This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.
Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum – if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.
The discipline of pharmacology has used the isolated tissue bath system for over 150 years. The versatility of this system has allowed scientists across the world to characterize receptors and receptor signal transduction, with this knowledge forming the basis of therapies that have treated millions of individuals with diseases or disorders such as hypertension, heart failure, diabetes, gastrointestinal disease, bladder dysfunction, asthma, and swallowing disorders, to name just a few. To this day, the isolated tissue bath remains an important facet of drug development and basic research, as it allows the tissue to function as a tissue. In this JoVE lesson, a formal protocol is shared to demonstrate a visual and virtual experiment utilizing the data from an isolated tissue bath experiment that measures isometric contraction, which permits receptor characterization.
The primary advantage of this technique is that the tissue is living and functions as a whole tissue, with a physiological outcome (contraction or relaxation) that is relevant to the body. It is a synthesis of steps (drug-receptor interaction, signal transduction, second messenger generation, change in smooth muscle excitability, and change in tissue function). While other techniques allow study of each of these steps (e.g. radioligand binding for drug affinity, measurement of second messengers), the isolated tissue bath technique allows for integration of all these steps1. Another advantage is that retaining tissue function permits calculation of important pharmacological variables that are more meaningful in a tissue vs a cellular setting; it comes closer to how the drugs examined would work in the body as a whole.
NOTE: All procedures described in this paper are performed according to guidelines established by the Institutional Animal Care and Use Committee (IACUC) of Michigan State University.
1. System Preparation and Setup
2. Tissue Preparation
3. Tissue Placement in Bath
4. Setting Passive Tension
NOTE: Each tissue has a length (Lo) at which smooth muscle cells respond optimally. Preliminary experiments to determine the optimal stretching tension which achieves this length must be performed for each individual tissue type to be examined. The rat thoracic aorta has an optimal passive stretching tension of 4 g.
5. Equilibration and Drug Making
6. Initial Challenge
7. Experiment
NOTE: Prazosin, an alpha adrenergic receptor antagonist, will be introduced to the tissue bath chamber to shift the concentration response curve to phenylephrine (PE); this is a demonstrable antagonism.
8. Data Analysis
In terms of agonism, the relative efficacy (EMAX) and potency (EC50) of an agonist in a given tissue can be calculated and compared to responses of other agonists in the same tissue1. In our experiment, the rat thoracic aorta was incubated with either vehicle or the α1 adrenergic receptor antagonist prazosin (5 nM) for one hour prior to adding the α1 adrenergic agonist phenylephrine to the tissue bath and generating contraction (Figure 1).
Figure 2 presents modified results of Figure 1. The green responses have marked the maximum contraction achieved by PE marked as max, the ½ maximum contraction marked as such and then associated with the PE concentration that caused that response. Identifying these values is identifying the effective concentration of an agonist that achieves a half max (50%) response or EC50 value. This was donegraphically in this example.Computer software that uses logistic functions for sigmoidal curves can be used for curve fitting and calculation of EC50 values.
In generating and interpreting these results, it is important to use both low and high concentrations of agonist to create the concentration-response curve. Without enough points to show a stable baseline and maximum plateau, EC50 and EMAX can only be estimated. This was done graphically in this example. Computer software that uses logistic functions for sigmoidal curves can be used for curve fitting and calculation of EC50 values.
Figure 1: Tissue Bath Schematic. Cartoon representing a ring of tissue placed in the double-wall, water jacketed tissue bath. Buffer inflow and outflow are regulated by a 3-way glass stopcock integrated into the base of the chamber. O2/CO2 is bubbled in through an aerator that is sealed with a gasket to prevent leak of PSS or gas. Recirculation input is below the output to maintain proper recirculation and prevent formation of air pockets that would cause temperature disregulation.
Figure 2: Rat Thoracic Aorta + Endothelium. The figure above depicts four separate experiments (different animals and done by different investigators on different set ups as represented by different colors) to test the ability of prazosin to shift a PE-induced contraction. Prazosin (5 nM) clearly shifted PE-induced contraction curve rightward in a parallel fashion.
Figure 3: Rat Thoracic Aorta + Endothelium. Graphical estimation of EC50 values for PE in the absence (vehicle) and presence (prazosin) of antagonist. Green line (group C) only is marked.
Salt | MW (g/mole) | 5 Liters | FINAL mM |
(grams) | |||
NaCl | 58.45 | 37.99 | 130 |
KCl | 74.56 | 1.75 | 4.7 |
KH2PO4 | 136.1 | 0.8 | 1.18 |
MgSO4•7H20 | 246.5 | 1.45 | 1.17 |
NaHCO3 | 84.21 | 6.25 | 14.9 |
Dextrose | 180.16 | 5 | 5.5 |
EDTA | 380 | 0.05 | 0.03 |
Table 1: Normal Physiological Salt Solution (PSS) Recipe. Contents of the bicarbonate-buffered PSS used in this experiment. Included are the molecular weights of all salts and the amount added to 5 L of dH2O to achieve the desired concentration.
