**2. Materials and methods**

118 Neuroendocrinology and Behavior

blood vessel.

et al., 2006).

CCK and gastrin possess identical 5 aminoacids at their C-terminals that are the biologically active part of both hormones. The dissimilarities in their potency and physiological activity are determined by the different positions of the *Tyr*-residue in the molecules of both peptides. When the *Tyr*-residue is in the 6th position, the peptide (gastrin) strongly potentiates the gastric secretion, its stimulating effect on the gallbladder contractions and pancreating secretion being much weaker. With the *Tyr*-residue in the 7th position, CCK

Immunohistochemical studies have shown that CCK is synthesized in the mucosal endocrine cells type I and type K of the small intestine, and in the endocrine cells type A of the pancreas. CCK-immunoreactivity has been also identified in the vagus nerve. Cholecystokinin is so called "brain-gut" neuropeptide – it is also produced by enteric

 The food intake in the small intestine is the main physiological stimulus for the CCK release – masts, proteins and aminoacids are the most powerful stimulants among the foods. The plasma CCK concentration increases from 1-2 pmol/l to 6-8 pmol/l after feeding (Cantor, 1986). Cholecystokinin plays a key role in facilitating digestion within the small intestine – this peptide stimulates delivery into the small intestine of digestive enzymes from the pancreas and bile from the gallbladder. Recently it was shown that CCK-8 can reduce food

Mechanisms of secretion of cholecystokinin group peptides into the gastro-intestinal tract are as follows: a) Endocrine mechanism – the peptide is released by the endocrine cell in a blood vessel and is afterwards transported by the circulation to the effector cell; b) Paracrine mechanism – the peptide is released by the endocrine cell in the intracellular space, reaching afterwards the effector cell by diffusion; c) Neurotransmitter mechanism – the peptide is released by the nerve terminal in the synaptic cleft and affects afterwards the activity of the effector neuron; d) Neuroendocrine mechanism – the peptide is released by the neuron in a

The peptide hormone CCK realizes its effects via binding to specific receptors localized on the cell membranes of the target organs. Two types of cholecystokinin receptors have been characterized so far: CCKA and CCKB which are approximately 50 % homologous (Dufresne

CCKA (gastro-intestinal) receptors. They prevail in the peripheral target organs (pancreas, gallbladder, small intestine), as well as in the vagus nerve afferent fibres, mediating the pancreatic enzyme secretion and the gallbladder and ileum motility (Crawley & Corwin, 1994; Xu et al., 2008). CCKA-receptors have also been identified in some brain regions where they take part in the modulation of dopaminergic neurotransmission, and in the regulation

CCKB (brain) receptors. They have been identified in various brain structures, as their number is largest in the cortex, hippocampus and limbic structures (Hokfelt et al., 1985). CCKB receptors, similar or identical to the peripheral gastrin receptors, have been

markedly enhances the gallbladder motility and the pancreatic secretion.

intake by capsaicin-insensitive, nonvagal mechanisms (Zhang & Ritter, 2012).

neurons, and is widely and abundantly distributed in the brain.

of food behavior - satiety effect (Lieverse et al., 1995).

demonstrated in peripheral organs, too.

Wistar rats of both sexes weighting 200–250 g were used. The experiment was carried out in accordance with the national regulations and DIRECTIVE 2010/63/EU of the European Parliament and of the Council of 22 September 2010 concerning the protection of animals

used for scientific and experimental purposes. The animals were anesthetized with Nembutal 50 mg/kg intraperitoneally and exsanguinated. Abdominal cavity was opened and longitudinal strips from different parts of gastro-intestinal tract, urinary bladder and uterus horn were dissected out. The isolated organs were transferred immediately in cold Krebs solution 3 °C), containing following composition in mM): NaCl—118.0, KCl—4.74, NaHCO3—25.0, MgSO4—1.2, CaCl2—2.0, KH2PO4—1.2 and glucose 11.0.

The Effects of Some Neuropeptides on Motor Activity of Smooth Muscle Organs in Abdominal and Pelvic Cavities 121





**Figure 1.** Smooth muscle contraction (SMC) - graph and parameters. Fmax – maximal force of the SMC; Fmax/2 – half of maximal force of the SMC; t0 – start of SMC. t 0 = 0; thc – half-contraction moment; tpeak – the moment of the maximum peak Fmax; thr – the moment when the curve decreases to Fmax/2.

