JPM Vol. 30, No. 2 October 1993:111-115

An in vitro Pharmacological Model of Vascular Smooth Muscle

Tahir Hussain and S. Jamal Mustafa

Department of Pharmacology, School of Medicine, East Carolina University, Greenville, North Carolina, U.S.A.

In order to develop an in vitro model of vascular tissue for pharmacological studies, seg- ments of porcine coronary arteries were incubated in culture media under sterile conditions in cell culture incubator at 37°C with 95% 02 + 5% COz. After 3 days of incubation, changes in isometric tension were measured in vascular rings and compared with the fresh tissue. KC1 (lo-75 mM) and prostaglandin Fz= (l-20 PM) produced a similar concentration- dependent contraction in the incubated and fresh arteries. The concentration-dependent relaxation curves produced by 2-chloroadenosine ( low8 to 10e4 M) and isoproterenol (lo-* to 1O-5 M) were unaltered in the incubated tissue versus fresh. Similarly, the relaxation responses to forskolin and sodium nitroprusside (10e8 to 10e5 M) were unaffected in the incubated arteries. The relaxations produced by substance P (lo-” to 10m8 M) and bradyki- nin (lo-’ M&the endothelium-dependent agents-were also unaltered in the incubated rings versus fresh. Therefore, we conclude that after the incubation of porcine coronary artery for 3 days, the contraction/relaxation responses to various agonists acting through different mechanisms were unaltered in porcine coronary artery. This in vitro model of vascular smooth muscle provides a potential for pharmacological and toxicological studies.

Keywords: 2-Chloroadenosine; Isoproterenol; Substance P; Coronary artery

Introduction

Preservation of isolated vascular and nonvascular tissues for pharmacological studies has been a focus of several investigations in the past decade. Cryopres- ervation of the tissues has been proven to be a useful technique (Muller-Schweinitzer, 1988; Schoeffter and Muller-Schweinitzer, 1990; Ku et al., 1992). Using this technique, we note the blood vessels were frozen slowly to -70°C and stored for several weeks at - 190°C while being immersed in fetal calf serum con- taining 1.8 M dimethyl sulfoxide. In this technique sev- eral major biochemical properties of the tissue have been shown to be well maintained after freezing/thaw- ing (Muller-Schweinitzer and Tapparelli, 1986). Re- cently, using the cryopreservation, we note pig coro- nary artery has been shown to maintain the relaxation responses to various agonists. However, the contrac- tile responses to 5-HT and PGFz~ were found to be significantly diminished in the stored coronary arteries

Address reprint requests to Dr. S. Jamal Mustafa, Department of Pharmacology, School of Medicine, East Carolina University, Greenville, NC 27858 U.S.A.

Received April 30, 1993; revised and accepted July 26, 1993.

Journal of Pharmacological and Toxicological Methods 30. 1 I I-115 (1993) 0 1993 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010

compared to fresh (Schoeffter and Muller-Schwei- nitzer, 1990). Endothelium-dependent relaxation re- sponses were also greatly impaired in the stored artery. Cryopreservation as suggested by Muller-Schweinitzer (1986) offers the potential to store the tissue in an inac- tive metabolic state for subsequent pharmacological studies. However, a vascular smooth muscle (coronary artery) model has not been developed yet where the tissue can be stored in the metabolically active state without any significant loss in functional (contraction/ relaxation) responses. This report describes an in vitro model for preserving the porcine coronary artery in culture media at 37°C without significant alteration in the contraction and the relaxation responses to various agonists including endothelium-dependent agents. Fur- thermore, this model has been proven to be useful in studying the regulation of adenosine receptor-messen- ger system(s) in the coronary artery (Hussain and Mus- tafa, 1993).

Materials and Methods

Tissue Preparation and Incubation Procedure

Porcine hearts were obtained from local abattoirs and transported to the laboratory in oxygenated

1056-8719/93/$6.00

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Krebs-Henseleit solution at 4°C within 30 min of sacri- fice. The left anterior descending and circumflex coro- nary arteries were dissected from each heart, cleaned of fat and connective tissue, and cut into small seg- ments under sterile conditions. The segments were di- vided into two groups. Group 1 consisted of “fresh arteries” which were used immediately for organ bath studies. The second group (group 2), which consisted of “incubated arteries,” were maintained in tissue cul- ture flasks containing Dulbecco’s modified Eagle’s me- dium (DMEM) with 7.5% fetal bovine serum, 7.5% donor horse serum, 3.70 g/L NaHC03, 100 p,g of strep- tomycin, 100 U/mL of penicillin, and 0.25 pg/mL of amphotericin B. The arteries were incubated in a cell culture incubator at 37°C with 5% CO, + 95% O2 sup- ply. The incubation medium was changed every day.

