Assays Using Electrically Unstimulated Tissue

As stated in Subheading 3.2., even when bioassays are to be performed without electrical stimulation, all tissues are first subjected to an equilibration procedure that involves electrical stimulation (Subheading 3.2.3.). Bioassays without electrical stimulation are used, for example, to measure the ability of cannabinoids to modulate responses to ^-adrenoceptor and P2X receptor agonists that act directly on the smooth muscle to induce contractions. Care must be taken in such experiments to avoid the tissues becoming desensitized to these agents, particularly when high concentrations are used. Two protocols have been used in this laboratory to investigate interactions between cannabi-noids and an ^-adrenoceptor agonist (phenylephrine, methoxamine or noradrenaline) or a P2X receptor agonist (P, y-methyleneadenosine 5'-triphosphate) as described in Subheadings 3.4.1. and 3.4.2. Both protocols initially involve the addition of a submaximal dose, A, of the selected agonist to the organ bath after which the resultant contractile response of the tissue is recorded. The agonist is then quickly washed out to avoid tissue desensitization. This procedure serves to identify responsive tissues, since occasionally a tissue will fail to contract in response to an to ^-adrenoceptor or P2X receptor agonist. Additionally, in Protocol 2 (Subheading 3.4.2.) this initial response is compared with a subsequent contractile response induced by a second addition of this agonist (dose A) that on this occasion is made in the presence of a cannabi-noid. How data from electrically unstimulated experiments are analyzed is discussed in Subheading 3.4.3. Examples of results obtained from such experiments can be found elsewhere (11,12).

3.4.1. Protocol 1

After establishing whether the tissues are responding to the ^-adrenoceptor or P2X receptor agonist (see Subheading 3.4.), either a cannabinoid or its vehicle is added to the organ bath, after which (time interval Y) a cumulative concentration-response curve is constructed for the chosen ^-adrenoceptor or P2X receptor agonist. This is achieved by adding the next dose to the bath as soon as the contractile response to the previous dose is complete, a protocol that reduces the risk of desensitization and that results in a dose cycle of about 2 min and in the production of a complete concentration-response curve within about i0 min (see also Fig. 3). To avoid desensitization problems, it is advisable not to attempt to obtain more than one concentration-response curve from any one tissue.

3.4.2. Protocol 2

The objective of this protocol is to establish the effect of a cannabinoid on the contractile response to a dose, A, of an ^-adrenoceptor or P2X receptor

Fig. 3. The electrically unstimulated protocols described in Subheading 3.4.

agonist. This involves measuring either the effect of a single dose of a cannabi-noid (Protocol 2a; see Subheading 3.4.2.1.) or the effects of a range of cannabi-noid doses (Protocol 2b; see Subheading 3.4.2.2.) on the contractile response to dose A of the ^-adrenoceptor or P2X receptor agonist. Additionally, the effect of a cannabinoid can be measured in the presence of an antagonist (Protocol 2c; see Subheading 3.4.2.3.). Protocols 2a, 2b, and 2c are depicted in Fig. 3.

3.4.2.1. Protocol 2a

The first step is to add the submaximal dose, A, of the selected contractile agonist. This is washed out as soon as a full contraction has been induced. One dose of the cannabinoid under investigation (or its vehicle) is now added and, after the period of time Y the submaximal dose A of the agonist is once again applied. The resultant contractile response is compared with the response that was elicited at the beginning of the experiment.

3.4.2.2. Protocol 2b

This section describes the protocol for investigating a range of cannabinoid doses on the contractile response to the submaximal dose A of the ^-adrenoceptor or P2X receptor agonist. As can be seen from Fig. 3, this protocol is an extended version of protocol 2a, described in Subheading 3.4.2.1. Thus, after the second contractile response to dose A of the ^-adrenoceptor or P2X receptor agonist has been determined, the cannabinoid and the contractile agonist are immediately washed out and another (higher) dose of cannabinoid then added. After the period of time Y dose A of the contractile agonist is once again applied to the organ bath, the response measured, and the bath contents immediately washed out. This cycle can be continued with progressively higher doses of cannabinoid.

3.4.2.3. Protocol 2c

This protocol is used to determine the ability of an antagonist to oppose cannabinoid-induced alterations of ^-adrenoceptor or P2X receptor agonist-induced contractions. As in Protocols 2a and 2b, the response to a submaximal dose, A, of the selected contractile agent is first determined. Then, a cannabinoid antagonist or its vehicle is added to the organ bath. After sufficient time, X, has elapsed for the antagonist to reach its pharmacological target, a cannabi-noid or its vehicle is added to the organ bath. As shown in Fig. 3, dose A of the a1-adrenoceptor or P2X receptor agonist is applied again after the period of time Y has elapsed and its contractile effect measured. This is followed by a bath wash-out. If required, further additions can now be made of the selected contractile agent and of progressively higher doses of cannabinoid as described for Protocol 2b (see Subheading 3.4.2.2.). The antagonist must then be replaced after each bath wash-out.

3.4.3. Data Analysis of Assays Using Electrically Unstimulated Tissues

Using a 0.5-g weight to calibrate the system, the amplitudes of evoked contractions are converted into the amount of applied tension that would be required in order to produce an equivalent increase. How the data are then analyzed depends on whether Protocol 1 or 2 was used. Subheading 3.4.3.1. discusses data analysis for Protocol 1, and Subheadings 3.4.3.2. to 3.4.3.4. data analysis for protocols 2a, 2b, and 2c.

3.4.3.1. Data Analysis for Protocol 1

This involves measuring the height of the contraction induced by each dose of the ^-adrenoceptor or P2X receptor agonist in the presence of a cannabinoid or its vehicle. In the mouse vas deferens, the addition of either an a1-adreno-ceptor or P2X receptor agonist results in a transient contraction followed by relaxation, even when there is no wash-out. Extra care is therefore needed to ensure that a measure is obtained of the maximal increase in tension produced by each dose of the contractile agonist. The size of each contraction should be measured from a point on the baseline just prior to the start of the induced contraction. Contractile responses are expressed in g tension, as discussed in Subheading 3.4.3.

3.4.3.2. Data Analysis of Protocol 2a

The initial contraction produced by the submaximal dose A of the chosen contractile agonist is determined. This is then compared to the contraction produced by a second addition of dose A of the same contractile agent, performed after addition to the organ bath of the cannabinoid (or its vehicle). Contractile responses are expressed in g tension (Subheading 3.4.3.), and comparisons can be made using Student's two-tailed t-test for paired data.

3.4.3.3. Data Analysis of Protocol 2b

When the effects of multiple doses of a cannabinoid on the response to the single dose A of the contractile agent are investigated, the size of each contraction produced in the presence of the cannabinoid can be compared to the size of the initial contraction evoked in the absence of the cannabinoid. This may be achieved by normalizing the data such that, for example, each contractile response elicited in the presence of the cannabinoid is expressed as a percentage of the initial contractile response, normalized to 100%. The effects of different cannabinoid doses can be compared with the initial contraction by performing a one-way analysis of variance (ANOVA) followed by a post hoc test such as Dunnett's test.

3.4.3.4. Data Analysis of Protocol 2c

The method used for analyzing the effect of a cannabinoid in the presence of an antagonist in electrically unstimulated tissues is essentially the same as the method used to analyze data obtained from experiments performed using Protocol 2a (Subheading 3.4.3.2.).

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