**4. Biological activities of BRPs**

In the BPP/CNP precursor of *Bothrops jararaca*, the pentapeptide BPP-5a [QKWAP] that was used as template for the development of the antihypertensive drug captopril, is found duplicated, *i.e.*, there are two copies of the same peptide in a single precursor protein. It is believed that this peptide might have a special importance in the venom of snakes belonging to the *Bothrops* genus, since it is also found repeated three times in isoform 1 [GenBank Acc. No. AY310914.1], and four times in isoform 2 [Genbank Acc. No. AY310915.1] of the precursors isolated from *Bothrops jararacussu* venom glands (Figure 5). In fact, BPP-5a is a potent poten‐

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

**Figure 5. Partial nucleotide and amino acid sequences of the BPP/CNP of precursor from***Bothrops jararacussu* **(isoform 1 and 2).** Shaded in grey, the amino acid sequences of the C-type natriuretic peptide (CNP). Sequences of new putative BPPs are shown in green, and the underlined sequences correspond to other previously known BPPs. In red, the pentapeptide BPP-5a that was found in duplicate in the pit viper precursor and, triplicate and quadruplicate in the isoforms 1 and 2, respectively, of the precursor from *Bothrops jararacussu*. Symbol "M" represents the initial me‐

The discovery of the potential inhibitory action of BPPs on ACE brought a great interest in these natural peptides, since the importance of ACE in blood pressure control and the urge to

develop a therapy for cardiovascular disease, as hypertension, was iminent [2].

**3. BRPs as structural model for drug development**

thionine and (-) the stop codon.

Applications

128

tiator of the BK effects in isolated guinea pig ileum, and also *in vivo* [29].

### **4.1. Interference of BRPs in the renin-angiotensin and kallikrein-kinin system**

The ACE (EC 3.4.15.1) is mainly expressed in vascular endothelium in epithelial cells of the proximal tubules of the kidney, brain, and intestinal cells [60]. This enzyme is responsible for conversion of angiotensin I (Ang I) to angiotensin II (Ang II), and for the degradation of BK. Therefore, this enzyme has roles in both renin-angiotensin and kallikrein-kinin system [61].

The renin-angiotensin system (RAS) is composed by a set of peptides, enzymes, and receptors, that are involved in the control of the extracellular fluid and blood pressure [62]. The formation of the effector peptide of this system occurs initially by the action of the renin released by the kidneys [62] that acts on the angiotensinogen produced in the liver [63]. This leads to the generation of the decapeptide Ang I, which then is cleaved by ACE to form the octapeptide Ang II, a potent antihypertensive molecule [64]. Ang II actions is mediated by the angiotensin receptors AT1 and AT2. The binding of Ang II to the AT1 receptor triggers several cellular processes, among them vasoconstriction, protein synthesis, cell growth, regulation of renal function, and electrolyte balance [65]. Ang II also acts as a neurotransmitter and as a neuro‐ regulador, modulating the central control of the blood pressure, influencing the sympathetic activity, salt appetite, and thirst [65].

The kallikrein-kinin system (KKS) is a metabolic cascade in which the tissue and plasma kallikrein release vasoactive kinins from both high and low molecular weight kininogens. The nonapeptide BK, derived from the cleavage of the high molecular weight kininogen by kallikrein, is the major plasma kinin playing a role in the KKS [66].

Kinins are involved in various physiological and pathological processes, including vasodila‐ tion, increased vascular permeability, release of plasminogen activator of tissue type (t-PA),

and nitric oxide (NO) and arachidonic acid metabolism, mainly due to their ability to activate endothelial cells [66]. Thus, the kinins participate in the physiologically regulation of blood pressure, cardiac and renal functions, and also in pathological processes as inflammation [66].

The several pharmacological activities of kinins are mediated basically by the their binding to two types of specific receptors (B1 and B2 receptors), prior to their fast metabolization by various peptidases [67].

Actions such as vasodilation and hypotension are mediated by the B2 receptor by releasing of NO, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF). On the other hand, the actions mediated by the B1 receptors include important roles in angiogenesis, inflammation, and septic shock [68]. Moreover, unlike B2 receptor, B1 receptor is not consti‐ tutively expressed, and its expression is induced by mediators of inflammation in conditions of injury [68].

The primarily responsible for the degradation of BK are the peptidases (zinc metallopeptidas‐ es) including ACE [67]. Since the early 90's, it is well known that somatic ACE has two active sites, the N-terminus (N-site) and the C-terminus (C-site) active sites [69]. Although *in vitro*, the two active sites are equally effective to convert Ang I to Ang II, as well as to degrade the BK into BK1-7 and BK1-5 [70], the N-site is several times more effective to hydrolyze other bioactive peptides, such as the AcSDKP, a negative regulatory factor for differentiation and proliferation of hematopoietic stem cells [71].