Organ Bath Experiments

The fresh and the incubated arteries were cut into rings approximately 4 mm in length. The rings were mounted in IO-mL organ baths filled with Krebs-Henseleit solution and oxygenated with 95% 02 + 5% CO:! (pH 7.4, 37°C). The composition of Krebs-Henseleit buffer was (mM) as follows: NaCl, 118; KCI, 4.8; MgS04, 1.2; KH2P04, 1.2; NaHC03, 25; CaC12, 2.5; and glucose, 11. Changes in isometric tension were measured with force transducers (Grass FT .03) connected to Sensormedics dynographs (R 611). Rings were equilibrated for 1 h under an initial tension of 2 g (determined separately from the length tension relationship); the buffer being changed every 15 min. After equilibration, rings were challenged with 35 mM KC1 until constant reproducible contractions were achieved. The integrity of endothelium was as- sessed by the ability of bradykinin (lop7 M) to relax the KCI-contracted coronary rings. After achieving sustained and reproducible contractions with 35 mM KCI, various agonists were added in a cumulative fash- ion to the bath to obtain concentration response curves. The contracting agents-KC1 and prostaglan- din FZc,-were added on the basal tone and the devel- oped tension was measured in grams. The relaxation responses to CAD, isoproterenol, SNP, and forskolin were determined in 35 mM KCI-precontracted rings. The data are represented as percentage relaxation of decrease in KCI-induced coronary tone. Agonist addi- tions were made only after the previous response had stabilized.

Functional (contraction/relaxation) responses to all agonists were studied after 3 days of incubation and compared to the fresh coronary rings.

Chemicals 2-Chloroadenosine and isoproterenol were pur-

chased from Research Biochemicals Inc. (Wayland,

JPM Vol. 30. No. 2 October 1993:111-115

MA). Forskolin, sodium nitroprusside, bradykinin, substance P, DMEM culture media, fetal bovine serum, donor horse serum, antibiotics, and antimy- cotic mixture were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were of the highest purity available and purchased from Sigma or Fisher (Pittsburgh, PA).

Results

KC1 (lo-75 mM) and prostaglandin Fza (l-20 FM) produced a concentration-dependent contraction in the coronary rings. The contraction response curves were similar in incubated and fresh vascular rings (Figure 1).

Relaxation responses to adenosine and B-adrenergic receptor agonists, known to act directly on the vascular smooth muscle, were studied on rings from both fresh

Figure 1. KCl- (upper) and prostaglandin Fz,- (lower) in- duced contraction in fresh and 3-day-old incubated coronary artery. The tensions generated by agonists are represented as means + SE of 8-10 vascular rings.

4 a

g 3

5 2

O-O FRESH

0-O INCUBATED

20 40 60 SO

KCI [mM]

O-O FFiESH

0-e INCUBAl’EB

T. HUSSAIN AND S. J. MUSTAFA 113 IN VITRO MODEL OF VASCULAR TISSUE

and incubated arteries precontracted with submaximal concentration of KC1 (35 mM) (Figure 2). The dose-response relaxation curves produced by 2-chlo- roadenosine ( 10s8 to lop4 M) and isoproterenol (lo-* to 10V5 M) were unaltered in the incubated rings com- pared to fresh. Similarly, the relaxation responses to forskolin and sodium nitroprusside (lo-* to lOa M) (Figure 3), direct activators of membrane-bound ade- nylate and soluble guanylate cyclases, respectively, were not altered after incubation of coronary artery rings. Adenylate cyclase assays were done in the mem- branes according to the method described earlier from this laboratory (Cushing et al., 1991). The basal activity of adenylate cyclase was not significantly different in incubated arteries (72 + 5 CAMP pmol/mg protein/20 min) compared to fresh (74.5 -+ 8.3 CAMP pmol/mg protein/20 min).

Substance P, an endothelium-dependent agent, pro- duced concentration-dependent (lo-‘* to 10m8 M) re- laxation in 35mM KC1 precontracted rings (Figure 4,

Figure 2. 2-Chloro~enosine- (upper) and isoproterenol- (ISOP) (lower) mediated relaxation in 35mM KCI-con- tracted arterial rings from fresh and incubated tissue. The data represent the means + SE of 8-12 vascular rings.

O-

25-

Figure 3. Relaxation produced by sodium nitroprusside (SNP) (upper) and forskolin (lower) in 35-mM KCl-precon- tracted fresh and incubated vascular rings. The values repre- sent means ? SE of lo-12 vascular rings.

