**1.4. Angiotensin II**

The octapeptide Angiotensin II (Ang II) is the main effector of the renin-angiotensin system (RAS). Ang II is generated in circulation or locally in tissues in the kidney, blood vessels, heart, and brain and etc.

The signal transduction mechanism for AT1 receptors is well known. AT1 receptors are distributed in adult tissues including blood vessel, heart, kidney, adrenal gland, liver, brain, and lung. These receptors activate phospholipase A2, phospholipase C, phospholipase D and L-type Ca2+ channels and inhibiting the adenylyl cyclase (Shokei & Hiroshi, 2011).

AT2 receptors are ubiquitously expressed in developing fetal tissues, suggesting a possible role of these receptors in fetal development and organ morphogenesis. AT2 receptors expression rapidly decreases after birth, and in the adult. Expression of these receptors are limited mainly to the uterus, ovary, certain brain nuclei, heart, and adrenal medulla. In various cell lines, AT2 receptors activated protein tyrosine phosphatase was shown to inhibit cell growth or induce programmed cell death (apoptosis) (Kim and Awao, 2011).

Ang II has a multifunctional role. It is general regulator of salt and water metabolism, thirst, sympathetic outflow and vascular smooth muscle cell tone. As a result Ang II acts as a principal controller of long term regulation of blood pressure (Robertson, 2005; Watanabe et al., 2005). Later, Ang II was found to exert long-term effects on tissue structure, including cardiac hypertrophy, vascular remodeling, and renal fibrosis (Watanabe et al., 2005)**.**

The key effector of peripheral renin-angiotensin system (RAS) - Ang II in circulation does not cross blood-brain barrier. Therefore it interacts on brain regions that lack the blood-brain barrier as circumventricular areas, organum vasculosum laminae terminalis, where Ang II stimulates salt appetite, thirst and vasopressin secretion (Fitts et al., 2000). Also, it influences neuronal activity in area postrema and takes part in central regulation of blood pressure (Otsuka et al., 1986).

Many immunohistochemical studies demonstrate the distribution of all components of RAS - angiotensinogen, Ang I, Ang II and renin in several brain regions of rats. Immunoreactivity for Ang II was detected in neurons and vessels in the brainstem, cerebellum, hypothalamus, basal ganglia, thalamus and cortex while for angiotensinogen and Ang I were found in neurons of the hypothalamic nuclei in rats (McKinley et al., 2003; Von Bohlen et al., 2006). AT1 receptor binding sites with higher density were localized in the lamina terminalis, hypothalamic paraventricular nucleus and the nucleus tractus solitaries, lamina terminalis and the subfornical organ. The median preoptic nucleus also contains membranes rich in AT1 receptors. All regions that are included in the regulation of cardiovascular functions as caudal ventrolateral medulla, and the midline raphe, also have AT1receptors (Allen et al., 1999; Lenkei et al., 1997). In the midbrain -in the lateral parabrachial nucleus, substantia nigra and periaqueductal gray AT1 receptors are presented from moderate to high densities (Lenkei et al., 1997). These localizations of RAS brain components show that Ang II is involved in the regulations of thirst, drinking, facilitating vasopressor effects and secretion of vasopressin, adrenocorticotrophic and luteinizing hormones. Ang II also stimulates secretion of neurotransmitters such as noradrenaline and 5-hydroxytryptamine (5-HT) and inhibits acetylcholine release. The brain RAS appears to be also an important modulator of the blood pressure circadian rhythm and it influences renal renin release.

Recent findings demonstrate that central effects of Ang II contribute to facilitated learning and enhance associative memory and learning possibly with differential effects on acquisition, storage and recall. Brain RAS is involved in the development of affective disorders and Ang II has a modulating effect of on anxiety (Georgiev & Yonkov, 1985).

RAS receptors alterations have been found in some neurodegenerative disorders - Parkinson's and Huntington's disease (Ge & Barnes, 1996). The number of AT1 receptors in caudate nucleus, putamen and substantia nigra was significantly decreased in Parkinson's disease patients in comparison to controls. In Huntington's disease patients, AT1receptors was found to be slightly decreased in putamen (Ge & Barnes, 1996). AT2 receptors that are localized in caudate nucleus was decreased in Parkinson's and increased in Huntington's disease patients. The receptor alterations were considerable; therefore the authors have concluded that brain RAS seems to decisively contribute to the pathology of the dopaminergic nigrostriatal pathway in these patients and may be a novel therapeutic target for neurodegenerative disorders (Savaskan, 2005).

## **1.5. Cholecystokinin**

116 Neuroendocrinology and Behavior

**1.4. Angiotensin II** 

heart, and brain and etc.

(Otsuka et al., 1986).

