**2.** *In Vitro* **pharmacology of blood vessels**

Pharmacology of isolated blood vessel allows the researcher to investigate the mechanisms of effect of spasmogens or vasodilatory substances. Most studies use the isolated vessel ring preparation in the organ bath, studying removed segments from the grafts during surgery. This technique only requires basic pharmacological equipment, i.e. isolated organ baths, transducers, recorder system etc. An important advantage of this method is that the vessel segment is studied in the organ bath and concentration-response curves for each vasoactive substances to be obtained under controlled conditions without extrinsic neural factors, circulating hormones interacting, blood flow or shear stress. Therefore, dose and response relationships to drugs, either vasoconstrictor or vasodilator substances, can be assessed more readily and accurately than is possible than in vivo experiments. This methodology also enabled agents to be compared with each other, and combinations of vasoactive drugs to be tested [10,11-13]. In vitro measurement of response of vascular preparations may help to researcher to predict what can happen, not what does actually happen in integrative and complicated in vivo conditions. However, isolated organ bath methods cannot identify the actual cause of in vivo spasm. The next challenge is to determine in the body what combination of factors, i.e. extrinsic neural factors, circulating hormones interacting, blood flow or shear stress, influencing passive distension from arterial wall are present the vessel with spasm.

Isolated organ bath technique is a standard research approach which requires basic pharma‐ cological equipment (Figure 1). Segments of human arteries obtained from patients undergo‐ ing CABG surgery are placed in oxygenated physiological solution, i.e. Krebs-Henseleit solution etc., at room temperature and transferred immediately to the laboratory. The arteries are dissected from adhering fat and connective tissue then cut into 3-4 mm length rings. The strips are mounted in an organ bath, containing physiological solution, on a L-shaped brace for tension measurement along the former circumferential axis. The solution is gassed with % 95 O2 and % 5 CO2 at 37 ºC. Changes in arterial tensions are recorded isometrically by a forcedisplacement transducer by using a recording system, preferably a computer software. The segments are allowed to equilibrate under final resting force of 1-2 g for at least 1 to 1.5 h and they were washed every 10-20 minutes. After the equilibration period, arterial strips were challenged with a vasoconstrictor, i.e. phenylephrine, prostaglandin F2α or potassium chloride (KCl) to test the viability of the vessel. After an additonal 30 min of equilibration period with repeated washing every 10 min, the tissues are challenged with increasing cumulative concentrations of the vasoconstrictor substance to be tested and responses are recorded.

Surgeons have studied graft pharmacology by measuring the effects of vasodilators on blood flow through arterial grafts before they were attached to the heart [9]. Pharmacologists have also joined the study of graft pharmacology by evaluating endothelial and smooth muscle function of bypass grafts using their standard in vitro method, the isolated vessel ring preparation in the organ bath. However, results from these in vitro studies need to be carefully extrapolated to the clinical situations, where the conditions of the arterial grafts are compli‐ cated. Even so, the organ bath method can provide very useful information about the effects

Several vasodilators have been tested and various antispastic methods have been suggested to prevent graft spasm; including papaverine, phenoxybenzamine, calcium antagonists and nitrates etc. Choice of a pharmacological agent to overcome the vasospasm encountered in the arterial grafts must be on the basis of pharmacological studies. Accordingly, current state of knowledge based on experiments to study the pharmacological effect of a number of vaso‐ constrictor and vasodilator substances and the practical application of this knowledge can be

Pharmacology of isolated blood vessel allows the researcher to investigate the mechanisms of effect of spasmogens or vasodilatory substances. Most studies use the isolated vessel ring preparation in the organ bath, studying removed segments from the grafts during surgery. This technique only requires basic pharmacological equipment, i.e. isolated organ baths, transducers, recorder system etc. An important advantage of this method is that the vessel segment is studied in the organ bath and concentration-response curves for each vasoactive substances to be obtained under controlled conditions without extrinsic neural factors, circulating hormones interacting, blood flow or shear stress. Therefore, dose and response relationships to drugs, either vasoconstrictor or vasodilator substances, can be assessed more readily and accurately than is possible than in vivo experiments. This methodology also enabled agents to be compared with each other, and combinations of vasoactive drugs to be tested [10,11-13]. In vitro measurement of response of vascular preparations may help to researcher to predict what can happen, not what does actually happen in integrative and complicated in vivo conditions. However, isolated organ bath methods cannot identify the actual cause of in vivo spasm. The next challenge is to determine in the body what combination of factors, i.e. extrinsic neural factors, circulating hormones interacting, blood flow or shear stress, influencing passive distension from arterial wall are present the vessel with spasm.

