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Final Accepted Version C-00463-2003.R1 Characterisation of T-type calcium current and its contribution to electrical
activity in the rabbit urethra
J. E. Bradley, U. A. Anderson, S. M. Woolsey, K. D. Thornbury, N. G. McHale & M. A. Hollywood Smooth Muscle Group, Department of Physiology, The Queen's University of Belfast, 97Lisburn Road, Belfast, BT9 7BL, N. Ireland.
Name and address for correspondence: Mark HollywoodSmooth Muscle GroupDepartment of PhysiologyThe Queen's University of Belfast97 Lisburn RoadBelfastBT9 7BL Tel: 02890 272069Fax: 02890 331838Email: m.hollywood@qub.ac.ukWeb: www.smoothmusclegroup.org Key words: Spontaneous activity, pacemaking, smooth muscle Final Accepted Version C-00463-2003.R1 Freshly dispersed smooth muscle cells were studied at 37oC using the amphotericin B perforated patch configuration 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 calcium currents, were recorded. When steady state inactivation curves for the L current were fitted with a Boltzmann equation, this yielded a V1/2 of –41 ± 3 mV. In contrast, the T current inactivated with a V1/2 of –76 ± 2 mV.
The L currents were reduced in a concentration dependent manner by nifedipine (IC50 = 225 ± 84 nM), Ni2+ (IC50= 324 ± 74 mM) and mibefradil (IC50= 2.6 ± 1.1 mM) but were enhanced when external Ca2+ was substituted with Ba2+. The T current was little affected by nifedipine at concentrations below 300 nM but was increased in amplitude when external Ca2+ was substitution with Ba2+. Application of either Ni2+ and mibefradil reduced the T current with an IC50 = 7 ± 1 mM and 40 nM respectively.
To assess the contribution of T current to electrical activity we examined the effects of Ni2+ and mibefradil on spontaneous electrical activity recorded with intracellular electrodes from strips of rabbit urethra. The spontaneous electrical activity consisted of complexes comprising a series of spikes superimposed upon a slow spontaneous depolarisation (SD). Inhibition of T current reduced the frequency of these SD, but had no effect on either the number of spikes per complex or the amplitude of the spikes. In contrast, application of nifedipine failed to significantly alter the frequency of the SD but reduced the number and amplitude of the spikes in each complex.
Final Accepted Version C-00463-2003.R1 A number of studies have demonstrated that urethral myogenic tone is critically dependent on the influx of Ca2+ across the cell membrane, since inhibition of L type calcium channels or removal of external Ca2+ reduces tone significantly in rat, humans and pig urethra in vitro [25, 3, 1]. Recent evidence suggests that influx through T type calcium channels may contribute to the maintenance of urinary continence since application of Ni2+ can reduce urethral tone in the rat [25]. A previous study from our laboratory [10] demonstrated the presence of two components of inward calcium current in human proximal urethral myocytes which possessed electrophysiological properties typical of T and L channels observed in a variety of cell types (for reviews see [15 & 22]). Although the biopsy samples used by Hollywood et al. (2003) were sufficient to allow the isolation of single cells, their small size precluded the detailed examination of the contribution of T currents to spontaneous electrical activity in the human urethra using In this study, we first confirmed the presence of T type Ca2+ currents in isolated rabbit urethral myocytes and characterised its electrophysiological and pharmacological properties.
Having demonstrated that we could selectively block this current with appropriate concentrations of mibefradil or Ni2+, we assessed the contribution of T current to spontaneous electrical activity of the rabbit urethra using intracellular microelectrodes. Preliminary accounts of this work have been reported to the Physiological Society [2].
Final Accepted Version C-00463-2003.R1 MATERIALS & METHODS
Strips of tissue, 0·5 cm in width were isolated from male and female New Zealand white rabbits, cut into 1 mm3 pieces and stored in Hanks Ca2+ free solution for 30 min prior to cell dispersal.
Occasionally, the tissue was stored overnight in Ca free Hank's at 4˚C before cell dispersal.
Tissue pieces were incubated in dispersal medium containing (per 5 ml) of Ca -free Hanks solution (see solutions): 15 mg collagenase (Sigma type 1A), 1 mg protease (Sigma type XXIV), 10 mg bovine serum albumin (Sigma) and 10 mg trypsin inhibitor (Sigma) for 10-15 mins at 37˚C.
Tissue was then transferred to Ca2+-free Hanks solution and stirred for a further 15-30 min to release single smooth muscle cells. These cells were plated in petri dishes containing 100 mM Ca Hank's solution and stored at 4˚C for use within 8 hours.
Perforated Patch Recordings from Single Cells
Currents were recorded using the perforated patch configuration of the whole cell patch clamp technique [11]. This circumvented the problem of current rundown encountered using the conventional whole cell configuration. The cell membrane was perforated using the antibiotic amphotericin B (600µg/ml). Patch pipettes were initially front-filled by dipping into pipette solution and then back filled with the amphotericin B containing solution. Pipettes were pulled from borosilicate glass capillary tubing (1.5mm outer diameter, 1.17mm inner diameter; Clark Medical Instruments) to a tip of diameter approximately 1-1.5µm and resistance of 2-4 MΩ.
Voltage clamp commands were delivered via an Axopatch 1D patch clamp amplifier (Axon Instruments) and membrane currents were recorded by a 12 bit AD/DA converter Final Accepted Version C-00463-2003.R1 (Axodata 1200 or Labmaster – Scientific Solutions) interfaced to an Intel computer running pClamp software. During experiments, the dish containing the cells was continuously perfused with Hanks solution at 36 ± 1˚C. Additionally the cell under study was continuously superfused by means of a custom built close delivery system with a pipette of tip diameter 200 µm placed approximately 300 µm from the cell. The Hanks solution in the close delivery system could be switched to a drug-containing solution with a dead space time of less than 5 seconds. In all experiments ‘n' refers to the number of cells studied and each experimental set usually contained samples from a minimum of 4 animals. Summary data is presented as the mean ± S.E.M. and statistical comparisons were made on raw data using Students' paired t test, taking p<0.05 level as significant.
