The human NK1 tachykinin receptor in the astrocytoma cell line U 373 MG was characterized using selective agonists and antagonists described for this receptor in the rat. Specific [3H]substance P binding sites were present on cell homogenates, whereas [3H]neurokinin A or [3H]-senktide binding sites were absent. The binding was saturable and reversible. The binding of [3H]substance P was inhibited by very low concentrations of [L-Pro9]substance P and [Sar9,Met(O2)11]substance P; septide was approximately 1,000-fold less potent. The most potent peptide antagonist was trans-4-hydroxy-1-(1H-indol-3-ylcarbonyl)-L-prolyl-N-methyl-N-(phe nylmethyl)-L- tyrosineamide. The rank order of potency for the nonpeptide antagonists was (S,S)-CP 96,345 > (+/-)-CP 96,345 > (+/-)-2-chlorobenzylquinuclidinone > (R,R)-CP 96,345 > RP 67580 > RP 68651. In [3H]-inositol-labeled cells, substance P stimulated phosphatidylinositol turnover. A good correlation was found when the abilities of NK1 receptor agonists for stimulating inositol phosphate production and for inhibiting [3H]substance P binding were compared. Similarly, the binding and functional assays were well correlated for the antagonists. As a result of its high sensitivity and selectivity, the U 373 MG cell line thus appears an excellent tool for investigating the pharmacology of the human NK1 receptor.

1 This study was undertaken to compare the potency and selectivity of the nonpeptide (RP 67580, ( ± )-CP-96,345 and its chloro-derivative [( ± )-cis-3-(2-chlorobenzylamino)-2-benzhydrylquinuclidine] (CP-C1)) and peptide (GR 71,251 and spantide) neurokinin, (NK,) 6 Several experiments indicated that the antagonists were highly selective for NKI receptors. RP 67580 did not modify the noradrenaline-evoked activation of phospholipase C in cortical astrocytes; when used at 10-1 M all antagonists had no or only little affinity for NK2 or NK3 binding sites and did not block the NKA (10-i M)-induced activation of phospholipase C in the hamster urinary bladder (a selective NK2 test). 7 In conclusion, RP 67580 appears to be a potent NK, antagonist in the mouse and the rat. Results obtained with (±)-CP-96,345 confirm the lower potency of this compound in these two species when compared with reported data obtained in the guinea-pig or man.

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In the human astrocytoma cell line U 373 MG, application of substance P (SP) leads to a transient increase in cytosolic calcium concentration and to a biphasic current response in voltage-clamped cells. Using these two functional assays we have characterized pharmacologically the SP response in U 373 MG cells. SP and [L-Pro9]SP displayed high potencies in both assays with EC50 values of 2.5 x 10(-9) M and 1 x 10(-9) M on calcium responses and 1 x 10(-9) M and 5 x 10(-9) M on ion current responses, respectively. The high potency of SP and [L-Pro9]SP as well as the low potency of [Lys5,MeLeu9,N-Leu10]neurokinin A(4-10) and the inactivity of senktide demonstrate the NK1-type pharmacology of these responses. Furthermore, the NK1 antagonists (+/-)-CP 96,345, its chloro analogue, (+/-)-cis-3-(2-chlorobenzylamino)-2-benzhydrylquinuclidine, and RP 67580 were potent antagonists of both SP responses. For the calcium mobilization induced by SP (10(-7) M), the IC50 values for the three antagonists were 4 x 10(-10) M, 4 x 10(-9) M, and 9 x 10(-9) M, respectively, whereas on the current response evoked by SP (10(-8) M), the IC50 values were 8 x 10(-9) M, 2.4 x 10(-8) M, and 1.2 x 10(-7) M, respectively. Despite differences in the absolute IC50 values obtained with both techniques, the relative potencies of the three antagonists correlate fairly well.(ABSTRACT TRUNCATED AT 250 WORDS)

The early stages of the carcinogenic process induced by aflatoxin B1 (AFB1) in rat liver during 24 weeks of feeding and the resulting tumours have been studied with respect to cytokeratin (CK) expression. A previously uncharacterized monoclonal antibody, MRCTU/J1, has been shown to recognize rat CK18 and together with antibodies against human CK8, 18 and 19, has been used to examine the possible lineage of tumour cells and also to identify the altered foci that might be most relevant to tumorigenesis. Results suggested that AFB1-induced transformation in liver may occur in more than one cell type, since tumours with the normal hepatocyte CK pattern and those with bile duct or oval cell CK phenotype were identified. Additionally, hepatocytes with a bile duct CK phenotype appeared during the early stages of carcinogenesis. The in vivo pattern of CK expression also appeared to be maintained in one normal and one hepatoma-derived cell line. Overexpression of CKs (particularly of CK19) was a much more selective marker for altered foci, compared to gamma-glutamyltranspeptidase, and was more consistently expressed at high levels in tumours, suggesting that it might be a more reliable way of identifying those cells involved in the transformation process.