EFFECTS OF CHOLINERGIC DRUGS ON URETERAL FUNCTION IN ANESTHETIZED DOGS

0022-5347/04/1724-1520/0 THE JOURNAL OF UROLOGY® Copyright © 2004 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 172, 1520 –1523, October 2004 Printed in U.S.A.

DOI: 10.1097/01.ju.0000132564.27665.fb

EFFECTS OF CHOLINERGIC DRUGS ON URETERAL FUNCTION IN ANESTHETIZED DOGS YOSHITAKA TOMIYAMA,* ISAO WANAJO, YOSHINOBU YAMAZAKI, MASAMI KOJIMA AND NOBUO SHIBATA From the Pharmacology Research and Development, Kissei Pharmaceutical Co., Ltd., Nagano, Japan

ABSTRACT

Purpose: We evaluated the effects of the nonselective muscarinic receptor agonist carbachol (CCh) and its antagonist atropine on ureteral function in anesthetized dogs. Materials and Methods: Drug effects were evaluated on elevated pressure in a completely obstructed ureter and peristalsis in its partially obstructed fellow ureters as well as on intravesical isovolumetric pressure. Results: CCh (0.1 to 1.0 ␮g/kg intravenously) dose dependently decreased elevated pressure and peristalsis in completely and partially obstructed ureters, respectively, and increased intravesical isovolumetric pressure. On the other hand, atropine (0.1 to 1.0 mg/kg intravenously) had no significant effects on these 3 variables. Prior administration of atropine (1.0 mg/kg intravenously) completely inhibited the described CCh induced effects. Conclusions: Our results demonstrate that in anesthetized dogs cholinergic receptor stimulation has a suppressive effect on ureteral pressure and peristalsis in obstructed ureters, in contrast to its activation of bladder smooth muscle. KEY WORDS: ureter, bladder, dog, cholinergic agents, cholinergic antagonists

Cholinergic nerves are known to be present in the urinary tract and known to regulate their motility. It has been observed that stimulation by cholinergic agonists induces ureteral contractions in isolated ureteral preparations1⫺6 and in anesthetized animals.7, 8 In sharp contrast, we recently found that the nonselective muscarinic agonist carbachol (CCh) decreased the tonic contraction induced by KCl in isolated ureteral preparations from dogs.9 Unfortunately there is sparse published information on the effects of cholinergic agents on ureteral function in anesthetized animals. We evaluated the effects of cholinergic drugs (CCh and the nonselective muscarinic antagonist atropine) on completely and partially obstructed ureters in anesthetized dogs. MATERIALS AND METHODS

Animals. This study conformed to current Japanese law. Male beagle dogs (Nihon Nosan Kogyo, Yokohama, Japan) weighing 8.2 to 11.2 kg were maintained under a 12-hour light-dark cycle with free access to water and standard laboratory food until the day of the experiment. Measurement of ureteral and intravesical pressures. Each dog was anesthetized with sodium pentobarbital (30 mg/kg intravenously) and artificial ventilation was maintained using a volume limited ventilator (SN-480 –3, Shinano Seisakusho, Tokyo, Japan) at 20 ml/kg and 15 strokes per minute. The dog was placed on a heating pad to minimize heat loss, and the left and right ureters were identified via a middle abdominal incision. Figure 1 shows our experimental model. Cannulas (Atom, Tokyo, Japan) (6Fr, inner diameter and length 0.67 mm and 6 cm, respectively) were inserted into the right and left lower ureters from the ureterovesical junctions to measure intraureteral pressure (IUP). The left ureteral cannula (6Fr) was connected to a T

