Institute of Veterinary Pharmacology (R.W., M.M., D.S.H., G.S.),
Department of Urology (B.W., U.E.S., H.D.), University of Bern, Bern,
Switzerland; and Environmental Toxicology Program, National Institute
of Environmental Health Sciences (C.J.P.), Research Triangle Park,
North Carolina
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Introduction |
Pharmacological
relaxation of the ureter smooth muscle would facilitate the treatment
of ureter stone colic and possibly enhance stone passage as well as
prepare the ureter for easier endoscopic access. Although considerable
studies have been made, a specific and potent agent to relax ureters
has not yet been found. KATP channel openers are
well known to achieve smooth muscle relaxation.
Physiologically, decreased cellular concentrations of ATP, i.e., during
metabolic stress and hypoxia or ischemia, cause opening of
KATP channels and subsequently induce a
hyperpolarized state of the cell membrane. The response of the
KATP channel to metabolic challenge is regulated
by adenylate kinase phosphotransfer, which amplifies metabolic signals
(Carrasco et al., 2001
; Zingman et al., 2001). The
KATP channel complex functions not only as a
K+ conductance but also as an enzyme regulating
nucleotide-dependent channel gating through an intrinsic ATPase
activity of the sulfonylurea receptor subunit, an ATP binding cassette
protein (Bienengraeber et al., 2000).
The hyperpolarization of the cell membrane leads to relaxation of
the smooth muscle cell through several mechanisms such as prevention of
depolarization-induced Ca2+ entry, inhibition of
the agonist-induced increase of inositol-1,4,5-trisphophate, or
reduction of Ca2+ sensitivity of the contractile
elements in smooth muscle cells (Quast, 1993; Quayle et al., 1997). The
K+ channel opener and NO donor nicorandil was the
first synthetic compound reported to open KATP
channels and thereby induce relaxation of vascular smooth muscle by
hyperpolarization (Furukawa et al., 1981; Taira, 1989).
The ureteral smooth muscle relaxing effect of the
K+ channel openers such as cromakalim has been
tested successfully (Klaus et al., 1990; Maggi et al., 1994; de Moura
and de Lemos, 1996). KATP channels can be
functionally suppressed by K+ channel modulators
such as glibenclamide (Quayle et al., 1997). Klaus et al. (1989 and
1990) reported that nicorandil leads to hyperpolarization and
subsequently to relaxation of ureteral smooth muscle of rabbit, guinea
pig, and human. However, nicorandil also contains a nitrate moiety and
produces smooth muscle relaxation by release of NO (Holzmann, 1983;
Taira, 1989).
It has been shown that NO mediates relaxation of smooth muscle by
various mechanisms. cGMP levels are elevated through stimulation of the
soluble GC (Holzmann, 1983). A high level of cGMP may provoke different
effects in a smooth muscle cell such as activation of KATP channels (Murphy and Brayden, 1995) or
activation of KCa channels (Robertson et al.,
1993). NO is generated in the cell by NO synthase (NOS). NO is known to
originate either from constitutive NOSs, such as endothelial NOS and
neuronal NOS, or from inducible NOS (Moncada and Higgs, 1995).
Endothelial NOS was first identified in vascular endothelium and
recently has also been found to be produced in pig calyceal urothelium,
where it is thought to modulate the adjacent smooth muscle cells and
therefore ureteral peristalsis (Iselin et al., 1999). NOS-containing
nerves have been found in the lower third of the pig ureter as well as
in the pig and human intravesical ureter and are thought to be involved
in ureteral peristalsis through a neurogenic pathway (Iselin et al.,
1996, 1997). Inducible NOS, which was first identified in macrophages (Moncada and Higgs, 1995), should also be considered as a possible source of NO in ureteral tissues.
cGMP seems to be a link between the mechanism of NO and the
K+ channel openers. Hernandez et al. (1997)
reported a relaxing effect due to NO in the isolated intravesical
ureter of the pig. This effect is mediated by a GC-dependent mechanism
that seems to facilitate the opening of glibenclamide-sensitive
KATP channels. Iselin et al. (1996) showed that
NO induced an elevation of cGMP in isolated pig ureteral smooth muscle.
