55 research outputs found
Ca 2+ signalling in urethral interstitial cells of Cajal
Interstitial cells of Cajal (ICC) in the urethra have been proposed as specialized pacemakers that are involved in the generation of urethral tone and therefore the maintenance of urinary continence. Recent studies on freshly dispersed ICC from the urethra of rabbits have demonstrated that pacemaker activity in urethra ICC is characterized by spontaneous transient depolarizations (STDs) under current clamp and spontaneous transient inward currents (STICs) under voltage clamp. When these events were simultaneously recorded with changes in intracellular Ca 2+ (using a Nipkow spinning disk confocal microscope) they were found to be associated with global Ca 2+ oscillations. In this short review we will consider some of these recent findings regarding the contribution of intracellular Ca 2+ stores and Ca 2+ influx to the generation of pacemaker activity in urethral ICC with particular emphasis on the contribution of reverse Na + /Ca 2+ exchange (NCX)
Contribution of K v 2.1 channels to the delayed rectifier current in freshly dispersed smooth muscle cells from rabbit urethra
We have characterized the native voltage-dependent K + (K v ) current in rabbit urethral smooth muscle cells (RUSMC) and compared its pharmacological and biophysical properties with K v 2.1 and K v 2.2 channels cloned from the rabbit urethra and stably expressed in HEK 293 cells (HEK Kv2.1 and HEK Kv2.2 ). RUSMC were perfused with Hanks' solution at 37°C and studied using the patch clamp technique with K + -rich pipette solutions. Cells were bathed in 100 nM penitrem A (Pen A) to block large conductance Ca 2+ -activated K + (BK) currents and depolarized to +40 mV for 500 ms to evoke K v currents. These were unaffected by margatoxin, κ-dendrotoxin or α-dendrotoxin (100 nM, n=3-5), but were blocked by stromatoxin-1 (ScTx, IC 50 ~130 nM), consistent with the idea that the currents were carried through K v 2 channels. RNA was detected for K v 2.1 K v 2.2 and the silent subunit K v 9.3 in urethral smooth muscle. Immunocytochemistry showed membrane staining for both K v 2 subtypes and K v 9.3 in isolated RUSMC. HEK Kv2.1 and HEK Kv2.2 currents were blocked in a concentration dependent manner by ScTx with estimated IC 50 values of ~150 nM (K v 2.1, n=5) and 70 nM (K v 2.2, n=6). The mean V 1/2 of inactivation of the USMC K v current was – 56±3 mV (n=9). This was similar to the HEK Kv2.1 current (–55 ± 3 mV, n=13) but significantly different from the HEK Kv2.2 currents (-30 ± 3 mV, n=11). Action potentials (AP) evoked from RUSMC studied under current clamp mode were unaffected by ScTx. However when ScTx was applied in the presence of Pen A, the AP duration was significantly prolonged. Similarly, ScTx increased the amplitude of spontaneous contractions threefold, but only after Pen A application.
These data suggest that K v 2.1 channels contribute significantly to the K v current in RUSMC
The effect of high [K(+)]o on spontaneous Ca(2+) waves in freshly isolated interstitial cells of Cajal from the rabbit urethra.