Thus, ACE inhibitors as BPPs inhibit not only the generation of Ang II, but also potentiate the effects of BK, by inhibiting its degradation. Therefore, the physiological effects of the angio‐ tensin system are decreased (since there is no formation of Ang II), and the physiological effects of KKS are potentiated (due to inhibition of the BK degradation). In contrast, the BIPs, most known as helokinestatins, inhibit KKS by blocking the B2 receptor (Figure 6).

the ACE N-terminal active site. In the same way, besides the weak BK potentiation effects of BPP-11e, it is also not among the best inhibitors of ACE, and no preference for any of the active

**Figure 6.** Schematic representation of ACE roles on the renin-angiotensin and kallikrein-kinin systems, and the poten‐ tial sites for interference by BRPs (BPPs and BIPs). A) *Conversion of angiotensin I into angiotensin II*, 2) *BK degradation* 3) *ACE inhibition by BPPs*. 4) *B2 receptor antagonism by BIPs*. Physiological effects on the renin-angiotensin system mediated by AT1 receptors include vasoconstriction, sodium and water retention, release of aldosterone, increased sympathetic nerve activity, among others, while those mediated by AT2 receptors include cell differentiation, vasodila‐ tion, among others. The effects on the kallikrein-kinin system, mediated by kinins action on B2 receptor include vaso‐ dilation and hypotension via release of NO, prostacyclins and endothelium-derived hyperpolarizing factor (EDHF). Due to the ACE inhibition by BPPs, the physiological effects of angiotensin system are decreased (with no formation of an‐ giotensin II) and the physiological effects of kallikrein-kinin are potentiated (by inhibition of BK degradation). In con‐

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Later on, in 2007, molecular studies of the antihypertensive activity of the BPPs, namely BPP-7a and BPP-10c, brought noteworthy information on the molecular mechanism underlying the action of these peptides at cellular level. In fact, these BPPs have a strong and sustained antihypertensive activity in awake spontaneously hypertensive rats (SHRs), but they do not prevent the formation of Ang II from Ang I *in vivo*, showing that they do not need to affect the physiological functions of ACE to promote the decrease of the blood pressure in these animals. Furthermore, for BPP-10c, we have also shown that the dose necessary to produce the antihypertensive effect is lower than that required to inhibit ACE *in vivo* [77], suggesting the participation of other putative targets determining this particular pharmacological effect.

This finding was reinforced by the studies conducted to clarify the biological distribution of BPP-10c using a I125 labeled analog, which showed that this peptide accumulated in various rat organs such as brain, liver, testis, and kidney, even after pre-saturation of the potential

This stimulated us to conduct studies aiming to identify new potential molecular targets for snake BPPs. So, it was shown that at least three BPPs, namely BPP-10c, BPP-12b, and BPP-13a, are able to bind to the enzyme argininosuccinate synthase (AsS) modulating positively its

active sites of ACE with a specific inhibitor of this enzyme, namely captopril [78].

sites of ACE was observed for this peptide [29].

trast, BIPs action on B2 receptor blocked BK effects. Adapted from [132-134].

activity [20, 75].

### **4.2. Mechanisms of action underlying the antihypertensive effect of BRPs**

Although ACE inhibition is a relevant mechanism to explain the activity of most BPPs, and despite of their high primary sequence similarity [53, 72], as previously suggested, the BPPs show remarkable wide variety of mechanistic pathways that could explain the antihyperten‐ sive activity of BPPs at molecular level [13, 14, 19, 20, 22, 23, 29, 73-76]. Definitely the biological effects of BPPs and the consequent pharmacological importance of their activity are not limited to and it cannot be explained solely based on their ability to inhibit ACE [2].

The differences were first observed when comparing the selectivity of the BPPs encoded by the neuronal BPP/CNP precursor protein [*e.g.* BPP-5a, BPP-10c, BPP-11e, BPP-12b, and BPP-13a] [14] by the different active sites of the somatic ACE and the corresponding biological activity of these peptides evaluated by their ability to potentiate the contractile effect triggered by BK in isolated guinea pig ileum. For instance, the BPP-5a was shown to be much less effective ACE inhibitor compared to BPP-13a, although presenting one of the most potent potentiator effects of BK in *ex vivo* experiments. In contrast, BPP-10c is an excellent selective inhibitor of the C-terminal active site of somatic ACE, and its BK potentiating effect is very similar to that observed for both BPP-5a and BPP-12b, which were shown to be selective for Venom Bradykinin-Related Peptides (BRPs) and Its Multiple Biological Roles http://dx.doi.org/10.5772/52872 131

and nitric oxide (NO) and arachidonic acid metabolism, mainly due to their ability to activate endothelial cells [66]. Thus, the kinins participate in the physiologically regulation of blood pressure, cardiac and renal functions, and also in pathological processes as inflammation [66]. The several pharmacological activities of kinins are mediated basically by the their binding to two types of specific receptors (B1 and B2 receptors), prior to their fast metabolization by

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

Actions such as vasodilation and hypotension are mediated by the B2 receptor by releasing of NO, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF). On the other hand, the actions mediated by the B1 receptors include important roles in angiogenesis, inflammation, and septic shock [68]. Moreover, unlike B2 receptor, B1 receptor is not consti‐ tutively expressed, and its expression is induced by mediators of inflammation in conditions

The primarily responsible for the degradation of BK are the peptidases (zinc metallopeptidas‐ es) including ACE [67]. Since the early 90's, it is well known that somatic ACE has two active sites, the N-terminus (N-site) and the C-terminus (C-site) active sites [69]. Although *in vitro*, the two active sites are equally effective to convert Ang I to Ang II, as well as to degrade the BK into BK1-7 and BK1-5 [70], the N-site is several times more effective to hydrolyze other bioactive peptides, such as the AcSDKP, a negative regulatory factor for differentiation and

Thus, ACE inhibitors as BPPs inhibit not only the generation of Ang II, but also potentiate the effects of BK, by inhibiting its degradation. Therefore, the physiological effects of the angio‐ tensin system are decreased (since there is no formation of Ang II), and the physiological effects of KKS are potentiated (due to inhibition of the BK degradation). In contrast, the BIPs, most