The objective of our study was to investigate the effects of nicorandil
and PKF 217-744b
[(3S,4R)-N-(3.4-dihydro-2.2- dimethyl-3-hydroxy-6-(2-methylpyridin-4-yl)-2H-1-benzopyran)-3-pyridinecarboxy-amid (PKF)] in vivo and in vitro on the human and pig ureter. PKF (Fig. 1) is a new K+
channel opener (Manley et al., 2001), which has been shown to antagonize the ozone-induced hyperactivity in guinea pig airway smooth
muscle (Buchheit et al., 1998).
| |
Materials and Methods |
In Vitro Experiments on Pig Ureteral Tissue.
Pig ureteral
tissue was taken 4 to 11 cm distal from the kidney hilus approximately
20 min post mortem at the local slaughterhouse. Immediately after
removal, the tissue was immersed in 4°C Krebs-Henseleit solution of the following composition: 118.4 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 2.5 mM CaCl2, 25 mM NaHCO3, 1.2 mM
KH2PO4, and 11.1 mM glucose
(Uchida et al., 1994). The smooth muscle tube of the ureter was cut
into rings of 0.5 to 1 cm in length and suspended in an organ bath for
isometric tension recording (organ bath of SCHULER type 809 B, Hugo
Sachs Electroniks, March, Germany). The organ bath chambers were
perfused with 95% O2 and 5%
CO2, the pH was 7.5, and the temperature was
maintained at 37°C. Isometric forces were recorded using a force
transducer type 351 (Hugo Sachs Electroniks). The preparations were
equilibrated for up to 1.5 h to maintain spontaneous contractions.
During the equilibration time, preload tension was maintained at 1 g, and the chambers were flushed every 20 min. Specimens that did not
show any spontaneous activity after 1.5 h of equilibration (50%)
were discarded.
As soon as spontaneous contractions remained stable in frequency and
amplitude, a concentration-response curve was determined with
nicorandil (3-30 µM). Recordings of contraction frequency were
obtained before (predrug) and after nicorandil were added to the organ
bath. The parameters were also recorded after washout of the compound (postdrug).
The antagonists, glibenclamide (0.1 and 1 µM, n = 8 for each concentration) and methylene blue (MB; 10 µM,
n = 8), were added to the organ bath 20 min before
nicorandil (10 µM, n = 8) was administered. Distilled
water was used to perform the vehicle experiments (n = 8). Each preparation was used for a single application of either
distilled water or nicorandil, with or without glibenclamide or MB.
The time during which spontaneous contractions occurred were
unpredictable and could last anywhere from 15 min to 5 or more hours.
Therefore, activity was evaluated again after washout of the drug and
only the preparations that showed reversibility of the drug effect
after washout were accepted for statistical evaluation. A decrease in
motility of about 10% between predrug and postdrug values was
considered acceptable.
In Vitro Experiments in Human Ureter Rings.
Human ureter
specimen were taken during cystectomy (n = 13) or
nephrectomy (n = 5) from 7 male and 11 female patients
(mean age 63 ± 12.1 years) performed at the Department of
Urology, University of Bern, Switzerland. Transportation, storage, and
preparation were as described for pig ureteral tissue. During the
equilibration time of 114 ± 33.6 min, preload tension was 2 g, and the chambers were flushed every 20 min. Spontaneous contractions
were rare in the isolated specimens of the human ureter. Therefore,
contractions were evoked by electrical field stimulation, and the
amplitude of contractions was evaluated. Electrical field stimulation
was started after 10 min of equilibration with two platinum electrodes (Hugo Sachs Electroniks) placed parallel to the ureter preparations. The stimulation pattern included trains of 300 ms at an interval of
200 s and impulses of 200 mA with a duration of 6 ms at a
frequency of 50 Hz applied by a Grass stimulator (S88; Grass
Instruments, Quincy, MA). After equilibration, when contraction
amplitude had reached a steady state, nicorandil (3-60 µM) and PKF
(0.03-0.3 µM) were administered at intervals of 20 min in a
cumulative manner.
