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Keywords:

  • Ureter;
  • bioassay;
  • comparison;
  • contraction

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Objective

To determine the optimal contractile response of isolated ureters to inflammatory mediators and neurotransmitters by evaluating four common methods of ureteric suspension.

Materials and methods

Ureters from adult domestic swine were placed immediately in Krebs buffer and cut into 4–5 mm segments. Four methods of suspension were compared: (i) an unopened ring segment suspended horizontally; (ii) a spirally cut segment (lumen cut open at a 45° and suspended end-to-end); (iii) an open longitudinal segment; and (iv) a closed longitudinal segment. All segments were placed in individual water-jacketed tissue baths containing Krebs buffer, the frequency of contraction measured using a force transducer and registered on a polygraph. The sensitivity of all four segments was tested by measuring the tension and frequency in response to increasing frequencies of electric field stimulation, and by a cumulative concentration–response curve to carbachol.

Results

Ureteric segments responded with an increased frequency of contraction depending on the intensity of stimulus to both electric field stimulation and carbachol. However, there were no significant differences in spontaneous levels of contraction, sensitivity or maximal response among the methods of suspension in response to electric field stimulation or carbachol.

Conclusion

These results indicate that all four methods of suspending the ureteric segments produce contractile responses sensitive enough to study the action of various neurotransmitters.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The response of isolated tissue is a useful method to investigate the role of inflammatory mediators and neurotransmitters in the physiology of various animal and human tissues [1]. The increased availability of human ureteric segments from surgery has enabled in vitro investigation of many of the major neurotransmitters and their receptors. However, sufficient human ureteric segments are not always available to allow their suspension as classical intact segments. In addition, the variability of the methods of suspension among different laboratories often invalidates the comparison of data.

To study ureteric responses to inflammatory mediators and neurotransmitters, the sensitivity of the various methods of ureteric suspension must be determined. Traditionally, ureteric segments have been suspended as horizontal rings or longitudinal strips [2[3][4][5][6]–7]. The musculature has been believed to be in a single layer, arranged in a tight helix, becoming more longitudinal as the ureter enters the bladder [8]. Recently, other investigators have described the musculature as a mesh-like structure, with muscular bundles orientated in varying directions [9[10]–11]. Similarly, it has been reported that muscular bundles occur in two layers, an outer layer of circular bundles and an inner layer of longitudinal bundles [12]. Morita et al. [13] investigated the anatomical structure of the smooth muscle layers of the midportion of the canine ureter. By studying transverse sections, they re-affirmed that the outer muscular bundles are orientated in a circular fashion while the inner bundles are orientated longitudinally. Based on these findings, they have used a spiral-cut segment of ureter in their tissue-bath assays, suggesting that this segment preserves more continuous muscular bundles and therefore can generate increased spontaneous contraction [13,14]. Unfortunately, the availability of long, viable human ureteric segments for use in research is limited and often precludes the use of spiral-cut segments. Typically, the segment would be suspended longitudinally in these cases. The purpose of the present study was to determine the responsiveness of isolated ureter to electric field stimulation (EFS) and a muscarinic agonist (carbachol) when suspended as a ring, spiral or longitudinal segment.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Preparation of ureteric segments

Segments of ureter were obtained from freshly killed domestic swine. The entire urinary tract was removed and placed immediately in aerated (95% O2 and 5% CO2 ) Krebs buffer (pH 7.4) of the following composition (mmol/L): NaCl, 119; NaH2PO4 , 1; KCl, 4.7; CaCl2 , 2.5; MgCl2 , 0.5; NaHCO3 , 25; and glucose, 25. The ureters were then isolated and the peri-ureteric fat removed; a mid-ureteric segment was isolated and divided into 4–5 mm long segments. Ureteric segments that were tested as rings were suspended between two stainless-steel stirrups (Fig. 1). The spiral segment was cut through the lumen at 45° until the lumen was completely opened. The ends of the segment were tied with thread to a force transducer and tissue holder, respectively. Longitudinal segments were suspended in two ways: the lumen was cut open and the segment suspended by opposite corners, or the lumen was left closed and the segment suspended by piercing opposite corners of the ureteric wall and tying thread through the piercing. The ends of the segments were tied with thread to a force transducer and tissue holder. All segments were kept in Krebs solution at a constant 37°C while being cut and prepared. All segments were suspended in water-jacketed tissue baths containing aerated Krebs buffer (37°C) between platinum-plate electrodes connected to a Grass S48 electric stimulator (Grass Instruments, Quincy, MA, USA). The tissues were attached to force displacement transducers (Grass FT-O3) and changes in tension displayed on polygraphs (Grass Model 79). The initial tension was set at 4 g, based on preliminary experiments (Table 1). Tissues were allowed to equilibrate for 1 h, during which the tissue bath content was replaced with fresh buffer and the tension re-adjusted every 15 min. Baseline tension was maintained at a consistent level throughout the experiment. The initial frequency (spontaneous levels) of contraction was identified after 1 h of equilibration.