(Diéguez C et al., 2010; Sangiao-Alvarellos et al., 2010)

regulates glucose homeostasis (Sun et al., 2007).

the hypothalamus. There are reductions in fatty acid synthase (FAS) mRNA expression both in the ventromedial nucleus of the hypothalamus and whole hypothalamus, as well as in FAS protein and activity. CPT1 activity is also reduced. Chronic ghrelin treatment does not promote AMPK-induced changes in the overall fluxes of hypothalamic fatty acid metabolism in normal rats and this effect is independent of ghrelin status. In addition, ghrelin plays a dual time-dependent role in modulating hypothalamic lipid metabolism.

A reciprocal relationship exists between ghrelin and insulin, suggesting that ghrelin

The octapeptide Angiotensin II (Ang II) is the main effector of the renin-angiotensin system (RAS). Ang II is generated in circulation or locally in tissues in the kidney, blood vessels,

The signal transduction mechanism for AT1 receptors is well known. AT1 receptors are distributed in adult tissues including blood vessel, heart, kidney, adrenal gland, liver, brain, and lung. These receptors activate phospholipase A2, phospholipase C, phospholipase D and L-type Ca2+ channels and inhibiting the adenylyl cyclase (Shokei & Hiroshi, 2011).

AT2 receptors are ubiquitously expressed in developing fetal tissues, suggesting a possible role of these receptors in fetal development and organ morphogenesis. AT2 receptors expression rapidly decreases after birth, and in the adult. Expression of these receptors are limited mainly to the uterus, ovary, certain brain nuclei, heart, and adrenal medulla. In various cell lines, AT2 receptors activated protein tyrosine phosphatase was shown to inhibit cell growth or induce programmed cell death (apoptosis) (Kim and Awao, 2011).

Ang II has a multifunctional role. It is general regulator of salt and water metabolism, thirst, sympathetic outflow and vascular smooth muscle cell tone. As a result Ang II acts as a principal controller of long term regulation of blood pressure (Robertson, 2005; Watanabe et al., 2005). Later, Ang II was found to exert long-term effects on tissue structure, including

The key effector of peripheral renin-angiotensin system (RAS) - Ang II in circulation does not cross blood-brain barrier. Therefore it interacts on brain regions that lack the blood-brain barrier as circumventricular areas, organum vasculosum laminae terminalis, where Ang II stimulates salt appetite, thirst and vasopressin secretion (Fitts et al., 2000). Also, it influences neuronal activity in area postrema and takes part in central regulation of blood pressure

Many immunohistochemical studies demonstrate the distribution of all components of RAS - angiotensinogen, Ang I, Ang II and renin in several brain regions of rats. Immunoreactivity for Ang II was detected in neurons and vessels in the brainstem, cerebellum, hypothalamus, basal ganglia, thalamus and cortex while for angiotensinogen and Ang I were found in

cardiac hypertrophy, vascular remodeling, and renal fibrosis (Watanabe et al., 2005)**.**

In 1928 Ivy and Oldberg extracted from dog duodenal mucosis a substance which injected intravenously contracted the gallbladder. The authors named this substance cholecystokinin (CCK). Later, Harper & Raper (1943) found a compound in this extract that stimulated the pancreatic secretion and called it pancreozymin. Purifying both hormones and determing their amino-acid sequention, Mutt (1980) proved them to be the same linear polypeptide, containing 33 amino-acid residues, and proposed the hybrid name "cholecystokininpancreozymin". Different CCK forms have been shown to exist: CCK-58, CCK-39, CCK-33, CCK-27, CCK-12, CCK-8, CCK-4, all of them containing a bioactive C-terminal. CCK-58 and CCK-39 are precursors of CCK-33 and by the degradation of the latter the shorter forms are obtained. Cholecystokinin octapeptide (CCK-8) is the most active one and is most widely spread in the gastro-intestinal tract and in the central nervous system. A cholecystokinin analogue, named caerulein, has been isolated from the skin of the frog *Hyla caerulea*.

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 markedly enhances the gallbladder motility and the pancreatic secretion.

The Effects of Some Neuropeptides on Motor Activity of Smooth Muscle Organs in Abdominal and Pelvic Cavities 119

Cholecystokinin is a major peptide hormone in the gut and a major peptide transmitter in the brain. Its synthesis requires endoproteolytic cleavage of proCCK at several mono- and dibasic sites by prohormone convertases. On one hand cholecystokinin is a classical gut hormone and a growth factor for the pancreas. On the other, the CCK gene is expressed also in large quantities in cerebral and peripheral neurons from where CCK peptides are released as potent neurotransmitters and modulators. Accordingly, cerebral CCK defects have been associated with major neuropsychiatric diseases such as anxiety, schizophrenia

CCK is also a key component of the aggression facilitating circuitry in the brain *(*Luo et al., 1998), and it is released during inter-male fighting (Becker et al., 2001). In addition to its many aversive motivational/emotional effects, CCK also plays a role in more positively valenced motivational states, such as mating (Dornan & Malsbury, 1989; Markowski & Hull, 1995), drug