Although ACE inhibition is a relevant mechanism to explain the activity of most BPPs, and despite of their high primary sequence similarity [53, 72], as previously suggested, the BPPs show remarkable wide variety of mechanistic pathways that could explain the antihyperten‐ sive activity of BPPs at molecular level [13, 14, 19, 20, 22, 23, 29, 73-76]. Definitely the biological effects of BPPs and the consequent pharmacological importance of their activity are not limited

The differences were first observed when comparing the selectivity of the BPPs encoded by the neuronal BPP/CNP precursor protein [*e.g.* BPP-5a, BPP-10c, BPP-11e, BPP-12b, and BPP-13a] [14] by the different active sites of the somatic ACE and the corresponding biological activity of these peptides evaluated by their ability to potentiate the contractile effect triggered by BK in isolated guinea pig ileum. For instance, the BPP-5a was shown to be much less effective ACE inhibitor compared to BPP-13a, although presenting one of the most potent potentiator effects of BK in *ex vivo* experiments. In contrast, BPP-10c is an excellent selective inhibitor of the C-terminal active site of somatic ACE, and its BK potentiating effect is very similar to that observed for both BPP-5a and BPP-12b, which were shown to be selective for

known as helokinestatins, inhibit KKS by blocking the B2 receptor (Figure 6).

**4.2. Mechanisms of action underlying the antihypertensive effect of BRPs**

to and it cannot be explained solely based on their ability to inhibit ACE [2].

various peptidases [67].

proliferation of hematopoietic stem cells [71].

of injury [68].

Applications

130

**Figure 6.** Schematic representation of ACE roles on the renin-angiotensin and kallikrein-kinin systems, and the poten‐ tial sites for interference by BRPs (BPPs and BIPs). A) *Conversion of angiotensin I into angiotensin II*, 2) *BK degradation* 3) *ACE inhibition by BPPs*. 4) *B2 receptor antagonism by BIPs*. Physiological effects on the renin-angiotensin system mediated by AT1 receptors include vasoconstriction, sodium and water retention, release of aldosterone, increased sympathetic nerve activity, among others, while those mediated by AT2 receptors include cell differentiation, vasodila‐ tion, among others. The effects on the kallikrein-kinin system, mediated by kinins action on B2 receptor include vaso‐ dilation and hypotension via release of NO, prostacyclins and endothelium-derived hyperpolarizing factor (EDHF). Due to the ACE inhibition by BPPs, the physiological effects of angiotensin system are decreased (with no formation of an‐ giotensin II) and the physiological effects of kallikrein-kinin are potentiated (by inhibition of BK degradation). In con‐ trast, BIPs action on B2 receptor blocked BK effects. Adapted from [132-134].

the ACE N-terminal active site. In the same way, besides the weak BK potentiation effects of BPP-11e, it is also not among the best inhibitors of ACE, and no preference for any of the active sites of ACE was observed for this peptide [29].

Later on, in 2007, molecular studies of the antihypertensive activity of the BPPs, namely BPP-7a and BPP-10c, brought noteworthy information on the molecular mechanism underlying the action of these peptides at cellular level. In fact, these BPPs have a strong and sustained antihypertensive activity in awake spontaneously hypertensive rats (SHRs), but they do not prevent the formation of Ang II from Ang I *in vivo*, showing that they do not need to affect the physiological functions of ACE to promote the decrease of the blood pressure in these animals. Furthermore, for BPP-10c, we have also shown that the dose necessary to produce the antihypertensive effect is lower than that required to inhibit ACE *in vivo* [77], suggesting the participation of other putative targets determining this particular pharmacological effect.

This finding was reinforced by the studies conducted to clarify the biological distribution of BPP-10c using a I125 labeled analog, which showed that this peptide accumulated in various rat organs such as brain, liver, testis, and kidney, even after pre-saturation of the potential active sites of ACE with a specific inhibitor of this enzyme, namely captopril [78].

This stimulated us to conduct studies aiming to identify new potential molecular targets for snake BPPs. So, it was shown that at least three BPPs, namely BPP-10c, BPP-12b, and BPP-13a, are able to bind to the enzyme argininosuccinate synthase (AsS) modulating positively its activity [20, 75].

The AsS is the rate-limiting step enzyme responsible for providing the substrate for the nitric oxide synthase (NOS) that produces NO [79, 80]. Guerreiro and colleagues also demonstrated that blood pressure decrease promoted in SHRs by BPP-10c administration is due to the increased bioavailability of L-arginine required for the production of NO [20], which is a potent vasodilator agent [81]. Later it was demonstrated that other BPPs also induces NO production to determine the antihypertensive effect [21, 75].

Arterial baroreflex is one of the most important regulatory mechanisms in the cardiovascular system, mainly by triggering a coordinated sympathetic and parasympathetic tone response

Venom Bradykinin-Related Peptides (BRPs) and Its Multiple Biological Roles

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133

The CNS is connected to the heart through two different groups of nerves, the parasympathetic and sympathetic systems. Stimulation of parasympathetic nerves determines the decrease of HR, of contraction force of atrial muscle, and of conduction of impulses through the atrioven‐ tricular node, and at the same time, it also causes the increase of the time delay between the atrial and ventricular contraction, and the reduction of blood flow through the coronary arteries, which maintains the nutrition of the myocardium. All these effects can be summarized by saying that the parasympathetic stimulation decreases all the activities of the heart. On the other hand, the stimulation of sympathetic nerves has exactly the opposite effects on the heart, leading to an increased HR, increased contraction force, and increased blood flow through the