image

Figure 1. The four different methods of ureteric segment cuts and suspension used. The figures on the left illustrate the cut of the segments and those on the right illustrate their suspension.

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Table 1.  Preliminary data to determine optimal initial tension applied to the ureter to promote contraction. The values expressed are the mean (sem) ureteric contractions per 5 min when various initial tensions were applied. There was no significant difference in spontaneous contraction rate among any of the preparations. However, there was a trend in longitudinal segments suggesting that 4 g of tension was optimal. The results from all three preparations suggest that increasing initial tension beyond 4 g reduced the contraction rate Thumbnail image of

Responsiveness of ureteric segments

All tissues were stimulated with square EFS pulses at 10 V, 3 ms duration and 1–64 Hz; the contraction effects were registered on the polygraph. The frequency of stimulation was increased in geometric increments to 64 Hz. Frequency was not increased beyond 64 Hz because this proved fatal to the tissues in preliminary experiments. After completing the frequency-response curve, the buffer in all tissue baths was replaced with fresh Krebs and a basal level of contraction measured. A concentration–response curve to carbamylcholine chloride (carbachol, Sigma Chemical Co., St Louis, MO, USA) was then obtained by cumulative exposure to this agonist [15]. The concentration–response curve began with 0.1 nmol/L carbachol and was terminated with the addition of 1 mmol/L carbachol, because the frequency of contraction decreased at this concentration. All tissues were treated with 1 mmol/L BaCl2 (Sigma Chemical Co.) to obtain the maximum contractile response.

After each stimulus, the frequency of contraction was evaluated every 5 min until it stabilized; the maximum contraction rate to each stimulus was identified. To normalize variances between tissues, the responses to the frequency-response and concentration–response curve are expressed as the percentage increase over the spontaneous or basal levels. The effective frequency of EFS that produced half the maximal effect (EF50 ) and the effective concentration of carbachol that produced half the maximal effect (EC50 ) were determined. All values are presented as the mean (sem) and were compared using the Kruskall–Wallis nonparametric analysis of variance [16]; differences where P<0.05 were declared statistically significantly different.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

All segments showed an increasing frequency of contraction that was dependent on the intensity of the stimulus, as illustrated by a representative polygraph tracing of the contractile response of a ureteric segment to EFS (Fig. 2). The number of bursts of contraction within the period did not increase, but the duration of the burst and number of contractions within the burst increased with increasing intensity. The increase in the duration of the burst did not necessarily correlate with the increase in contractions within the burst. Also, at high levels of stimulation, the duration of the burst often superseded the silent period between bursts, and therefore precluded determining the duration of the burst. Thus the number of contractions within all the bursts in a 5-min period were counted.

image

Figure 2. A typical polygraph tracing representing the 32 total ureters used in this study, measuring contractions in ureteric segments when stimulated with EFS. There was spontaneous contraction after a 1-h equilibration period. The tissues were then exposed to increased frequency of EFS as indicated. No significant change occurred in the strength of the contractions.

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All ureteric segments used showed spontaneous levels of contraction (Table 2) but there was no significant difference in the spontaneous level of contraction between the different methods of suspension (P=0.96). All ureteric segments responded to EFS with an increased rate of contraction dependent on the intensity of the frequency of stimulation. There was no significant difference between the responses of the preparations to EFS when measured as the absolute increase in frequency of contraction (Fig. 3a), maximum response to EFS or the EF50 (P=0.41; Table 2). All ureteric segments showed a basal contraction rate after the termination of the frequency-response curve to EFS (Table 2), but there was no significant difference in the basal level of contraction among the different methods of suspension (P=0.76).

