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In vitro or in vivo receptor binding Where does the truth lie.

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In Vitro or In Vivo Receptor Binding:
Where Does the Truth he?
Studies of receptor binding can be performed under
both in vitro and in vivo conditions. In vitro studies of
tissue homogenates represent the classic approach by
which quantitative measurement of the number of
binding sites and their affinity for a particular ligand
are performed. These measurements must be carried
out under strict equilibrium conditions which, of
course, can easily be achieved in vitro. Use of tissue
slices rather than tissue homogenates for in vitro analysis can improve anatomical localization and can, if desired, be conducted with the same quantitative precision. Interpretations of these in vitro studies usually
make the tacit assumption that such data reflect the in
vivo condition despite rather obvious differences.
In vivo studies strive to more accurately evaluate
true in vivo receptor function. Following intravenous
administration of a radioligand, tissue samples from experimental animals or positron emission tomographic
(PET) images from human subjects are analyzed. In
contrast to in vitro studies, in vivo studies occur
under nonequilibrium conditions. These are brought
about by factors that affect the concentration of free
radioligand in a region of interest, including nonspecific binding of the tracer in blood and brain, bloodbrain-barrier permeability, and blood flow, as well as in
vivo metabolism of the tracer that can cause various
radiolabeled metabolites to appear in both blood and
tissue. As a result, quantitative estimates of the number of receptors and their affinity cannot be achieved
from a single measurement in time. Rather, multiple
measurements are needed to delineate the time course
of radioactivity resulting from the radioligand in both
the region of interest and the blood after correction for
the presence of labeled metabolites. These data must
then be analyzed with an appropriate mathematical
model relating brain and blood radioactivity measurements to measurements of receptor physiology. Inferences drawn from qualitative in vivo measurements
based on single-tissue autoradiograms or PET images
must be viewed with extreme caution despite their
intuitive visual appeal. Unfortunately, this sort of inference is the rule rather than the exception.
In this issue of the Annals, Bennett and Wooten El]
provide an important insight into the difficulty in interpreting in vivo and in vitro receptor binding studies. They used three different techniques (in vitro
homogenates, in vitro tissue slices, and in vivo tissue samples) to measure binding of tritium-labeled
spiperone (a dopamine receptor ligand with very high
affinity for the D2 dopamine receptor) in the striatum
of rats with unilateral lesions of the nigrostriatal pathways. Animals with such lesions have previously been
shown to exhibit behavior consistent with the presence
of “supersensitive” dopamine receptors. This experimental model is analogous in many ways to Parkinson’s
disease. In vitro receptor binding studies on striatal
membrane preparations from such animals, as well as
striatal nuclei from brains of subjects with Parkinson’s
disease, have demonstrated increased binding consistent with the presence of an increased number of receptors or receptors with an increased affinity for the
ligand, or both. One would anticipate similar results
from in vivo binding studies. The data of Bennett and
Wooten, however, demonstrate a disparity between
in vivo and in vitro measurements. Their in vitro results with homogenates of striatal membrane preparation showed significant changes in both affinity and
receptor concentrations on the side of the lesion, consistent with previous investigations. However, both in
vitro tissue slices and striatal tissue samples obtained
following the in vivo administration of the radioligand
revealed absolutely no changes.
How are we to interpret such discrepant findings?
The task is not easy. One interpretation, that of Bennett and Wooten, is that the in vitro behavior of receptors in tissue homogenates simply does not accurately
reflect in vivo behavior. Clearly, in vitro homogenation
of tissue drastically disrupts normal anatomy and may
account for the fact that many more apparent receptors
are labeled and that radioligand dissociates much more
rapidly than in vivo. However, in vitro autoradiography also failed to demonstrate any apparent change.
It is, therefore, difficult to argue that the difference can
be explained simply on the basis of a difference between in vivo and in vitro behavior of the radioligand.
Likewise, it is difficult to argue that compounding variables in the in vivo experiments, such as experimentally induced variability in the delivery and removal of
radioligand from the tissues or the presence of
radiolabeled metabolites, obscure the changes. How
then could the changes have been missed by in vitro
autoradiography as well as by in vivo tissue sampling?
There is an alternate explanation. Neither in vitro
autoradiography nor in vivo tissue sampling were used
in a truly quantitative fashion. A single time point was
examined in the in vivo experiment, and only relative
differences between denervated and normal striata
were recorded in the in vitro autoradiography experiments. Examination of the quantitative data from Bennett and Wooten's in vitro tissue homogenate experiments (see their Fig 1) reveals that induced changes
were complex, involving changes in both receptor
numbers and affinity. The amount of radioligand retained in tissue at equilibrium is the product of the
binding affinity of the receptor and the total number of
receptors. Mintun and associates [3} have referred to
this product as the binding potential (binding potential 4
number of receptors (Bmm)x affinity (K/'). The average binding potential calculated by Klotz analysis { 31
using the in vitro quantitative data is almost twice as
high in the lesion-affected side compared with the control side (i.e., lesioned, 0.75; control, 0.46). A similar
calculation cannot be made from either the in vitro
autoradiography or in vivo tissue-sampling data. If this
interpretation of the experiments of Bennett and
Wooten is correct, it provides strong support for the
routine implementation of reliable quantitative strategies for the analysis of data from both in vitro autoradiograms, and for in vivo experiments employing
either experimental animals or human subjects with
PET. Regardless of their interpretation, the data of
Bennett and Wooten serve as a singularly important
example that we must understand our tools before we
can hope to understand our results.
Joel S. Perlmutter, M D
Marcus E. Raichle, M D
Washington University School of Medicine
St. Louis, MO 63110
We thank John Katzenellenbogan, PhD, for useful discussions
1. Bennett JP Jr, Wooten GF: Dopamine denervation does not alter
in vivo 3H-spiperone binding in rat striatum: implications for
external imaging of dopamine receptors in Parkinson's disease.
Ann Neurol 19378-383, 1986
2. Klotz IM, Hunston DL: Properties of graphical representations
of multiple classes of binding sites. Biochemistry 10:3065-3069,
197 1
3. Mintun MA, Raichle ME, Kilbourn MR, et al: A quantitative
model for the in vivo assessment of drug binding sites with positron emission tomography. Ann Neurol 15:217-227, 1984
Editorial: Perlmutter and Raichle: R e c e p t o r Binding
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