**3.1. Urinary bladder**

The coordination of smooth muscle activity of the urinary tract in the process of the urine evacuation is regulated by complex, and not yet fully understood interactions between neural and hormonal control mechanisms (Dixon et al., 1997).

The urinary bladder is innervated by three groups of peripheral nerves: sacral parasympathetic (Helm et al., 1982; Crowe & Burnstock, 1989; Gabella & Uvelius, 1990; Lasanen et al., 1992; Uvelius & Gabella, 1998), thoracolumbar sympathetic (Downie, 1981; Feher & Vajda, 1981) and sacral sensory (De Groat & Booth, 1993). According to the secreted mediator in the neural terminals innervation is classified as cholinergic, adrenergic, and non-adrenergic, non-cholinergic (NANC) (Callahan & Creed, 1986). In humans detrusor neurotransmission is exclusively cholinergic (Andersson et al., 1982; Sibley, 1984; Chen et al., 1994), while its adrenergic innervation is sparse, nonuniform, and it is considered nonessential for micturition function (de Groat & Booth, 1984; Janig & McLachlan, 1987).

A lot of different neuropeptides have been found to be synthesized, stored and released from organs in the lower urinary tract (LUT). Some of them are secreted from the peripheral neural terminals of the autonomic nervous system – vasoactive intestinal peptide (VIP), tachykinins (substance P), neuropeptide Y, calcitonin gene-related peptide, neurokinin A. Others which act by para- and autocrine mechanisms as angiotensin II are locally synthesized. The exact function of many of these peptides has not been fully established, however they may play role in a sensory and efferent innervation (de Groat & Kawatani, 1985; Burnstock, 1990; Maggi, 1991) or serve as neuromodulators in the ganglia or at the neuromuscular junctions. Their actions have been thought to include mediation of the micturition reflex activation, smooth muscle contraction, potentiation of efferent neurotransmission and changes in vascular tone and permeability (Maggi, 1991, 1995; Hernandez et al., 2006).

A few hormones with systemic circulation – vasopressin, oxytocin, are influencing bladder function (Uvelius B. et al., 1990; Romine & Anderson, 1985). These also include a newly discovered peptide ghrelin, which is secreted from the gastrointestinal tract, and stimulates contractions of smooth muscles of blood vessels and gastro-intestinal tract (Tack et al., 2006; Wiley & Davenport, 2002)

#### *Angiotensin II and urinary bladder*

There is insufficient information on the effect of Ang II on non-cardiovascular organ smooth muscles (Touyz & Berry, 2002). The interactions of the Ang II with the urinary bladder are of particular interest regarding the genesis and treatment of the disorders of the micturition. The physiological effects of Ang II on the function of the urinary bladder and the transduction mechanisms which mediate it have not been fully elucidated. Ang II and its precursor Ang I cause dose-dependent contractions of muscle strips from rat urinary bladder (Andersson et al., 1992). According to experimental data of Anderson and coauthors Ang II acts as a modulator in neurotransmission in the urinary bladder (Andersson & Arner, 2004). There are research data confirming that Ang II carries out its physiological effects by binding to membrane AT1 receptors (Tanabe et al.,1993), whose number on the surface of detrusor smooth muscle cells is regulated by the dietary content of sodium and potassium and the age of experimental animals (Weaver-Osterholtz et al., 1996; Szigeti et al., 2005). AT1 receptors activate PLC, dihydropyridine-sensitive Ca2+-channels and inhibit adenylilcyclase, thus reducing intracellular cAMP concentration (Chiu et al., 1994).

122 Neuroendocrinology and Behavior

be statistically signicant.

**3.1. Urinary bladder** 

**3. Results and discussions** 

octapeptide was then injected i.v. at increasing doses every 30 min. Atropine or

Obtained data were processed by the statistical program Statistica 6.1, StaSoft, Inc. and presented as mean ± standard error. A P-value less than or equal to 0.05 was considered to

The coordination of smooth muscle activity of the urinary tract in the process of the urine evacuation is regulated by complex, and not yet fully understood interactions between