The bladder and urethra were removed from both male and female rabbits immediately after they had been killed by lethal injection of pentobarbitone. The most proximal 3 cm of the urethra was removed and placed in Krebs solution. This was then opened up, the urothelium removed and the preparation pinned out on a silicon rubber base and superfused with Krebs solution at 35-37oC.
To prevent spontaneous contractions from dislodging impalements, tissues were incubated with 5 mM wortmannin. Smooth muscle cells were impaled with glass microelectrodes filled with 3M KCl and had a resistance of 80 - 120 MW. Transmembrane potentials were recorded with a standard electrometer (IE 251, Warner Instruments) and stored on a PC running Stratchclyde Electrophysiology Software (WinEDR v2.3.3). Drugs were superfused through the bath and left for at least 10 minutes before washout.
Final Accepted Version C-00463-2003.R1 The composition of the solutions used was as follows (in mM): (1) Hanks solution 129.8 Na , 5.8 K , 135 Cl-, 4.17 HCO3-, 0.34 HPO4 , 0.44 H2PO4-, 1.8 Ca , 0.9 Mg , 0.4 SO4 , 10 glucose, 2.9 sucrose and 10 HEPES, pH adjusted to 7.4 with NaOH. (2) Cs+ perforated patch pipette solution 133 Cs , 135 Cl-, 1.0 Mg , 0.5 EGTA, 10 HEPES, pH adjusted to 7.2 with CsOH. (3) Ba2+ substituted Hanks solution 129.4 Na , 5.4 K , 135 Cl-, 4.17 HCO3-, 1.8 Ba , 0.9 Mg , 10 glucose, 2.9 sucrose and 10 HEPES, pH adjusted to 7.4 with NaOH. (4) Krebs solution 146.2 Na+, 5.9 K+, 133.3 Cl-, 25 HCO3-, 1.2 H2PO4-, 2.5 Ca , 1.2 Mg and 11 glucose, pH maintained at 7.4 by bubbling with 95% O2, 5% CO2.
Drugs Used
The following drugs were used: Amphotericin B (Sigma), NiCl2 (Sigma), nifedipine (Tocris), Mibefradil (A gift from Roche), Wortmannin (Sigma).
Stock solutions of NiCl2 (concentration 0.1 M) were made up in H20. Mibefradil and nifedipine (1 mM) were made up in ethanol. All drugs were then diluted to their final concentrations in Hanks solution. Drug vehicles had no effect on the currents studied.
Final Accepted Version C-00463-2003.R1 Using our dispersal procedure, both interstitial cells and smooth muscle cells could be reliably isolated from the rabbit urethra as described previously [23]. In the present study we focused on studying the smooth muscle cells, which were unbranched, spindle shaped, contracted in response to either depolarising pulses or application of noradrenaline (10 mM) but did not show sponataneous electrical activity. Under identical recording conditions to previous studies [21], more than 70% of rabbit urethral myocytes possessed both low voltage activated (LVA) and high voltage activated (HVA) calcium currents but rarely possessed calcium activated-chloride currents (<5%). On the basis of the electrophysiological and pharmacological data presented below we will henceforth refer to the HVA current as L current and the LVA current as T current. Interestingly, when cells were maintained at room temperature ( 22oC) only L-type currents were apparent (data not shown). For this reason all experiments were carried out at 35- 37oC. We observed considerable variation in the amplitude of T current between cells. In 34 cells, a depolarising step from –100 mV to -40 mV evoked a current that had a peak amplitude of –57 ± 6 pA (range –10 pA to –149 pA).
Effect of altering holding potential on currents
Figure 1 A shows representative traces taken from an experiment in which a cell was held at potentials of either –100 mV (left trace) or –60 mV (middle trace) and then depolarised from –70 mV through to 0 mV for 500 ms in 10 mV steps. When cells were held at –100 mV and then depolarised, inward currents activated at –60 mV. In contrast, when the same cell was held at –60 mV (Figure 1A, middle trace), and the voltage steps repeated, inward currents now activated at –40 mV. To isolate the currents unmasked by holding the cell at –100 mV, difference currents Final Accepted Version C-00463-2003.R1 were obtained by subtracting the currents obtained at holding potentials of –60 mV from those at –100 mV and are shown in the right panel of Figure 1A. These difference currents activated at - 60 mV, were maximal at –30 mV and reversed at approximately 20 mV. Figure 1B shows an IV plot for the current obtained from this cell at holding potentials of –100 mV (filled circles) and - 60 mV (open circles) and the associated difference currents (filled squares). A clear ‘shoulder' was apparent at negative potentials on the IV curve when the cell was held at –100 mV. In contrast, when the cell was held at –60 mV the ‘shoulder' was absent. Subtraction of these two currents unmasked an inward current that activated at –60 mV, peaked at –30 mV and reversed at To compare the steady state activation of both components of current, activation curves were constructed from currents evoked from cells held at –60 mV and difference currents obtained by subtraction as described above. The filled circles and squares in Figures 2B and 2D show summary data points obtained from the IV relationships in 7 cells, that were used to construct steady state activation curves for L and T current respectively. When these data were fitted with a Boltzmann equation of the form I/Imax =1/{1 + exp[-K(V - V1/2 )]} where K is the slope factor and V1/2 is the voltage at which there is half maximal activation. This yielded a V1/2 of –19 ± 1 mV for L current compared to –34 ± 3 mV for the T current. These data are consistent with the idea that two components of inward current were present in rabbit urethral myocytes.
