Stephen Legon

Comparison of the expression of calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) with CGRP and adrenomedullin binding in cell lines.

The calcitonin receptor‐like receptor (CRLR) and specific receptor activity modifying proteins (RAMPs) together form receptors for calcitonin gene‐related peptide (CGRP) and/or adrenomedullin in transfected cells. There is less evidence that innate CGRP and adrenomedullin receptors are formed by CRLR/RAMP combinations. We therefore examined whether CGRP and/or adrenomedullin binding correlated with CRLR and RAMP mRNA expression in human and rat cell lines known to express these receptors. Specific human or rat CRLR antibodies were used to examine the presence of CRLR in these cells. We confirmed CGRP subtype 1 receptor (CGRP1) pharmacology in SK‐N‐MC neuroblastoma cells. L6 myoblast cells expressed both CGRP1 and adrenomedullin receptors whereas Rat‐2 fibroblasts expressed only adrenomedullin receptors. In contrast we could not confirm CGRP2 receptor pharmacology for Col‐29 colonic epithelial cells, which, instead were CGRP1‐like in this study. L6, SK‐N‐MC and Col‐29 cells expressed mRNA for RAMP1 and RAMP2 but Rat‐2 fibroblasts had only RAMP2. No cell line had detectable RAMP3 mRNA. SK‐N‐MC, Col‐29 and Rat‐2 fibroblast cells expressed CRLR mRNA. By contrast, CRLR mRNA was undetectable by Northern analysis in one source of L6 cells. Conversely, a different source of L6 cells had mRNA for CRLR. All of the cell lines expressed CRLR protein. Thus, circumstances where CRLR mRNA is apparently absent by Northern analysis do not exclude the presence of this receptor. These data strongly support CRLR, together with appropriate RAMPs as binding sites for CGRP and adrenomedullin in cultured cells.

Detection of human probombesin mRNA in neuroendocrine (small cell) carcinoma of the lung. In situ hybridization with cRNA probe.

The production of human bombesin (gastrin‐releasing peptide), a peptide with mitogenic action, is a recognized feature of neuroendocrine (small cell) carcinoma of the lung. However, immunostaining of bombesin is not always possible in these tumors, probably because of poor storage mechanisms or rapid release of hormone. Molecular biological analysis of the gene encoding human bombesin has revealed the DNA sequence of human pro‐bombesin. We have used in situ hybridization to study the expression of the human bombesin gene at the cellular level in small cell carcinoma of the lung. Probombesin cDNA was subcloned in pSP64 vector, linearized with Bam HI and transcribed in the presence of phosphorus 32 (32P)‐cytosine triphosphate (CTP) and SP6 polymerase. The cRNA probe was applied to tissue sections (from six cases of small cell carcinoma of the lung, freshly fixed in 4% paraformaldehyde), cell culture preparations (two different cell lines of small cell carcinoma), and cytologic specimens (smears of cells from three different cases of small cell carcinoma). Hybridization of probombesin mRNA was detected in tumor cells in all samples. Specificity of the signal was determined by control experiments, including the use of a probe which has a sequence identical to probombesin mRNA. Our results provide evidence for the expression of the bombesin gene in small cell carcinoma of the lung at a cellular level and show that probombesin mRNA is highly expressed in these tumors.

Plasma membrane calcium pump expression in human placenta and small intestine.

To identify the forms of the plasma membrane calcium pump present in tissues that transport calcium, cDNA from human placenta and proximal small intestine was amplified by the polymerase chain reaction using a pair of mixed primers based on all the known human and rat plasma membrane calcium pump sequences. Clones were identified from the two human forms HPMCA1 and HPMCA4, but no new sequences were found in either tissue. RNA blots probed with HPMCA1 showed two bands in both tissues; probing with HPMCA4 gave a single, larger species. In placenta, HPMCA4 was the more abundant form and similar expression was found in full-term and second-trimester placentas. In contrast, in the small intestine, HPMCA1 was more abundant, suggesting that calcium absorption is not associated with any one specific isoform in calcium transporting cells.