It was observed that BPP-11e causes a slight reduction in MAP, but surprisingly with a strong reduction in HR [75], suggesting a BPPs action in specialized muscle cells located in the sinoatrial region (pacemaker) of the heart, which is a special region of the heart that controls the cardiac frequency [92]. Although the heart has its own intrinsic control systems, it can operate under neural influences, therefore effectiveness of the cardiac action can be significantly modified by regulatory pulses from the CNS [92]. Thus there is also possible that the BPP-11e has an effect on the stimulation of the parasympathetic system and/or in the decreasing of the sympathetic system stimulation, leading to a reduction of the HR and a slight decrease in MAP,

A more detailed study of the BPPs effects on the CNS was performed for the BPP-10c, in which intracerebroventricular administration was shown to produce similar effects to those observed for higher doses injections of this peptide by intravenous route. In our interpretation, this data suggested the involvement of the CNS in the pathway underlying these biological effects [74].

Aiming to explain tbe BPPs effects on CNS, *Lameu et al*. have also conducted studies to demonstrate that the BPP-10c acts through activation of an unidentified Gi/o-coupled receptor present in neuronal cells, and that this effect was independent of both ACE inhibition and B2 receptor activation. Peptide–receptor binding resulted in the activation of calcium influx and release of intracellular calcium by calcium-induced calcium release (CICR) mechanism, which was shown to involve the activation of the ryanodine- or IP3-sensitive calcium stores and also the inhibition of adenylate cyclase [74]. However the specific target GPCR could not be

On the other hand, affinity chromatography, using immobilized BPP-10c, associated with mass spectrometric and immunoblot analyses, allowed the identification of two important targets of BPP-10c, namely the AsS in the kidney cytosol [20] and the synapsin in the brain (Figure 7) [93]. AsS, together with argininosuccinate lyase (AsL), is part of the urea cycle in the liver and of the arginine-citrulline cycle, the major source of arginine in the renal cells and citrulline– NO cycle, which is the main source of NO in other cells, including endothelial and neuronal

on the heart and vessels [85-90].

blood vessels [87, 91].

identified yet.

cells [94].

observed after *in vivo* injection of this peptide [75].

Moreover, at least for the BPP-5a-induced NO production, the involvement of both B2 receptor and mACh-M1, without any involvement of AsS, was recently demonstrated [23]. BPP-13a induces NO production through a mechanism that involves activation of subtype M3 mus‐ carinic receptor (mACh-M3), triggering the raise of the free intracellular calcium concentration ([Ca2+]*<sup>i</sup>* ) that is able to activate NOS and to provide the substrate for NO production by modulating the AsS activity [75].

Both BPP-11e and BPP-12b do not stimulate NO production, but the [Ca2+]*<sup>i</sup>* mobilization assays suggest that these peptides are agonists of a membrane receptor involved in the release of EDHF, and other functions involving the modulation of gene expression and activation of different NOS enzymes is expected [82]. As BPP-12b modulates positively AsS activity only at very high concentrations, this should not be its main mechanism of action [75].

Since the BPP-9a has ACE as main target for its biological actions, based on its potent inhibitory activity against this enzyme also showing selectivity for the C-terminal active site [73], we suggest that it is possible to suggest this pathway to explain the antihypertensive effect and BK potentiation of BPP-9a (teprotide). Moreover, it has no effect on the AsS induced intracel‐ lular calcium and it also does not interfere with NO production.

Apparently all BPPs share the ability to decrease arterial pressure [21, 56, 75, 77, 83], through the amplitude of the antihypertensive effect caused by BK, each related peptides is different. But, unfortunately, the mechanisms of action of other BPPs are still less understood up to now [75].

### **4.3. Peripheral and central biological activities of BPPs**

Changes in mean arterial pressure (MAP) promoted by some BPPs are accompanied by a significant reduction in heart rate (HR) [23, 75, 77] rather than by an HR increase, as it would be expected by the response of the baroreceptors to the hypotension [84]. The fact is that *in bolus* injections of BPPs decrease both MAP and HR of awake SHRs, and BPPs expression in the same precursor protein of a brain expressed peptide as CNP suggests a CNS role for these peptides. In fact, recently it was shown that the BPP-10c is able to promote the release of the neurotransmitters GABA and glutamate, which are known to participate in the regulation of cardiac and vascular autonomic systems, leading to decline MAP and HR of SHRs [22]. According to Lameu and collaborators, BPP-10c-induced decrease of MAP results from this BRP-induced interference in the autonomic nervous system, provoking subsequent changes in HR and baroreflex control [22, 74].

Arterial baroreflex is one of the most important regulatory mechanisms in the cardiovascular system, mainly by triggering a coordinated sympathetic and parasympathetic tone response on the heart and vessels [85-90].

The AsS is the rate-limiting step enzyme responsible for providing the substrate for the nitric oxide synthase (NOS) that produces NO [79, 80]. Guerreiro and colleagues also demonstrated that blood pressure decrease promoted in SHRs by BPP-10c administration is due to the increased bioavailability of L-arginine required for the production of NO [20], which is a potent vasodilator agent [81]. Later it was demonstrated that other BPPs also induces NO production

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

Moreover, at least for the BPP-5a-induced NO production, the involvement of both B2 receptor and mACh-M1, without any involvement of AsS, was recently demonstrated [23]. BPP-13a induces NO production through a mechanism that involves activation of subtype M3 mus‐ carinic receptor (mACh-M3), triggering the raise of the free intracellular calcium concentration

suggest that these peptides are agonists of a membrane receptor involved in the release of EDHF, and other functions involving the modulation of gene expression and activation of different NOS enzymes is expected [82]. As BPP-12b modulates positively AsS activity only at