The duration of the tonic contraction was dened from the beginning of the contraction,

For a more correct and acurate comparison between the parts of the gastrointestinal tract was performed normalization of the different intervals as a relative part of total length of the process Txn=Tx/chr). As a result the following normalized parameters were obtained: Thcn,

The gall bladder pressure *in vivo* was recorded in six conscious dogs weighing 18 to 20 kg. The animals were starved for 18 hours and were then anesthetized with chloralose (90 mg/kg i.v.). Laparotomy was performed through upper midline abdominal incision. Gall bladder bile was aspirated with a syringe. The bile volume was between 30 and 40 ml. A small balloon mounted on the top of a polyethylene catheter was introduced into the gall bladder. Experiments were started four weeks after surgery and were carried out twice a week at of at least two-day intervals. The dogs were deprived of food, but were given water *ad libitum* for 18 hours before each trial. The balloon was filled with 1 ml distilled water and connected through a catheter to a pressure transducer. The changes in gall bladder pressure were measured in mmHg. The first 40-min records were used as controls. Cholecystokinin

tpeak and the moment when the curve decreases to Fmax/2 (Thr = thr - tpeak);

maximum peak of the SMC (T(c-hc) = tpeak-thc)

the maximum peak (Tc = t peak - t 0);

(Tchr = thr – t0).

until the amplitude fell to 50%.

Tcn, T(c-hc)n, Thrn.

Longitudinal strips approximately 2 mm wide, 0.5 mm thick and 8 mm long) were dissected following the direction of the muscle bundles. The two ends of each strip were tied with silk ligatures. The distal end was connected to the organ holder; the proximal end was stretched and attached to a mechano-electrical transducer FSG-01 (Experimetria, Ltd., Hungary) via a hook. The preparations were mounted in organ baths TSZ-04/01, containing Krebs solution, pH 7.4, continuously bubbled with Carbogen (95% O2, 5% CO2). The organ baths were mounted in parallel above an enclosed water bath, maintaining the solution temperature at 37 °C. Each smooth muscle strip was initially stretched to a tension of 1.0 g followed by 90 minutes of equilibration. During this period, the smooth muscle strips were replenished with fresh Krebs solution at 15-th min, 60-th min and 75-th min.

After initial period of adaptation, they were treated with the solution of different neuropeptides, and the obtained responses were registered.

The phasic contractions of the smooth muscles before application of neuropeptide and the changes of motor activity, expressed as tonic contractions, relaxations or lack of reaction after treatment were recorded. The contractile activity signals were transduced by mechanical-force sensor, amplified, digitized and recorded using ISOSYS ADVANCED digital acquisition software, produced by Experimetria Ltd., Hungary.

#### *Chemicals and drugs*

Ang II (Sigma-Aldrich), vasopressin (Sigma-Aldrich), ghrelin (PolyPeptide Group) synthetic octapeptide of cholecystokinin (Squibb, USA), acetylcholine chloride (Sigma-Aldrich), hexamethonium chloride (Sigma-Aldrich) were solubilized in bidistillated water. All reagents for the preparation of Krebs solution were purchased from Sigma-Aldrich.

#### *Data analysis and statistic processing*

The recorded force-vs.-time curves permit determination of amplitudes and integrated force of contraction, the latter represented by the area under the curve (AUC), as well as defining of time parameters. Data acquisition and the initial conversion of the experimental data for the later analysis was performed with KORELIA – Processing software (Yankov, 2010). For later analysis, evaluation and identification was used KORELIA-Dynamics program. (Yankov, 2006; Yankov, 2011).

Following time parameters were examined Figure 1:



120 Neuroendocrinology and Behavior

*Chemicals and drugs* 

*Data analysis and statistic processing* 

(Yankov, 2006; Yankov, 2011).