Concentration in bath | Addition of agonist made to bath |
1 x 10-9 M | 50 ml 1 x 10-6 M |
3 x 10-9 M | 100 ml 1 x 10-6 M |
1 x 10-8 M | 35 ml 1 x 10-5 M |
3 x 10-8 M | 100 ml 1 x 10-5 |
1 x 10-7 M | 35 ml 1 x 10-4 M |
3 x 10-7 M | 100 ml 1 x 10-4 M |
1 x 10-6 M | 35 ml 1 x 10-3 M |
3 x 10-6 M | 100 ml 1 x 10-3 M |
1 x 10-5 M | 35 ml 1 x 10-2 M |
3 x 10-5 M | 100 ml 1 x 10-2 M |
1 x 10-4 M | 35 ml 1 x 10-1 M |
Table 2: Tissue Bath Additive Agonist Concentrations. The amount of agonist to be added to each bath to properly construct cumulative concentration-response curves. The desired bath concentration is achieved through addition of sequential amounts of increasing concentrations of agonist. Each addition takes into account the concentration of agonist already present in the bath. This is done to minimize the increase in volume in the tissue bath that would result from using increasing volumes of a single concentration of agonist.
Measurement of isometric force as a research tool is over 150 years old, but it continues to be the prototypical technique for receptor characterization in contractile tissues2. The power of this technique is in its simplicity and versatility: by recording responses elicited by increasing concentrations of an agonist in the presence or absence of an antagonist, a myriad of information can be derived about the pharmacological characteristics of each drug and the receptor to which it binds3-5. Experiments of this type also garner information about the competitive versus non-competitive nature of the antagonists used, as well as receptor heterogeneity and non-specific drug effects6-8. Thus, with simple permutations to this isolated tissue bath experiment, a relatively complete pharmacological profile of the receptors that mediate agonist-induced muscle contraction can be generated.
In terms of agonism, the relative efficacy (EMAX) and potency (EC50) of an agonist in a given tissue can be calculated and compared to responses of other agonists in the same tissue1. In our experiment, the rat thoracic aorta was incubated with either vehicle or the α1 adrenergic receptor antagonist prazosin (5 nM) for one hr prior to adding the α1 adrenergic agonist phenylephrine to the tissue bath and generating contraction. Figure 2 presents modified results of Figure 1. The green responses have been marked with the maximum contraction achieved by PE marked as max, the ½ maximum contraction marked as such and then associated with the PE concentration that caused that response. Identifying these values is identifying the effective concentration of an agonist that achieves a half max (50%) response or EC50 value.
Unfortunately, using agonist-dependent parameters alone to determine receptor binding characteristics can be complicated9. Ideally, additional experiments using receptor antagonists allow the calculation of two important parameters that are key to defining the interaction between a drug and a receptor: the -log10 of the antagonist dissociation constant (pKB) and the -log10 of the molar antagonist concentration necessary to elicit two-fold rightward shift in the concentration-response curve (pA2)10. Both KB and pA2 gain their usefulness from the fact that they are agonist-independent values, and remain constant even between different tissues11. From the data in Figure 2, pKB can be calculated using the following equation and based on EC50 values calculated elsewhere:
EC50 in the absence of prazosin = 2 x 10-8 M
EC50 in the presence of prazosin = 7 x 10-6 M
Solve for KB in the following equation:
log (dr-1) = log [B] – log KB
Where:
[B] = antagonist concentration, or 5 x 10-9 M. log (5 x 10-9 M) = -8.3
dr = dose ratio of EC50 value in presence of antagonist/EC50 in absence. If there is a rightward shift, this value will be greater than one. Thus:
dr = 7 x 10-6 M/2 x 10-8 M or 700 x 10-8 M/2 x 10-8 M = 700/2 = 350
Substituting for [B] and dr:
log (350-1) = -8 – log KB
2.54 = -8 – log KB
pKB = 10.54
This value for prazosin is consistent with the values obtained when prazosin interacts with an α1adrenergic receptor12,13, suggesting that the adrenergic receptor mediating contraction to phenylephrine is the α1 adrenergic receptor.
Several steps are critical to the success of these experiments. Tissues must remain in PSS after dissection to prevent loss of tissue viability. Any isometric muscle preparation has an optimal length-tension relationship that generates maximal force against passive tension14,15. For the experiment described in this protocol, optimal passive tension was previously determined by briefly adding increments of 0.5 g of passive tension to the tissue. The tissue should begin without tone and then after 30 min of equilibration, the tissue was challenged with a maximal concentration of KCl (80 mM), which generated active tone. Then, the tissue was washed and allowed to return to baseline tone. After 15 min at baseline, another 0.5 g of passive tension was placed and this process repeated until a plateau of active tension was achieved with additional passive tension. In preliminary experiments, 4 g total of passive tension was determined to achieve maximal active tension generation in aortic rings, and thus this total amount of tension is placed on the rings prior to equilibration. Procedurally, it’s best to zero prior passive tension application, but can be done at any time prior the experiment. Over-stretching tissues, at any point in the experiment or dissection, will negatively impact tissue viability and experimental outcomes. This is most imperative when placing tissues in the bath, as this step has the highest likelihood of excessive stretch. Adequate washing, in number and duration is required for reproducible effects. A second challenge too soon after an initial challenge, or before tissues have returned to baseline tension, will result in aberrant responses. If studying reversible antagonists/inhibitors, tissues should not be washed prior to agonist addition, as the inhibitor concentration will be decreased.