Interstitial cells of Cajal (ICC) act as putative pacemaker cells in the rabbit urethra. Pacemaker activity in ICC results from spontaneous global Ca(2+) waves that can be increased in frequency by raising external [K(+)]. The purpose of this study was to elucidate the mechanism of this response. Intracellular [Ca(2+)] was measured in fluo-4-loaded smooth muscle cells (SMCs) and ICC using a Nipkow spinning disk confocal microscope. Increasing [K(+)]o to 60 mmol/L caused an increase in [Ca(2+)]i accompanied by contraction in SMCs. Raising [K(+)]o did not cause contraction in ICC, but the frequency of firing of spontaneous calcium waves increased. Reducing [Ca(2+)]o to 0 mmol/L abolished the response in both cell types. Nifedipine of 1 μmol/L blocked the response of SMC to high [K(+)]o, but did not affect the increase in firing in ICC. This latter effect was blocked by 30 μmol/L NiCl2 but not by the T-type Ca(2+) channel blocker mibefradil (300 nmol/L). However, inhibition of Ca(2+) influx via reverse-mode sodium/calcium exchange (NCX) using either 1 μmol/L SEA0400 or 5 μmol/L KB-R7943 did block the effect of high [K(+)]o on ICC. These data suggest that high K(+) solution increases the frequency of calcium waves in ICC by increasing Ca(2+) influx through reverse-mode NCX
LINGO1 is a regulatory subunit of large conductance, Ca<sup>2+</sup>-activated potassium channels
Executive functions in preschool children with aggressive behavior: impairments in inhibitory control
The question whether executive function (EF)
deficits in children are associated with conduct problems
remains controversial. Although the origins of aggressive
behavior are to be found in early childhood, findings from
EF studies in preschool children with aggressive behavior
are inconsistent. The current study aimed to investigate
whether preschool children with aggressive behavior show
impairments in EF. From a population-based sample, 82
preschool children who were showing aggressive behavior
as indicated by scores at or above the 93rd percentile on the
Aggressive Behavior Scale of the CBCL 1 1/2-5 were
selected. These children with aggressive behavior were
matched on IQ to a group of typically developing control
children (N=99). Six neuropsychological tasks were administered
to assess set shifting, inhibition, working memory
and verbal fluency. A factor analysis was conducted which
yielded one clear factor: inhibition. Aggressive preschool children showed poorer performance on this inhibition
factor than control children and boys performed worse on
this factor than girls. This association between aggressive
behavior and inhibition deficits was maintained after controlling
for attention problems. In addition, gender differences
in all EFs measured were found with boys exhibiting
more impairment in EF than girls. These findings demonstrate
that preschool children with aggressive behavior show
impairments in inhibition, irrespective of attention problems
Characterization of norepinephrine-evoked inward currents in interstitial cells isolated from the rabbit urethra.
Freshly dispersed interstitial cells from the rabbit urethra were studied by using the perforated-patch technique. When cells were voltage clamped at -60 mV and exposed to 10 microM norepinephrine (NE) at 80-s intervals, either large single inward currents or a series of oscillatory inward currents of diminishing amplitude were evoked. These currents were blocked by either phentolamine (1 microM) or prazosin (1 microM), suggesting that the effects of NE were mediated via alpha(1)-adrenoceptors. NE-evoked currents were depressed by the blockers of Ca(2+)-activated Cl(-) currents, niflumic acid (10 microM), and 9-anthracenecarboxylic acid (9-AC, 1 mM). The reversal potential of the above currents changed in a predictable manner when the Cl(-) equilibrium potential was altered, again suggesting that they were due to activation of a Cl(-) conductance. NE-evoked currents were decreased by 10 microM cyclopiazonic acid, suggesting that they were dependent on store-released Ca(2+). Inhibition of NE-evoked currents by the phospholipase C inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (100 microM) suggested that NE releases Ca(2+) via an inositol 1,4,5-trisphosphate (IP(3))-dependent mechanism. These results support the idea that stimulation of alpha(1)-adrenoceptors releases Ca(2+) from an IP(3)-sensitive store, which in turn activates Ca(2+)-activated Cl(-) current in freshly dispersed interstitial cells of the rabbit urethra. This elevates slow wave frequency in these cells and may underlie the mechanism responsible for increased urethral tone during nerve stimulation
Cajal beyond the gut : interstitial cells in the urinary system – towards general regulatory mechanisms of smooth muscle contractility ?
Interstitial cells of Cajal (ICC), similar to GI pacemakers have
been identified throughout the urinary system. Although each part
of the system serves a different function, ranging from peristalsis
of the ureters, storage of urine by the bladder, and a sphincteric
action by the urethra, they share a common mechanism in being
able to generate phasic myogenic contractions. Even the urethra,
often considered to be a ‘tonic’ smooth muscle, achieves an apparently
sustained contraction by averaging numerous small asynchronous
‘phasic’ contractions. This activity can occur in the
absence of any neural input, implying the presence of an intrinsic
pacemaker. Intracellular microelectrode recordings from urethral
muscle strips reveal electrical slow waves similar to those of the GI
tract. To study this further, we isolated single cells from rabbit urethra
and found not only smooth muscle cells (SMC), but a second
cell type comprising ~10% of the total. The latter cells were
branched and non-contractile and closely resembled intestinal
ICC. Electrophyiological studies revealed that, while the isolated
smooth muscle cells were electrically quiescent, the ‘ICC’ fired
electrical slow waves similar to those observed in the whole tissue.