tube. One end of this T tube was connected to a pressure transducer (Biotrans, GE Marquette Medical Systems, Tokyo, Japan) and measuring system (AP-601G and RPM6008, Nihon Kohden, Tokyo, Japan). The other end was attached to the 27 gauge needle and a PE20 cannula (Becton Dickinson, Parsippany, New Jersey) (inner diameter and length 0.38 mm and 40 cm, respectively) to create partial obstruction. Urine overflowed through the PE20 cannula. The urethra between the bladder and prostate was ligated, as were the 2 ureterovesical junctions. A cannula (6 Fr) was inserted into the bladder body to measure intravesical isovolumetric pressure (IVP). Initial bladder pressure was adjusted to about 3.5 mm Hg by warm saline instillation to create the partially filled condition. The free ends of the right ureteral and bladder cannulas were each directly connected to the pressure transducer. In this way the right and left ureters became a completely obstructed ureter (COU) and a partially obstructed ureter (POU), respectively. Continuously maintained ureteral peristalsis was observed in the POU. To examine the effects of drugs on ureteral peristalsis we used the IUP record for the POU. The effects of a given drug on ureteral peristalsis were evaluated by summing the total amplitude of all contractions occurring during a 1-minute period (mm Hg ⫻ contractions per minute). The right femoral artery was cannulated and arterial blood pressure (BP) was recorded via a pressure transducer and measuring system as described, with heart rate (HR) determined from the arterial pulse wave by a cardiotachometer (AT-601G, Nihon Kohden). IUP, IVP, BP and HR were recorded on a thermowriting rectigraph (WS-681G, Nihon Kohden). Experimental protocol. When IUP in the COU had increased to a plateau, drug solution (various concentrations) was injected intravenously every 15 minutes. IUP, IVP, BP and HR were recorded for 60 minutes after the first drug administration. Drugs. The drugs used were CCh, atropine hemisulfate (Sigma Chemical Co., St. Louis, Missouri), sodium pentobarbital (Dainippon Pharmaceutical Co., Ltd., Osaka, Japan)

Accepted for publication April 16, 2004. Study guidelines received institutional laboratory animal committee approval. * Correspondence: Pharmacology Research and Development, Kissei Pharmaceutical Co., Ltd., 4365–1, Kashiwabara, Hotaka, Nagano 3998304, Japan (telephone: ⫹81–263-82– 8820; FAX: ⫹81–263-81–1045; e-mail: yoshitaka [email protected]). 1520

CHOLINERGIC DRUGS AND URETERAL FUNCTION

FIG. 1. Ureteral obstruction model in anesthetized dog. One end of T tube was attached to 27 gauge needle and PE20 cannula to create partial obstruction in left ureter. Initial bladder pressure was adjusted to about 3.5 mm Hg by warm saline instillation to create partially filled condition.

and sodium heparin (Aventis Pharma Japan, Tokyo, Japan). CCh and atropine were dissolved and diluted in saline. Data analyses. All results are expressed as the mean ⫾ SEM. Data were analyzed using Dunnett’s multiple comparison test to compare pre-drug and post-drug administration values with p ⬍0.05 considered statistically significant. RESULTS

In terms of the levels of IUP, ureteral peristalsis, IVP, mean BP (MBP) and HR recorded before drug administration there were no significant differences between the CCh and atropine treated groups (see table). In our preliminary experiments CCh and atropine had hardly any effects on unobstructed ureteral pressure or peristalsis in anesthetized dogs. Therefore, we evaluated the effects of these drugs on COU and POU. Effects of CCh on IUP and IVP. Figure 2 shows typical IUP tracings in COU and POU as well as IVP after intravenous administration of 1.0 ␮g/kg CCh in an anesthetized dog. Group data indicated that CCh dose dependently decreased elevated IUP in COU (fig. 3, A). Although the highest dose of CCh (1.0 ␮g/kg) produced increased peristaltic amplitude immediately after intravenous administration in POU (fig. 2), CCh (0.1 to 1.0 ␮g/kg intravenously) induced dose related transient inhibition of the sum of peristalsis (amplitude ⫻ frequency) in POU (fig. 3, B) and decrease in MBP (fig. 3, D). On the other hand, CCh increased IVP in a dose dependent manner (fig. 3, C). CCh decreased HR slightly but the effect was not significant vs the pre-administration value (fig. 3, E). Effects of atropine on IUP and IVP. Figure 4 shows typical IUP tracings in COU and POU as well as IVP after intravenous administration of 1.0 mg/kg atropine in an anesthetized dog. Group data revealed that the lowest dose of atropine (0.1 mg/kg intravenously) tended to induce slight decreases in IUP in COU and ureteral peristalsis (fig. 5, A and B), while the highest dose (1.0 mg/kg intravenously) tended slightly to decrease IVP, MBP and HR (fig. 5, C to E). However, no significant differences were noted vs pre-administration values. Intravenous administration of 1.0 mg/kg atropine comData before drug administration in 4 experiments