The urinary bladder is innervated by three groups of peripheral nerves: sacral parasympathetic (Helm et al., 1982; Crowe & Burnstock, 1989; Gabella & Uvelius, 1990; Lasanen et al., 1992; Uvelius & Gabella, 1998), thoracolumbar sympathetic (Downie, 1981; Feher & Vajda, 1981) and sacral sensory (De Groat & Booth, 1993). According to the secreted mediator in the neural terminals innervation is classified as cholinergic, adrenergic, and non-adrenergic, non-cholinergic (NANC) (Callahan & Creed, 1986). In humans detrusor neurotransmission is exclusively cholinergic (Andersson et al., 1982; Sibley, 1984; Chen et al., 1994), while its adrenergic innervation is sparse, nonuniform, and it is considered non-

essential for micturition function (de Groat & Booth, 1984; Janig & McLachlan, 1987).

A lot of different neuropeptides have been found to be synthesized, stored and released from organs in the lower urinary tract (LUT). Some of them are secreted from the peripheral neural terminals of the autonomic nervous system – vasoactive intestinal peptide (VIP), tachykinins (substance P), neuropeptide Y, calcitonin gene-related peptide, neurokinin A. Others which act by para- and autocrine mechanisms as angiotensin II are locally synthesized. The exact function of many of these peptides has not been fully established, however they may play role in a sensory and efferent innervation (de Groat & Kawatani, 1985; Burnstock, 1990; Maggi, 1991) or serve as neuromodulators in the ganglia or at the neuromuscular junctions. Their actions have been thought to include mediation of the micturition reflex activation, smooth muscle contraction, potentiation of efferent neurotransmission and changes in vascular tone

A few hormones with systemic circulation – vasopressin, oxytocin, are influencing bladder function (Uvelius B. et al., 1990; Romine & Anderson, 1985). These also include a newly discovered peptide ghrelin, which is secreted from the gastrointestinal tract, and stimulates contractions of smooth muscles of blood vessels and gastro-intestinal tract (Tack et al., 2006;

There is insufficient information on the effect of Ang II on non-cardiovascular organ smooth muscles (Touyz & Berry, 2002). The interactions of the Ang II with the urinary bladder are of

hexamethonium was administered before cholecystokinin.

neural and hormonal control mechanisms (Dixon et al., 1997).

and permeability (Maggi, 1991, 1995; Hernandez et al., 2006).

Wiley & Davenport, 2002)

*Angiotensin II and urinary bladder* 

Our experiments show that the administration of Ang II solution to the smooth muscle stirps induce tonic contractions, in confirmation of the findings from other researchers concerning its effect on urinary bladder contractile activity. The increased amplitude of contraction following the administration of Ang II in the presence of increased extracellular Ca2+ provided evidence of additive synergism (Hadzhibozheva et al., 2009; Tolekova et al., 2010). The blockade of AngII-induced tonic contraction after the administration of blockers of T-type Ca2+ -channels unequivocally showed the role of transmembrane Ca2+ -influx in the initiation of smooth muscle contraction (Ilieva et al., 2008). The Ang II bindings to its membrane receptors, leads to activation of phospholipase C, which results in formation of inositol triphosphate, which triggers release of Ca2+ from sarcoplasmatic reticulum (SR). It's also well known that Ang II causes calcium-induced calcium release in smooth-muscle cells. Angiotensin II causes depolarization and opening of VDCC, providing additional Ca2+ influx from the extracellular fluid (Seki et al., 1999). When moving inside the cell, Ca2+ binds to ryanodine receptors and triggers supplementary Ca2+ release from SR stores (Berridge, 2008). In the experiment we applied specific inhibitor to this particular calcium-induced Ca2+ release mechanism. The resulting lack of tonic contraction suggested that this signaling pathway, leading to intracellular calcium increase is of greater importance for the development of detrusor muscle contraction than the inositol triphosphate pathway. Our experimental data also showed that the increase of calcium in extracellular fluid produced additive synergistic effect on Ang IImediated contraction of detrusor smooth muscle strips.

The circulating AngII is formed in blood under influence of angiotensin–converting enzyme (ACE). During the last decade a lot of new facts that significantly broaden our knowledge of the RAS have been accumulated. Local tissue RAS was found in the blood vessels, heart, kidneys, small intestines, pancreatic tissue, liver, ovaries and brain (Paul at al., 2006). Other enzymes involved in RAS and physiologicaly active metabolites of Ang I and Ang II were also found (Waldeck et al., 1997; Miyazaki & Takai, 2006).

