| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
TEACHING IN THE LABORATORY
1 Laboratoire de Physiopathologie de la paroi arterielle, Croissance et Cancer, EA2103, Faculté de Médecine, 37032 Tours, France
2 Laboratoire de Nutrition, Croissance et Cancer, EA2103, Faculté de Médecine, 37032 Tours, France
3 Departement de Physiologie Animale, Faculté des Sciences, Université de Tours, 37200 Tours, France
Abstract
The laboratory exercise described in this article used a simple preparation and a straightforward protocol to illustrate how the neurotransmitter norepinephrine (NE) induces an increase of tension in an artery. This was a practical class designed for undergraduate students of the University of Tours. The students performed several protocols to understand how NE acts to contract aortic ring vessels, which sources of calcium are mobilized, and whether the calcium sensitivity of the contractile regulatory apparatus is involved. The design of this exercise allowed students to participate actively in an exercise demonstrating that many mechanisms are involved and act additively to allow arterial tone to develop. Furthermore, the students were introduced to an isolated organ chamber technique that is used to study cellular mechanisms of many tissues and that is still important for smooth muscle research.
Key words: vascular contraction; arterial pressure; smooth muscle cell; teaching
The physiology of the vascular smooth muscle cells (VSMCs) is a rapidly changing and complex area of research that has seen recent significant advances impacting on diverse scientific fields. VSMC physiology links cardiac physiology with vascular physiology. For example, aortic pressure oscillations are due to cardiac cycles, and cardiac metabolism is linked to systemic artery (coronary) flow rate.
In the vasculature, the radius of the blood vessel is altered by the contraction of VSMCs. Thus it is possible to predict the effect of stimuli on the vessel and then to extrapolate to blood pressure. One way to demonstrate VSMC contraction is to attach artery ring segments to a force transducer and record changes in tension. These responses can subsequently be used to predict the effects of agents on blood pressure.
We conduct this laboratory exercise each year as part of a cardiovascular physiology course that is required for undergraduate students of our university. This laboratory exercise provides students with an opportunity to examine methods by which physiological data are generated with the use of a relatively easy vascular ring segment preparation. This exercise complements a laboratory exercise designed to demonstrate electrical activity in the cardiovascular system (7a). Teaching such techniques to undergraduate students is important because these skills are needed both in fundamental studies and in applied studies performed in pharmaceutical companies. This exercise is most effective when performed after treatment of related topics on systemic circulation in lectures. These include the anatomy of vessels, the structure and electrophysiological features of smooth muscle cells, excitation-contraction coupling, and the regulation of the contractile apparatus.
In this laboratory exercise, the students perform seven protocols to demonstrate how norepinephrine (NE) contracts VSMCS. They initially check for the absence of a functional endothelium (4), and they observe a tonic contraction. They demonstrate the existence of excitation-contraction coupling (14). They then determine the source of calcium (intracellular/extracellular) involved in the NE contraction. Finally, they generate a diagram representing the different pathways involved in the NE contraction.
This laboratory exercise was inspired by the laboratory exercise described by Gonzalez et al. (5), which was focused on the modulatory role of the endothelium on NE-induced contraction.
MATERIALS AND METHODS
Aortic ring preparation.
In this study, all the protocols were conducted according to the ethical standards of the Ministère Français de l’Agriculture for the care and use of laboratory animals. On the day of the laboratory exercise, one heparinized guinea pig (300 g) was anesthetized with pentobarbital sodium (50 mg/kg ip) and exsanguinated. Thoracic aortic segments were isolated, cleaned of adventitia, and cut into rings. The endothelium was removed by gently rubbing the lumen with the tip of a forceps. Six aortic rings, each 5 mm long (for six groups of two students) were mounted on stainless steel hooks and suspended in a 30-ml jacketed tissue bath with physiological salt solution (PSS) maintained at 37°C and aerated with 95% O2-5% CO2 (Fig. 1).
|
NE was prepared at 10-6 M in the presence of ascorbic acid, and the solution was placed in an amber bottle. Nifedipine was prepared in alcohol, and the final concentration of solvent was <0.1%. A high extracellular K+ solution was prepared by addition of 110 mM KCl (K110) by substitution of NaCl to preserve osmolarity. An external solution without calcium (0Ca) was prepared by omitting calcium and by adding 1 mM EGTA [pCa (= -log10[Ca], where p is potential) calculated to be >9]. The pH was then adjusted to 7.4 using NaOH.
Isometric tension recording.
Isometric force generation was recorded with Grass transducers (FT03; Quincy, MA) connected to an amplifier (Gilles Pinal) and chart recorders (Linseis, Advantec) (Fig. 1A). Artery rings were preloaded to 3 g, which induced passive tension to detect the maximum active tension. After the preload, the aortic rings were equilibrated (stress relaxation) for 1 h. The preload period of 1 h is necessary to fully stretch the intermediate elastic filament to observe contraction. A contraction of 1 g or greater to NE is considered viable.