In Vivo Experiments in Pigs.
All animals in this study
received humane care in compliance with the laws about the care and use
of laboratory animals in Switzerland.
Male and female pigs (n = 28) were anesthetized with
halothane, after premedication with ketamine and xylazine
hydrochloride. One catheter was inserted into the carotid artery to
measure arterial blood pressure, and a second catheter was placed into
the jugular vein to administer infusions and drugs. A double lumen
catheter was inserted through the lateral abdominal wall into each
renal pelvis. The tip of one lumen was placed in the renal pelvis
allowing perfusion of the upper urinary tract, the tip of the other
lumen was placed in the mid-portion of the ureter to measure
intraluminal pressure. Contractions in both ureters, arterial blood
pressure, and heart rate (ECG) were monitored continuously and recorded during the entire experiment using a Hellige SMR 821 (Hellige GmbH,
Freiburg, Germany). Perfusion of each ureteropelvic unit with saline at
a stable rate of 0.5 ml/min was performed to serve as controls or
baseline frequency and amplitude of ureteral contractions.
In experiments with intravenous drug administration, vehicle as well as
increasing drug doses (nicorandil, 0.003-1 mg/kg, n = 7; PKF, 0.001-0.1 mg/kg, n = 6) were given as a bolus
at intervals of 5 to 10 min. The frequency and amplitude of ureteral
contractions were evaluated for 5 min after the intravenous drug
administration. It should be noted that intravenous PKF and nicorandil
abolished the frequency of spontaneous contractions completely at the
doses of 0.1 and 1 mg/kg, respectively. Therefore, contraction
amplitude was only analyzed as long as three-fourths of the experiments contained contractions.
In experiments with topical drug administration, one ureter was first
perfused with the vehicle followed by drug solutions (nicorandil,
0.03-30 mg/ml, n = 9; PKF, 0.001-1 mg/ml,
n = 6) increasing the concentration every 15 to 20 min.
The other ureter was perfused with saline. The frequency and amplitude
of ureteral contractions were evaluated from minute 5 to minute 10 from
the beginning of the perfusion.
Drug effects are shown in the graphs as percentages of control values
(100%), showing contraction frequency and amplitude together with the
cardiovascular parameters.
Chemicals.
Nicorandil was donated by Merck (Dietikon,
Switzerland), and this compound was dissolved in distilled water for
the in vitro experiments or in saline for the in vivo experiments.
PKF was provided by Novartis (Basel, Switzerland). PKF was dissolved in
phosphate-buffered saline, pH 7.4 (1 mg/ml) (in vivo) or in distilled
water containing 0.6 mg/ml tartaric acid (in vitro), and further
dilution was done with saline (in vivo) or distilled water (in vitro).
For the in vitro experiments, glibenclamide, donated by Hoechst
(Frankfurt, Germany), was dissolved in 1-methyl-2-pyrrolidon (Dr. Grogg
Chemie AG) and further diluted with distilled water. The pH was
adjusted to 7.5 by adding sodium hydroxide. For the in vivo
experiments, glibenclamide was dissolved in dimethyl sulfoxide (with a
final concentration of 2.5%) and further diluted with phosphate-buffered saline (pH 7.4) and saline served as the vehicle (1:1).
MB [3,7-bis (dimethylamino)phenazathionium chloride], purchased from
Dr. Grogg Chemie AG, was dissolved in distilled water (in vitro
experiments). For the in vivo experiments, MB for intravenous injection was purchased from the Inselspital Bern (Bern,
Switzerland) and dissolved in saline. Ingredients (pro analysi) for
Krebs-Henseleit solution were all purchased at Dr. Grogg Chemie AG.
Statistics.
In the in vitro experiments on pig and human
ureter tissue, Friedman's analysis of variance and Wilcoxon signed
rank test were used for corresponding pairs, the Mann-Whitney
U test was used for noncorresponding pairs. A p
value of 0.05 or less was considered statistically significant.