Final Accepted Version C-00463-2003.R1 Voltage dependence of inactivation
To assess the voltage dependence of inactivation of both currents, a standard double pulse protocol was employed. Figure 2A shows a typical experiment in which a cell was held at conditioning potentials ranging from –110 to 0 mV for 2 s prior to stepping to the test potential of 0 mV for 500 ms to evoke the peak L current. Most inactivation of this current occurred over the potential range of –60 to –30 mV. The inactivation data were fitted with a Boltzmann equation described above where V1/2 is the voltage at which there is half maximal inactivation. The L current inactivated with a V1/2 of –41 ± 3 mV.
We next examined the inactivation of the T current by applying the same conditioning potentials as above but stepping to –40 mV to elicit T current with minimal activation of L current. Figure 2C shows a typical example of voltage dependent inactivation obtained using the protocol shown. In contrast to the L current, the T current was almost completely inactivated when conditioning potentials were more positive than –40 mV. Figure 2D (open squares) shows summary data obtained from 5 cells. When these data were fitted with the above Boltzmann equation (solid line) a V1/2 of –76 ± 2 mV was obtained. The grey solid lines in Figure 2D show the Boltzmann fits to the activation/inactivation curves for the L current for comparison.
Pharmacology of the inward currents.
Having demonstrated that two components of inward current could be isolated on the basis of their voltage dependence of activation and inactivation, we next wanted to test if each of these currents possessed a different pharmacological profile. We first wished to exclude the possibility that the negatively activating current was similar to the novel, Ba2+ sensitive, voltage dependent Final Accepted Version C-00463-2003.R1 cation current described previously [14]. The inset of Figure 3A shows the protocol used to evoke both components of inward current. Cells were held at –100 mV and stepped to –40 mV for 250 ms to evoke T current and then depolarised to –50 mV for 500 ms to inactivate most of the remaining T current. The cell was further depolarised to 0 mV for 250 ms to evoke peak L- type calcium current. When Ca2+ was substituted with equimolar (1.8 mM) Ba2+, the amplitude of the T current was increased slightly but the rate of inactivation was little affected. In contrast the L current was increased in amplitude and its time dependent inactivation was slowed. Figures 3B and 3C show summary bar charts for 6 cells in which the amplitude and the time constant (t) of inactivation respectively, of the T and L currents were recorded before (open bars) and during (hatched bars) substitution of external Ca2+ with equimolar Ba2+. Under control conditions the T current recorded at –40 mV was –37 ± 10 pA and this was significantly increased to -59 ± 16 p A in the presence of 1.8 mM Ba2+ (p<0.05). Although the amplitude of the T current was enhanced by Ba2+, the time constant of inactivation was little affected (t =19 ± 2 ms in Ca2+ compared to 16 ± 2 ms in Ba2+, n.s). In contrast, the time constant of inactivation of the peak L current was increased from 24 ± 3 ms to 68 ± 7 ms (p<0.05) in the presence of Ba2+ and the amplitude increased from -265 ± 69 pA in 1.8 mM Ca2+ to -386 ± 77 pA in 1.8 mM Ba2+ (p<0.05).
Effect of Nifedipine
We examined the effect of the L-type calcium channel antagonist nifedipine on both components of inward current. The inset of Figure 4A shows the protocol used to evoke both T and L current. Figure 4A shows currents obtained before and during application of increasing concentrations of nifedipine. Application of nifedipine at concentrations up to 300 nM decreased Final Accepted Version C-00463-2003.R1 the amplitude of the L current by 90 % whereas the T current was only reduced by 10%. The ability of nifedipine to discriminate between both components of inward current is reflected in the summary data shown in Figure 4B. The concentration effect curves illustrating the effects of nifedipine on T (open circles) and L current (closed circles) were constructed from data obtained from 5 cells. When the data for the L were fitted with a Langmuir equation of the form Idrug/Icontrol =1/[1 + ([drug]/IC50)] where IC50 of is the half-maximal effective dose, this yielded an IC50 of 225 ± 84 nM. In contrast, the data obtained from steps to –40 mV (open circles) could not be easily fitted with a Langmuir equation. For example, while 3 nM nifedipine reduced the T current by 10%, the next 3 concentrations had little further effect. This reduction by 30 nM nifedipine presumably reflects its effect on a small component of L current present at –40 mV.
Effect of Ni2+
To test the possibility that the currents activated at negative potentials were carried through T type calcium channels, we next examined the effects of a variety of concentrations of Ni2+ on both components of current. The inset of Figure 5A shows the typical double pulse protocol used to activate T and L current at –40 mV and 0 mV respectively. Figure 5A shows the effects of increasing concentrations of Ni2+ on both currents. Although Ni2+ reduced both currents in a concentration dependent manner, the T current was more sensitive to Ni2+ and was reduced by 80% in the presence 30 mM Ni2+. In contrast, the same concentration of Ni2+ only reduced the L- Final Accepted Version C-00463-2003.R1 type current by 25 %. Figure 5B shows summary concentration effect curves for the effect of Ni2+ on T current (open circles) and L current (filled circles) in 5 cells. When the data were fitted with the Langmuir equation above, this yielded an IC50 for the T current of 7 ± 2 mM compared to 324 ± 74 mM for the L-type current.
