**3.2. Gastro-intestinal tract**

#### *Angiotensin II*

126 Neuroendocrinology and Behavior

*Ghrelin* 

of AVP action on smooth muscle contractions.

contractions mediated by these peptides.

*Does Ghrelin have an effect on a urinary bladder?* 

muscle cells of the rat bladder a similar effect takes place.

due to the manifestation of this mechanism (Tolekova et al., 2010).

muscle membrane may contribute additionally to the differences in the computed parameters and shape of the contraction graphic. There are existing data about the effect of AVP on the potassium channels of brain cells after fluid percussion brain injury indicating that AVP inhibited activity of the KATP and KCa channels (Armstead, 2001). It is known that the urinary bladder smooth muscle cells have a number of potassium channels, including ATP-dependent K channels and Ca2+-dependent K channels (Petkov et al., 2001). Interaction with those channels could be a possible explanation for the prolonged duration

On the other hand, Ang II stimulates the activity of L/T-type voltage dependent calcium channels in vascular smooth muscle cells (Lu et al., 1996). We can suggest that in the smooth

The increase of concentration of the extracellular Ca2+ exerts a synergistic effect on Ang IIand AVP-mediated contractions. The raise of the amplitude of contraction is a consequence of increased transmembrane calcium influx due to the higher electrochemical gradient. As a result the intracellular calcium concentration is maintained at the higher level than the level of the resting state. There is evidence that this pattern of variations in calcium concentration contributes to the development of the mechanism of "locking" of the smooth muscle cells (Tanaka et al., 2008). We suppose that the above mentioned significant difference in AUC is

The endocrine effects of the peptide ghrelin on various organs and systems are not well examined; however it is known that it stimulates the motility of digestive tract (Tack et al., 2006). On the vascular smooth muscle it exercises a dilatatory influence which is comparable to that caused by adrenomeduline (Wiley & Davenport, 2002). Binding of ghrelin to the its membrane receptors in some tissues triggers signal transduction mechanism via Gq protein and results in activation of PLC and release of IP3 and Ca2+ (Davenport et al., 2005). There are no data in the literature regarding the effects of ghrelin on urinary bladder smooth muscle. The presence of ghrelin receptors on the membranes of detrusor smooth muscle cells is not proven yet. Therefore it is interesting to investigate whether and how ghrelin affects the bladder detrusor and if so by which signal transduction mechanism. Moreover, there is not existing published comparison between the effects of AVP and Ang II on detrusor contractile activity as well as effects of calcium and ghrelin on the smooth-muscle

The receptors for ghrelin described in the literature mediate their activity with activation of PLC and subsequent increase in concentration of intracellular calcium (Davenport et al., 2005). Therefore, the application of ghrelin on muscle strips of urinary bladder would lead to the occurrence of tonic contractions. During the experiments we found no statistically significant changes in contractile activity after application of ghrelin as compared to the

*Role of extracellular calcium for Ang II- and AVP-mediated contractions of smooth muscle cells* 

Angiotensin II has potent contractile effect on smooth muscles in the gastro-intestinal tract (GIT). The question for the exact effects of Ang II on GIT remains still opened. Local RAS or parts of it had been found in rat rectum (De Godoy et al., 2006), rat small intestine, and in the guinea pig gall bladder (Leung et al., 1993). The role of Ang II had been confirmed in the development of diseases such as the gastro-esophageal reflux (Fändriks, 2010), incontinence of internal anal sphincter (De Godoy et al., 2006; Rattan et al., 2003), and Crohn's disease (Fändriks, 2010; Wang et al, 1993) as well as other inflammatory and motility disorders of the GIT (Fändriks, 2010).