In Vivo Experiments.
The absolute values of the different
parameters were evaluated with the Wilcoxon signed rank test using the
computer program Statview (version 5.0; SAS Institute, Inc., Cary, NC).
The results were considered significant for p < 0.05.
Concentration-response curves were calculated from the log
concentration-effect curves using a Hill equation and estimation via
least-squares method. All data were converted to percentage of response
against controls. The underlying equation for Hill function is
|
(1)
|
where Vmax is the maximal
attainable response, K is the half-effective concentration
(EC/ED50, i.e., the concentration/dose yielding
half the maximum effect), and the exponent 
describes the shape of
the function (Hill coefficient). When the response was a reduction, the
value of Vmax represented the maximal
reduction in response. In one case, the response was mixed, with an
initial climb and eventual drop. This response was modeled using a
noncompetitive response curve based on the Hill equation of the form
|
(2)
|
where D is given by eq. 1,
VmaxA is the maximal drop in response,
KA is the concentration yielding half
the maximal response (EC/ED50A), and 
is the Hill
coefficient for the reduction phase of the concentration-response
curve. Statistical significance of any comparisons made on the basis of
this model (e.g., testing to see if the Hill coefficient equals 1) is
made using the Wald statistic. Confidence bounds presented for
parameters in the Hill model are also based upon the Wald statistic
(Portier et al., 1993).
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Results |
Effects of Nicorandil and Inhibition by Glibenclamide and MB on
Spontaneous Contractions of Isolated Pig Ureter in Vitro.
About
50% of the preparations showed spontaneous activity, typically 0.5 to
1.5 h after having been placed in the organ bath chamber. The
frequencies observed ranged from 0.1 to 5.4 contractions per minute.
Contractions of the individual ureter rings did not follow a uniform
pattern. Regular rhythmical motility with a constant amplitude as well
as periodical clusters of contractions with shifts in amplitude were
seen. Predrug activity and the reversible inhibitory effect of
nicorandil on an isolated pig ureter ring are shown in Fig.
2.
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Fig. 2.
Original recording of the effect of 30 µM
nicorandil and subsequent washout on the spontaneous contractions of an
isolated pig ureter ring in vitro. Concentration-response relationship
of nicorandil (3-30 µM, n = 8) was calculated
for the frequency of spontaneous contractions in isolated pig ureter
rings in vitro. The results are presented as mean ± S.E.M. of the
percentage of inhibition of predrug controls.
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Nicorandil at concentrations of 3 to 30 µM (n = 8)
decreased the frequency of spontaneous ureteral contractions. The
decrease was concentration-dependent and significant at 10 µM (to
33 ± 8%, p < 0.01) and 30 µM (to 8 ± 4%, p < 0.001) when compared with predrug and vehicle
controls. Calculation of the EC50 as well as
predrug activity and the inhibitory effect of nicorandil on an isolated
pig ureter ring are shown in Fig. 2. No significant effect was seen in
vehicle experiments (n = 8).
Pretreatment with the KATP antagonist
glibenclamide inhibited the effect of 10 µM nicorandil
(n = 8). The inhibition of the frequency of
contractions caused by 10 µM nicorandil was reduced from 33 ± 8% of controls to 44 ± 8 and 87 ± 4% of controls in the
presence of glibenclamide concentrations of 0.1 µM (n = 8) and 1 µM (n = 8), respectively (Fig.
3). The antagonistic effect of
glibenclamide was only significant at the higher concentration (p < 0.001). These effects were also significant when
compared with predrug controls (p < 0.01). No
significant effect was seen in vehicle experiments. Pretreatment with
MB (10 µM) (n = 8) significantly (p < 0.05) inhibited the nicorandil (10 µM) effect on the frequency of
contractions from 33 ± 8 to 64 ± 10% of controls.
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Fig. 3.