A lot of recent studies have shown that Ang II acts as a cytokine and growth-like factor. (Kim & Iwao, 2000; Touyz & Berry, 2002). It regulates the smooth muscle mass in the bladder wall in normal and pathological conditions. Chronic bladder outlet obstruction causes changes in smooth muscle mass and connective tissue both in humans' disease and

under experimental conditions in rats (Yamada et al., 2009). The application of ACE blocking substances significantly reduce the quantity of the newly synthesized collagen in the bladder, which is an indirect indicator of the effects of the local RAS on developing pathologic hypertrophic changes in the bladder. Phull and coauthors (2007) showed that applying angiotensin receptor anatagonists, reduced urethral resistance on rat models with stress urinary incontinence.

The Effects of Some Neuropeptides on Motor Activity of Smooth Muscle Organs in Abdominal and Pelvic Cavities 125

shows contractions with approximately equal amplitude, but with integrated muscle force significantly increased after the application of AVP (Figure 2, Table 1) (Tolekova et al., 2010).

**AVP** 1,55±0,16 761,29±113,3 28±4 99,6±15 127,4±18 255,3±35 382,7±43

**Ang II** 1,73±0,26 115,13±20,7 12,6±1,6 19,7±5 32,3±3,3 61,5±13,6 93,7±13,3

**Figure 2.** Angiotensin II- and AVP – induced contractions after processing of signals with specialized

For detailed study and comparison of the tonic contractions under the influence of the two peptides, we are using time parameters, described in Yankov (2011) (Figure 1, Table 1), generated after signal processing with specialized software (Yankov, 2010). The similar parameters were used for investigation of the contractions of the skeletal muscles (Raikova & Aladjov, 2004). Our research results show that Ang II causes contractions with a shorter duration (shorter time for reaching Fmax/2 and Fmax). The AVP-induced response reaches significant AUC value at the expense of the lower rate of increased and decreased contractile activity (longer duration of Thr and Tchr). The greater length of AVP contraction time is mainly due to longer T(c-hc) period. It is particularly interesting that despite acting through the same transduction pathways, Ang II and AVP cause tonic contractions with different measured force. We assume that this difference, especially the higher speed of relaxation, might be related to the specific Ang II metabolism in bladder cells. Production of Ang III and Ang 1-7 may be a cause of the faster rate of relaxation (Varagic et al., 2008). The interactions of the two peptides – Ang II and AVP with the ion channels of the smooth

software.

**Table 1.** Force and time-parametes means±SE) of Ang II- and AVP-induced contractions.

**Fmax.g) AUCgs) Thc s) Tc-hc) Tc s) Thr s) Tchrs)** 

The all main components of RAS – Ang I, Ang II and ACE are found inside bladder tissues (Weaver-Osterholtz et al., 1996). Tissue levels of Ang I and Ang II were higher than circulating levels, which confirms the existence of local synthesis in the urinary bladder, despite the lower measured activity of the angiotensin-converting enzyme, insitu is relatively This fact supports the hypothesis of auto- and paracrine actions of Ang II. It has been shown that Ang I also causes contraction of smooth muscle cells of the bladder, through the interaction with AT1 receptors.

Ang II -mediated contraction is not completely blocked after administration of ACE inhibitor (Lindberg et al., 1994). These facts indicate the existence of alternative pathways for the synthesis of Ang II. Urata and co-authors show that in the heart of the main enzyme converting Ang I to Ang II is a serine protease human himase (Urata et al., 1990). Andersson and co-authors found that application of enalaprilat not fully block contraction of urinary bladder detrusor stripts induced by Ang I (Andersson et al., 1992). This means that the remaining contractile effect is due to separate mechanism of Ang II formation.

#### *Vasopressin*

Besides its vasoconstrictor activity, AVP is involved in the modulation of the intrinsic smooth muscle activity of the urinary system. Vargiu and coauthors demonstrate that AVP increases the contractile activity of upper urinary tract that is most pronounced in small calices, which are the main pacemaker of the urinary tract and is mediated by V1 receptors (Vargiu et al., 2004). It remains unaffected by the blockade of sodium channels with tetrodotoxin. However, the application of nifedipine and L-type calcium channels blockers reduced spontaneous and AVP-induced activity of smooth muscles of the upper urinary tract (Vargiu et al., 2004). This demonstrates the importance of transmembrane calcium influx for the contractile activity of smooth muscle of the lower urinary tract.