Most of the effects of Ang II concerning the smooth muscle contractile activity of GIT are associated with AT1 receptors (Fändriks, 2010; Fan et al., 2002; Hawcock & Barnes, 1993; Rattan et al., 2003). AT2 receptors are also described in GIT (Fändriks, 2010; Fan et al., 2002; De Godoy et al., 2006; Hawcock & Barnes, 1993; Ewert et al., 2003; Leung et al., 1993; De Godoy et al., 2002). Although different signaling pathways have been assumed, for example activation of various phosphatases, cGMP -NO system etc. (Ewert et al., 2003; Dinh et al., 2001), their actual signal transduction is not quite elucidated. AT2 receptors are associated with the exchange of water and salts, sodium hydrogen carbonate secretion in the duodenum (Fändriks, 2010) and the secretion of nitric oxide in pig's jejunum (Ewert et al., 2003). The significance of AT2 receptors for GIT motility has not been established yet. It is supposed that they have the opposite effect of AT1 receptors (Gallinat et al., 2000), but as a factor for the smooth muscle relaxation they had been proved only for the internal anal sphincter (De Godoy et al., 2006; De Godoy & Rattan, 2005).

The Effects of Some Neuropeptides on Motor Activity of Smooth Muscle Organs in Abdominal and Pelvic Cavities 129

**Figure 3.** Normalized time-parameters of contractile activity of different GIT segments, induced by

the muscle response to Ang II along the rat intestine, which confirms previous studies of Ang II - provoked intestinal contractions (Ewert et al., 2006). In the literature there are evidences about the uneven distribution of the Ang II receptors in most tissues of the adult organism (Steckelings et al., 2010). Regarding GIT there has been described an unequal density of AT1 receptors (Fändriks, 2010), which could explain the obtained results. From the other side, the duodenal contraction has the smallest amplitude, which strongly differentiates the reaction of the duodenum from the other GIT segments. This could be due to low density of duodenal Ang II receptors and with a local production of NO by the

There had been established several transduction pathways of Ang II- induced SMC (Dinh et al., 2001; Romero et al., 1998). The modulating effect of Ang II on different ion currents also had been reported (Chorvatova et al., 1996; Romero et al., 1998). According to Thc and Tc parameters, there is a marked difference between the stomach from the one side, and the intestines from the other. All of the studied intestinal segments showed similar speed of contraction, while the duration of the stomach reaction was far longer. That data suggest a possible transduction pathway for SMC of the stomach, different than in the others GIT

Some authors consider possible competitive interactions between AT1 and AT2 receptors in smooth musculature of the intestine, which supports some previous statements that the

Angiotensin II. 1-Thcn, 2-T(c-hc)n and Thrn.

duodenal mucosa (Aihara et al., 2005).

segments.

There is not enough information in the literature, regarding to the comparative characteristics of Ang II - induced responses from the various segments of GIT. Dose dependent curves, which are commonly used as a method for studying the provoked smooth muscle contractions (Fändriks, 2010; Fan et al., 2002; Hawcock & Barnes, 1993; Leung et al., 1993; Park et al., 1973), could give information about the effective doses and maximal responses, but not a data for other important characteristics of the smooth muscle contractions. The different phases of the contraction in the various segments of the GIT, were not clarified and analyzed by application of a time-parameter analysis, as it was made in the study of the skeletal muscle contraction (Raikova & Aladjov, 2004). For comparison and detailed analysis of Ang II contractile effects of Ang II on the different segments of longitudinal strips from rat GIT we are using again time-parameters.

The amplitudes and integral muscle force of different segments from GIT in our experimental study showed marked correlation (r=0.88, p<0.005). The duodenal muscle strip demonstrated the lowest amplitude and smallest integral force of contraction - 0.55±0.13 g, 41.43±15.52 gs. The amplitudes of the registered angiotensin II-induced contraction from stomach (1.14±0.13 g), jejunum (1.11±0.14 g) and ileum (1.09±0.16 g) are similar and there are not statistically significant differences between them. But stomach integral force (178.09±19.63 gs) is significantly greater than that of duodenum and other intestines and is equally powerful as that of the colon. Under influence of Angiotensin II, rectum developed highest amplitude of contraction 4.74±0.65 g and most powerful integral force - 328.43±75.23 gs.