Isolated organ bath technique is a standard research approach which requires basic pharma‐ cological equipment (Figure 1). Segments of human arteries obtained from patients undergo‐ ing CABG surgery are placed in oxygenated physiological solution, i.e. Krebs-Henseleit solution etc., at room temperature and transferred immediately to the laboratory. The arteries are dissected from adhering fat and connective tissue then cut into 3-4 mm length rings. The strips are mounted in an organ bath, containing physiological solution, on a L-shaped brace

of vasoactive substances in the arterial grafts.

**2.** *In Vitro* **pharmacology of blood vessels**

outlined as following sections:

252 Artery Bypass

**Figure 1.** A schematic diagram of a human arterial ring preparation in an organ bath.

Each cumulative concentration is applied after the relaxation to previous concentration reached to a plateau. Vasoconstrictor substance -evoked responses are usually expressed as percentage of the maximum response in each corresponding tissue. Vasodilator agents are studied by establishing concentration-relaxation curves after precontracting the segments with a vasoconstrictor, i.e. phenylephrine, prostaglandin F2α or potassium chloride (KCl). The relaxation is usually expressed as a percentage of the precontracting force. Potency, ie, sensitivity of the vessel to a drug is calculated as EC50 values (the concentrations of vasocon‐ strictor required to produce 50 % of the calculated maximum response). EC50 value is used to determine pEC50 value (negative log10 of the EC50 value). This value can differ considerably with the nature of the agent used for precontraction of the vessel and the amount of contraction that a particular concentration of vasoconstrictor substance will develop. The degree of relaxant effect of a dilator on a vessel precontracted by a particular vasoconstrictor agent, namely functional antagonism, is reflected by pEC50 value. Another important value is the maximal efficacy (Emax) which reflects the range of maximal response to the drug at high concentration.

**Vasoconstrictors** Vascular Smooth Muscle

Endothelin ETA, ETB ETB

Norepinephrine α1, α<sup>2</sup> α<sup>2</sup> Methoxamine α<sup>1</sup> … Phenylephrine α<sup>1</sup> … Dopamine α1\*\*\* …

5-HT 5-HT<sup>2</sup> 5-HT1D TxA2 \* TP TP (?)\*\*

TxA2 \* TP TP (?)\*\* PGF2α FP FP (?)\*\*

Histamine H (H1, H2) H<sup>1</sup>

Acetylcholine M3? M2

Angiotensin II AII AII

Potassium …

\* TxA2 is also considered as one of the endothelium-derived contracting factors; it is also derived from platelets.

\*\*\* Dopamine also affects α1 and α<sup>2</sup> receptors, exist in cardiac and bronchial cells respectively, it causes vasoconstriction

**Table 1.** Vasoconstrictors and their Receptors Involved in Vascular Smooth Muscle; Vasodilators in which Mediate

Vasopressin (ADH) V1\*\*\*\* …

EDCFs

α-Adrenoceptor agonists

Platelet-derived substances

Substances released from mast cells and basophils

Muscarinic receptor agonists

Renin-angiotensin system

\*\* TP and FP receptors in endothelial cells to be clarified.

\*\*\*\* Mainly effective in renal medulla, it also enhances sympathetic constriction, EDCFs = Endothelium-derived contracting factors, ADH = antidiuretic hormone.

Depolarizing agent

at high dose.

Relaxation via Endothelium.

Prostanoids

Contraction

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A special method that measures the individual length-tension relationship curve for each vessel segment, cut to a precise length, has been developed [10]. This method, called as normalization technique, sets passive distension of the vessel segment to correspond with that caused transmural pressure experienced in vivo. The principal is to establish individual lengthtension exponential curves for each vessel by relating the isometric tension, obtained from strain gauge transducers, with the corresponding diameter. This technique has been conti‐ nously used by several researchers for studying CABG pharmacology [10,14-16].