There are notable advantages and disadvantages to the isolated tissue bath assays.
Disadvantages: Tissues may experience different degrees of damage during surgical removal or placement of rings on hooks. Since the endothelial cell is the inner lining of the aortic ring, care must be taken on the placement of the ring on hooks so as not to damage this cell layer. Tissues may also have distinctly different lengths of time in which they are viable in the tissue bath, and this has to be determined; not all tissues are the same. Along these lines, tissues may change in their responsiveness throughout the day such that time controls become a necessary control for every experiment. A good example of this is the guinea pig trachea that improves in contractility maximum by 100% during a typical 8 hr experiment. Drugs that are poorly soluble in water may precipitate out in the PSS. Finally, cumulative and non-cumulative additions of a drug that is an agonist could result in different outcomes if receptor desensitization to the agonist occurs; angiotensin II is one such drug which shows rapid tachyphylaxis.
Advantages: One of the primary advantages of tissue bath experiments is that it is real time; one can see the experiment as it unfolds and can rapidly make conclusions and plan next steps, as well as troubleshoot during an experiment. An experiment takes a day to do. Multiple tissues can typically be prepared from one animal such that an animal can serve as its own control, and that adds strength to an experiment. One also can isolate the tissue from other factors so as to test a relatively pure response of the tissue to the drug. In vitro experiments such as the tissue bath system also allow use of a small amount of drug compared to an in vivo experiment.
The basic experimental design described herein can be extensively modified to allow for the recording of additional parameters or the introduction of other external stimuli. For example, addition of electrodes allow for electric field stimulation of innervating nerves16,17. With the addition of thermal or pH probes, the effects of temperature and pH on contractile responses can also be measured18,19. Similarly, oxygen can be substituted partially or in full with N2 to evaluate hypoxia induced effects. Furthermore, the same basic principles of isometric contractility measurement used in this video can be used to develop systems that allow concomitant measurement of isometric tension development and changes in intracellular calcium20. Signal transduction is also readily studied, since systems exist that can rapidly freeze a tissue sample during a response, such that the activity of a signal transduction pathway system can be verified biochemically.
The variation of equipment that can be used to do this is enormous. This whole system, either hand constructed or automated, can be purchased from multiple different companies. The tissue baths and tissue holders used in this protocol were hand-blown (tissue baths) and hand constructed (holders) by an in-house Michigan State University machine shops.
The authors have nothing to disclose.
The Watts Laboratory through the years, and Dr. Marlene Cohen and Kathy Schenck for teaching us this assay over two decades ago. NIGMS R25GM074119 supported development of this teaching module for a Short Course in Integrative and Systems Pharmacology held on the campus of MSU from 2005 to 2013.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
LabChart Software | ADInstruments | 7.2 | |
PowerLab (4 channel) | ADInstruments | ML760 | |
QuadBridge (4 channel) | ADInstruments | ML112 | ML112 |
Grass Adapter Cable | ADInstruments | MLAC11 | MLAC11 |
Grass Force-Displacement Transducer | Grass Instrument Co | FT03 | FT03 |
Grass Transducer Cable | Grass Instrument Co | TAC-7 REV-1 | |
BNC to BNC Cable | ADInstruments | MLAC01 | |
IsoTemp 2100 | Fisher Scientific | IC-2100 | |
Tissue Bath | Multiple Sources | ||
Physiological Salt Solution | PSS | ||
Braided Silk Suture | Harvard Apparatus | 51-7615 | SP104 |
Ring Stand | Humboldt MFG Co | H-2122 7 | |
Dissecting Dishes | Handmade with Silicone | ||
Tygon Tubing | VWR Scientific | 63010-100 | R-3603 |
Hose Clamps | Cole-Parmer Instrument Co | 06832-08 | SNP-8 |
50ml Muscle Bath | Eberhartglass Blowing | Custom | |
250ml Warming Chambers | Eberhartglass Blowing | Custom | |
Gas Dispersion Tube | Ace Glass | 7202-06 | 7202-02 |
Micrometer | |||
Custom Stands | |||
Three-Prong Clamps | VWR International | Talon | |
S-Connector | VWR International | Talon | |
Tissue Hooks | Hand Made in House | Custom | |
Tissue Dissection | |||
Leica Stereomicroscope MZ6 | Leica | 10447254 | |
Stereomaster Microscope Fiber-Optic Light Source | Fisher Scientific | 12562-36 | 12562-36 |
Culture Petri Dish | Pyrex | 7740 Glass | |
Sylgard Silicone Elastomer | Dow Corning | Sylgard 170 Kit | |
Vannas Scissors | George Tiemann & Co | 160-150 | 160-150 |
Splinter & Fixation Forceps | George Tiemann & Co | 160-55 | 160-55 |