The analysis of time parameters of Ang II-mediated contractions indicated that the gastric response to Ang II required more time to develop: the time to reach Thc and Tc parameters was 29.09± 2.53 s and 78.18 ± 5.87 s respectively. This tendency for a slower progress of the reaction was maintained during the whole contraction of the stomach and its Tchr was 224.90 ± 18.45s. All of the registered intestinal contractions showed similar values for Thc and Tc parameters. For the remaining two time - parameters - Thr and Tchr, the results from the intestinal contractions were analogous, with exception of the ileac contraction, which Thr (106.33±9.89 s) and Tchr (141.08±9.48 s) were significantly prolonged. After normalization of time-parameter, it was shown that jejunum and colon have similar pattern of contractions and relaxation (Figure 3). The relaxation takes one half of the process and the first and the second part of the contraction are with almost identical proportion, The other parts of GIT stomach, duodenum and rectum have almost similar pattern of contractions and relaxations. Only ilium differs from these two groups. The relative part of its relaxation was 0.75 from whole duration of process. Application of the time parameters clearly shows the presence of bilateral symmetry in the responses of the gastrointestinal tract.

The amplitude comparison of the Ang II-induced contractions divides the isolated smooth muscle preparations into two groups. The stomach and the small intestines form one group, and the large intestines form another. It is obvious that the large intestines are more sensitive to Ang II and react with more powerful contractions. There is a gradual increase in

factor for the smooth muscle relaxation they had been proved only for the internal anal

There is not enough information in the literature, regarding to the comparative characteristics of Ang II - induced responses from the various segments of GIT. Dose dependent curves, which are commonly used as a method for studying the provoked smooth muscle contractions (Fändriks, 2010; Fan et al., 2002; Hawcock & Barnes, 1993; Leung et al., 1993; Park et al., 1973), could give information about the effective doses and maximal responses, but not a data for other important characteristics of the smooth muscle contractions. The different phases of the contraction in the various segments of the GIT, were not clarified and analyzed by application of a time-parameter analysis, as it was made in the study of the skeletal muscle contraction (Raikova & Aladjov, 2004). For comparison and detailed analysis of Ang II contractile effects of Ang II on the different segments of

The amplitudes and integral muscle force of different segments from GIT in our experimental study showed marked correlation (r=0.88, p<0.005). The duodenal muscle strip demonstrated the lowest amplitude and smallest integral force of contraction - 0.55±0.13 g, 41.43±15.52 gs. The amplitudes of the registered angiotensin II-induced contraction from stomach (1.14±0.13 g), jejunum (1.11±0.14 g) and ileum (1.09±0.16 g) are similar and there are not statistically significant differences between them. But stomach integral force (178.09±19.63 gs) is significantly greater than that of duodenum and other intestines and is equally powerful as that of the colon. Under influence of Angiotensin II, rectum developed highest amplitude of

The analysis of time parameters of Ang II-mediated contractions indicated that the gastric response to Ang II required more time to develop: the time to reach Thc and Tc parameters was 29.09± 2.53 s and 78.18 ± 5.87 s respectively. This tendency for a slower progress of the reaction was maintained during the whole contraction of the stomach and its Tchr was 224.90 ± 18.45s. All of the registered intestinal contractions showed similar values for Thc and Tc parameters. For the remaining two time - parameters - Thr and Tchr, the results from the intestinal contractions were analogous, with exception of the ileac contraction, which Thr (106.33±9.89 s) and Tchr (141.08±9.48 s) were significantly prolonged. After normalization of time-parameter, it was shown that jejunum and colon have similar pattern of contractions and relaxation (Figure 3). The relaxation takes one half of the process and the first and the second part of the contraction are with almost identical proportion, The other parts of GIT stomach, duodenum and rectum have almost similar pattern of contractions and relaxations. Only ilium differs from these two groups. The relative part of its relaxation was 0.75 from whole duration of process. Application of the time parameters clearly shows the presence of

The amplitude comparison of the Ang II-induced contractions divides the isolated smooth muscle preparations into two groups. The stomach and the small intestines form one group, and the large intestines form another. It is obvious that the large intestines are more sensitive to Ang II and react with more powerful contractions. There is a gradual increase in

sphincter (De Godoy et al., 2006; De Godoy & Rattan, 2005).

longitudinal strips from rat GIT we are using again time-parameters.

contraction 4.74±0.65 g and most powerful integral force - 328.43±75.23 gs.

bilateral symmetry in the responses of the gastrointestinal tract.