Effect of nicorandil on the contraction frequency of
porcine ureter rings in vitro: vehicle alone ( ,
n = 8), 10 µM nicorandil ( ,
n = 8), 10 µM nicorandil after preincubation with
0.1 µM ( , n = 8), and 1 µM glibenclamide
( , n = 8), 10 µM nicorandil after
preincubation with 10 µM MB
( ,
n = 8). The results are presented as mean ± S.E.M. in percentage of predrug controls.  , statistically
significantly different from predrug control.
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Effects of Nicorandil and PKF on Evoked Contractions (Field
Stimulation) of Isolated Human Ureter in Vitro.
In electrically
stimulated tissues of the human ureter (n = 6),
nicorandil concentrations of 10, 30, and 60 µM resulted in a
concentration-dependent decrease in the amplitude of contractions to
93 ± 13% (p < 0.05), 57 ± 14%
(p < 0.05), and 22 ± 9% (p < 0.001) of controls. Washout reversed the nicorandil effect to 82 ± 11% of controls. No significant effect was seen in vehicle experiments.
PKF concentrations of 0.03, 0.1, and 0.3 µM decreased and finally
abolished the amplitude of electrically induced contractions on human
ureter (n = 5) to 69 ± 13% (p < 0.001), 4 ± 2% (p < 0.001), and 0 ± 0%
(p < 0.001). Washout could not reverse the PKF effect. No significant effect was seen in vehicle experiments.
The dose-response curves for nicorandil and PKF are shown in Fig.
4. Calculations of
EC50 and Vmax
showed that PKF was more potent than nicorandil in isolated human
ureter by the factor 810, but no significant difference was observed in
the maximal effect (Table 1).
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Fig. 4.
Comparison of the concentration-response curves of
PKF (0.03-0.3 µM, n = 6) and nicorandil (3-60
µM, n = 6) on the amplitude of electrically
induced contractions in human ureter rings in vitro. The results are
presented as mean ± S.E.M. of the percentage of inhibition of
predrug controls.
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TABLE 1
EC50 levels and Vmax of nicorandil and PKF
on frequency of contractions of isolated pig ureter preparations and on
amplitude of contraction of isolated human ureter preparations
ED50 levels and Vmax of nicorandil and PKF
on frequency of contractions in the ureters of anaesthetized pigs.
Basic contractions of specimens of isolated pig ureters and ureters of
anaesthetized pigs were spontaneous, whereas in the human ureter
preparations, contractions were evoked by electrical field stimulation.
EC50/ED50, their upper and lower confidence limits
(CL), were obtained by applying empirical fitting method, as described
under statistical analysis.
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Effect of Nicorandil and PKF on the Ureter of Anesthetized Pigs in
Vivo.
Nicorandil given intravenously (0.003-1 mg/kg,
n = 7) decreased the frequency of contractions
significantly to 4 ± 3% of controls as shown in Fig.
5A. Contraction amplitude was unaffected.
A slight but significant increase of heart rate and a significant
decrease in mean arterial blood pressure was seen (Fig. 5A).
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Fig. 5.
Effects of nicorandil on frequency and amplitude of
ureteral contractions, blood pressure and heart rate in anesthetized
pigs. A, i.v. administration of nicorandil (0.003-1 mg/kg,
n = 7), glibenclamide + nicorandil, and MB + nicorandil; B, perfusion of the ureter with nicorandil (0.03-30 mg/ml,
n = 9). Glibenclamide was administered i.v. during
perfusion of the ureter with 30 mg/ml nicorandil. The results are
presented as mean ± S.E.M. in percentage of predrug controls.
, p < 0.05, compared with vehicle; #,
p < 0.05, compared with highest dose (A) or
concentration (B) of nicorandil.
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Intravenous administration of glibenclamide (0.2 mg/kg) and MB (0.1 mg/kg) had no significant effect on the nicorandil-induced decrease in
the frequency of contractions or fall in arterial blood pressure. The
heart rate remained slightly but significantly increased (Fig. 5A).
