Martina Schulz

The cholecystokinin2-receptor mediates calcitonin secretion, gene expression, and proliferation in the human medullary thyroid carcinoma cell line, TT.

Gastrin-induced release of calcitonin from medullary thyroid carcinomas (MTC) is based on the expression of the cholecystokinin(2)-receptor (CCK(2)R) in these tumors. Recently, we have shown that the CCK(2)R is expressed not only in MTC but also in C-cells within the normal thyroid gland. The functions of the CCK(2)R in MTC and C-cells are largely unknown. We therefore explored the effects of gastrin-induced CCK(2)R stimulation in the highly differentiated MTC cell line, TT. CCK(2)R expression in TT-cells is detectable by RT-PCR as well as immunocytochemistry. Stimulation of the CCK(2)R by gastrin induces immediate release of calcitonin from TT-cells. Moreover, quantitative (LightCycler) RT-PCR demonstrates that gastrin stimulates transcription of the calcitonin and chromogranin A genes in TT-cells. TT-cell proliferation, assessed by counting of viable cells and (3)H-thymidine uptake, is markedly increased by gastrin. This effect is inhibited by the CCK(2)R-specific antagonist L-365,260. Our findings suggest physiological functions for the CCK(2)R in calcitonin-secretion and gene expression as well as a pathophysiological role in MTC proliferation. CCK(2)R antagonists might have therapeutic potential in these tumors.

Evidence for CCKB receptors in the guinea-pig kidney: localization and characterization by [125I]gastrin binding studies and by RT-PCR

Two types of receptors for gastrin and cholecystokinin (CCK) have been identified in the gastrointestinal tract and in the central nervous system: CCKA and CCKB receptors. Here we report evidence for the expression of CCKB receptors in the guinea-pig kidney. Specific binding sites for [125I]gastrin were detected in sections of the guinea-pig kidney: Binding was saturable, pH-, temperature- and time-dependent, and specific for gastrin-related peptides. The potencies for inhibition of binding of [125I]gastrin were CCK-8 > gastrin 17-I > CCKB receptor antagonist L-365,260 > des(SO3)CCK-8 > CCKA receptor antagonist L-364,718. Autoradiography demonstrated specific [125I]gastrin binding to medullary collecting ducts and to a much lesser extent to glomeruli, but not over other structures. CCKB receptor cDNA fragments were amplified by RT-PCR from total kidney, isolated tubuli and from tissues known to express CCKB receptors such as stomach and brain. The kidney might therefore be a previously unidentified site of action for gastrin and cholecystokinin-related peptides.

CHARACTERIZATION OF CCK RECEPTORS IN STOMACH SMOOTH MUSCLE : EVIDENCE FOR TWO SUBTYPES

Cholecystokinin (CCK) and related peptides such as gastrin are important regulators of gastric smooth muscle contraction. Several studies have shown that these effects of CCK and gastrin are mediated by CCK(B) receptors. However, recent studies suggest the expression of an additional CCK receptor subtype distinct from CCK(B) receptors in this tissue. This study was designed to distinguish between CCK(A) and CCK(B) receptors on guinea-pig stomach smooth muscle cells and to evaluate these cells for additional receptor subtypes. We cloned these receptors by hybridization screening of a guinea-pig smooth muscle cDNA library using 32P random primed labeled cDNA probes from the recently cloned rat CCK(A) and CCK(B) receptor coding regions. In addition to clones representing the CCK(B) subtype, clones of CCK(A) receptor subtype, but no additional CCK receptor subtypes, could be identified. All isolated clones displayed highly homologous nucleotide sequences in comparison to previously characterized CCK(A) and CCK(B) receptors from different species. The results of cDNA hybridization at different levels of stringency and Southern blot analysis using guinea-pig genomic DNA suggest that it is unlikely that additional CCK receptors despite CCK(A) and CCK(B) receptors exist in stomach smooth muscle.