Effect of Mibefradil
We next assessed the effects of mibefradil on both currents in order to find a concentration that could selectively inhibit the T current. Figure 6A shows the typical effects of 0.1 mM and 0.3 mM mibefradil on T and L currents. Although 0.1 mM mibefradil reduced the T current by 80%, it had no effect on L current. Even when the concentration of mibefradil was increased to 0.3 mM the L current was reduced by only 20% but the T current was abolished. Figure 6B shows summary concentration effect curves, which illustrate the selective blockade of T current by mibefradil at concentrations below 0.3 mM. When the data for the L current (filled circles) was fitted with a Langmuir equation described above, this yielded an IC50 of 2.6 ± 1.1 mM. Although the T current data (open circles) could be fitted with a Langmuir to yield an IC50 of 40 nM, the absence of data at concentrations below 0.1 mM made it difficult to accurately determine the IC50.
However, it is clear from this data that the IC50 for mibefradil on T current is approximately 3 orders of magnitude less than that of the L current.
Having established that 0.1 mM mibefradil could selectively block the T current at –40 mV without any effect on the L current, we examined its effects on the current voltage relationships from cells held at –100 mV. Figure 7A shows currents elicited by a series of depolarising steps from –70 mV through to 0 mV before (left panel) and during (middle panel) application of Final Accepted Version C-00463-2003.R1 mibefradil. The right panel shows the mibefradil sensitive current which activated at –50 mV, inactivated rapidly, and peaked at –20 mV. Figure 7B shows a summary of 6 similar experiments in which the mean currents before (filled circles) and during (open circles) mibefradil application were plotted. Difference currents (filled squares) were obtained by subtraction of the currents before and during mibefradil application. The mibefradil sensitive current possessed characteristics typical of T current, since it activated at –50 mV and peaked at -20 mV.
To assess the contributions of both currents to spontaneous electrical activity in the urethra we examined the effects of Ni2+ mibefradil and nifedipine on strips of rabbit urethra impaled with intracellular microelectrodes. The mean resting membrane potential was –50 ± 0.5 mV (n=185 from 86 animals), which was close to the peak T window current (see Figure 2). We found considerable variation in the resting membrane potential and type of electrical activity recorded between successive impalements, even in the same tissue. These observations are consistent with the idea that the urethra possesses a heterogenous population of cells that may contribute to electrical activity [23]. The first type of activity that was observed in 16% of impalements consisted of single rapid spikes that were followed by large after-hyperpolarisations. The second type of activity was observed in 10% of impalements and consisted of slow waves similar to those recorded by Hashitani et al (1996). In 74% of impalements, we observed spontaneous activity which consisted of spike complexes and is illustrated in Figures 8 and 9. These complexes were comprised of a series of spikes superimposed upon a spontaneous depolarisation (SD). The complexes occurred at a mean frequency of 5 ± 0.4 min-1 (range = 2 Final Accepted Version C-00463-2003.R1 min-1 to 12 min-1) and had on average 5 ± 0.3 spikes per complex (range 2 to 16 spikes per complex). The mean amplitude of the SD observed in spike complexes was 10 ± 0.5 mV (range = 3 mV to 27 mV) and the duration of the complex was 2.1 ± 0.1 s (range = 0.1 s to 7.4 s). Cells which showed this type of activity had a mean resting membrane potential of –51 ± 0.6 mV (n=137, range = -34 mV to –70 mV). Since the majority of impalements demonstrated the latter type of activity, we concentrated our efforts on examining the effects of L and T channel blockers on cells that showed spike complex activity. To quantify the effects of drugs on this activity we measured 4 parameters, the number of SDs, the number of spikes during each SD, the maximum amplitude of the spikes and the resting membrane potential.
Effect of Ni2+ on spontaneous activity.
Figures 8A and B show a typical example of the effects of Ni2+ on spontaneous activity. In this example the SD fired at a frequency of 3 min-1. Application of 30 mM Ni2+ (Figure 8B) decreased the frequency of SD to 2 min-1 but failed to affect either resting membrane potential, the amplitude of the spikes within the complex or the number of spikes per SD (compare inset of Figure 8A with 8B). Figures 8C-E shows a summary of SD frequency, spike amplitude and the number of spikes/SD recorded in 8 cells before (open bars) and during 30 mM Ni2+ (hatched bars). These data confirm that neither spike amplitude or the number of spikes/SD were significantly altered. Although application of Ni2+ had no effect on resting membrane potential (- 54 ± 2 mV before compared to –54 ± 2 mV during Ni2+), the SD frequency was significantly reduced from 5.2 ± 1.4 min-1 to 3.6 ± 0.8 min-1 (p<0.05).
Effect of mibefradil on spontaneous activity.
Final Accepted Version C-00463-2003.R1 Having demonstrated that 100 nM mibefradil could selectively block T current, we examined its effects on SDs. Figure 9A shows a recording of spontaneous activity prior to application of mibefradil. The inset shows a typical SD on an expanded time scale. In this example SDs occurred at a frequency of 5 min–1. In the presence of mibefradil (Fig. 9B) the frequency of SDs was reduced to 3 min-1 but there was no significant change in spike amplitude, resting membrane potential or the number of spikes per SD (compare inset of Figure 9A with 9B). Figures 9C-E summarise the mean frequency of SDs, spike amplitude and number of spikes per SD in 6 cells before (open bars) and during (hatched bars) application of 100 nM mibefradil. Although the frequency of SDs was significantly reduced from 3.8 ± 0.8 to 2.8 ± 1 min (P<0.05) none of the other parameters were significantly altered. Similarly, resting membrane potential was unchanged by mibefradil (-53 ± 1 mV before, compared to –52 ± 1 mV during).
Effect of nifedipine on spontaneous activity.