Nicorandil given topically (0.03-30 mg/ml, n = 9)
significantly decreased the frequency of ureteral contractions to
44 ± 21% of controls as shown in Fig. 5B. No significant effect
was seen on the amplitude of contractions (data not shown). After
perfusion with saline, the effect on frequency could be reproduced by a repeated perfusion with nicorandil (30 mg/ml). This effect was reversed
by intravenous glibenclamide at a concentration of 0.2 mg/kg in the
treated ureter but not in the control ureter.
In the saline-perfused ureter the frequency of contractions declined
significantly as illustrated in Fig. 5B. Again the amplitude of
contractions was not affected by nicorandil (data not shown). In
contrast to the intravenous administration, arterial blood pressure
showed no significant decrease and no effect was seen on heart rate
(Fig. 5B).
PKF given intravenously (0.001-0.1 mg/kg, n = 6)
significantly decreased and finally abolished the frequency of
contractions in a dose-dependent fashion, as shown in Fig.
6A. The ED50 for the initial increase in the frequency of contractions and the ED50A for decrease obtained at higher
doses of PKF are shown in Fig. 7, and the
estimates for both ED50 and
Vmax are given in Table 1. Therefore,
an increase of this effect was observed at low doses of PKF, as
illustrated in Fig. 7. PKF had no significant effect on the amplitude
of contractions at doses from 0.001 to 0.01 mg/kg (Fig. 6A). Heart rate
remained stable whereas the mean arterial blood pressure decreased
significantly to 33 ± 5% of controls. The effects of PKF were
not significantly antagonized by glibenclamide at a dose of 0.2 mg/kg
(Fig. 6A).
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Fig. 6.
Effects of PKF on frequency and amplitude of ureteral
contractions, blood pressure, and heart rate in anesthetized pigs. A,
i.v. administration of PKF (0.001-0.1 mg/kg, n = 6) and glibenclamide + PKF; B, perfusion of the ureter with PKF
solution (0.001-1 mg/ml, n = 6). Glibenclamide was
administered i.v. during perfusion of the ureter with 1 mg/ml PKF. The
results are presented as mean ± S.E.M. in percentage of predrug
controls. , p < 0.05, compared with the
vehicle; #, p < 0.05, compared with highest dose
(A) or concentration (B) of PKF.
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Fig. 7.
Dose-response relationship of PKF was calculated for
the frequency of contractions registered in the ureter of anesthetized
pigs. Intravenous administration of PKF (0.001-0.1 mg/kg,
n = 6) increased the frequency of contractions at
low doses and decreased the frequency of contractions at higher doses
in pig ureter in vivo. The solid line shows the plot of the combined
increased and decreased effects for all doses used in these
experiments. The dashed line illustrates the calculated
ED50 and Vmax for the increase
of the frequency of contractions (activation), and the dotted line
shows the effect and the calculated ED50A and
VmaxA for the decreasing effect
(suppression, see Eq. 2). The results are presented as mean ± S.E.M of the percentage of difference to predrug controls.
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PKF applied topically (0.001-1 mg/ml, n = 6)
significantly decreased the frequency of contractions as shown in Fig.
6B. No significant effect was observed on the amplitude of contractions (data not shown). Glibenclamide (0.2 mg/kg) given intravenously could
significantly, but not completely, restore the frequency of
contractions that had been decreased by topical PKF at a concentration of 1 mg/ml. The contralateral saline-perfused ureter showed a significant concentration-dependent decrease in the frequency of
contractions (Fig. 6B). No significant decrease in the amplitude of
contractions was seen (data not shown). In contrast to intravenous administration, topical PKF had no effect on the mean arterial blood
pressure or heart rate (Fig. 6B).
Calculation of the EC50 value for the frequency
of contractions showed that topical PKF is about 1000 times more potent
than topical nicorandil as shown in Table 1.
| |
Discussion |
In the present study, the K+ channel opener
and NO donor nicorandil decreased the frequency of contractions in
isolated pig ureteral smooth muscle preparations. Glibenclamide and MB
inhibited the effects of nicorandil in the isolated porcine ureter. In
human ureteral rings the amplitude of contractions induced by electric field stimulation was suppressed by nicorandil and PKF in vitro. All in
vitro effects of nicorandil and PKF were concentration-dependent.