There is a data that shows the presence of V1-receptors in smooth muscle of the urinary bladder, who's binding to AVP leads to activation of the inositol-triphosphate (IP3) pathway, similarly to binding to Ang II (Crankshaw, 1989; Dehpour et al., 1997). It was found that the removal of extracellular calcium prevents the effects of AVP, suggesting it possible involvement in the activation process (Crankshaw, 1989). In our experiments AVP application to the bladder detrusor smooth muscle strips stimulates powerful tonic contractions. This result supports currently available information on this issue (Uvelius et al., 1990).

#### *Comparison of the effects of Ang II and AVP on contractile activity*

Ang II and AVP, accomplish their effects through the formation of the second messenger IP3. The comparison of the independent effects of Ang II and AVP on urinary bladder strips


shows contractions with approximately equal amplitude, but with integrated muscle force significantly increased after the application of AVP (Figure 2, Table 1) (Tolekova et al., 2010).

**Table 1.** Force and time-parametes means±SE) of Ang II- and AVP-induced contractions.

124 Neuroendocrinology and Behavior

stress urinary incontinence.

*Vasopressin* 

through the interaction with AT1 receptors.

under experimental conditions in rats (Yamada et al., 2009). The application of ACE blocking substances significantly reduce the quantity of the newly synthesized collagen in the bladder, which is an indirect indicator of the effects of the local RAS on developing pathologic hypertrophic changes in the bladder. Phull and coauthors (2007) showed that applying angiotensin receptor anatagonists, reduced urethral resistance on rat models with

The all main components of RAS – Ang I, Ang II and ACE are found inside bladder tissues (Weaver-Osterholtz et al., 1996). Tissue levels of Ang I and Ang II were higher than circulating levels, which confirms the existence of local synthesis in the urinary bladder, despite the lower measured activity of the angiotensin-converting enzyme, insitu is relatively This fact supports the hypothesis of auto- and paracrine actions of Ang II. It has been shown that Ang I also causes contraction of smooth muscle cells of the bladder,

Ang II -mediated contraction is not completely blocked after administration of ACE inhibitor (Lindberg et al., 1994). These facts indicate the existence of alternative pathways for the synthesis of Ang II. Urata and co-authors show that in the heart of the main enzyme converting Ang I to Ang II is a serine protease human himase (Urata et al., 1990). Andersson and co-authors found that application of enalaprilat not fully block contraction of urinary bladder detrusor stripts induced by Ang I (Andersson et al., 1992). This means that the

Besides its vasoconstrictor activity, AVP is involved in the modulation of the intrinsic smooth muscle activity of the urinary system. Vargiu and coauthors demonstrate that AVP increases the contractile activity of upper urinary tract that is most pronounced in small calices, which are the main pacemaker of the urinary tract and is mediated by V1 receptors (Vargiu et al., 2004). It remains unaffected by the blockade of sodium channels with tetrodotoxin. However, the application of nifedipine and L-type calcium channels blockers reduced spontaneous and AVP-induced activity of smooth muscles of the upper urinary tract (Vargiu et al., 2004). This demonstrates the importance of transmembrane

calcium influx for the contractile activity of smooth muscle of the lower urinary tract.

result supports currently available information on this issue (Uvelius et al., 1990).

*Comparison of the effects of Ang II and AVP on contractile activity* 

There is a data that shows the presence of V1-receptors in smooth muscle of the urinary bladder, who's binding to AVP leads to activation of the inositol-triphosphate (IP3) pathway, similarly to binding to Ang II (Crankshaw, 1989; Dehpour et al., 1997). It was found that the removal of extracellular calcium prevents the effects of AVP, suggesting it possible involvement in the activation process (Crankshaw, 1989). In our experiments AVP application to the bladder detrusor smooth muscle strips stimulates powerful tonic contractions. This

Ang II and AVP, accomplish their effects through the formation of the second messenger IP3. The comparison of the independent effects of Ang II and AVP on urinary bladder strips

remaining contractile effect is due to separate mechanism of Ang II formation.

