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)

T- and L-type Ca 2+ currents in freshly dispersed smooth muscle cells from the human proximal urethra

The purpose of the present study was to characterise Ca 2+ currents in smooth muscle cells solated from biopsy samples taken from the proximal urethra of patients undergoing surgery for bladder or prostate cancer. Cells were studied at 37°C using the amphotericin B perforated-patch onfiguration of the patch-clamp technique. Currents were recorded using Cs + -rich pipette solutions to block K + currents. Two components of current, with electrophysiological and pharmacological properties typical of T- and L-type Ca 2+ currents, were present in these cells. When steady-state inactivation curves for the L current were fitted with a Boltzmann equation, this yielded a VÎ of _45 ± 5 mV. In contrast, the T current inactivated with a VÎ of _80 ± 3 mV. The L currents were reduced in a concentration-dependent manner by nifedipine (ED50 = 159 ± 54 nM) and Ni 2+ (ED50 = 65 ± 16 mM) but were enhanced when external Ca 2+ was substituted with Ba 2+ . The T current was little affected by TTX, reduction in external Na + , application of nifedipine at concentrations below 300 nM or substitution of external Ca 2+ with Ba 2+ , but was reduced by Ni 2+ with an ED50 of 6 ± 1 mM. When cells were stepped from _100 to _30 mV in Ca 2+ -free conditions, small inward currents could be detected. These were enhanced 40-fold in divalent-cation-free solution and blocked in a concentration-dependent manner by Mg 2+ with an ED50 of 32 ± 16 mM. These data support the idea that human urethral myocytes possess currents with electrophysiological and pharmacological properties typical of T- and L-type Ca 2+ currents

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

Back-illuminated electron multiplying technology: The world's most sensitive CCD for ultra low-light microscopy

ABSTRACT The back-illuminated Electron Multiplying Charge Coupled Device (EMCCD) camera stands to be one the most revolutionary contributions ever to the burgeoning fields of low-light dynamic cellular microscopy and single molecule detection, combining extremely high photon conversion efficiency with the ability to eliminate the readout noise detection limit. Here, we present some preliminary measurements recorded by a very rapid frame rate version of this camera technology, incorporated into a spinning disk confocal microscopy set-up that is used for fast intracellular calcium flux measurements. The results presented demonstrate the united effects of: (a) EMCCD technology in amplifying the very weak signal from these fluorescently labelled cells above the readout noise detection limit, that they would otherwise be completely lost in; (b) back-thinned CCD technology in maximizing the signal/shot noise ratio from such weak photon fluxes. It has also been shown how this innovative development can offer significant signal improvements over that afforded by ICCD technology. Practically, this marked advancement in detector sensitivity affords benefits such as shorter exposure times (therefore faster frame rates), lower dye concentrations and reduced excitation powers and will remove some of the barriers that have been restricting the development of new innovative low-light microscopy techniques

Prenatal exposures and exposomics of asthma

This review examines the causal investigation of preclinical development of childhood asthma using exposomic tools. We examine the current state of knowledge regarding early-life exposure to non-biogenic indoor air pollution and the developmental modulation of the immune system. We examine how metabolomics technologies could aid not only in the biomarker identification of a particular asthma phenotype, but also the mechanisms underlying the immunopathologic process. Within such a framework, we propose alternate components of exposomic investigation of asthma in which, the exposome represents a reiterative investigative process of targeted biomarker identification, validation through computational systems biology and physical sampling of environmental medi

Spontaneous Electrical Activity in Sheep: Mesenteric Lymphatics

Background: It has recently become apparent that the lymph pump is an electrical entity thatrivals the heart in complexity. Many interesting currents have been demonstrated by voltage clamping isolated lymphatic smooth muscle cells, but until now the role of these currents in the intact syncitium has not been studied. Methods and Results: Intracellular microelectrode recordings were made from smooth muscle of sheep mesenteric lymphatics to investigate the electrophysiological basis of lymphatic pumping. Approximately 50% of the vessels exhibited spontaneous electrical activity, varying from regular oscillations in membrane potential to spike complexes. Spike complexes generally consisted of one or more action potentials superimposed on a slower depolarization or ‘plateau’ phase and were often preceded by a slow diastolic depolarization or ‘pre-potential’. Norepinephrine (5 uM) induced depolarizing events in quiescent preparations. Both agonist-induced oscillations and spike complexes were attenuated or completely abolished by 2-aminoethoxydiphenyl borate (2-APB); 10–100 uM). Cesium (1 mM) reduced the frequency of spontaneous firing by approximately 30% by flattening the pre-potential phase. In addition to having a negative inotropic effect, 10 mM Cs+ also caused gradual membrane depolarization and prolonged the plateau. 1 uM nifedipine abolished spontaneous events while tetrodotoxin (TTX; 0.5–1 uM) decreased the amplitude and maximum dV/dt of the spike upstroke or stopped activity completely. Spontaneously active segments of lymphatic vessel were inhibited by the chloride channel blocker, anthracene-9-carboxylic acid (9-AC; 250 uM-1 mM) suggesting that I Cl(Ca) plays a significant role in the generation of spontaneous activity in this tissue. Penitrem-A (0.1 uM) did not affect resting membrane potential but increased action potential amplitude and prolonged the plateau, suggesting that calcium-activated potassium current does not make a significant contribution to resting membrane conductance but is important in membrane repolarization following calcium influx during the action potential. In contrast 4-aminopyridine (4-AP; 5 uM) caused significant membrane depolarization, suggesting the existence of an active 4-AP-sensitive current at rest. Conclusions: These results demonstrate that the currents found in isolated voltage-clamped cells from sheep mesenteric lymphatics do play a significant role in the shaping of spontaneous electrical activity of the intact syncitium

Characterization of outward K(+) currents in isolated smooth muscle cells from sheep urethra.

The perforated-patch technique was used to measure membrane currents in smooth muscle cells from sheep urethra. Depolarizing pulses evoked large transient outward currents and several components of sustained current. The transient current and a component of sustained current were blocked by iberiotoxin, penitrem A, and nifedipine but were unaffected by apamin or 4-aminopyridine, suggesting that they were mediated by large-conductance Ca(2+)-activated K(+) (BK) channels. When the BK current was blocked by exposure to penitrem A (100 nM) and Ca(2+)-free bath solution, there remained a voltage-sensitive K(+) current that was moderately sensitive to blockade with tetraethylammonium (TEA; half-maximal effective dose = 3.0 +/- 0.8 mM) but not 4-aminopyridine. Penitrem A (100 nM) increased the spike amplitude and plateau potential in slow waves evoked in single cells, whereas addition of TEA (10 mM) further increased the plateau potential and duration. In conclusion, both Ca(2+)-activated and voltage-dependent K(+) currents were found in urethral myocytes. Both of these currents are capable of contributing to the slow wave in these cells, suggesting that they are likely to influence urethral tone under certain conditions

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