**Figure 3.** Normalized time-parameters of contractile activity of different GIT segments, induced by Angiotensin II. 1-Thcn, 2-T(c-hc)n and Thrn.

the muscle response to Ang II along the rat intestine, which confirms previous studies of Ang II - provoked intestinal contractions (Ewert et al., 2006). In the literature there are evidences about the uneven distribution of the Ang II receptors in most tissues of the adult organism (Steckelings et al., 2010). Regarding GIT there has been described an unequal density of AT1 receptors (Fändriks, 2010), which could explain the obtained results. From the other side, the duodenal contraction has the smallest amplitude, which strongly differentiates the reaction of the duodenum from the other GIT segments. This could be due to low density of duodenal Ang II receptors and with a local production of NO by the duodenal mucosa (Aihara et al., 2005).

There had been established several transduction pathways of Ang II- induced SMC (Dinh et al., 2001; Romero et al., 1998). The modulating effect of Ang II on different ion currents also had been reported (Chorvatova et al., 1996; Romero et al., 1998). According to Thc and Tc parameters, there is a marked difference between the stomach from the one side, and the intestines from the other. All of the studied intestinal segments showed similar speed of contraction, while the duration of the stomach reaction was far longer. That data suggest a possible transduction pathway for SMC of the stomach, different than in the others GIT segments.

Some authors consider possible competitive interactions between AT1 and AT2 receptors in smooth musculature of the intestine, which supports some previous statements that the magnitude of the response to Ang II depends on the expression of both receptors (Ewert et al., 2006). It had been demonstrated that only AT1 receptors are relevant for the maximum response in ANG-induced contractions (Hawcock & Barnes, 1993; Fändriks, 2010; Fan et al., 2002; Rattan et al., 2003; Ewert et al., 2006; Fändriks, 2010). Despite the existing assumptions that stimulation of AT2 receptors may have the opposite effect than that of AT1 (Gallinat et al., 2000), the role of AT2 receptors for the relaxation phase of the SMC in GIT is not examined. The importance of AT2 receptors for the relaxation of the rectum has been described only (De Godoy et al., 2006; De Godoy & Rattan, 2005). Regarding the time parameters for relaxation, the stomach again showed the slowest response. In this case the ileum indicated significantly prolonged reaction compared to the other intestinal segments. The reason for that difference may be the complete absence or the low density of AT 2 receptors in the ileum (Fändriks, 2010).

The Effects of Some Neuropeptides on Motor Activity of Smooth Muscle Organs in Abdominal and Pelvic Cavities 131

contraction. The higher values of the absolute and normalized time – parameters for this interval are the evidence. The difference in the total muscle mass of the preparations significantly contributes for these distinctive force parameters. It is worth noted, that the time-parameters (absolute and normalized) of Ang II – mediated bladder and rectal SMC, with the exception of T(c-hc) parameter, do not indicate significant differences (Figure 4). This proves the suggestion that in the urinary bladder and rectum the Ang II - mediated contractions are developed by similar mechanisms. Moreover, this assumption is an indirect evidence for an approximately equal density of Ang II receptors in these two organs. The uniformity of response to Ang II is supported by the fact that it the rectum a local reninangiotensin system has also been established (De Godoy & Rattan, 2006). It could be considered again that the locally generated metabolites of Ang II contribute for this pattern

**Figure 4.** Normalized time-parameters of urinary bladder and rectum Ang II – induced contractions.

Dose-dependent effects of AVP on gastro-intestinal tract from different species were observed, but regarding the rectal musculature the information is insufficient and

All of the normalized time-intervals were calculated as a relative part from Tchr.

*Does AVP have an importance for the motility of the gastro-intestinal tract?* 

of the contraction process.