To assess the contribution of L current to spontaneous electrical activity, we examined the effects of 100 nM nifedipine on spike complexes. Even though this concentration would be only be expected to block 40 % of the L current, it was chosen because it should have little effect on T current (see figure 4). In contrast to the effects of T current blockade, application of nifedipine, had no significant effect on the frequency of SDs (7.4 ± 1.7 min-1 before compared to 7.0 ± 2 min1 during nifedipine, n=8). However, application of nifedipine significantly reduced both the amplitude and frequency of the spikes. In eight experiments the amplitude of the spikes was reduced from 44 ± 4 mV before to 16 ± 6 mV (p<0.05) during application of nifedipine and the Final Accepted Version C-00463-2003.R1 number of spikes per SD was reduced from 4.2 ± 1 spikes/complex to 0.7 ± 0.3 spikes/complex during nifedipine (p<0.05).
Final Accepted Version C-00463-2003.R1 The results of this study demonstrate the presence of two components of calcium current with characteristics typical of T and L currents found in a variety of lower urinary tract smooth muscles, including human bladder, guinea-pig bladder and human urethra [27, 5, 10]. Peres-Reyes [22] has recently provided a review on T currents and demonstrated that they, like the currents described in the present study, could be readily discriminated from L currents on the basis of their activation at relatively low voltages (between –80 mV and –60 mV), inactivation over negative voltage ranges (V1/2 between –90 and –70 mV), equal permeability to Ca2+ and Ba2+, relative insensitivity to dihydropyridines and sensitivity to mibefradil and Ni2+.
The electrophysiological characteristics of both T and L currents in rabbit urethra were remarkably similar to those found in the human urethra [10]. Thus, the T currents in both cell types could be evoked at potentials positive to –70 mV and inactivated with a V1/2 of –76 mV (rabbit) compared to –80 mV (human urethral myocytes, [10]). In both rabbit and human myocytes, the L currents activated at –40 mV, inactivated more slowly than the T currents and their V1/2 of inactivation was –41 mV and –45 mV respectively. Similarly, the reversal potential of the T current in both cell types was 20 mV whereas the L currents reversed at 45 mV.
Differences between T and L current reversal potentials have been noted in other studies [26] and are presumably due to the fact that T channels pass significant outward currents at potentials positive to +20 mV in the presence of normal external Ca2+ [8].
It is interesting to note that although the sensitivity of the L currents to nifedipine in rabbit and human urethra myocytes were similar (IC50 of 159 nM and 225 nM respectively), their was a significant difference in the sensitivity to Ni2+. The L current in human urethral myocytes was Final Accepted Version C-00463-2003.R1 approximately 5 fold more sensitive to Ni2+ (IC50 = 65 nM; [10] compared to IC50 = 324 nM in the rabbit). These data suggest that there may be subtle differences in the molecular species encoding the L channels in both cell types, although this would require confirmation.
Although both human and rabbit urethral myocytes possess T current, some studies have failed to demonstrate T current in smooth muscle cells isolated from the urethra of different species [28]. Whether this reflects species variation or perhaps differences in recording conditions is unclear at present. However, it is interesting to note that T current amplitude is significantly influenced by temperature [20, 21, 22]. In the present study we found that reducing the bath temperature from 35ºC to 22ºC practically abolished the T current evoked by a step from –100 mV to –40 mV and reduced the L current evoked from a holding potential of –50 to 0 mV by 50% (n=3, data not shown). Given the temperature sensitivity of the T current, it is essential that studies are carried out at body temperature before the presence of T current can be excluded.
Substitution of external Ca2+ with Ba2+ greatly enhanced the amplitude and slowed the inactivation of the current evoked by a step from –50 mV to 0 mV. Interestingly, the amplitude of the current evoked by a step –100 mV to –40 mV was also increase in Ba2+, unlike the novel, voltage dependent, non-selective cation current demonstrated in murine colonic myocytes [14].
This effect on T current could be due to contaminating L current, however, since the time constant of inactivation of the T current was little affected, this seems unlikely. These data suggest that Ba2+ may permeate T channels in urethral myocytes more easily than Ca2+ as has been demonstrated in both native T channels [12] and reexpressed a1H subunits [18].
Final Accepted Version C-00463-2003.R1 The sensitivity of the rabbit urethral T current to Ni2+ (IC50 7 mM) was also similar to that described in human urethral myocytes [10]. The low concentration of Ni2+ required to block the T currents in urethral myocytes suggests that the pore forming subunit in these myocytes may be a1H, since these have been shown to be approximately 10 fold more sensitive to Ni2+ than the a1G and a1i subunits recently cloned [13, 16]. We also examined the effects of the T channel blocker mibefradil [19] and found that it could dramatically reduce the T current at concentrations as low as 100 nM, whilst having little effect on L current. Although we did not use low enough concentrations of mibefradil to produce a full concentration effect curve for its effects on T current, it is clear from the data presented, that the IC50 is < 100 nM, which is in agreement with that reported for T channels recorded in Ca2+ containing solutions [22].