In anesthetized pigs, intravenous and topical administration of
nicorandil and PKF decreased the frequency of ureteral contractions. This is the first time that ureteral smooth muscle relaxation by
nicorandil and PKF have been investigated in vivo.
The effects of nicorandil in smooth muscle cells have been reported to
be mediated by a K+ channel activation and a NO
donor-associated effect. However, the interactions of these two
pathways are still unclear. According to Perez-Vizcaino et al. (1998),
K+ channel opening and GC stimulation are
independent pathways that induce additive relaxation in isolated piglet
pulmonary and mesenteric arteries. Hein and Kuo (1999) investigated the
effect of adenosine on the porcine coronary arterioles. They proposed
that at low concentrations, adenosine selectively opens endothelial
KATP channels, while higher concentrations
directly stimulate the KATP channels in the
smooth muscle. In the endothelium, the activation of
KATP channels results in synthesis and release of
NO, which subsequently activates smooth muscle-soluble GC leading to
vasodilatation. The opening of smooth muscle KATP
channels causes vasodilatation without production of NO (Hein and Kuo,
1999). In the isolated superior mesenteric artery bed of the rat,
removal of NO and blockade of adenosine receptors potentiate the
vasorelaxant effects of K+ channel openers. The
modulating effect of NO seems to be mediated by cGMP (McCulloch and
Randall, 1996).
Our present findings of nicorandil-induced inhibition of the frequency
of spontaneous contractions in isolated ureter preparations confirmed
the in vitro results previously reported (Klaus et al., 1989, 1990).
Other K+ channel openers such as cromakalim and
levcromakalim have been shown to decrease ureter motility in several in
vitro studies (Klaus et al., 1989, 1990; Kontani et al., 1993b; Maggi
et al., 1994; de Moura and de Lemos, 1996). Although relaxation of
airway smooth muscle in guinea pigs has been previously reported
(Buchheit et al., 1998), this is the first investigation showing that
PKF decreased the contraction amplitude of ureteral smooth muscle in
vitro on isolated human ureter rings and contraction frequency in vivo
on ureters of anesthetized pigs. PKF was more potent and showed a
higher efficacy compared with nicorandil in vitro on isolated human
ureteral tissue and in vivo on ureters of anesthetized pigs (Table 1;
Fig. 4).
Although the effect of nicorandil and PKF were concentration- and
dose-dependent in the in vitro as well as in the in vivo experiments, a
steep fall in the frequency of contractions was seen. Nicorandil
applied to isolated porcine ureter tissue at a concentration of 3 µM
decreased the frequency of contractions only to 92% of control values
whereas a 30 µM concentration significantly decreased contractions
and almost inhibited them completely. This steep slope of the dose- or
concentration-response curve makes a classical binding of ligands to
the corresponding receptor questionable. On the other hand according to
Zhu (1993), the presence of a large fraction of spare receptors in a
tissue results in a rather steep increase in the corresponding receptor response.
Intravenous administration of PKF at lower doses induced an initial
increase in the frequency of contractions in the ureter of the
anesthetized pig. The significant decrease in the frequency of
contractions seen at higher doses indicates a mixed effect of this
substance. This mixed effect of PKF could have been missed in the in
vitro experiments on human ureter rings due to the fact that frequency
could not be evaluated using electric field stimulation. A comparison
of the calculated ED50 of nicorandil and the
calculated ED50A (suppression) of
PKF shows that PKF was more potent in reducing the frequency of
contractions than nicorandil in vivo.
Whether the effects of nicorandil and PKF on the ureters of
anesthetized pigs resulted from influencing the activity of single smooth muscle cells or a superimposed pacemaker system remains unclear.
