Roger K. Craig

Human and rat α-CGRP but not calcitonin cause mesenteric vasodilatation in rats

Abstract A single gene encodes both calcitonin and the calcitonin gene-related peptide (CGRP). Human and rat α-CGRP were compared with sodium nitroprusside in the rat and rabbit isolated mesenteric vascular preparation perfused at constant flow. In the presence of the vasoconstrictor noradrenaline (10 −5 M), rat α-CGRP was about 10 times as potent as either human α-CGRP or sodium nitroprusside as a vasodilator in the rat mesenteric vasculature. In the rabbit mesenteric vasculature the order of potency was rat α-CGRP > human α-CGRP > sodium nitroprusside. Human and salmon calcitonin showed no vasodilator activity at doses 100 times greater than human α-CGRP. These results show that human and rat α-CGRP are potent vasodilators in the mesenteric vasculature, an effect not mimicked by the alternative gene product, the plasma calcium lowering hormone calcitonin.

Cardiovascular effects of human and rat CGRP compared in the rat and other species

In the anaesthetised rat, human and rat CGRP (calcitonin gene-related peptide) which differ by 4 out of 37 amino acids, when given intravenously, lowered blood pressure and increased heart rate. The effects of human CGRP were unaltered by either propranolol or by mepyramine plus cimetidine. In the rat isolated perfused heart the peptides decreased coronary perfusion pressure and evoked a tachycardia. The latter effect was not seen in the rabbit isolated heart, although human CGRP increased coronary flow. The two peptides were equipotent at increasing the rate and force of contraction in the rat isolated right atrium, effects unaltered by propranolol. In the guinea-pig isolated atrium, rat CGRP was 10 times as potent as a chronotropic agent than as an inotrope, unlike human CGRP which was equipotent. In conclusion, human and rat CGRP probably acted directly on the cardiovascular system to produce their qualitatively similar effects.

Human α- and β-CGRP and rat α-CGRP are coronary vasodilators in the rat

Abstract The effects of the recently described human α-calcitonin gene-related peptide (CGRP), human β-CGRP and rat α-CGRP have been compared with those of the vasodilator sodium nitroprusside, on the rat and rabbit isolated heart. Hearts were perfused at constant flow and [Arg 8 ]-vasopressin was used to increase coronary perfusion pressure. In the rat heart, the order of potency for evoking cumulative dose-dependent falls in perfusion pressure was human β-CGRP>rat α-CGRP>human α-CGRP>sodium nitroprusside. In the same preparations the three CGRPs (but not sodium nitroprusside) elicited cumulative dose-related increases in heart rate. In the rabbit heart the order of potency for vasodilatation was rat α-CGRP>human α-CGRP>sodium nitroprusside. In marked contrast to results from the rat, neither rat α-CGRP nor human α-CGRP altered heart rate in the rabbit isolated heart. These results show that human α- and β-CGRP and rat α-CGRP are vasodilators in the coronary vasculature, but that there is species variation as CGRP had a positive chronotropic effect in the rat heart but not in the rabbit heart.

Postjunctional inhibition of contractor responses in the mouse vas deferens by rat and human calcitonin gene-related peptides (CGRP)

1 The effects of rat and human α‐calcitonin gene‐related peptide (CGRP) were compared in the mouse and rabbit isolated vas deferens preparation contracted by either field stimulation or acetylcholine. 2 The peptides were about equipotent at inhibiting twitch responses of the mouse vas deferens to field stimulation at 0.2 Hz (IC50 12 ± 4 nM and 15 ± 3 nM, rat and human α‐CGRP respectively). Rat α‐CGRP was less potent at inhibiting responses to 10 Hz than to either 0.2 Hz or 1.0 Hz stimulation. The potency of rat α‐CGRP at 1.0 Hz was unaltered by halving the calcium concentration of the Krebs solution. 3 The inhibitory effect of human α‐CGRP was not antagonized by either propranolol (300 nM) or idazoxan (300 nM), although in the same tissues these latter two drugs reduced responses to isoprenaline and clonidine respectively. 4 Rat α‐CGRP (100 nM) and human α‐CGRP (1.0 μM) did not alter the uptake of [3H]‐noradrenaline (30 nM) into mice isolated vasa deferentia. Rat α‐CGRP (3–100 nM) did not alter the fractional release per pulse (1.0 Hz, 100 pulses) of tritium from vasa preloaded with [3H]‐noradrenaline, although at the same time the peptide inhibited responses of the smooth muscle to field stimulation. 5 Rat and human α‐CGRP were equipotent at inhibiting contractions of the mouse vas deferens evoked by acetylcholine although the peptides were less potent than against twitch responses. 6 In the rabbit vas deferens neither rat nor human α‐CGRP (3 nM‐1 μM) inhibited either twitch responses or acetylcholine contractions. 7 These results suggest that rat and human α‐CGRP inhibit contractor responses of the mouse vas deferens not by interference with adrenergic mechanisms, but through postjunctional (possibly CGRP) receptors. A similar mechanism may underlie effects of CGRP in other tissues. The rabbit vas deferens appears to lack the CGRP ‘receptors’.

Characterisation of an mRNA encoding a human ribosomal protein homologous to the yeast L44 ribosomal protein

We describe the isolation and characterisation of a full-length cDNA sequence (pZH-21) of a human ribosomal protein (rp) mRNA isolated from a cDNA library constructed from the human ZR-75-1 mammary tumour cell-line. The predicted protein is highly basic and shows 72% homology at the amino acid (aa) level with yeast rp L44. Comparative RNA blotting of ZR-75-1 poly(A)+ RNA isolated from cells cultured in the presence of the anti-oestrogen tamoxifen demonstrates the presence of a number of mRNA species whose concentration is elevated co-ordinately 5-6-fold in the presence of 17beta-oestradiol. Insulin in the presence of tamoxifen, also enhanced rp mRNA levels suggesting increased levels are a reflection of cell proliferation as opposed to specific hormonal regulation. Genomic analysis demonstrates the presence of a family of related human sequences, and homology with rat and guinea pig rp genes, but not yeast DNA. The conservation of rp aa sequence, in the absence of detectable homology at the nucleotide (nt) level, points to an important common functional role of the L44 protein in ribosome structure and function in man and yeast.