The pattern of electrical activity recorded with intracellular microelectrodes from the whole rabbit urethra was complex, consisting of 3 main patterns of phasic activity- spike activity, slow waves and spike complexes. The heterogeneity of electrical activity observed in the present study was not unexpected, given that recordings were made from the whole urethra which consists of circular and longitudinal smooth muscle layers, in which both ICC and smooth muscle cells are present. At present we do not know if the different types of activity observed reflect different properties or populations of cells within the smooth muscle layers. However, given the similarities between the electrical activity recorded with intracellular electrodes and that observed in isolated urethral cells, it is tempting to speculate that the slow waves, spikes and spike complexes represent recordings from distinct cell populations. The first type of activity resembled the single fast spikes recorded from strips of urethra previously [2] and were similar to the single action potentials that can evoked from rabbit urethral myocytes with brief Final Accepted Version C-00463-2003.R1 depolarisations [23]. The slow wave activity recorded in the minority of impalements, bore a striking resemblance to the spontaneous activity observed in isolated rabbit urethra ICC under current clamp [23] and to the intracellular recordings from circular muscle by Hashitani et al. (1996). This suggests that slow wave activity is from urethral ICC or cells that are well coupled to these. The third type of activity that was recorded in 74% of impalements from the urethra, resembled neither the rapid fast spikes or the slow waves recorded from isolated smooth muscle cells and ICC respectively. These spike complexes comprised a series of rapid action potentials, superimposed upon a SD. We believe that this type of activity represents the electrical activity recorded from ‘follower' smooth muscle cells that are located ‘downstream' to the pacemaker cells, as is the case in the GI tract [6,7]. However, until we can confirm that the different patterns of electrical activity represent recordings from different populations of cells within the urethra, these ideas are will remain speculative. Future experiments will be directed towards repeating the elegant experiments of Dickens et al. (1999) on the urethra, to address these issues.
The range of membrane potentials recorded using intracellular microelectrodes was also quite variable (from –34 mV to –70 mV) even in different impalements of the same tissue. It is unlikely that any T current would be available in cells that had membrane potentials of –34 mV but as figure 2D suggests, significant T current would be available at more hyperpolarised membrane potentials. Given that the peak of the T window current was close to the mean resting membrane potential recorded with intracellular microelectrodes ( -50 mV), it is possible that sufficient T current would be present to contribute to electrical activity.
The ability of both mibefradil and Ni2+ to alter spontaneous activity in strips of urethra suggest that sufficient T current was available to contribute to electrical activity even at membrane Final Accepted Version C-00463-2003.R1 potentials of -50 mV. When we selectively blocked the T current, it was clear that neither the amplitude of the spikes nor the number of spikes per SD were altered. However, both drugs significantly reduced the frequency of SDs. These data suggest that the T current in the urethra contributes to the modulation of SD frequency but contributes little to spike generation.
However, we can not discount the possibility that these effects are due to blockade of ionic conductances other than T current, since mibefradil has been shown to block K+, Na+ and Cl+ channels [22]. However the concentrations of Ni2+ and mibefradil used in the present study are thought to be relatively specific for T current [22].
Another alternative explanation is that mibefradil and Ni2+ mediate their effects by altering the pacemaking mechanisms in urethral IC. However, one might expect that if the pacemaker mechanism depended on T current, then urethral IC slow wave frequency should show some voltage dependence. However, as Sergeant et al. [24] demonstrated, the frequency of slow waves in urethral IC was little affected by voltage. Another possible explanation of our results is the T current may contribute to the propagation of the pacemaking depolarisations throughout the urethra. A number of studies on the gastrointestinal tract have demonstrated that conductances with properties similar to T currents are present in ICC [16] and that these currents contribute to the propagation of the pacemaking signal throughout the bulk smooth muscle via the ICC [28].
At present, we can not exclude the possibility that urethral IC express T currents, or that they are essential for the propagation of the pacemaking depolarisations in the urethra. However, the presence of T current in urethral smooth muscle, suggests that it could facilitate the propagation of the pacemaking depolarisations. Since the T current activates at negative membrane potentials, relatively small depolarisations (presumably supplied by urethral IC) would be required to Final Accepted Version C-00463-2003.R1 elevate the membrane potential sufficiently to activate T currents, bring the membrane potential into the threshold range for L current activation and thus set off a series of spike complexes. Such a mechanism could contribute to the modulation of the electrical activity in the urethra and thus Final Accepted Version C-00463-2003.R1 The authors would like to thank Action Medical Research, the Wellcome Trust and the RalphShackman Trust for funding this study.
Final Accepted Version C-00463-2003.R1 1. Brading A. F. (1999). The physiology of the mammalian outflow tract. Exp. Physiol. 84:
2. Bradley, J. E. Anderson, U. A. Woolsey, S. M. McHale. N.G., Thornbury K.D. & Hollywood. M. A. (2003) Characterisation of T type calcium current and its role in spontaneous activity in rabbit urethral smooth muscle. J. Physiol. (in press) 3. Bridgewater M., MacNeil H. F. and Brading A. F. (1993) Regulation of tone in pig urethral smooth muscle. J. Urol. 150: 223-228
4. Callahan SM, Creed KE. (1985). The effects of oestrogens on spontaneous activity and responses to phenylephrine of the mammalian urethra. J Physiol. 358:35-46.
5. Chow KY, Wu C, Sui GP, Fry CH. (2003). Role of the T-type Ca2+ current on the contractile performance of guinea pig detrusor smooth muscle. Neurourol Urodyn; 22(1):77- 6. Cousins HM, Edwards FR, Hickey H, Hill CE, & Hirst G.D. S. (2003). Electrical coupling between the myenteric interstitial cells of Cajal and adjacent muscle layers in the guinea-pig gastric antrum. J Physiol. 550: 829-844.
7. Dickens EJ, Hirst GD, Tomita T. (1999) Identification of rhythmically active cells in guinea- pig stomach. J Physiol. 514: 515-31.
8. Fukushima, Y & Hagiwara, S (1985). Currents carried by monovalent cations through calcium channels in mouse neoplastic B lymphocytes. J Physiol. 358: 255-284.
9. Hashitani H, Van Helden DF, Suzuki H. (1996). Properties of spontaneous depolarizations in circular smooth muscle cells of rabbit urethra. Br J Pharmacol. 118:1627-1632.