**Figure 2.** Angiotensin II- and AVP – induced contractions after processing of signals with specialized software.

For detailed study and comparison of the tonic contractions under the influence of the two peptides, we are using time parameters, described in Yankov (2011) (Figure 1, Table 1), generated after signal processing with specialized software (Yankov, 2010). The similar parameters were used for investigation of the contractions of the skeletal muscles (Raikova & Aladjov, 2004). Our research results show that Ang II causes contractions with a shorter duration (shorter time for reaching Fmax/2 and Fmax). The AVP-induced response reaches significant AUC value at the expense of the lower rate of increased and decreased contractile activity (longer duration of Thr and Tchr). The greater length of AVP contraction time is mainly due to longer T(c-hc) period. It is particularly interesting that despite acting through the same transduction pathways, Ang II and AVP cause tonic contractions with different measured force. We assume that this difference, especially the higher speed of relaxation, might be related to the specific Ang II metabolism in bladder cells. Production of Ang III and Ang 1-7 may be a cause of the faster rate of relaxation (Varagic et al., 2008). The interactions of the two peptides – Ang II and AVP with the ion channels of the smooth

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 of AVP action on smooth muscle contractions.

The Effects of Some Neuropeptides on Motor Activity of Smooth Muscle Organs in Abdominal and Pelvic Cavities 127

spontaneous activity. The effects of ghrelin are displayed only when it is applied in combination with other peptides – Ang II or AVP. In combination with Ang II, ghrelin reduces its contractile effect on the bladder (Ilieva et al., 2008, a). The combination of ghrelin with AVP leads to similar yet significantly less manifested decrease, especially in the AUC. Based on these results we can assume that the urinary bladder possesses receptors for ghrelin, different from those in the digestive tract, with respect to the intracellular signaling mechanism to which they are coupled. The significant reduction in the amplitude of Ang IIinduced contraction as well as the partial reduction of AVP-provoked contraction after ghrelin application could be explained by the interaction between signal transduction pathways by which the both peptides act. To our knowledge, this is the first *in vitro* study demonstrated the inhibitory effect of ghrelin on bladder motor activity. Our results were confirmed by Matsuda et coauthors (2011). They showed in experiments in vivo that intracerebroventricular

It is likely that ghrelin acts through the second messenger cAMP. Activation of this signal pathway causes relaxation of smooth muscles by decreasing the activity of miosinkinase and stimulating Ca2+-efflux. This effect of ghrelin could be explained with interactions between the two types of transduction pathways, which have opposite effects (Rasmussen &

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

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

administration of Ghrelin increases bladder capacity dose dependently.

Rasmussen, 1990; Churchill, 1985).

**3.2. Gastro-intestinal tract** 

the GIT (Fändriks, 2010).

*Angiotensin II* 

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

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

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

#### *Ghrelin*

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 contractions mediated by these peptides.

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

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 spontaneous activity. The effects of ghrelin are displayed only when it is applied in combination with other peptides – Ang II or AVP. In combination with Ang II, ghrelin reduces its contractile effect on the bladder (Ilieva et al., 2008, a). The combination of ghrelin with AVP leads to similar yet significantly less manifested decrease, especially in the AUC.

Based on these results we can assume that the urinary bladder possesses receptors for ghrelin, different from those in the digestive tract, with respect to the intracellular signaling mechanism to which they are coupled. The significant reduction in the amplitude of Ang IIinduced contraction as well as the partial reduction of AVP-provoked contraction after ghrelin application could be explained by the interaction between signal transduction pathways by which the both peptides act. To our knowledge, this is the first *in vitro* study demonstrated the inhibitory effect of ghrelin on bladder motor activity. Our results were confirmed by Matsuda et coauthors (2011). They showed in experiments in vivo that intracerebroventricular administration of Ghrelin increases bladder capacity dose dependently.

It is likely that ghrelin acts through the second messenger cAMP. Activation of this signal pathway causes relaxation of smooth muscles by decreasing the activity of miosinkinase and stimulating Ca2+-efflux. This effect of ghrelin could be explained with interactions between the two types of transduction pathways, which have opposite effects (Rasmussen & Rasmussen, 1990; Churchill, 1985).
