Bronwyn A. Evans

Expression of β3‐adrenoceptor mRNA in rat brain

The reverse transcription/polymerase chain reaction was used to demonstrate β3‐adrenoceptor mRNA in rat brain regions. Levels were highest in hippocampus, cerebral cortex and striatum and lower in hypothalamus, brainstem and cerebellum.

Expression of β3‐adrenoceptor mRNA in rat tissues

1 This study examines the expression of β3‐adrenoceptor messenger RNA (β3‐AR mRNA) in rat tissues to allow comparison with atypical β‐adrenoceptors determined by functional and radioligand binding techniques. 2 A reverse transcription/polymerase chain reaction protocol has been developed for determining the relative amounts of β3‐AR mRNA in rat tissues. 3 Measurement of adipsin and uncoupling protein (UCP) mRNA was used to examine all tissues for the presence of white and brown adipose tissue which may contribute β3‐AR mRNA. 4 The β3‐AR mRNA is expressed at high levels in brown and white adipose tissue, stomach fundus, the longitudinal/circular smooth muscle of both colon and ileum, and colon submucosa. There was substantial expression of adipsin in colon submucosa and moderate expression in fundus, suggesting that in these regions at least some of the β3‐AR signal may be contributed by fat. Pylorus and colon mucosa showed moderate levels of β3‐AR mRNA with lower levels of adipsin. Ileum mucosa and submucosa showed low but readily detectable levels of β3‐AR. 5 Expression of adipsin in rat skeletal muscles coupled to very low levels of β3‐AR mRNA indicates that the observed β3‐AR may be due to the presence of intrinsic fat. β3‐AR mRNA was virtually undetectable in heart, lung and liver. These results raise the possibility that the atypical β‐AR demonstrated by functional and/or binding studies in muscle and in heart is not the β3‐AR. 6 By use of two different sets of primers for amplification of β3‐AR cDNA, no evidence was found for differential splicing of the mRNA in any of the tissues examined. 7 The detection of β3‐AR mRNA in the gut mucosa and submucosa suggests that in addition to its established roles in lipolysis, thermogenesis and regulation of gut motility β3‐AR may subserve other functions in the gastrointestinal tract. The absence of β3‐AR mRNA in rat heart or its presence with adipsin in skeletal muscle suggests that atypical β‐adrenoceptor responses in heart and skeletal muscle are unlikely to be mediated by β3‐AR.

Characterization of β-adrenoceptor mediated smooth muscle relaxation and the detection of mRNA for β1-, β2- and β3-adrenoceptors in rat ileum

Functional and molecular approaches were used to characterize the β‐AR subtypes mediating relaxation of rat ileal smooth muscle. In functional studies, (−)‐isoprenaline relaxation was unchanged by CGP20712A (β1‐AR antagonist) or ICI118551 (β2‐AR antagonist) but shifted by propranolol (pKB=6.69). (±)‐Cyanopindolol, CGP12177 and ICID7114 did not cause relaxation but antagonized (−)‐isoprenaline relaxation. BRL37344 (β3‐AR agonist) caused biphasic relaxation. The high affinity component was shifted with low affinity by propranolol, (±)‐cyanopindolol, tertatolol and alprenolol. CL316243 (β3‐AR agonist) relaxation was unaffected by CGP20712A or ICI118551 but blocked by SR58894A (β3‐AR antagonist; pA2=7.80). Enhanced relaxation after exposure to forskolin and pertussis toxin showed that β3‐AR relaxation can be altered by manipulation of components of the adenylate cyclase signalling pathway. The β1‐AR agonist RO363 relaxed the ileum (pEC50=6.18) and was blocked by CGP20712A. Relaxation by the β2‐AR agonist zinterol (pEC50=5.71) was blocked by SR58894A but not by ICI118551. In rat ileum, β1‐, β2‐ and β3‐AR mRNA was detected. Comparison of tissues showed that β3‐AR mRNA expression was greatest in WAT>colon=ileum>cerebral cortex>soleus; β1‐AR mRNA was most abundant in cerebral cortex>WAT>ileum=colon>soleus; β2‐AR mRNA was expressed in soleus>WAT>ileum=colon>cerebral cortex. These results show that β3‐ARs are the predominant β‐AR subtype mediating rat ileal relaxation while β1‐ARs may produce a small relaxation. The β2‐AR agonist zinterol produces relaxation through β3‐ARs and there was no evidence for the involvement of β2‐ARs in relaxation despite the detection of β2‐AR mRNA.

Genes for the alpha and gamma subunits of mouse nerve growth factor are contiguous.

Here we describe the structure and linkage of genes encoding the alpha and gamma subunits of mouse nerve growth factor (NGF). These genes are members of the highly homologous glandular kallikrein multigene family. Together with the beta subunit, the alpha and gamma proteins constitute the high mol. wt. (7S) form of NGF isolated from mouse submandibular gland. The gamma subunit is an active serine protease and is thought to cleave pro‐beta‐NGF to generate the mature growth factor. The alpha subunit has no detectable proteolytic activity, but is essential for the stable formation of 7S NGF. Lack of enzyme activity of the alpha subunit can be attributed, at least in part, to the deletion of 15 nucleotides in a highly conserved coding region which is normally involved in the activation of serine proteases from their inactive zymogen form.