Since the BPP-9a has ACE as main target for its biological actions, based on its potent inhibitory activity against this enzyme also showing selectivity for the C-terminal active site [73], we suggest that it is possible to suggest this pathway to explain the antihypertensive effect and BK potentiation of BPP-9a (teprotide). Moreover, it has no effect on the AsS induced intracel‐

Apparently all BPPs share the ability to decrease arterial pressure [21, 56, 75, 77, 83], through the amplitude of the antihypertensive effect caused by BK, each related peptides is different. But, unfortunately, the mechanisms of action of other BPPs are still less understood up to

Changes in mean arterial pressure (MAP) promoted by some BPPs are accompanied by a significant reduction in heart rate (HR) [23, 75, 77] rather than by an HR increase, as it would be expected by the response of the baroreceptors to the hypotension [84]. The fact is that *in bolus* injections of BPPs decrease both MAP and HR of awake SHRs, and BPPs expression in the same precursor protein of a brain expressed peptide as CNP suggests a CNS role for these peptides. In fact, recently it was shown that the BPP-10c is able to promote the release of the neurotransmitters GABA and glutamate, which are known to participate in the regulation of cardiac and vascular autonomic systems, leading to decline MAP and HR of SHRs [22]. According to Lameu and collaborators, BPP-10c-induced decrease of MAP results from this BRP-induced interference in the autonomic nervous system, provoking subsequent changes

Both BPP-11e and BPP-12b do not stimulate NO production, but the [Ca2+]*<sup>i</sup>*

lular calcium and it also does not interfere with NO production.

**4.3. Peripheral and central biological activities of BPPs**

in HR and baroreflex control [22, 74].

very high concentrations, this should not be its main mechanism of action [75].

) that is able to activate NOS and to provide the substrate for NO production by

mobilization assays

to determine the antihypertensive effect [21, 75].

modulating the AsS activity [75].

([Ca2+]*<sup>i</sup>*

Applications

132

now [75].

The CNS is connected to the heart through two different groups of nerves, the parasympathetic and sympathetic systems. Stimulation of parasympathetic nerves determines the decrease of HR, of contraction force of atrial muscle, and of conduction of impulses through the atrioven‐ tricular node, and at the same time, it also causes the increase of the time delay between the atrial and ventricular contraction, and the reduction of blood flow through the coronary arteries, which maintains the nutrition of the myocardium. All these effects can be summarized by saying that the parasympathetic stimulation decreases all the activities of the heart. On the other hand, the stimulation of sympathetic nerves has exactly the opposite effects on the heart, leading to an increased HR, increased contraction force, and increased blood flow through the blood vessels [87, 91].

It was observed that BPP-11e causes a slight reduction in MAP, but surprisingly with a strong reduction in HR [75], suggesting a BPPs action in specialized muscle cells located in the sinoatrial region (pacemaker) of the heart, which is a special region of the heart that controls the cardiac frequency [92]. Although the heart has its own intrinsic control systems, it can operate under neural influences, therefore effectiveness of the cardiac action can be significantly modified by regulatory pulses from the CNS [92]. Thus there is also possible that the BPP-11e has an effect on the stimulation of the parasympathetic system and/or in the decreasing of the sympathetic system stimulation, leading to a reduction of the HR and a slight decrease in MAP, observed after *in vivo* injection of this peptide [75].

A more detailed study of the BPPs effects on the CNS was performed for the BPP-10c, in which intracerebroventricular administration was shown to produce similar effects to those observed for higher doses injections of this peptide by intravenous route. In our interpretation, this data suggested the involvement of the CNS in the pathway underlying these biological effects [74].

Aiming to explain tbe BPPs effects on CNS, *Lameu et al*. have also conducted studies to demonstrate that the BPP-10c acts through activation of an unidentified Gi/o-coupled receptor present in neuronal cells, and that this effect was independent of both ACE inhibition and B2 receptor activation. Peptide–receptor binding resulted in the activation of calcium influx and release of intracellular calcium by calcium-induced calcium release (CICR) mechanism, which was shown to involve the activation of the ryanodine- or IP3-sensitive calcium stores and also the inhibition of adenylate cyclase [74]. However the specific target GPCR could not be identified yet.

On the other hand, affinity chromatography, using immobilized BPP-10c, associated with mass spectrometric and immunoblot analyses, allowed the identification of two important targets of BPP-10c, namely the AsS in the kidney cytosol [20] and the synapsin in the brain (Figure 7) [93]. AsS, together with argininosuccinate lyase (AsL), is part of the urea cycle in the liver and of the arginine-citrulline cycle, the major source of arginine in the renal cells and citrulline– NO cycle, which is the main source of NO in other cells, including endothelial and neuronal cells [94].

> AsS is a ubiquitously expressed enzyme, present in many tissues, including brain and kidney [94]. *In vivo* BPP-10c administration in SHRs animal models results in increase of plasma arginine level [20] and augmented NO production in brain tissues, as well as in neuronal and

Venom Bradykinin-Related Peptides (BRPs) and Its Multiple Biological Roles

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NO is generated in the citrulline-NO cycle by NO synthase (NOS) using L-arginine as a substrate. Three isoforms of NOS have been described: Ca2+-dependent endothelial (eNOS) and neuronal (nNOS) isoforms [95] and inducible NOS. The expression and activity of the latter are induced by inflammatory stimuli, independent of the cytosolic Ca2+ concentra‐

NO is mainly involved in the regulation of local and systemic vascular resistance in sodium balance, and hence in blood pressure control [97], since it is one of the smooth muscle relaxing factors released by the endothelium, which diffuses to the adjacent smooth muscle cells

Nevertheless, NO has been attributed to other various functions, including non-cholinergic and non-adrenergic smooth muscle relaxation, reduction of arterial pressure, and signal transmission in the CNS [100]. NO-mediated actions in the CNS include central vascular regulation [101] and baroreflex control of HR [102]. Antihypertensive activity, based on the facilitated release of both GABA and glutamate in the CNS and NO production, is suggested to result in the diminished transmission of sympathetic tone to the periphery [101, 103].