In conclusions the observed differences in the Ang II - induced gastro-intestinal contractions may be due to:


#### *Ang II – provoked rectal response. Comparison with the urinary bladder response*

The application of Ang II on the rectal preparation caused a development of expressed tonic contraction, which amplitude and integral muscle force were significantly greater than those of the bladder. The higher amplitude is achieved at the expense of the second half of the contraction. The higher values of the absolute and normalized time – parameters for this interval are the evidence. The difference in the total muscle mass of the preparations significantly contributes for these distinctive force parameters. It is worth noted, that the time-parameters (absolute and normalized) of Ang II – mediated bladder and rectal SMC, with the exception of T(c-hc) parameter, do not indicate significant differences (Figure 4). This proves the suggestion that in the urinary bladder and rectum the Ang II - mediated contractions are developed by similar mechanisms. Moreover, this assumption is an indirect evidence for an approximately equal density of Ang II receptors in these two organs. The uniformity of response to Ang II is supported by the fact that it the rectum a local reninangiotensin system has also been established (De Godoy & Rattan, 2006). It could be considered again that the locally generated metabolites of Ang II contribute for this pattern of the contraction process.

130 Neuroendocrinology and Behavior

receptors in the ileum (Fändriks, 2010).

maximal response - expressed by Thr.

different phases of SMC.

may be due to:

magnitude of the response to Ang II depends on the expression of both receptors (Ewert et al., 2006). It had been demonstrated that only AT1 receptors are relevant for the maximum response in ANG-induced contractions (Hawcock & Barnes, 1993; Fändriks, 2010; Fan et al., 2002; Rattan et al., 2003; Ewert et al., 2006; Fändriks, 2010). Despite the existing assumptions that stimulation of AT2 receptors may have the opposite effect than that of AT1 (Gallinat et al., 2000), the role of AT2 receptors for the relaxation phase of the SMC in GIT is not examined. The importance of AT2 receptors for the relaxation of the rectum has been described only (De Godoy et al., 2006; De Godoy & Rattan, 2005). Regarding the time parameters for relaxation, the stomach again showed the slowest response. In this case the ileum indicated significantly prolonged reaction compared to the other intestinal segments. The reason for that difference may be the complete absence or the low density of AT 2

In conclusions the observed differences in the Ang II - induced gastro-intestinal contractions

 Variation in the Ang II receptor subtypes distribution. Regarding GIT there has been described an unequal density of Ang II receptors. This uneven distribution of the receptors could explain the differences in the amplitude and duration of Thc of SMC. Counteraction between Ang II receptor subtypes. Competing actions between Ang II receptors have been discussed in the smooth musculature of rat small intestine. The relative receptor expression is a determinant of the magnitude of response to Ang II. This might be of importance for the duration of muscle contraction after reaching the

 Activation of various transduction pathways. There is data that Ang II can modulate ionic conductance in distinct tissues. The different duration of the interval between Thc and Tc, as well as Thr may be due to the involvement of some membrane ion channels. Presence of local rennin - angiotensin system and formation of numerous of active angiotensin derivates. It is proven that most tissues are the source, target and degradation site of Ang II. Furthermore, local rennin - angiotensin system or parts of it had been found in rat rectum and rat small intestine. This is another possibility which

could explain the obtained data about the phase of relaxation and force of SMC. The use of time - parameters significantly contributes to the analysis of the contraction process and permits a good comparison of the Ang II – induced responses. Presentation of the time parameters as part of the total contraction normalization) gives an idea for

 The obtained results provide a direction for further research work on Ang II-mediated contractions of GIT and for clarifying the exact role of the AT1 and AT2 receptors in the

The application of Ang II on the rectal preparation caused a development of expressed tonic contraction, which amplitude and integral muscle force were significantly greater than those of the bladder. The higher amplitude is achieved at the expense of the second half of the

the development of the process in the different time intervals.

*Ang II – provoked rectal response. Comparison with the urinary bladder response* 

**Figure 4.** Normalized time-parameters of urinary bladder and rectum Ang II – induced contractions. All of the normalized time-intervals were calculated as a relative part from Tchr.

*Does AVP have an importance for the motility of the gastro-intestinal tract?* 

Dose-dependent effects of AVP on gastro-intestinal tract from different species were observed, but regarding the rectal musculature the information is insufficient and

controversial (Ohlsson et al., 2006). AVP has been shown to increase the gastric and duodenal motility in humans and rabbits (Ohlsson et al., 2006; Li et al., 2007), as well as colonic peristalsis, but the expression of the AVP receptors in intestine has not been examined yet (Ohlsson et al., 2006). Some authors have demonstrated that AVP increase the gastro-intestinal motility via the oxytocin OT1 receptors, but the experiment is only for stomach and duodenum from rabbits (Li et al., 2007).