Ectopic synthesis of high-Mr calcitonin by the BEN lung carcinoma cell line reflects aberrant proteolytic processing.

Cloning and nucleotide sequence analysis of the human calcitonin mRNA from the BEN lung carcinoma cell line, a cell line known to secrete high‐M r forms of calcitonin, showed no difference in the Coding region at the nucleotide level compared with calcitonin mRNA isolated from medullary thyroid carcinoma which secretes calcitonin monomer. Therefore, the secretion of high‐M r forms of calcitonin reflects the absence or limited activity of proteolytic processing enzymes within the secretory pathway of this cell line. In all other respects, as judged by RNA blotting and S1 mapping, calcitonin/α‐CGRP expression was identical to that found in medullary thyroid carcinoma, including the differential use of an alternative splice donor site within intron 1. The BEN cell line also produces low levels of α‐CGRP mRNA and secretes CGRP antigenic determinants. Analysis of plasma CGRP levels in 12 patients with anaplastic lung carcinoma showed elevated levels in 11 of these, suggesting that CGRP may be an important diagnostic marker for this disease.

The construction, identification and characterisation of plasmids containing human alpha-lactalbumin cDNA sequences.

We describe the cloning of double-stranded cDNA synthesized from lactating human mammary gland total poly(A)-containing RNA, into the EcoRI site of the plasmid pAT153. Nine recombinants were shown to contain alpha-lactalbumin cDNA sequences as determined by positive hybridisation translation of complementary RNA. Restriction enzyme maps were determined for six of these. Alignment of the restriction map with the known amino acid sequence of human alpha-lactalbumin provided evidence that two plasmids, designated phO-53 and phB-35, contained the complete coding sequence of the primary translation product (pre-alpha-lactalbumin). Hybridisation studies using purified human, monkey and guinea-pig alpha-lactalbumin cDNA demonstrated that greater nucleotide sequence divergence has occurred within the rodents than the primates, and that rodent alpha-lactalbumin mRNAs retain regions of homology with primate alpha-lactalbumin mRNAs.

Loss of transcriptional repression contributes to the ectopic expression of the calcitonin/α-CGRP gene in a human lung carcinoma cell line

The calcitonin/α‐CORP (CT/CGRP) gene is ectopically expressed in a wide variety of neoplasia. We have investigated the molecular mechanisms responsible for this ectopic expression in the human cell line BEN, which is derived from a poorly differentiated squamous cell lung carcinoma. We show that a trans‐acting factor which represses expression of the CT/CGRP gene in HeLa cells is absent or inactive in BEN cells, and have localised the repressor binding site to a 53 bp fragment 1500 bp upstream of the transcription start site.

α1-antitrypsin and serum albumin mRNA accumulation in normal, acute phase and ZZ human liver

α1‐Antitrypsin and albumin mRNA levels of 4 human livers were assessed using a newly sequenced cDNA clone of the carboxyterminal third of α1‐antitrypsin and a previously cloned albumin cDNA sequence. The relative concentration of α1‐antitrypsin mRNA was the same in poly(A)‐containing RNA isolated from acute phase (MM) and α1‐antitrypsin deficient (ZZ) individuals. In the acute phase liver relative to the normal (MM) liver, total RNA extracts showed a marked decrease in albumin mRNA concentration but no increase in α1‐antitrypsin mRNA. The ZZ liver showed decreased total and poly(A)‐containing RNA content but the same proportion of α‐antitrypsin to albumin mRNA as in the normal (MM) liver. This supports other evidence that ZZ α1‐antitrypsin deficiency is due to a defect in polypeptide processing (secretion) rather than a deficiency in mRNA accumulation.

Hormonal regulation of specific gene expression

A FEBS Advanced Course (No. 53) was held September 1%22,19?8 at The Mjd~e~x Hospital Medical School an the ?-Iormonal Reg~a~o~ of Specific Gene Expression’. The course coincided wi& the celebrations to mark the 50th Anniversary of the Foundation of the Courtauld Institute of Biochemistry. A Symposium was held on the first day at which the speakers surveyed various aspects of the subject for the comparative nonexpert. The subsequent course lasted two and a half days and consisted of talks by invited speakers and shorter contributions from participants. As with the last caurse on a related but different subject [FEBS Letters (1976) 72,215-2261 we, as organizers, were asked to produce a survey of the papers presented and of the discussion and in particular to provide a bibliography that would serve as an entree to the field. Rather than try to provide a blow by blow account we have tried to pinpoint the present status of the various aspects af the subject. All the major contributors have had an opportunity to comment on a draft report and we hope that there is a reasonabie concensus concerning the views expressed. The plan of the course was to consider first the progress that has been made in developing cell cultures that have either been shown to respond to hormones or have the potential of doing so. There followed several sessions in which current work on various hormones was considered in turn. Particular emphasis was placed on the use of in vitro systems, either cell cultures or explants. In the last session the contribution of current developments in recombinant DNA research to endocrinology was reviewed. There are two broad approaches to an understanding of gene expression in eukaryotes. Either one can utilize systems in which a few well-defined prateins are produced in response to specific stimuli, or one can study systems in which the specific genes of interest are reiterated to such a degree that they may be isolated by physical means alone. The present course was almost entirely confined to examples of the first of the two approaches.