Treatments with BPP-10c also induced an increase in AsS gene expression [22]. In contrast, nNOS was not found differentially expressed in the brains of SHRs treated with BPP-10c compared to vehicle-treated animals. On the other hand, the gene expression levels of eNOS, similarly to those of AsS, were found increased in the brains of SHRs animals treated with BPP-10c [22]. This data is in line with the results obtained by Kishi and colleagues [104] who were able to show that the overexpression of eNOS in the rostral ventrolateral medulla and the nucleus of the solitary tract of hypertensive rats results in reduced systolic arterial pressure

However, the specificity of NO reactions with neuronal targets is determined in part by the precise localization of NOS within the cell. The targeting of NOS to discrete nuclei of neurons, mediated by adapter proteins, allowed to suggest that both synapsin and NOS participate of

Knowing that the BPP-10c binds to synapsin in the CNS, we hypothesized the formation of a quaternary complex upon binding of BPP-10c with synapsin. The formation of this complex would direct the reactions of NO in neural targets, which would be determined in part by the

We were able to show that BPP-10c is capable to induce intracellular Ca2+ signaling that involves the activation of GPCRs, NO production, and release of neurotransmitters, such as GABA and glutamate [22, 74]. The amino acid glutamate is the major excitatory neurotrans‐ mitter in the CNS of mammals, whereas GABA is the main mediator of sympathetic inhibitory currents. Both glutamate and GABA play key roles in the control of cardiovascular function

a ternary complex, which changes in the subcellular localization of NOS [105].

location of this complex and by targeting NOS to specific sites of neurons [105].

endothelial cells [20, 22].

promoting vasodilatation [98, 99].

tion [96].

and reduced HR.


**Figure 7. Synapsin binds to BPP-10c**. (**A**) SDS-PAGE analysis of brain rat cytosolic proteins submitted to HiTrap-BPP-10c affinity chromatography. Rat brain cytosol preparation to affinity chromatography using the HiTrap NHS-acti‐ vated HP resin to which BPP-10c was immobilized by chemical conjugation **M (KDa)**, molecular mass markers; **lane 1**, protein eluted by competition using 5 mg of BPP-10c; **lane 2**, protein eluted with 100 mM glycine, 0.5 M NaCl pH 3.0 (elution buffer: by lowering the pH). (**B**) protein identification by mass spectrometric analysis of the bands enclosed in the box in the panel **A**. The 74-kDa major protein that bounds to BPP-10c was identified as synapsin, by trypsin diges‐ tion and peptide mass fingerprint analysis.

AsS is a ubiquitously expressed enzyme, present in many tissues, including brain and kidney [94]. *In vivo* BPP-10c administration in SHRs animal models results in increase of plasma arginine level [20] and augmented NO production in brain tissues, as well as in neuronal and endothelial cells [20, 22].

NO is generated in the citrulline-NO cycle by NO synthase (NOS) using L-arginine as a substrate. Three isoforms of NOS have been described: Ca2+-dependent endothelial (eNOS) and neuronal (nNOS) isoforms [95] and inducible NOS. The expression and activity of the latter are induced by inflammatory stimuli, independent of the cytosolic Ca2+ concentra‐ tion [96].

NO is mainly involved in the regulation of local and systemic vascular resistance in sodium balance, and hence in blood pressure control [97], since it is one of the smooth muscle relaxing factors released by the endothelium, which diffuses to the adjacent smooth muscle cells promoting vasodilatation [98, 99].

Nevertheless, NO has been attributed to other various functions, including non-cholinergic and non-adrenergic smooth muscle relaxation, reduction of arterial pressure, and signal transmission in the CNS [100]. NO-mediated actions in the CNS include central vascular regulation [101] and baroreflex control of HR [102]. Antihypertensive activity, based on the facilitated release of both GABA and glutamate in the CNS and NO production, is suggested to result in the diminished transmission of sympathetic tone to the periphery [101, 103].

Treatments with BPP-10c also induced an increase in AsS gene expression [22]. In contrast, nNOS was not found differentially expressed in the brains of SHRs treated with BPP-10c compared to vehicle-treated animals. On the other hand, the gene expression levels of eNOS, similarly to those of AsS, were found increased in the brains of SHRs animals treated with BPP-10c [22]. This data is in line with the results obtained by Kishi and colleagues [104] who were able to show that the overexpression of eNOS in the rostral ventrolateral medulla and the nucleus of the solitary tract of hypertensive rats results in reduced systolic arterial pressure and reduced HR.

However, the specificity of NO reactions with neuronal targets is determined in part by the precise localization of NOS within the cell. The targeting of NOS to discrete nuclei of neurons, mediated by adapter proteins, allowed to suggest that both synapsin and NOS participate of a ternary complex, which changes in the subcellular localization of NOS [105].