Table 2.  Response of ureteric segments to EFS or stimulation with carbachol. There was no significant difference among the four methods of suspension in spontaneous or basal contraction rate, maximal contraction rate, EF50 or EC50 , as indicated by the Kruskall–Wallis P value. Values are mean (sem) for eight replicates Thumbnail image of
image

Figure 3. The a, frequency-response and b, concentration–response curves of isolated ureteric segments suspended by the four methods (longitudinal open cut, dark green; longitudinal closed, light green; ring, light red; spiral, dark red) when stimulated with a, EFS and b, carbachol. Values are expressed as a percentage above spontaneous levels to normalize variability between ureters. Error bars indicate the sem; there was no significant difference among the four methods of suspension (n=8).

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All segments showed increased contraction in response to carbachol, similar to that occurring in response to EFS. There was no significant difference between the responses of the preparations when measured as an absolute increase in frequency of contraction (Fig. 3b), maximum response to carbachol or the EF50 (Table 2). The maximal contractions per minute in response to BaCl2 were 16.2 (1.2), 16.4 (2.0), 15.9 (1.7), and 16.9 (2.4) for ring, spiral, closed longitudinal and open longitudinal segments, respectively, indicating that the different preparations presented similar maximal frequencies.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

These results indicate there is no significant difference in sensitivity, maximal response or spontaneous levels of contraction among the various methods of suspending the ureteric segments when subjected to EFS or carbachol. As EFS induces contraction through the release of various mediators from the tissue and carbachol stimulates contraction via muscarinic receptors, all four suspension methods are suitable for analysing ureteric responses to a variety of stimuli. These results are in contrast to a previous report by Morita et al. [13] who, using canine ureter, showed that spiral segments were more responsive to noradrenaline than were longitudinal or ring segments. In addition, that report indicated that ring and longitudinal segments had very low levels of spontaneous contraction, caused by disruption of the muscular bundles, which is avoided using the spiral-cut method. The present results indicate that, in swine ureter, all four preparations showed spontaneous contraction, and responded equally well to EFS and carbachol. Similarly, previous experiments from our laboratory have shown that segments of human ureter suspended as rings show spontaneous contraction and respond to neurotransmitters [17].

Other investigators have used longitudinal or ring suspensions to determine ureteric response to various stimuli. Sahin et al. [2], using human ureters suspended as rings, investigated the effect of nifedipine and verapamil on contraction and found spontaneous contractility that was terminated by the addition of the calcium antagonists. Triguero et al. [3] used sheep ureters suspended as longitudinal strips to study the effect of the nitric oxide synthase (NOs) inhibitor NG-nitro-l-arginine and determined that NOs inhibitors did not change the activity of the ureteric strips. Similarly, Hertle et al. [7] investigated human megaureters suspended as longitudinal strips and reported megaureters to be more sensitive to noradrenaline than normal ureters. In addition, Angelo-Khattar et al. [4] used human ureter suspended as rings or longitudinal strips and reported the presence of spontaneous contractility that was terminated with indomethacin. Potenzoni et al. [18] identified the presence of spontaneous contraction in human ureter suspended longitudinally while investigating the effect of rociverine on ureteric contractility, and found this anticholinergic to inhibit contraction.

Another factor when evaluating ureteric responses is the species of the ureter. The reports mentioned used human or sheep ureters to obtain spontaneous contractility. The present results indicate that swine ureters also show spontaneous contractility, in contrast to the work of Morita et al. [13] with canine ureter. This may suggest that the swine ureter provides a better model for the study of ureteric physiology than the canine ureter. Comparative studies have shown swine and sheep ureters to have more similarities histologically to human ureter than to the canine ureter [19].

In summary, the present results indicate that the contractile responses to EFS and carbachol are predictable and quantifiable using isolated swine ureter, regardless of the method of suspension. These results will enable investigators to perform contraction studies using limited ureteric segments with no need for spiral segments. Further studies in human and canine ureters are indicated.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Ureter specimens were provided by Robert D. Neubauer and Manuel Viedma of Johnsonville Foods Inc. (Watertown, WI 53094, USA).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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