The basis of this difference was the presence of a large pacemaker
current involving the activation of calcium-activated Cl - channels
by spontaneous intracellular Ca 2+ waves. These, in turn, have been
shown to be modulated by neurotransmitters such as nitric oxide,
noradrenaline and ATP, thus providing a possible mechanism
whereby neural regulation of the urethra, as well as spontaneous
tone, may be mediated via ICC
Activation of Ca2+-activated Cl- current by depolarizing steps in rabbit urethral interstitial cells.
Interstitial cells were isolated from strips of rabbit urethra for study using the amphotericin B perforated-patch technique. Depolarizing steps to -30 mV or greater activated a Ca2+ current (ICa), followed by a Ca2+-activated Cl- current, and, on stepping back to -80 mV, large Cl- tail currents were observed. Both currents were abolished when the cells were superfused with Ca2+-free bath solution, suggesting that Ca2+ influx was necessary for activation of the Cl- current. The Cl- current was also abolished when Ba2+ was substituted for Ca2+ in the bath or the cell was dialyzed with EGTA (2 mM). The Cl- current was also reduced by cyclopiazonic acid, ryanodine, 2-aminoethoxydiphenyl borate (2-APB), and xestospongin C, suggesting that Ca2+-induced Ca2+ release (CICR) involving both ryanodine and inositol 1,4,5-trisphosphate receptors contributes to its activation
Role of mitochondria in modulation of spontaneous Ca2+ waves in freshly dispersed interstitial cells of Cajal from the rabbit urethra.
Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit pacemaker activity that results from spontaneous Ca(2+) waves. The purpose of this study was to investigate if this activity was influenced by Ca(2+) uptake into mitochondria. Spontaneous Ca(2+) waves were recorded using a Nipkow spinning disk confocal microscope and spontaneous transient inward currents (STICs) were recorded using the whole-cell patch clamp technique. Disruption of the mitochondrial membrane potential with the electron transport chain inhibitors rotenone (10 microm) and antimycin A (5 microm) abolished Ca(2+) waves and increased basal Ca(2+) levels. Similar results were achieved when mitochondria membrane potential was collapsed using the protonophores FCCP (0.2 microm) and CCCP (1 microm). Spontaneous Ca(2+) waves were not inhibited by the ATP synthase inhibitor oligomycin (1 microm), suggesting that these effects were not attributable to an effect on ATP levels. STICs recorded under voltage clamp at -60 mV were also inhibited by CCCP and antimycin A. Dialysis of cells with the mitochondrial uniporter inhibitor RU360 (10 microm) also inhibited STICS. Stimulation of Ca(2+) uptake into mitochondria using the plant flavonoid kaempferol (10 microm) induced a series of propagating Ca(2+) waves. The kaempferol-induced activity was inhibited by application of caffeine (10 mm) or removal of extracellular Ca(2+), but was not significantly affected by the IP(3) receptor blocker 2-APB (100 microm). These data suggest that spontaneous Ca(2+) waves in urethral ICC are regulated by buffering of cytoplasmic Ca(2+) by mitochondria
Pacemaker activity in urethral interstitial cells is not dependent on capacitative calcium entry.
The aim of the present study was to investigate the properties and role of capacitative Ca(2+) entry (CCE) in interstitial cells (IC) isolated from the rabbit urethra. Ca(2+) entry in IC was larger in cells with depleted intracellular Ca(2+) stores compared with controls, consistent with influx via a CCE pathway. The nonselective Ca(2+) entry blockers Gd(3+) (10 microM), La(3+) (10 microM), and Ni(2+) (100 microM) reduced CCE by 67% (n = 14), 65% (n = 11), and 55% (n = 9), respectively. These agents did not inhibit Ca(2+) entry when stores were not depleted. Conversely, CCE in IC was resistant to SKF-96365 (10 microM), wortmannin (10 microM), and nifedipine (1 microM). Spontaneous transient inward currents were recorded from IC voltage-clamped at -60 mV. These events were not significantly affected by Gd(3+) (10 microM) or La(3+) (10 microM) and were only slightly decreased in amplitude by 100 microM Ni(2+). The results from this study demonstrate that freshly dispersed IC from the rabbit urethra possess a CCE pathway. However, influx via this pathway does not appear to contribute to spontaneous activity in these cells
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