Following time parameters were examined Figure 1:

contraction (SMC) and half-contraction moment (Thc = t hc - t 0);

used for scientific and experimental purposes. The animals were anesthetized with Nembutal 50 mg/kg intraperitoneally and exsanguinated. Abdominal cavity was opened and longitudinal strips from different parts of gastro-intestinal tract, urinary bladder and uterus horn were dissected out. The isolated organs were transferred immediately in cold Krebs solution 3 °C), containing following composition in mM): NaCl—118.0, KCl—4.74,

Longitudinal strips approximately 2 mm wide, 0.5 mm thick and 8 mm long) were dissected following the direction of the muscle bundles. The two ends of each strip were tied with silk ligatures. The distal end was connected to the organ holder; the proximal end was stretched and attached to a mechano-electrical transducer FSG-01 (Experimetria, Ltd., Hungary) via a hook. The preparations were mounted in organ baths TSZ-04/01, containing Krebs solution, pH 7.4, continuously bubbled with Carbogen (95% O2, 5% CO2). The organ baths were mounted in parallel above an enclosed water bath, maintaining the solution temperature at 37 °C. Each smooth muscle strip was initially stretched to a tension of 1.0 g followed by 90 minutes of equilibration. During this period, the smooth muscle strips were replenished

After initial period of adaptation, they were treated with the solution of different

The phasic contractions of the smooth muscles before application of neuropeptide and the changes of motor activity, expressed as tonic contractions, relaxations or lack of reaction after treatment were recorded. The contractile activity signals were transduced by mechanical-force sensor, amplified, digitized and recorded using ISOSYS ADVANCED

Ang II (Sigma-Aldrich), vasopressin (Sigma-Aldrich), ghrelin (PolyPeptide Group) synthetic octapeptide of cholecystokinin (Squibb, USA), acetylcholine chloride (Sigma-Aldrich), hexamethonium chloride (Sigma-Aldrich) were solubilized in bidistillated water. All

The recorded force-vs.-time curves permit determination of amplitudes and integrated force of contraction, the latter represented by the area under the curve (AUC), as well as defining of time parameters. Data acquisition and the initial conversion of the experimental data for the later analysis was performed with KORELIA – Processing software (Yankov, 2010). For later analysis, evaluation and identification was used KORELIA-Dynamics program.


reagents for the preparation of Krebs solution were purchased from Sigma-Aldrich.

NaHCO3—25.0, MgSO4—1.2, CaCl2—2.0, KH2PO4—1.2 and glucose 11.0.

with fresh Krebs solution at 15-th min, 60-th min and 75-th min.

digital acquisition software, produced by Experimetria Ltd., Hungary.

neuropeptides, and the obtained responses were registered.


**Figure 1.** Smooth muscle contraction (SMC) - graph and parameters. Fmax – maximal force of the SMC; Fmax/2 – half of maximal force of the SMC; t0 – start of SMC. t 0 = 0; thc – half-contraction moment; tpeak – the moment of the maximum peak Fmax; thr – the moment when the curve decreases to Fmax/2.

The duration of the tonic contraction was dened from the beginning of the contraction, until the amplitude fell to 50%.

For a more correct and acurate comparison between the parts of the gastrointestinal tract was performed normalization of the different intervals as a relative part of total length of the process Txn=Tx/chr). As a result the following normalized parameters were obtained: Thcn, Tcn, T(c-hc)n, Thrn.

The gall bladder pressure *in vivo* was recorded in six conscious dogs weighing 18 to 20 kg. The animals were starved for 18 hours and were then anesthetized with chloralose (90 mg/kg i.v.). Laparotomy was performed through upper midline abdominal incision. Gall bladder bile was aspirated with a syringe. The bile volume was between 30 and 40 ml. A small balloon mounted on the top of a polyethylene catheter was introduced into the gall bladder. Experiments were started four weeks after surgery and were carried out twice a week at of at least two-day intervals. The dogs were deprived of food, but were given water *ad libitum* for 18 hours before each trial. The balloon was filled with 1 ml distilled water and connected through a catheter to a pressure transducer. The changes in gall bladder pressure were measured in mmHg. The first 40-min records were used as controls. Cholecystokinin

octapeptide was then injected i.v. at increasing doses every 30 min. Atropine or hexamethonium was administered before cholecystokinin.

The Effects of Some Neuropeptides on Motor Activity of Smooth Muscle Organs in Abdominal and Pelvic Cavities 123

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).

mediated contraction of detrusor smooth muscle strips.

also found (Waldeck et al., 1997; Miyazaki & Takai, 2006).

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 II-

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

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

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 be statistically signicant.