Students were divided into six groups of two students to promote active participation. Initially, they prepared the PSS solution and the other solutions required for the exercise. In this way, they learned how to make solutions at correct concentrations. For example, students used alcohol to dissolve the nifedipine and make a stock solution as concentrated as possible so that very little alcohol carried over into the final PSS solution (<0.1%). Second, they mounted their own rings, and then the teacher performed the preload to/with 3 g.
The week before the laboratory experiments, we gave students a description (two pages) of the experiments that would be done during the laboratory exercise. Thus they had advance knowledge of all of the drugs and their effects. Furthermore, just before they performed the experiments we reexplained the effects of these drugs.
All of the figures shown in this article were obtained by the students during the class. They are shown in the article as they broadly appeared to the students.
EXPERIMENTAL PROTOCOLS
Protocol 1: mechanical aortic response to NE and endothelium removal test.
The purpose of this first protocol is to observe the contraction of the aortic rings after the addition of 10-6 M NE. The type of contraction is a tonic contraction that develops slowly and is characterized by fast and slow kinetics (Fig. 2A). When the amplitude of the contraction reaches the steady state, the students add 10-6 M acetylcholine in the bath solution to check that the endothelium was effectively removed from the aortic rings (4). The absence of significant relaxation shows that the arteries were denuded of the endothelium (Fig. 2A). Acetylcholine could not have induced a relaxation by releasing NO or other vasodilatators that would have attenuated the amplitude of the contraction (7).
|
1-adrenergic receptors that activate phospholipase C, which generates inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG), which activates protein kinase C (PKC). This leads to the release of calcium from the intracellular stores and the transmembrane influx of calcium through receptor-operated channels (ROC) and voltage-operated channels (VOC) (10).
Protocol 2: evidence that two excitation couplings coexist in the aortic rings.
A high concentration of extracellular K+ (K110) produces a sustained contraction (Fig. 2B). The resting membrane potential of the VSMCs is largely (but not exclusively) determined by the ratio between the intracellular concentration and the extracellular concentration of K+ and is thus close to equilibrium potential for K+ (EK) (13). An increase of the extracellular concentration of K+ close to the intracellular concentration of K+ shifts the resting membrane potential toward 0 mV (EK = 0 mV) and consequently causes the complete depolarization of the membrane. This depolarization activates VOC such as L-type calcium channels and induces contraction by increasing the intracellular calcium concentration. The rise of the calcium in the cytosol is instantaneous (within milliseconds), without the prolonged delay associated with the activation of the phosphatidylinositol cascade (12). This is a typical contraction obtained by activation of electromechanical coupling (14).
The addition of NE induces a further contraction. This contraction, in completely depolarized VSMCs (note that this information was communicated to the students during the laboratory exercise), represents the "pure" pharmacological coupling (14) (Fig. 2B).
This protocol demonstrates that, in the aortic VSMCs, two couplings can be activated to induce contraction. These two mechanisms can operate simultaneously to produce a contractile response to NE.
Protocol 3: NE aortic response in presence of nifedipine.
In the presence of nifedipine, an L-type calcium channel blocker, the aortic resting tone is not changed (Fig. 3A). This indicates that the L-type calcium channel is not activated in resting conditions. An addition of NE in the bath solution induces a contraction with an amplitude (steady-state amplitude) similar to the one obtained in the absence of this blocker (Fig. 2A). This shows that NE-induced contraction is due mainly to pharmacological coupling and that VOCs are not activated. Nevertheless, the absence of effect of NE on VOCs and on the resting membrane potential cannot be definitively eliminated, because an absence of activation of electromechanical coupling is still feasible, for example by an increase of PNa and PCl (inducing depolarization) which is canceled by an increase of K permeability (PK) (inducing hyperpolarization).
|
Washing the aortic rings with a solution containing calcium induces a transient contraction (Fig. 3B). This contraction depends on the entry of calcium through calcium channels such as store-operated cation channels (SOCs), which are activated by depletion of intracellular calcium from the sarcoplasmic reticulum (3, 10). Then these channels can also be activated during NE contraction to produce, with ROCs, a long-lasting tonic contraction tonic.
Protocol 5: aortic response to caffeine.