The Effects of Some Neuropeptides on Motor Activity of Smooth Muscle Organs in Abdominal and Pelvic Cavities 133

**Figure 5.** CCK-8 (2.5; 5 and 10 ng/kg i.v.)-induced gallbladder pressure before (A) and after atropine (B)

g/kg i.v.) or hexamethonium (C) (1 mg/kg i.v.). Means ± S.E.M. of 12 experiments in 6 conscious

There is substantial evidence for the involvement of AVP in conditions of uterine hyperactivity. Even more, it has been shown that the human myometrium is more sensitive

Ang II at concentration of 1 µmol induced tonic contraction with maximum amplitude of 6.00 ± 0.22 g and an integral force of muscle contraction of 1150.00 ± 614.70 gs. AVP applied

(20 

dogs are presented, *P* < 0.01.

to AVP than to oxytocin (Bossmar et al., 2007).

In our study, the application of AVP does not significantly alter the characteristics of the spontaneous phasic contractile activity of gastro-intestinal segments except this of stomach. This could be explained with the absence of AVP receptors type V1, which are present in the urinary bladder. In rectal musculature V2 receptors could be presented – in such a case, the rectum as a terminal department of gastro-intestinal tract shows analogy with the distal and the collecting tubules of the kidneys. This is still an assumption that remains to be investigated.

## *Gallbladder*

The mechanical activity of the gallbladder of conscious dogs consisted of spontaneous rhythmic contractions with a frequency of 2 to 5 cpm. Fluctuations of the tone were also observed. Bolus injection of cholecystokinin octapeptide i.v. produced a dose-dependent increase in gallbladder pressure (Figure 5A). Atropine decreased gallbladder pressure and reduced or even abolished the cholecystokinin action (Figure 5B). Hexamethonium led to gallbladder relaxation and also greatly reduced the gallbladder response to cholecystokinin octapeptide (Figure 5C).

The mechanisms through which atropine or hexamethonium inhibit cholecystokininproduced gallbladder contractions under in vivo conditions are not understood. One possible explanation might be that the excitatory effect of cholecystokinin on the gallbladder is mediated by acetylcholine release from cholinergic neurons at pre- and post- ganglionic level. Another possibility is that atropine and/or hexamethonium are able to block the release of endogenous cholecystokinin from the endocrine cells or neurons. This suggestion is supported by the fact that vagotomy abolish gallbladder response to cholecystokinin after acidification (Fried et al., 1983), infusion of fat into the duodenum (Magee et al., 1984) or after drinking water (Sundler et al., 1977). The release of CCK in the circulation in response to fat or other meals is also reduced after vagotomy or atropine. It is also possible that atropine or hexamethonium blockade of cholinergic input to the gallbladder may unmask the release of neuronal inhibitory influence which could then compete with the release of CCK. Such inhibitory agents could be somatostatin and vasoactive intestinal peptide (Lenz et al., 1993; Milenov et al., 1995).

#### *Uterus*

It has been reported that in the uteri of a number of species, local production of Ang II and the enzymes for its synthesis are present. Besides the proven contractile effect of Ang II on the uterine arteries, research in this area showed that myometrium is also sensitive to the effect of this octapeptide (Keskil et al., 1999).

investigated.

octapeptide (Figure 5C).

et al., 1993; Milenov et al., 1995).

effect of this octapeptide (Keskil et al., 1999).

