Receptors for thyrotropin-releasing hormone (TRH) in the rat brain and the pituitary are heterogenous. The receptors were classified into four types according to the dissociation constant (KD). High-affinity receptors (KD less than 3 nM) are present in the pituitary, hypothalamus, amygdala, and limbic forebrain which contains the nucleus accumbens and the septum. Intermediate-affinity receptors (KD, 5-16 nM) are evidently present in the frontal cortex, hippocampus, striatum, thalamus, and the brainstem, but may also be present in other regions. Low-affinity TRH receptors (KD, 50-80 nM) are seen in the limbic forebrain, amygdala, and the hypothalamus. Very-low-affinity receptors (KD, 215 nM) exist in the pituitary. Experiments using DN-1417 (gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide citrate), a synthetic TRH analogue with a more potent central activity, indicated the presence of TRH receptors having a high affinity to DN-1417 at least in the limbic forebrain but not in the pituitary. This type of receptor is not labeled by [3H](3-methyl-histidine2)-TRH. Density of the TRH receptor is the highest in the pituitary and next highest in the amygdala.

The mode of action of cytochalasin B was examined in vitro using bile canaliculusenriched plasma membrane fractions isolated from rat liver. The pericanalicular microfilaments, which are mainly actin filaments and which are normally attached to the canalicular membranes, were dissociated from the membranes by cytochalasin B treatment. A microfilamentous network was found in the supernate of the cytochalasin-treated specimens and a number of polypeptides, of which a polypeptide corresponding in molecular weight to actin was a notable member . These results suggest that actin filaments become detached from the canaliculus membranes by cytochalasin B.The cytochalasins are widely used in biology and pathology as cytopharmacological agents because of their effects on the contractile functions of actin filaments (34) . Alteration of microfilament contractility is a general effect that has been observed in many cell types, but the functional sequelae vary with the specialized nature of the cell involved (4,18,36) . Accompanying these functional changes in cell motility, structural changes have been observed in the electron microscope . The actin filaments (F-actin), which are -5 nm in diameter, disappear and the regions normally occupied by the filaments contain granules and short filaments . It has generally been held that this structural change is a manifestation of disassembled or altered microfilaments (19,30) .Isolated bile canaliculus-enriched plasma membrane fractions of rat liver provide a useful experimental tool for the study of microfilament-membrane association. It has been shown by heavy meromyosin binding (7,24), by immunofluorescent staining, and by immunoelectron microscopic techniques (10, 12) that the pericanalicular microfilaments are mainly actin filaments, and that they remain attached to the canalicular membranes even after the homogenization and centrifugation procedures involved in the isolation of these fractions (6,21) . This model lends itself to an examination of the direct effects of cytochalasin B (CB) on plasma membraneassociated filaments in a cell-free system, and hence gets away from difficulties inherent in the interpretation of results in a more complex cellular system . MATERIALS AND METHODSThe bile canaliculus-enriched fractions of liver cell plasma membranes (BCM) were isolated from the livers of male Wistar rats, weighing -150 g, according to 524 a modification (6) of the method reported by Song et al . (29) . CB (Aldrich Chemical Co . Inc., Milwaukee, Wise .) was dissolved in dimethyl sulfoxide (DMSO) (3 mg of CB/0.1 ml of DMSO) and was diluted with 3 mM phosphatebuffered saline (PBS) (pH 7.4) at an appropriate concentration . Each BCM fraction (2 .2 . mg average protein content) determined by the method of Lowry et al. (l6) was divided into two parts of equal weight . One part was incubated at room temperature for 90 minwith 1.5 ml of PBS containing CB and 0.2% DMSO . Studies were carried out with final CB concentrations of 2.2 x 10-5 M and 2.2 x 10 -6 M. The other pa...

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