 0), using osmotic minipumps surgically placed into the uterine lumen on Day 10 (n = 11 per group). Nonpregnant and pregnant mares served as negative and positive controls, respectively (n = 11 per group). Mares were defined as having prolonged CL function if plasma progesterone remained > 2.5 ng/ml and if ovulation did not occur on Days 9 to 30. Corpus luteal function was prolonged until Day 30 in 111 nonpregnant mares, 411 sham-treated mares, 611 E-17β-treated mares, 811 PGE2-treated mares, and 1111 pregnant mares. The incidence of prolonged CL function was similar (P=0.16) in the sham-treated and nonpregnant mares. The hypothesis that PGE2 would prolong CL function in nonpregnant mares was supported, since the incidence of prolonged CL function was higher (P=0.003) in PGE2-treated versus nonpregnant mares, tended to be higher (P=0.09) in PGE2-versus sham-treated mares, and was not lower (P=0.11) in PGE2-treated versus pregnant mares. The hypothesis that E-17β would prolong CL function in nonpregnant mares was not supported, since the incidence of prolonged CL function was not higher (P=0.34) in E-17β-versus sham-treated mares, and was lower (P=0.02) in E-17β-treated versus pregnant mares. These results demonstrate that intrauterine administration of a pharmacologic dose of PGE2 initiated prolonged CL function in nonpregnant mares. Further experiments are needed to confirm the role of conceptus secretion of PGE2 in CL maintenance, and to determine the mechanism of action of PGE2 within the equine reproductive tract.

Pharmacological characterisation of a re-entry model for atrial tachycardia in conscious dogs

We studied the effects of intravenously administered autonomic agents, the calcium channel blocker diltiazem, and Class I and III anti-arrhythmic agents on a re-entry model for atrial tachycardia in seven conscious dogs. The model was created by a Y-shaped incision in the intercaval region and front free wall of the right atrium. Acetylcholine (30 micrograms.kg-1) transiently but significantly shortened the cycle length of atrial tachycardia (CL) by 9 (SD 5)% while atropine (0.2 mg.kg-1) alone did not change or terminate the rhythm. Isoprenaline (4 micrograms.kg-1) briefly reduced CL, by 13(6)% whereas propranolol (1 mg.kg-1) alone significantly prolonged the CL and terminated the arrhythmia in 50% of the trials. Diltiazem (200 micrograms.kg-1) did not significantly change the CL. Class Ia agents procainamide (30 mg.kg-1) and quinidine (3.5 approximately 20 mg.kg-1, effective doses) increased CL by 60(30) and 43(28)% respectively and terminated the arrhythmias. Class Ib agents, phenytoin (5 and 10 mg.kg-1, cumulative doses), lignocaine (3.2 approximately 7 mg.kg-1), and mexiletine (6 approximately 20 mg.kg-1), prolonged CL by 31(14), 40(15), and 72(13)%; however, consistent conversion of the rhythm occurred only with mexiletine. Ic agents flecainide (0.5 approximately 3 mg.kg-1) and encainide (0.6 approximately 6.4 mg.kg-1) lengthened the CL by 58(17) and 75(43)% and converted the rhythm in all cases. Class III agents clofilium (0.5 mg.kg-1), d-sotolol (3.2 approximately 20 mg.kg-1), and N-acetylprocainamide (15 approximately 120 mg.kg-1) prolonged the CL by 14(4), 16(5), and 19(7)% and terminated the arrhythmia in all trials. The magnitudes of CL prolongation by the Class III agents were significantly smaller than those by the Class Ia, Ib, or Ic agents. These results show that in this re-entrant model for atrial tachycardia endogenous adrenergic but not cholinergic systems play a major role in maintaining impulse propagation and that calcium channel mediated slow inward currents do not participate in the impulse generation processes. Further, using CL as an index, the model serves as a useful tool for differentiating Class III from Class I anti-arrhythmic activities.

Inositol trisphosphate isomers, but not inositol 1,3,4,5-tetrakisphosphate, induce calcium influx in Xenopus laevis oocytes.