IUP (COU) (mmHg) Ureteral peristalsis (POU) (mm Hg ⫻ contractions/min) IVP (mm Hg) MBP (mm Hg) HR (beats/min)

Mean CCh Group ⫾ SEM

Mean Atropine Group ⫾ SEM

61.8 ⫾ 5.3 416.5 ⫾ 80.5

50.2 ⫾ 7.5 350.9 ⫾ 73.5

3.0 ⫾ 1.2 149.6 ⫾ 5.7 138.5 ⫾ 7.2

4.4 ⫾ 0.95 133.8 ⫾ 10.9 151.0 ⫾ 18.5

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FIG. 2. Typical tracings of effects of 1.0 ␮g/kg CCh intravenously on IUP in COU, IUP in POU and IVP in anesthetized dog. Arrowhead indicates drug injection.

pletely inhibited CCh (1.0 ␮g/kg intravenously) induced changes in all parameters (fig. 5). DISCUSSION

It has been shown that cholinergic stimulation induces contraction in canine isolated detrusor smooth muscle10 and also that it increases IVP in the anesthetized dog.11 The effects of cholinergic drugs have been commonly observed in the isolated ureter.1⫺6, 9 However, there are only a few reports of the effects of cholinergic drugs on ureteral function in in vivo experiments.7, 8 In this study we examined the effects induced by intravenous CCh and atropine administration on partially and completely obstructed ureters in the anesthetized dog. Effects of CCh. CCh induced responses in the urinary tract and cardiovascular system were completely blocked by prior administration of atropine (1.0 mg/kg intravenously) in this study. Therefore, they were produced via muscarinic stimulation. In this study we found that intravenous administration of CCh dose dependently decreased elevated IUP in COU and peristalsis in POU in anesthetized dogs (fig. 3). Our observations were almost in agreement with the report that parasympathetic nerve stimulations produced a decrease in ureteral peristaltic activities in anesthetized cats.7 We have previously reported that cholinergic stimulation induces relaxation, mainly via the M4 receptor, on tonic contraction induced by KCl in segments of the longitudinal muscle layer isolated from sites throughout the entire length of the canine ureter.9 Therefore, it seems highly likely that in the anesthetized dog the observed CCh induced suppression of elevated IUP in COU and peristalsis in POU were mediated via the stimulation of M4 receptors on ureteral smooth muscle. Previously we have noted that in the spiral smooth muscle layer of the canine ureter cholinergic stimulation enhanced spontaneous rhythmic contractions via the M3 receptor.9 However, this response was observed only in the lower ureter near the ureterovesical junction, where the cholinergic innervation is by far the richest in the whole ureter. The highest dose of CCh (1.0 ␮g/kg) tended to increase slightly the amplitude of peristalsis immediately after intravenous administration in POU (fig. 2). This observation was almost the same as that in the report showing that acetylcholine produced enhanced ureteral peristalsis within 1 minute after intravenous administration in anesthetized cats.7 However, intravenous administration of CCh continuously produced neither a further increase in elevated IUP in COU, nor enhanced peristalsis (amplitude ⫻ frequency) in POU (figs. 2 and 3). If M3 receptors, which mediate ureteral contractions,

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CHOLINERGIC DRUGS AND URETERAL FUNCTION

FIG. 3. Effects of intravenous injection of increasing doses of CCh (circles) on IUP in COU (A), peristalsis in POU (B), IVP (C), MBP (D) and HR (E) in anesthetized dog. Data represent mean ⫾ SEM of 4 experiments. Asterisk indicates p ⬍0.05 vs pre-administration value.