The origin of ureteral peristalsis in intact ureters is thought to be
from a main pacemaking area in the pelvicalyceal border which has the
steepest depolarization (Dixon and Gosling, 1973; Hannappel et al.,
1982; Hanke et al., 1992; Lammers et al., 1996). Klemm et al. (1999)
thoroughly investigated the cells underlying pacemaker activity in the
guinea pig upper urinary tract. Pacemaker oscillations of the membrane
potential, possibly generated by the so called "atypical smooth
muscle cells" postulated as pacemaker cells, were recorded in both
the pelvicalyceal junction and the proximal renal pelvis but never in
the distal renal pelvis or the ureter. Furthermore, he suggested that
ICC-like cells, cells morphologically similar to the intestinal
"interstitial cells of Cajal", in the renal pelvis and the
pelvicalyceal junction play an integrative role by acting as a conduit
for the electrical signals of the pacemaker cells to the smooth muscle
cells of the renal pelvis and the ureter (Klemm et al., 1999).
Indications of spontaneously active smooth muscle cells present in the
porcine urinary tract distal to the renal pelvis or the ureteropelvic junction have been reported before (Hannappel et al., 1982). In isolated ureter rings, the spontaneous activity could originate from
solitary pacemaker cells whose own spontaneous activity would be
subordinate to the pelvicalyceal pacemaking system in the intact urinary system. Solitary pacemaker cells, or the lack of these cells in
some preparations, could explain our observation that 50% of the
isolated pig ureteral preparations did not show any spontaneous activity.
In the present study, an inhibition of the nicorandil-induced
relaxation of the ureter by MB and glibenclamide was observed in
isolated pig ureters in vitro suggesting that the activity of
nicorandil is K+ channel- and cGMP-associated.
However, MB and glibenclamide did not inhibit the nicorandil effect on
the frequency and the amplitude of ureteral contractions in vivo. This
is probably due to the fact that the concentrations reaching the intact
ureters of the anesthetized animals were not sufficient to obtain the
effect seen in vitro. Although the antagonists in all in vivo
experiments were always administered intravenously and therefore
reached the ureteral smooth muscle systemically, technical problems in
dissolving glibenclamide and MB might be a reason for not achieving
effective drug concentrations in the blood and subsequently at the
receptor site. In experiments where nicorandil and PKF were
administered topically, the effects observed on the contralateral
solvent-perfused ureter strongly suggest absorption of the drug by the
urothelium resulting in a decrease in the frequency of contractions of
the contralateral ureter.
Our findings regarding the inability of glibenclamide to antagonize the
effects of K+ channel openers in the in vivo
experiments were in accordance with the results of Kontani et al.
(1993a) who investigated the effects of glibenclamide on the ureter of
anesthetized rats.
In summary, the results of our in vitro experiments on isolated pig
ureteral tissue gave evidence of a relaxing effect of nicorandil
mediated by soluble GC and an activation of KATP
channels. In the isolated specimens of human ureters, nicorandil and
PKF decreased the amplitude of contractions. The relaxing effect of nicorandil as well as PKF could be confirmed on the ureters of anesthetized pigs, but the effect of nicorandil and PKF could not be
inhibited by glibenclamide and MB in vivo.
Based on our findings, we conclude that both nicorandil and PKF may be
promising drugs for clinical approaches such as topical administration
to relax the ureter before ureteroscopy. In comparison with nicorandil,
PKF was more potent in inhibiting contractility of ureteral smooth
muscle in vivo and in vitro. In contrast to the topical administration,
intravenous administration may be limited by the detrimental side
effects on arterial blood pressure observed in our study. Therefore,
ureteral tissue-selective KATP channel openers
without adverse cardiovascular effects would be clinically advantageous
(Jahangir et al., 2001).
We thank Merck (Dietikon, Switzerland), Novartis (Basel,
Switzerland) and Hoechst (Frankfurt, Germany) for the generous donation of drugs. We are indebted to Prof. Ulrich Quast (Tuebingen, Germany) for fruitful discussions.
This paper was supported by the Swiss National Science
Foundation (to H.D., U.E.S., G.S.).
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Abstract
Introduction
8 M] was more potent than nicorandil
[EC50 = 4.38 × 10