Final Accepted Version C-00463-2003.R1 10. Hollywood MA, Woolsey S, Walsh IK, Keane PF, McHale NG, Thornbury KD. (2003). T- and L-type Ca2+ currents in freshly dispersed smooth muscle cells from the human proximal urethra. J Physiol. 550:753-764.
11. Horn R. and Marty A. (1988) Muscarinic activation of ionic currents measured by a new whole-cell recording method. J. Gen. Physiol. 92: 145-159
12. Huguenard JR & Prince DA. (1992) A novel T-type current underlies prolonged Ca(2+)- dependent burst firing in GABAergic neurons of rat thalamic reticular nucleus. J Neurosci. 13. Klockner U, Lee JH, Cribbs LL, Daud A, Hescheler J, Pereverzev A, Perez-Reyes E, Schneider T. (1999). Comparison of the Ca2+ currents induced by expression of three cloned alpha1 subunits, alpha1G, alpha1H and alpha1I, of low-voltage-activated T-type Ca2+ channels. Eur J Neurosci. 11:4171-4178.
14. Koh SD, Monaghan K, Ro S, Mason HS, Kenyon JL, Sanders KM. (2001) Novel voltage- dependent non-selective cation conductance in murine colonic myocytes. J Physiol. 533: 341-
15. Kotlikoff MI, Herrera G, Nelson MT. (1999) Calcium permeant ion channels in smooth muscle. Rev Physiol Biochem Pharmacol. 134:147-99.
16. Lee HK, Sanders KM. (1993). Comparison of ionic currents from interstitial cells and smooth muscle cells of canine colon. J Physiol. 460:135-52.
17. Lee JH, Gomora JC, Cribbs LL, Perez-Reyes E. (1999). Nickel block of three cloned T-type calcium channels: low concentrations selectively block alpha1H. Biophys J. 77:3034-3042.
Final Accepted Version C-00463-2003.R1 18. McRory JE, Santi CM, Hamming KS, Mezeyova J, Sutton KG, Baillie DL, Stea A, Snutch TP (2001). Molecular and functional characterization of a family of rat brain T-type calcium channels. J Biol Chem. 276(6): 3999-4011.
19. Mishra S.K, & Hermsmeyer K. (1994). Selective inhibition of T-type Ca2+ channels by Ro 40-5967. Circ Res; 75(1):144-148.
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21. Nobile M, Carbone E, Lux HD, Zucker H. (1990)Temperature sensitivity of Ca currents in chick sensory neurones. Pflugers Arch. 415(6): 658-663.
22. Perez-Reyes E. (2003) Molecular physiology of low-voltage-activated T-type calcium channels. Physiol. Rev. 83(1):117-161.
23. Sergeant, G. P., Hollywood, M.A., McCloskey, K.D., Thornbury, K.D. and McHale, N.G.
(2000). Specialised pacemaking cells in the rabbit urethra J Physiol 526: 359-366.
24. Sergeant GP, Hollywood MA, McCloskey KD, McHale NG, Thornbury KD. (2001) Role of IP3 in modulation of spontaneous activity in pacemaker cells of rabbit urethra. Am J Physiol (Cell). 280(5): C1349-1356.
25. Shafei. M., Thornbury, K.D., McHale, N.G., & Hollywood, M.A. (2003). Relative contributions of calcium influx and calcium stores to myogenic tone in the rat urethra. J 26. Staes M, Talavera K, Klugbauer N, Prenen J, Lacinova L, Droogmans G, Hofmann F, Nilius The amino side of the C-terminus determines fast inactivation of the T-type calcium channel alpha1G.(2001). J Physiol. 530:35-45.
Final Accepted Version C-00463-2003.R1 27. Sui GP, Wu C, Fry CH. (2001) Inward calcium currents in cultured and freshly isolated detrusor muscle cells: evidence of a T-type calcium current. J Urol: 165: 621-626.
28. Teramoto N, Yunoki T, Ikawa S, Takano N, Tanaka K, Seki N, Naito S, Ito Y. (2001). The involvement of L-type Ca(2+) channels in the relaxant effects of the ATP-sensitive K(+) channel opener ZD6169 on pig urethral smooth muscle. Br J Pharmacol. 134(7):1505-1515.
29. Ward SM, Baker SA, Faoite Ad A, Sanders KM.(2003). Propagation of slow waves requires IP3 receptors and mitochondrial Ca2+ uptake in canine colonic muscles. J Physiol; 549: 207-
Final Accepted Version C-00463-2003.R1 Figure 1. Characteristics of T and L currents in human urethral smooth muscle cells. A,
shows families of currents recorded from a cell held at –100 mV and –60 mV. The right panel shows the difference currents obtained by subtraction and demonstrates a negatively activating inward current that peaks at –30 mV. B, shows an IV plot taken from the same cell in which the peak inward currents were plotted when cells were depolarised from holding potentials of –100 mV (closed circles) and –60 mV (open circles). The filled squares show the difference currents that were obtained by subtraction.
Figure 2. Voltage dependent inactivation of L and T currents in human urethral smooth
muscle cells. A, shows a typical family of currents obtained by stepping to 0 mV for 200 ms
following application of a series of 2s conditioning potentials from –110 mV to +10 mV. B, shows summary data demonstrating the voltage dependence of inactivation (open circles) and activation curves (filled circles) of the L current. The continuous lines represent Boltzmann fits to the data. C, shows a typical inactivation profile for T current obtained by stepping to -40 mV for 200 ms following application of a series of 2s conditioning potentials from –110 mV to +10 mV. D, shows summary data demonstrating the voltage dependence of inactivation (open squares) and activation curves (filled squares) of the T current. The continuous black lines represent Boltzmann fits to the data. The continuous grey lines show the fits obtained for L current from the data in figure 2B for comparison.