FIG. 4. Typical tracings of effects of 1.0 mg/kg atropine intravenously on IUP in COU, IUP in POU and IVP in anesthetized dog. Arrowhead indicates drug injection.

are indeed limited to the lower part of the canine ureter, this may be why cholinergic stimulation hardly increased ureteral motility in our anesthetized dogs. It is well known that in the bladder of many species cholinergic stimulation induces detrusor contraction mainly via M3 receptors, which cause an increase in the intracellular Ca2⫹ concentration, and partly via M2 receptors, which decrease the intracellular level of cyclic adenosine monophosphate, a substance that produces relaxation of isolated blad-

FIG. 5. Effects of intravenous injection of increasing doses of atropine (triangles) and 1.0 ␮g/kg CCh intravenously on IUP in COU (A), peristalsis in POU (B), IVP (C), MBP (D) and HR (E) in anesthetized dog. Data represent mean ⫾ SEM of 4 experiments.

der smooth muscle.12 The nonselective muscarinic agonist bethanechol reportedly increases IVP in the anesthetized dog.11 In this study we observed that intravenous administration of CCh also increases IVP, again in the anesthetized dog. To judge from these data the CCh induced elevation in IVP that we observed was probably mediated by the stimulation of M3 and M2 receptors. In our anesthetized dogs intravenous administration of CCh dose dependently and strongly decreased MBP, probably due to vascular relaxation mediated by M3 receptor stimulation, and HR, probably mediated by a suppressive effect on the atria via M2 receptor stimulation).13 We could not evaluate the urinary tract effects of CCh at more than 1.0 ␮g/kg because it had powerful cardiovascular effects, as noted in our preliminary study. Effects of atropine. Atropine has no direct effects on canine isolated ureteral smooth muscle.9 In the current study intravenous atropine had no significant effects on elevated IUP in COU or on ureteral peristalsis in the anesthetized dog. This is in agreement with a recent report that intravenous atropine had no effect on ureteral function in conscious pigs.14 It is possible that the acetylcholine release from parasympathetic nerves was low or cholinesterase activity was high in the smooth muscle of the obstructed ureter in our anesthetized dogs. Atropine neither relaxes the detrusor muscle in vitro15 nor significantly lowers IVP, as shown in the current study. It may be that atropine decreases micturition pressure by antagonizing micturition stimuli arriving through the pelvic nerve, leading to augmented urine retention, but when bladder pressure is below a threshold pressure (the collecting phase for urine), this drug has no effect on IVP.

CHOLINERGIC DRUGS AND URETERAL FUNCTION

It is important to measure IUP and IVP under the drained bladder condition and confirm the relationship between the ureter and bladder in motility because it is possible that IVP affects IUP. Therefore, we require further investigation to observe the relationship between the ureter and bladder in terms of motility using advanced animal models. CONCLUSIONS

We report that intravenous administration of CCh decreased elevated IUP in COU and ureteral peristalsis in POU in the anesthetized dog. Thus, when ureteral motility is increased by obstruction, cholinergic stimulation has a suppressive effect on the obstructed ureter, in contrast to its activation of the bladder, at least in the anesthetized dog. Parasympathetic nerve stimulation produced contractions on detrusor to void urine11 and also on intravesical ureter to prevent vesicoureteral reflex in the voiding phase of the bladder.1, 4 Since it was highly possible that parasympathetic nerve stimulation suppressed ureteral motility to decrease the ureteral pressure and prevent urine transport through the ureter in the voiding phase of the bladder, it probably produced protection in the ureteral tissues.

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