*Uterus* 

*Gallbladder* 

stomach and duodenum from rabbits (Li et al., 2007).

controversial (Ohlsson et al., 2006). AVP has been shown to increase the gastric and duodenal motility in humans and rabbits (Ohlsson et al., 2006; Li et al., 2007), as well as colonic peristalsis, but the expression of the AVP receptors in intestine has not been examined yet (Ohlsson et al., 2006). Some authors have demonstrated that AVP increase the gastro-intestinal motility via the oxytocin OT1 receptors, but the experiment is only for

In our study, the application of AVP does not significantly alter the characteristics of the spontaneous phasic contractile activity of gastro-intestinal segments except this of stomach. This could be explained with the absence of AVP receptors type V1, which are present in the urinary bladder. In rectal musculature V2 receptors could be presented – in such a case, the rectum as a terminal department of gastro-intestinal tract shows analogy with the distal and the collecting tubules of the kidneys. This is still an assumption that remains to be

The mechanical activity of the gallbladder of conscious dogs consisted of spontaneous rhythmic contractions with a frequency of 2 to 5 cpm. Fluctuations of the tone were also observed. Bolus injection of cholecystokinin octapeptide i.v. produced a dose-dependent increase in gallbladder pressure (Figure 5A). Atropine decreased gallbladder pressure and reduced or even abolished the cholecystokinin action (Figure 5B). Hexamethonium led to gallbladder relaxation and also greatly reduced the gallbladder response to cholecystokinin

The mechanisms through which atropine or hexamethonium inhibit cholecystokininproduced gallbladder contractions under in vivo conditions are not understood. One possible explanation might be that the excitatory effect of cholecystokinin on the gallbladder is mediated by acetylcholine release from cholinergic neurons at pre- and post- ganglionic level. Another possibility is that atropine and/or hexamethonium are able to block the release of endogenous cholecystokinin from the endocrine cells or neurons. This suggestion is supported by the fact that vagotomy abolish gallbladder response to cholecystokinin after acidification (Fried et al., 1983), infusion of fat into the duodenum (Magee et al., 1984) or after drinking water (Sundler et al., 1977). The release of CCK in the circulation in response to fat or other meals is also reduced after vagotomy or atropine. It is also possible that atropine or hexamethonium blockade of cholinergic input to the gallbladder may unmask the release of neuronal inhibitory influence which could then compete with the release of CCK. Such inhibitory agents could be somatostatin and vasoactive intestinal peptide (Lenz

It has been reported that in the uteri of a number of species, local production of Ang II and the enzymes for its synthesis are present. Besides the proven contractile effect of Ang II on the uterine arteries, research in this area showed that myometrium is also sensitive to the

**Figure 5.** CCK-8 (2.5; 5 and 10 ng/kg i.v.)-induced gallbladder pressure before (A) and after atropine (B) (20 g/kg i.v.) or hexamethonium (C) (1 mg/kg i.v.). Means ± S.E.M. of 12 experiments in 6 conscious dogs are presented, *P* < 0.01.

There is substantial evidence for the involvement of AVP in conditions of uterine hyperactivity. Even more, it has been shown that the human myometrium is more sensitive to AVP than to oxytocin (Bossmar et al., 2007).

Ang II at concentration of 1 µmol induced tonic contraction with maximum amplitude of 6.00 ± 0.22 g and an integral force of muscle contraction of 1150.00 ± 614.70 gs. AVP applied

in the same concentration as Ang II induced tonic contractions with amplitude of 6.61 ± 0.39 g (n = 8) and an integral force of muscle contraction of 7245.00 ±901.00 gs. The duration of the AVP-induced responses was several times greater than those of Ang II and the recording of AVP-mediated contractions was stopped on the 30th minute without achievement of Tchr parameter.

The Effects of Some Neuropeptides on Motor Activity of Smooth Muscle Organs in Abdominal and Pelvic Cavities 135

search a closer connection between them in preparing the uterus for pregnancy and labor. Probably these two peptides act synchronously which potentiate their own effects (Douglas

Studies on rats show that AVP is more potent uterotonic agent than OT in non pregnant condition (Bossmar et al., 2007) and during parturition OT predominantly promotes uterine contractions, while AVP is more important for vasoconstriction, thus reducing the bleeding

The study of Ang II – and AVP – mediated uterine contractions contributes considerably for the revealing of the mechanisms that generate and modulate uterine activity. This could be

*Department of Physiology, Pathophysiology and Pharmacology, Medical Faculty, Trakia University,* 

*Department of General and Clinical Pathology, Medical Faculty, Trakia University, Stara Zagora,* 

This work was supported by Grant DDVU-02-24/2010 from the National Science Fund,

Aguilera G (1994) Regulation of pituitary ACTH secretion during chronic stress. Front

Aihara E, Kagawa S, Hayashi M & Takeuchi K (2005) ACE inhibitor and AT1 antagonist stimulate duodenal HCO3- secretion mediated by a common pathway - involvement of

Allen AM, MacGregor DP, McKinley MJ, Mendelsohn FA (1999) Angiotensin II receptors in

Andersson KE & Arner A (2004) Urinary bladder contraction and relaxation: physiology and

Sofia, Bulgaria and Grant MF - 1/2010 from Medical Faculty, Trakia University.