Caffeine penetrates the cell membrane to activate the release of calcium from the sarcoplasmic reticulum and induces a transient contraction (Fig. 4A). This experiment is performed in the absence of extracellular calcium (0Ca). Therefore, the contraction is dependent only on the release of calcium from the intracellular stores. The students wait no longer than 5 min with the 0Ca solution to prevent a decrease of calcium in the sarcoplasmic reticulum. Caffeine induces a transient contraction, which decreases as calcium is removed from the cytosol by calcium transporters (10). The tone reaches a steady state that is lower than the resting tone; then caffeine induces a further relaxation (Fig. 4A). This relaxation is due to another action of caffeine, which is to decrease the calcium sensitivity of the contractile regulatory apparatus by increasing protein kinase A (PKA) activity (12, 13). Indeed, caffeine acts as a 3',5'-cyclic-nucleotide phosphodiesterase inhibitor, which induces an increase of cAMP and then triggers the activation of PKA (6).
|
Protocol 6: aortic response to caffeine in the presence of high external concentration of K+.
A high external concentration of K+ induces contraction by activation of VOC and the consequent increase of the cytoplasmic calcium concentration (Fig. 4B). The application of caffeine induces a fast relaxation that returns to baseline or to lower levels. This experiment confirms that caffeine can not only release calcium from the sarcoplasmic reticulum but also be a powerful relaxant compound due to its phosphodiesterase inhibitor action.
Protocol 7: aortic response to phorbol 12,13-dibutyrate.
Previously in this article, it was found that the VSMCs of the aorta were able to develop and maintain maximum tonic contraction in response to NE. It was shown that the onset of the contraction was linked to an increase of cytosolic calcium concentration and calcium from the sarcoplasmic reticulum. However, NE also activates PKC, which increases the calcium sensitivity of the contractile regulatory apparatus (12, 13). PKC is reported in high concentration in VSMCs and can be directly activated physiologically by DAG and pharmacologically by the tumor-promoting agents, phorbol esters (8, 9). One of these agents often used is the phorbol 12,13-dibutyrate (PDB), which is a potent PKC activator in VSMCs (2, 11). PDB (0.2 x 10-6 M) induces a slowly developing and sustained contraction (Fig. 5). With the kinetics of PDB-induced contraction taken into account, it thus appears that the activation of PKC can be an effective pathway in the maintenance of VSMCs during NE-induced contraction in the aorta.
|
STUDENT EVALUATIONS
After the practical class experiments, the students took an exam designed to assess the perceived value of the laboratory demonstration. A typical example of such an exercise is given in Fig. 6. For this exam, it was postulated that the recording was a mechanical recording of guinea pig aortic rings, but students had forgotten to put that into the legend, so the students had to guess which modifications (in the bath solution) were performed at the level indicated by the arrows and the letter from a to q. It was possible to give several answers, but only one answer by letter was expected. Finally, the students had to speculate on a possible mechanism of action.
|
DISCUSSION
At the end of this laboratory exercise, students had to generate a diagram (Fig. 7) representing the different pathways involved in the NE contraction, and they had to label it as in Fig. 7. They found that the VSMCs of the aorta were able to develop and maintain maximum tonic contraction in response to NE. The onset of the contraction was linked to an increase of cytosolic calcium concentration and calcium from the sarcoplasmic reticulum (IP3). However, the maintenance of the contraction was due to increasing calcium sensitivity of the contractile regulatory apparatus by PKC and to calcium entry through ROC and SOC channels.
|
In summary, the laboratory protocols on the VSMC contraction described in this article reinforced for the students the concept of VSMC physiology. The protocols allowed the students to observe methods by which physiological data are generated by use of an isolated VSMC model, a preparation frequently used in animal physiological and pharmacological studies. This also provided the students the opportunity to interpret results. It was amazing to see the students’ faces when we told them that the role of the endothelium on acetylcholine inducing the relaxation was discovered by using the same experimental set-up that they were using.
These lab experiments can easily be set up with standard equipment found in most cardiovascular physiology laboratories.
Acknowledgments
We thank Maryse Pingaud for technical help to prepare classes, Chantal Boisseau for secretarial assistance, and Gilles Pinal for expertise in electronic devices. We thank Veronique Vandier and Dr. Prem Kumar for language correction. We also thank Gael Rochefort, Patrick Massoma and Rabii Benikdes for making Fig. 1.
Address for reprint requests and other correspondence: C. Vandier, Laboratoire de physiopathologie de la paroi artérielle, Faculté de Médecine, 2 bis Boulevard Tonnellé, 37032 Tours, France (E-mail: vandier{at}univ-tours.fr).
Received for publication December 31, 2001. Accepted for publication May 15, 2002.
REFERENCES
This article has been cited by other articles:
|
|
 |
|
  J.-Y. Le Guennec, C. Vandier, and G. Bedfer SIMPLE EXPERIMENTS TO UNDERSTAND THE IONIC ORIGINS AND CHARACTERISTICS OF THE VENTRICULAR CARDIAC ACTION POTENTIAL Advan Physiol Educ, September 1, 2002; 26(3): 185 - 194. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