#### **2.1. Vasoconstrictor and vasodilator agents**

Exogenous and endogenous vasoconstrictors are particularly important for vasoconstric‐ tion and its extreme form—vasospasm (Figure 2). Table 1 lists vasoconstrictor substances that are generally considered spasmogens for blood vessels and the receptors located on the cellular membrane of vascular smooth muscle, and of endothelium, which mediates vasodilatation. Most of these vasoconstrictor substances contract blood vessels through receptor-mediated mechanisms, i.e. internally secreted epinephrine and norepinephrine cause blood vessels to contract by stimulating α-adrenergic receptors on the vascular smooth muscle. Consequently, a selective α -receptor antagonist will be highly effective because the site of interaction is same. The contraction caused by epinephrine and nore‐ pinephrine is partly caused by depolarization of the tissue through voltage-operated cal‐ cium (Ca2+) channels (VOCC) and partly caused by calcium release from intracellular sources. Thus, this mechanism would be more resistant to functional antagonist nifedi‐ pine. On the other hand, increased extracellular K+ depolarizes smooth muscle membrane by closing of the hyperpolarizing K+ channels. This effect allows VOCC to open and in‐ tracellular [Ca2+ ] to rise, resulting in smooth muscle contraction. Therefore, a VOCC an‐ tagonist such as nifedipine would readily relax a tissue precontracted by potassium (K+ ).


\* TxA2 is also considered as one of the endothelium-derived contracting factors; it is also derived from platelets.

\*\* TP and FP receptors in endothelial cells to be clarified.

Each cumulative concentration is applied after the relaxation to previous concentration reached to a plateau. Vasoconstrictor substance -evoked responses are usually expressed as percentage of the maximum response in each corresponding tissue. Vasodilator agents are studied by establishing concentration-relaxation curves after precontracting the segments with a vasoconstrictor, i.e. phenylephrine, prostaglandin F2α or potassium chloride (KCl). The relaxation is usually expressed as a percentage of the precontracting force. Potency, ie, sensitivity of the vessel to a drug is calculated as EC50 values (the concentrations of vasocon‐ strictor required to produce 50 % of the calculated maximum response). EC50 value is used to determine pEC50 value (negative log10 of the EC50 value). This value can differ considerably with the nature of the agent used for precontraction of the vessel and the amount of contraction that a particular concentration of vasoconstrictor substance will develop. The degree of relaxant effect of a dilator on a vessel precontracted by a particular vasoconstrictor agent, namely functional antagonism, is reflected by pEC50 value. Another important value is the maximal efficacy (Emax) which reflects the range of maximal response to the drug at high

A special method that measures the individual length-tension relationship curve for each vessel segment, cut to a precise length, has been developed [10]. This method, called as normalization technique, sets passive distension of the vessel segment to correspond with that caused transmural pressure experienced in vivo. The principal is to establish individual lengthtension exponential curves for each vessel by relating the isometric tension, obtained from strain gauge transducers, with the corresponding diameter. This technique has been conti‐

Exogenous and endogenous vasoconstrictors are particularly important for vasoconstric‐ tion and its extreme form—vasospasm (Figure 2). Table 1 lists vasoconstrictor substances that are generally considered spasmogens for blood vessels and the receptors located on the cellular membrane of vascular smooth muscle, and of endothelium, which mediates vasodilatation. Most of these vasoconstrictor substances contract blood vessels through receptor-mediated mechanisms, i.e. internally secreted epinephrine and norepinephrine cause blood vessels to contract by stimulating α-adrenergic receptors on the vascular smooth muscle. Consequently, a selective α -receptor antagonist will be highly effective because the site of interaction is same. The contraction caused by epinephrine and nore‐ pinephrine is partly caused by depolarization of the tissue through voltage-operated cal‐ cium (Ca2+) channels (VOCC) and partly caused by calcium release from intracellular sources. Thus, this mechanism would be more resistant to functional antagonist nifedi‐ pine. On the other hand, increased extracellular K+ depolarizes smooth muscle membrane by closing of the hyperpolarizing K+ channels. This effect allows VOCC to open and in‐ tracellular [Ca2+ ] to rise, resulting in smooth muscle contraction. Therefore, a VOCC an‐ tagonist such as nifedipine would readily relax a tissue precontracted by potassium (K+

).

nously used by several researchers for studying CABG pharmacology [10,14-16].