PG, NO and bradykinin. J Physiol Pharmacol, 563: 391-406.

beneficial for a better understanding and control of myometrial dysfunction.

Anna Tolekova, Petya Hadzhibozheva, Tsvetelin Georgiev, Stanislava Mihailova

after delivery (Chan et al., 1996; Douglas et al.,2001).

Eleonora Leventieva-Necheva, Kiril Milenov and Reni Kalfin *Institute of Neurobiology, Bulgarian Academy of Sciences, Sofia, Bulgaria* 

et al.,2001).

**Author details** 

and Galina Ilieva

Maya Gulubova

*Bulgaria* 

*Stara Zagora, Bulgaria* 

**Acknowledgement** 

Neuroendocrinol.,15:321-50.

the human brain. Regul Pept, 79:1–7.

pathophysiology. Physiol Rev, 84: 935-986.

**5. References** 

Our experiment confirmed the contractile effect of these two peptides on the myometrium, which is in accordance with the results of other authors working on the same issues (Anouar et al., 1996; Chan et al., 1996; Keskil et al., 1999).

The contractions induced by both peptides have similar amplitudes, but they are with different duration and characteristics. The registered AVP - provoked uterine responses were found to have a sustained oscillating character Figure 6). When analyzed by mathematical modeling such contractions were recognized as underdamped process - the system tries to establish a stable level different from the baseline (Yankov, 2009). The differences in the developed contractions may be due to split of the classical or inclusion of additional transduction pathway for each of the studied peptides. Both of them have several main groups of receptors. The receptors for Ang II are AT1 and AT2 (De Gasparo et al., 2000), while the receptors for AVP are V1a, V1b and V2 (Petersen, 2006).

**Figure 6.** Original record of vasopressin-induced uterine contraction.

To establish the importance of these receptors for the uterine muscle contraction will be the subject of our next experiments. However, several interesting facts immerge:

First – the constrictor effect of Ang II is associated with AT1 receptors, but the uterus is one of the few organs with a. uterina inferior where AT2 receptors are predominant (Keskil et al., 1999). AT2 receptors are mainly regarded to oppose the effects of AT1 and cause dilation, blood pressure reduction, nitric oxide production (Hannan et al., 2003). Perhaps the significantly shorter phase of contraction and relaxation was due to their activation under the influence of Ang II in the uterus. Second – the constrictor effect of AVP is realized by V1a receptors which are found in uterine arteries. With regard to the contractile response of the myometrium, however, there are statements that the resulting contraction from the AVP influence is due to activation of other receptors, different from the mentioned above (Anouar et al., 1996). Some authors go even further and argue that AVP accomplish its effect on uterine musculature by OT receptors, which have big similarity with V1 receptors (Chan et al., 1996).

Considering that both peptides are released from supraoptic nuclei in the hypothalamus and that they have a powerful contractile effect on the smooth muscle, it is appropriate to search a closer connection between them in preparing the uterus for pregnancy and labor. Probably these two peptides act synchronously which potentiate their own effects (Douglas et al.,2001).

Studies on rats show that AVP is more potent uterotonic agent than OT in non pregnant condition (Bossmar et al., 2007) and during parturition OT predominantly promotes uterine contractions, while AVP is more important for vasoconstriction, thus reducing the bleeding after delivery (Chan et al., 1996; Douglas et al.,2001).

The study of Ang II – and AVP – mediated uterine contractions contributes considerably for the revealing of the mechanisms that generate and modulate uterine activity. This could be beneficial for a better understanding and control of myometrial dysfunction.