O-

2 25-

3 SO.

x

75-

1004 , 9 8 7 6 5

SNP [-log M]

FORSKOLIN [-log M]

1004 \ 1

8 7 6 5 4 2-CHLOROADENOSINE [-log M]

Figure 4. Ralaxation produced by substance P in 35mM KCI-precontracted fresh and incubated vascular rings. The values represent the means +. SE of 8-12 vascular rings.

f o-o FRESH

m-0 INCUBATED

1004 7 6

ISOP [-log M]

, 12 11 10 9 8

SUBTANCE P [-log M]

114 JPM Vol. 30, No. 2 October 1993:111-115

lower). The relaxation response in the incubated rings was similar to the fresh tissue.

Bradykinin- (lo-’ M) mediated endothelium-depen- dent relaxation response in the incubated artery (53.8% + 3.55%) was not significantly different from the fresh tissue (47.8% + 3.58%).

Discussion

In the present communication, relaxation/contrac- tion responses to various agonists acting through dif- ferent mechanisms were compared between incubated and fresh coronary rings. The contractile responses to KC1 and PGF*, and the relaxations produced by CAD, isoproterenol, forskolin, sodium nitroprusside, and substance P were unaltered in the incubated artery for 3 days.

Using cryopreservation of vascular tissue (Schoef- fter and Muller-Schweinitzer, 1990) there has been some success in maintaining the functional ability (con- traction/relaxation) of the tissues. However, the con- traction response to prostaglandin FZo! (Schoeffter and Muller-Schweinitzer, 1990) and the relaxation re- sponses to various agonists especially substance P, his- tamine, and isoproterenol (Ku et al., 1992) were signifi- cantly attenuated due probably to the freezing/thawing of the tissue. Our model does not involve any cryopres- ervation process that may be deleterious and would subsequently alter the metabolic activity of the tissue. This model is based on the simple technique of organ culture where the tissue is maintained in media with standard culture conditions.

KC1 and prostaglandin FZa are known to cause con- traction, respectively, through voltage-operated (Bol- ton, 1986; Rooke et al., 1984) and receptor-operated (Fukuo et al., 1986; Uski and Andersson, 1984) mecha- nisms. Unaltered contractile responses to these agents may suggest that the functional activity of the ionic/ receptor system(s) and the contractile apparatus were unaltered after incubating and storing the vascular tis- sue for 3 days.

Isoproterenol (P-adrenergic receptor agonist) and 2- chloroadenosine (adenosine receptor agonist) pro- duced a concentration-dependent relaxation which was unaltered in the incubated arteries compared with fresh. P-Receptors and adenosine receptors have been shown to generate CAMP by stimulating adenylate cy- clase via guanine nucleotide binding proteins (Inge- brestsen, 1980; Londos and Wolff, 1977; Cushing et al., 1991; Hussain and Mustafa, 1992). The biochemical events beyond the stimulation of adenylate cyclase also involve phosphorylation of many cytosolic pro- teins (Ingebrestsen, 1980) for the ultimate functional response. Furthermore, the direct stimulation of ade- nylate cyclase and thus generation of CAMP by for-

skolin (Daly et al., 1981) was also investigated. Like isoproterenol and adenosine, the relaxation response to forskolin was not changed in the incubated coronary artery compared to fresh. The basal activity of adenyl- ate cyclase measured in the membranes was also unal- tered by incubation. These results indicate that the ac- tivity of stimulatory receptor(s)-G-proteins-cyclase complex in the membrane and the subsequent mecha- nism(s) were not affected during the incubation. We have reported in an earlier study (Hussain and Mus- tafa, 1993) that the responses to KC1 and an adenosine analogue (Zchloroadenosine) were not significantly al- tered over 9 days of incubation.

Substance P and bradykinin are known to act on endothelium and release endothelium-derived releas- ing factor (NO) which, in turn, generates cGMP by activating soluble guanylate cyclase in smooth muscle and produces relaxation (Lionln et al., 1991). The re- laxation responses to substance P and bradykinin in the incubated arteries were not different from fresh tissue. The relaxation through direct activation of guanylate cyclase by sodium nitroprusside, an agent known to act directly on the enzyme (Gruetter et al., 1979), was not altered in the incubated artery. These data suggest that the endothelium-dependent relaxa- tion mechanism(s) were not impaired in the vascular tissue after incubation. In a recent report, canine femo- ral arterial rings were incubated in culture media over a period of 21 days (Berman and Williams, 1993). In this report, the histologic appearance of blood vessels including endothelial cells were unaltered during the period of incubation, and the functional responses were not measured.

In summary, the relaxation/contraction responses originating from the endothelium, smooth muscle membrane, or the cytosol were unaltered in porcine coronary artery after incubating in the culture media for 3 days. We are already using such a model in our laboratory to study the regulation of the adenosine re- ceptor system, and this will be useful for pharmacologi- cal and toxicological studies that require the mainte- nance of vascular tissue in a metabolically active state.

This study was supported by grant no. HL 27339 from NIH.

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