Knowing that the BPP-10c binds to synapsin in the CNS, we hypothesized the formation of a quaternary complex upon binding of BPP-10c with synapsin. The formation of this complex would direct the reactions of NO in neural targets, which would be determined in part by the location of this complex and by targeting NOS to specific sites of neurons [105].

**Figure 7. Synapsin binds to BPP-10c**. (**A**) SDS-PAGE analysis of brain rat cytosolic proteins submitted to HiTrap-BPP-10c affinity chromatography. Rat brain cytosol preparation to affinity chromatography using the HiTrap NHS-acti‐ vated HP resin to which BPP-10c was immobilized by chemical conjugation **M (KDa)**, molecular mass markers; **lane 1**, protein eluted by competition using 5 mg of BPP-10c; **lane 2**, protein eluted with 100 mM glycine, 0.5 M NaCl pH 3.0 (elution buffer: by lowering the pH). (**B**) protein identification by mass spectrometric analysis of the bands enclosed in the box in the panel **A**. The 74-kDa major protein that bounds to BPP-10c was identified as synapsin, by trypsin diges‐

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

Applications

134

tion and peptide mass fingerprint analysis.

We were able to show that BPP-10c is capable to induce intracellular Ca2+ signaling that involves the activation of GPCRs, NO production, and release of neurotransmitters, such as GABA and glutamate [22, 74]. The amino acid glutamate is the major excitatory neurotrans‐ mitter in the CNS of mammals, whereas GABA is the main mediator of sympathetic inhibitory currents. Both glutamate and GABA play key roles in the control of cardiovascular function

in the CNS [103]. Excitatory amino acid neurotransmitters, like glutamate and aspartate, generally cause pressure responses and tachycardia, while inhibitory amino acid neurotrans‐ mitters, namely GABA and glycine, are responsible for depressing bradycardia [104]. It is well established that the excitatory amino acid glutamate is considered the main neurotransmitter of primary afferent fibers of baroreceptors to the nucleus tractus solitarii (NTS) [106]. Further‐ more, an excitatory projection from NTS to the caudal ventrolateral medulla (CVLM) is an essential part of the circuit of baroreflex control. The CVLM communicates with the rostral ventrolateral medulla (RVLM) by secretion of GABA. In addition to GABAergic inhibition of RVLM, excitatory amino acids are also known to exert important roles in cardiovascular regulation [107]. These neurotransmitters can regulate vasodilatation through reduction of both sympathetic activity and baroreflex sensitivity control, by acting on regulation of both sympathetic and parasympathetic systems. Therefore, the augmented baroreflex sensitivity by i.v. injection of BPP-10c is attributted to the release of these neurotransmitters [22, 74]. These data is summarized in Figure 8.

The fact of BPPs decrease the HR does not mean that its action is limited to the CNS. Taking the example of BPP-10c, *in vivo* biodistribution studies showed a significant presence of this peptide in the brain, however accumulation was also observed in the rat kidney [78]. Consid‐ ering its high accumulation in kidneys with the fact that BPP-10c induces NO production in endothelial cells [20, 108] and the increase of plasma L-arginine level *in vivo* [20], we conclude that this peptide, and also potentially the other BPPs, may display both peripheral and central

Venom Bradykinin-Related Peptides (BRPs) and Its Multiple Biological Roles

http://dx.doi.org/10.5772/52872

137

Moreover, there might exhist BPPs with exclusive peripheral action. As suggested for BPP-9a that promotes discret decrease of MAP and does not affect HR [75], whose effects were possible to be explained solely based on the classical mechanism of action suggested for the BPPs, *i.e*.,

The pharmaceutical compositions for the applications in chornic-degenerative diseases and hypertension of the BPPs and their structural and/or conformational analogs, as well as the isolation and purification of BRPs secreted by snake venom glands are protected by the patents US20050031604 and US20080199503. The inventions further refer to pharmaceutical composi‐ tions that increase the biodisponibility and efficacy of BRPs peripheral and central biological activities. BRPs allowed the development a successful oral drug to treat human hypertension, but they also have the potential to become a drug by itself or a drug model to develop compouds devoted to treat central nervous system diseases, once pharmaceutical composi‐

**4.4. Mechanism of action that underlies the hyperalgesia and inflammatory responses**

For many years it has been known that BK is an inflammatory mediator involved in the nociceptive process [109]. BK and also the BRPs produce pain and hyperalgesia due to their

In particular, two novel BRPs named fulvonin [SIVLRGKAPFR] and cyphokinin [DTRPPGFTPFR] were recently described in wasp (*Cyphononyx fulvognathus*). They could be structurally and functionally considered as BRPs, since they both are able to inhibit ACE as well as to induce the hyperalgesic effect in living rats after intraplantar injection, mostly due to the agonist action of these peptides on distinct B2 or B1 receptors, respectively [10].

In the last few years, as presented here, it was possible to describe a number of new mechanisms of action for BRPs previously known only as potent ACE inhibitors. Taking this into account, many pharmaceutical applications could be possible suggested for these peptides solely based on the treatment of pathologies related with their targets, for instace

relying in the selective inhibition of the somatic ACE [73].

tions that allow efficient delivery *in vivo* of these BRPs is achieved.

**5. Potential and effective pharmaceutical applications**

**5.1. Application of BPPs to treat CNS disorders and hypertension**

ability to excite and/or sensitize nociceptors [10].

the somatic ACE, AsS and so on.

actions.