It was demonstrated that CCK is colocalized with dopamine in ventral striatal dopamine neurons (Hokfelt et al., 1980). Consistent with the neuronal colocalization and extensive overlap of expression between CCK and the dopaminergic system, CCK peptides have significant effects on dopamine mediated behaviors. Administration of CCK peptides exhibit many of the behavioral characteristics of antipsychotics including inhibition of conditioned avoidance responding (Cohen et al., 1982), inhibition of apomorphine-induced stereotypic behavior (Zetler, 1983), and inhibitionof amphetamine-induced hyperlocomotion (Schneider et al., 1983). Microinjection of CCK into the anterior nucleus accumbens inhibits dopamine release, inhibits dopamine-mediated behaviors and is blocked by a CCKA antagonist whereas injection into the posterior nucleus accumbens has the opposite effects and these effects are mediated by CCKB receptors (Vaccarino & Rankin, 1989, Crawley, 1992). Thus, it appears that different CCK-based circuitries in the brain can facilitate both negative and positive emotional processes. It is also interesting to note that selective CCKB agonists that cross the blood brain barrier such as pentagastrin and CCK-4 are used to induce panic attacks in clinical studies. Consequently, a CCK agonist for schizophrenia

CCK was the first gut hormone discovered to have anoretic effects. Its actions include inhibition of food intake, delayed gastric emptying, stimulation of pancreatic enzyme secretion, and stimulation of gall bladder contraction. These effects are mediated via binding to CCK receptors on the vagus nerve. CCK administration to humans and animals inhibits food intake by reducing meal size and duration. However, at high dose nausea and taste aversion have been detected making CCK an unlikely candidate for an anti-obesity

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

addiction (Crespi, 2000) and brain reward processes (Degen et al., 2001, Josselyn, 1996).

and satiety disorders (Crawley & Corwin, 1994; Liddle, 1997).

would need to be either nonselective or CCKA selective.

treatment.

**2. Materials and methods** 

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

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

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 blood vessel.

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 et al., 2006).

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

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 demonstrated in peripheral organs, too.

Cholecystokinin is a major peptide hormone in the gut and a major peptide transmitter in the brain. Its synthesis requires endoproteolytic cleavage of proCCK at several mono- and dibasic sites by prohormone convertases. On one hand cholecystokinin is a classical gut hormone and a growth factor for the pancreas. On the other, the CCK gene is expressed also in large quantities in cerebral and peripheral neurons from where CCK peptides are released as potent neurotransmitters and modulators. Accordingly, cerebral CCK defects have been associated with major neuropsychiatric diseases such as anxiety, schizophrenia and satiety disorders (Crawley & Corwin, 1994; Liddle, 1997).

CCK is also a key component of the aggression facilitating circuitry in the brain *(*Luo et al., 1998), and it is released during inter-male fighting (Becker et al., 2001). In addition to its many aversive motivational/emotional effects, CCK also plays a role in more positively valenced motivational states, such as mating (Dornan & Malsbury, 1989; Markowski & Hull, 1995), drug addiction (Crespi, 2000) and brain reward processes (Degen et al., 2001, Josselyn, 1996).

It was demonstrated that CCK is colocalized with dopamine in ventral striatal dopamine neurons (Hokfelt et al., 1980). Consistent with the neuronal colocalization and extensive overlap of expression between CCK and the dopaminergic system, CCK peptides have significant effects on dopamine mediated behaviors. Administration of CCK peptides exhibit many of the behavioral characteristics of antipsychotics including inhibition of conditioned avoidance responding (Cohen et al., 1982), inhibition of apomorphine-induced stereotypic behavior (Zetler, 1983), and inhibitionof amphetamine-induced hyperlocomotion (Schneider et al., 1983). Microinjection of CCK into the anterior nucleus accumbens inhibits dopamine release, inhibits dopamine-mediated behaviors and is blocked by a CCKA antagonist whereas injection into the posterior nucleus accumbens has the opposite effects and these effects are mediated by CCKB receptors (Vaccarino & Rankin, 1989, Crawley, 1992). Thus, it appears that different CCK-based circuitries in the brain can facilitate both negative and positive emotional processes. It is also interesting to note that selective CCKB agonists that cross the blood brain barrier such as pentagastrin and CCK-4 are used to induce panic attacks in clinical studies. Consequently, a CCK agonist for schizophrenia would need to be either nonselective or CCKA selective.

CCK was the first gut hormone discovered to have anoretic effects. Its actions include inhibition of food intake, delayed gastric emptying, stimulation of pancreatic enzyme secretion, and stimulation of gall bladder contraction. These effects are mediated via binding to CCK receptors on the vagus nerve. CCK administration to humans and animals inhibits food intake by reducing meal size and duration. However, at high dose nausea and taste aversion have been detected making CCK an unlikely candidate for an anti-obesity treatment.