Figure 3. Effects of Ba2+ on T and L currents. A, peak T and L currents were evoked by a step
from -100 mV to -40 mV for 250 ms. A step to -50 mV for 500 ms was applied to inactivate T current and this was followed by a further step to 0 mV to evoke peak L current in the same cell.
Substitution of external Ca2+ with equimolar Ba2+ increased the amplitude and slowed the decay of the current evoked by a step from –50 to 0 mV but had little effect on the amplitude of the current evoked by a depolarisation to –40 mV from a holding potential of –100 mV. B, shows a Final Accepted Version C-00463-2003.R1 summary barchart for 6 cells in which the peak amplitude of the T and L current was plotted before (open bars) and during (hashed bars) equimolar substitution of external Ca2+ with Ba2+. C, shows a summary barchart in which the time dependent inactivation of the T and L currents was plotted in before (open bars) and during (hashed bars) equimolar substitution of external Ca2+ Figure 4. Effect of nifedipine on T and L currents. A, The traces demonstrate the effect of
increasing concentrations of nifedipine on the peak T and L current. Note that 300 nM nifedipine practically abolished L current evoked by a step from –50 to 0 mV but had less effect on the peak T current evoked by a step from –100 to –40 mV. B, shows a summary of effects of a variety of nifedipine concentrations on the peak T (open circles) and L currents (closed circles).
The L current data were fitted with a Langmuir equation (continuous line, see text) and yielded an ED50 of 225 ± 84 nM. The T current data were not easily fitted with a Langmuir equation.
Figure 5. Effects of Ni2+ on T and L currents. A, peak T and L currents were evoked using the
protocol described in Figure 3A. Application of 10 mM Ni2+ reduced the amplitude of the T current by 60% but reduced the L current by only 15%. B, shows summary concentration effect curves obtained from 5 cells. When the data obtained for T current (open circles) and L current (filled circles) were fitted with the Langmuir equation (continuous lines), this yielded an IC50 of 7 ± 2 mM and 324 ± 74 mM respectively.
Figure 6. Effects of Mibefradil on T and L currents. A, peak T and L currents were evoked
using the protocol described in Figure 3A. Application of 100 nM mibefradil reduced the amplitude of the T current by 75% but no measurable effect on the L current. When the concentration of mibefradil was increased to 300 nM, the T current was abolished but the L current was reduced by only 20%. B, shows summary concentration effect curves obtained from 6 cells. When the data obtained for T current (open circles) and L current (filled circles) were Final Accepted Version C-00463-2003.R1 fitted with the Langmuir equation (continuous lines), this yielded an IC50 of 40 nM and 2.6 ± 1.1 mM respectively.
Figure 7. Application of 100 nM mibefradil blocks T current selectively. A, shows currents
obtained by depolarising from –70 mV through to 0 mV from a holding potential of –100 mV under control conditions (left panel) and during application of mibefradil (middle panel). The mibefradil-sensitive current (right panel) was obtained by subtraction. Panel B shows a summary IV for 6 cells obtained before (closed circles) and during (open circles) application of 100 n M mibefradil. The mibefradil -sensitive current (closed squares) was obtained by subtracting the middle panel from the right panel in A. This current activated at -50 mV and peaked at -20 mV whereas the mibefradil -resistant current (closed squares) activated at -40 mV and peaked at 0 Figure 8. Nickel slows SD frequency.
A, shows an example of spontaneous electrical activity recorded from the urethra. The inset shows a single complex on an expanded time scale. The complexes consisted of a spontaneous depolarisation upon which a number of spikes were superimposed. B, shows the reduction in frequency of SD in the presence of Ni2+ (30 mM), but as the inset suggests, Ni2+ application had no effect on the number of spikes during the SD. C, D and E shows summary bar charts for 8 experiments in which the effects of Ni2+ were assessed on SD frequency, the peak amplitude of the spikes and the number of spikes per SD respectively.
Figure 9. Selective blockade of T current with mibefradil reduces SD frequency. A shows
typical SD activity recorded from the urethra in the absence of drugs. The inset shows a typical complex recorded on an expanded timescale. B, shows the reduction in frequency of SD in the presence of mibefradil (100 nM). The inset demonstrates the lack of effect of mibefradil on the spikes. C, D and E shows summary barcharts for 6 experiments in which the effects of mibefradil Final Accepted Version C-00463-2003.R1 were assessed on SD frequency, the peak amplitude of the spikes and the number of spikes per SD respectively.
-60 HPDifference current Conditioning Potential (mV)
Conditioning Potential (mV)
T current L current [Nifed] nM
50 = 225 ± 84 nM [Ni2+] µM 103
L current: IC50 = 324 ± 74 µM T current: IC50 = 7 ± 2 µM 300 nM Mibefradil 100 nM Mibefradil 300 nM Mibefradil 100 nM Mibefradil L current: IC50 = 2.6 ± 1.1 µM T current: IC50 40 nM ControlMibefradil (100 nM) Nickel (30 µM) Mibefradil (100 nM) Mibefradil (100 nM)

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Removal of estrone from water by adsorption on zeolites with regeneration by direct uv photolysis

Worcester Polytechnic Institute Removal of Estrone From Water by Adsorption on Zeolites with Regeneration by Direct UV PhotolysisHuajing Wen John A. Bergendahl Robert W. ThompsonWorcester Polytechnic Institute, rwt@wpi.edu Follow this and additional works at: Suggested CitationWen, Huajing , Bergendahl, John A. , Thompson, Robert W. (2009). Removal of Estrone From Water by Adsorption on Zeolites withRegeneration by Direct UV Photolysis. Environmental Engineering Science, 26(2), 319-326.Retrieved from:

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