To investigate the mechanisms by which inositol phosphates regulate cytosolic free Ca2+ concentration ([Ca2+]c), we injected Xenopus oocytes with inositol phosphates and measured Ca2+-activated Cl- currents as an assay of [Ca2+]c. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) injection (0.1-10.0 pmol) induced an initial transient Cl- current (I1) followed by a second more prolonged Cl- current (I2). Both currents were Ca2+-dependent, but the source of Ca2+ was different. Release of intracellular Ca2+ stores produced I1, whereas influx of extracellular Ca2+ produced I2; Ca2+-free bathing media and inorganic calcium channel blockers (Mn2+, Co2+) did not alter I1 but completely and reversibly inhibited I2. Injection of the Ins(1,4,5)P3 metabolite, inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) (0.2-10.0 pmol) generated a Ca2+-dependent Cl- current with superimposed current oscillations that resulted from release of intracellular Ca2+, not Ca2+ influx. Injection of the Ins(1,3,4,5)P4 metabolite, inositol 1,3,4-trisphosphate (10.0 pmol), or the synthetic inositol trisphosphate isomer, inositol 2,4,5-trisphosphate (1.0-10.0 pmol), mimicked the effect of Ins(1,4,5)P3, stimulating an I1 resulting from release of intracellular Ca2+ and an I2 resulting from influx of extracellular Ca2+. The results indicate that several inositol trisphosphate isomers stimulate both release of intracellular Ca2+ and influx of extracellular Ca2+. Ins(1,3,4,5)P4 also stimulated release of intracellular Ca2+, but it was neither sufficient nor required for Ca2+ influx.

Length of pseudopregnancy and pattern of uterine protein release as influenced by time and duration of oestrogen administration in the pig.

Treatment of gilts with 5 mg oestradiol benzoate on Day 9.5, 11, 12.5, 14, 15.5 or Days 14-16 resulted in an interoestrous interval of about 30 days. Administration of oestradiol benzoate daily from Days 11 to 15 or two periods of treatment on Days 11 and 14 to 16 resulted in prolonging CL function beyond 60 days from the pre-treatment oestrus. Endometrial secretory response to oestrogen stimulation, based on the ability of oestrogen to release calcium and uterine protein into the lumen appears to occur after Day 10 of the oestrous cycle. The results suggest that maintenance of prolonged CL function appears to require two periods of oestrogen stimulation. The first period occurs on Day 11 when the endometrium has become responsive to oestrogen stimulation followed by a second prolonged increase in oestrogen stimulation after Day 14. These findings accord with the normal patterns of oestrogen released by pig blastocysts during early pregnancy.

The oestrous cycle of the mare and its uterine control.

SUMMARY The reproductive findings from a group of nonpregnant mares were studied. Oestrous cycle length averaged 20.6 days (range 13–34) excluding anoestrous periods, or 25 days (31–141) if included. Average oestrus length was 5.7 days (range 1–24) but from February to May it averaged 7.6 days (range 2–24) and from May to November 4.8 days (range 1–10). Seventy‐eight per cent of the mares ovulated within 48 hours prior to the end of oestrus, 10% were out of oestrus before ovulation occurred, while 76% of the ovulations occurred between 4 p.m. and 8 a.m. Follicles averaged 45 mm in size the day of ovulation and multiple ovulations occurred 25.5% of the time. Oestrus without associated ovulation was very uncommon in this group of mares, whereas ovulation without oestrus occurred in 6 of the 11 mares, including one mare who ovulated 32 of 34 times without oestrus. The CL were palpable for an average period of 8.9 days (range 1–18). On occasions, a hematoma formed within the ovulation site, reached a size of 10–12 cm in length and persisted beyond the next ovulation without affecting cycle length.Dioestrus averaged 15.4 days (range 6–25) excluding anoestrus, or 19.5 days (range 6–121) if anoestrus was included. Dioestrous ovulations unaccompanied by signs of oestrus and with the cervix pale, tight, dry and sticky occurred in 10 of the 11 mares. The CL formed following dioestrous ovuations were normal, but did not affect cycle length.A syndrome of spontaneous prolongation of the corpus luteum for 2 to 3 months was observed in 6 of the 11 mares. Oestrus was not manifested during this time, but considerable follicular activity and, in some instances, ovulation was observed. Hysterectomised mares and some mares with pyometra had prolonged CL and follicular activity with a few ovulating similar to mares with spontaneously‐prolonged CL. Other mares with pyometra had normal cyclic ovarian activity. Evidence suggests that the endometrium had been destroyed by the infection in the anoestrus mares with pyometra and, thus, was incapable of forming and/or releasing luteolytic factors. Experimental intrauterine inoculation of Streptococcus zooepidemicus during dioestrus reduced oestrous cycle length in 5 of 7 inoculations, whereas inoculations during oestrus failed to alter cycle length.