**2.1. Vasoconstrictor and vasodilator agents**

concentration.

254 Artery Bypass

\*\*\* Dopamine also affects α1 and α<sup>2</sup> receptors, exist in cardiac and bronchial cells respectively, it causes vasoconstriction at high dose.

\*\*\*\* Mainly effective in renal medulla, it also enhances sympathetic constriction,

EDCFs = Endothelium-derived contracting factors, ADH = antidiuretic hormone.

**Table 1.** Vasoconstrictors and their Receptors Involved in Vascular Smooth Muscle; Vasodilators in which Mediate Relaxation via Endothelium.

As stated above, vasodilator agents are usually studied by precontracting the vessel. The level of precontraction force should be chosen in the range of 60% to 80% of the maximum contrac‐ tion of that agent. The precontractile tone should reach to a plateau and remain stable during the experimental period. The precontraction may dissipate in a time-dependent manner. This may lead researcher to ascribe decreased tone due to added drug instead of spontenaous relaxation. Therefore, a parallel time control is necessary to show that the precontraction is stable [11,17,18].

regard to contractility and endothelial function. These differences, together with histological and anatomical diversity, may account for possible differences in the perioperative spasm.

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The contractility of IMA to vasoconstrictors has been studied extensively [10,13]. TxA2 is one of the several EDCFs, but it is also derived from platelets. Endothelin is also considered as one of the EDCFs. These two substances are two of the most potent vasoconstrictors known and they are very potent in IMA as well. Elevated plasma concentrations of ET [28] or TXA2 [29] have been found during cardiopulmonary bypass. Therefore,.these vasoconstrictors are prime

Some receptors on the smooth muscle of IMA have been characterized. For example, IMA is an α1-adrenoceptor-dominant artery with little α2- or β -function [30,31]. Other receptors functionally demonstrated in IMA are ETA, ETB [32], 5-HT [33], angiotensin [34],TP (throm‐ boxane-prostanoid) [35], vasopressin V1 receptors [36,37], and vasoactive intestinal peptide [38] receptors. Dopaminergic receptors have also been demonstrated in the IMA [39]. The

As stated above, some vasoconstrictors have been demonstrated as being vasorelaxant agents. 5-HT is an example of this type of vasoconstrictors and it directly contracts vas‐ cular smooth muscle through 5-HT2 receptors [40] and relaxes blood vessels through en‐ dothelial NO release, mediated by 5-HT1D receptors, [41] located in the endothelium. When endothelium is lost, perhaps also when it is damaged, platelets aggregate in the area where endothelium is denuded and release substances such as 5- HT (also TxA2) that strongly contract smooth muscle. Accordingly, studies have shown 5-HT does not strongly contract IMA with intact endothelium [13,42]. However, its contracting effect is

The endothelium-dependent relaxation exists in IMA [43]. It has also been demonstrated that vascular endothelial growth factor may induce endothelium-dependent relaxation in the human IMA [44]; the relaxation has recently been demonstrated to be mediated by both NO and PGI2 [45]. Further, physiological substances such as CRF induce both endo‐ thelium-dependent and -independent relaxation in the human IMA [46]. IMA releases both NO and EDHF [47]. Recent studies have demonstrated that the endothelium of the IMA releases more NO than the RA at both basal and stimulated levels [47]. Further, the IMA has a greater hyperpolarizing effect on bradykinin-stimulated release of EDHF than

In addition, receptors, for common stimuli of EDRF such as acetylcholine, bradykinin, and substance P are present in the endothelium of arterial grafts [15,48,49]. The vascular endothelial growth factor (VEGF)-induced, endothelium- dependent relaxation, mediated by both NO and prostacyclin in the IMA, has been shown mainly through the KDR (kinase insert domain) receptors, rather than Flt-1 (fms-like thyrosine kinase-1) receptors [45]. Most recently, corti‐ cortropin-releasing factor (CRF) receptors CRF1, CRF2α, and CRF2β have been shown to be present in the IMA [45]. The CRF urocortin- induced endothelium-dependent relaxation in the

IMA is likely through CRF receptors allocated in the endothelium of the IMA [50].

candidates as spasmogens for arterial grafts during CABG surgery.

agonists for these receptors may also be spasmogenic agents for the IMA.

unmasked when endothelium is denuded [13,42].

the RA does [47].