**Figure 8. Schematic representation of BPP-10c mechanism of action in the CNS**. According to [22, 74, 93], the pro‐ posed mechanism to explain the BPP-10c effects on the blood pressure (BP) and heart rate (HR) in spontaneously hy‐ pertensive rats (SHRs) was summarized in this figure. First, BPP-10c-induced [Ca2+]*<sup>i</sup>* elevations activates signal transduction pathways responsible for the increased nitric oxide synthase (NOS) activity and the expression of the en‐ zymes, namely endothelial NOS (eNOS) and argininosuccinate synthase (ASS). It also triggers the release of the neuro‐ transmitters GABA and glutamate. After the BPP-10c internalization by neuronal cells, this peptide binds to synapsin to control the release of GABA and glutamate, and to direct the NOS to discrete cores of the neurons. Furthermore, BPP-10c can positively activate the AsS functions to increase the levels of L-arginine. NO production due to increased concentration of L-arginine, NOS activation, and increased expression of eNOS and AsS should contribute for the re‐ lease of neurotransmitters and also for the regulation of autonomous activity. Likewise GABA and glutamate deter‐ mine the reduction of both blood pressure and heart rate, and the increases of the baroreflex sensitivity in SHRs.

The fact of BPPs decrease the HR does not mean that its action is limited to the CNS. Taking the example of BPP-10c, *in vivo* biodistribution studies showed a significant presence of this peptide in the brain, however accumulation was also observed in the rat kidney [78]. Consid‐ ering its high accumulation in kidneys with the fact that BPP-10c induces NO production in endothelial cells [20, 108] and the increase of plasma L-arginine level *in vivo* [20], we conclude that this peptide, and also potentially the other BPPs, may display both peripheral and central actions.

in the CNS [103]. Excitatory amino acid neurotransmitters, like glutamate and aspartate, generally cause pressure responses and tachycardia, while inhibitory amino acid neurotrans‐ mitters, namely GABA and glycine, are responsible for depressing bradycardia [104]. It is well established that the excitatory amino acid glutamate is considered the main neurotransmitter of primary afferent fibers of baroreceptors to the nucleus tractus solitarii (NTS) [106]. Further‐ more, an excitatory projection from NTS to the caudal ventrolateral medulla (CVLM) is an essential part of the circuit of baroreflex control. The CVLM communicates with the rostral ventrolateral medulla (RVLM) by secretion of GABA. In addition to GABAergic inhibition of RVLM, excitatory amino acids are also known to exert important roles in cardiovascular regulation [107]. These neurotransmitters can regulate vasodilatation through reduction of both sympathetic activity and baroreflex sensitivity control, by acting on regulation of both sympathetic and parasympathetic systems. Therefore, the augmented baroreflex sensitivity by i.v. injection of BPP-10c is attributted to the release of these neurotransmitters [22, 74]. These

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

**Figure 8. Schematic representation of BPP-10c mechanism of action in the CNS**. According to [22, 74, 93], the pro‐ posed mechanism to explain the BPP-10c effects on the blood pressure (BP) and heart rate (HR) in spontaneously hy‐

transduction pathways responsible for the increased nitric oxide synthase (NOS) activity and the expression of the en‐ zymes, namely endothelial NOS (eNOS) and argininosuccinate synthase (ASS). It also triggers the release of the neuro‐ transmitters GABA and glutamate. After the BPP-10c internalization by neuronal cells, this peptide binds to synapsin to control the release of GABA and glutamate, and to direct the NOS to discrete cores of the neurons. Furthermore, BPP-10c can positively activate the AsS functions to increase the levels of L-arginine. NO production due to increased concentration of L-arginine, NOS activation, and increased expression of eNOS and AsS should contribute for the re‐ lease of neurotransmitters and also for the regulation of autonomous activity. Likewise GABA and glutamate deter‐ mine the reduction of both blood pressure and heart rate, and the increases of the baroreflex sensitivity in SHRs.

]*i*

elevations activates signal

pertensive rats (SHRs) was summarized in this figure. First, BPP-10c-induced [Ca2+

data is summarized in Figure 8.

Applications

136

Moreover, there might exhist BPPs with exclusive peripheral action. As suggested for BPP-9a that promotes discret decrease of MAP and does not affect HR [75], whose effects were possible to be explained solely based on the classical mechanism of action suggested for the BPPs, *i.e*., relying in the selective inhibition of the somatic ACE [73].

The pharmaceutical compositions for the applications in chornic-degenerative diseases and hypertension of the BPPs and their structural and/or conformational analogs, as well as the isolation and purification of BRPs secreted by snake venom glands are protected by the patents US20050031604 and US20080199503. The inventions further refer to pharmaceutical composi‐ tions that increase the biodisponibility and efficacy of BRPs peripheral and central biological activities. BRPs allowed the development a successful oral drug to treat human hypertension, but they also have the potential to become a drug by itself or a drug model to develop compouds devoted to treat central nervous system diseases, once pharmaceutical composi‐ tions that allow efficient delivery *in vivo* of these BRPs is achieved.

### **4.4. Mechanism of action that underlies the hyperalgesia and inflammatory responses**

For many years it has been known that BK is an inflammatory mediator involved in the nociceptive process [109]. BK and also the BRPs produce pain and hyperalgesia due to their ability to excite and/or sensitize nociceptors [10].

In particular, two novel BRPs named fulvonin [SIVLRGKAPFR] and cyphokinin [DTRPPGFTPFR] were recently described in wasp (*Cyphononyx fulvognathus*). They could be structurally and functionally considered as BRPs, since they both are able to inhibit ACE as well as to induce the hyperalgesic effect in living rats after intraplantar injection, mostly due to the agonist action of these peptides on distinct B2 or B1 receptors, respectively [10].