Molecular cloning of the mouse angiotensinogen gene

We describe here the cloning, restriction mapping, and sequencing of the mouse angiotensinogen gene. The 5 flanking region contains consensus sequences for several hormone-responsive elements and viral-like enhancers within 750 bp of the cap site. The deduced amino acid sequence shows 87% identity with rat angiotensinogen, but there is a discrepancy in the number of cysteine residues in the mature protein among rat (n = 3), human (n = 4), and mouse (n = 4). Because angiotensinogen is homologous to other members of the serine protease inhibitor family, we aligned the putative reactive center of angiotensinogens from various species. This alignment shows that the inhibitor site in human angiotensinogen is different from its rodent counterpart, but the role of this sequence divergence in the pathogenesis of human disease remains to be established.

Functional and molecular evidence for β1‐, β2‐ and β3‐adrenoceptors in human colon

Relaxation of carbachol pre‐contracted human colonic muscle to (–)‐isoprenaline was examined in circular, longitudinal and taenia coli preparations to determine the β‐adrenoceptor subtypes involved. β1‐, β2‐ and β3‐Adrenoceptor mRNAs were also measured in colonic muscle and mucosa. (–)‐Isoprenaline caused relaxation of longitudinal smooth muscle preparations with pEC50=7.39±0.12, and this response was inhibited by both propranolol (0.1 μm, pKB 8.55±0.12) and the selective β1‐antagonist, CGP 20712A (0.1 μm, pKB 8.80±0.20), while the selective β2‐antagonist, ICI 118551 (0.1 μm) failed to inhibit isoprenaline relaxation consistently. (–)‐Isoprenaline caused relaxation of taenia coli with a pEC50 of 6.70±0.17. Propranolol (0.1 μm), CGP 20712A (0.1 μm) and ICI 118551 (0.1 μm) inhibited the isoprenaline response with similar low affinities (pKB values 7.93, 7.71 and 7.54, respectively). Carbachol pre‐contracted circular smooth muscle preparations failed to relax consistently to isoprenaline and these responses were not characterized. β1‐ and β2‐Adrenoceptor mRNAs were present in circular/longitudinal muscle samples and taenia coli samples, and lower levels were detected in mucosa. β3‐mRNA was also present in both muscle preparations but was not detected in human colonic mucosa. In summary, β1‐adrenoceptors are the predominant subtype mediating isoprenaline‐induced relaxation of the thin longitudinal smooth muscle of human colon, while β3‐receptors do not appear to be involved in these responses. However, β3‐adrenoceptors may play a role in relaxation of the taenia coli as conventional antagonist affinities are low. β3‐Adrenoceptor mRNA was present in taenia coli and circular/longitudinal smooth muscle but absent from human colonic mucosa.

Kallikreins, kinins and growth factor biosynthesis

Abstract The process of cleaving one protein with another is a very basic one in biology. In many organisms mature proteins are released from their precursor forms by the action of proteases. Proteolysis is used extensively in other important processes such as the coagulation cascades and in the biosynthesis of biologically active peptides. Peptide biosynthesis is characterized by the limited, specific digestion of substrates at a variety of types of cleavage sites. Analysis of the genes coding for glandular kallikreins, a group of presumptive prohormone processing enzymes, has enabled elucidation of the complete structure of the enzymes and determination of sites of gene expression. The physical properties of these enzymes and their interaction with their substrates provide a unique opportunity to analyse structure-function relationships.

Human prostate-specific antigen (APS) is a member of the glandular kallikrein gene family at 19q13

The amino acid sequence of human prostate-specific antigen (APS) suggests that it is a member of the glandular kallikrein subfamily of serine proteases. In the mouse, the kallikrein-like family is localized in a single locus on chromosome 7, while other serine proteases are distributed over a variety of different chromosomes. To investigate the physical relationship between the human kallikrein genes, we have used in situ hybridization and Southern analysis of a human x mouse somatic cell hybrid panel to map the APS gene to 19q13, concordant with the renal kallikrein KLK1 gene. This finding indicates that APS is a member of a human kallikrein-like gene family with analogous organization to that of the mouse.

The sheep erythropoietin gene: molecular cloning and effect of hemorrhage on plasma erythropoietin and renal/liver messenger RNA in adult sheep

Erythropoietin (Epo) production was studied in adult sheep. Nine ewes, body weight (BW) 39 +/- 1.3 kg, were hemorrhaged a volume of blood equivalent to 1.6% BW, and sampled at 0, 2, 4, 6, 24 h. Oxygen content (O2 CT) decreased by 2.7 +/- 0.6 ml/dl at 2 h. Plasma immunoreactive (IR) Epo was only significantly increased at 24 h, from 18.5 +/- 3.5 to 40 +/- 10.7 mU/ml (mean +/- SEM). A further 5 ewes were bled extensively (2793 +/- 82 ml) over 54 h, and killed for Epo mRNA determination. The O2 CT decreased from 12.3 +/- 1.6 to 4.1 +/- 0.6 ml/dl, and plasma Epo increased from 15 +/- 4 to 1675 +/- 287 mU/ml. The sequence of ovine Epo cDNA was derived from the kidney RNA of a severely bled sheep using reverse transcription/polymerase chain reaction (RT/PCR), and from an ovine Epo genomic clone. The cDNA encodes a peptide of 194 amino acids, including a 27 amino acid signal peptide. The deduced amino acid sequence of sheep Epo shows 82%, 78% and 80% homology with mature Epos of human, mouse and monkey, respectively. The gene structure resembles closely those of human and mouse, with 5 exons and 4 introns. The expression of the ovine Epo gene in tissues from normal and hemorrhaged sheep was analysed by a competitive RT/PCR method. Epo mRNA was difficult to detect in liver from normal sheep, but was detectable at 0.01-0.04 amol/microgram total RNA in kidney from normal sheep. In the kidneys of severely bled sheep, the Epo mRNA levels (per micrograms total RNA) increased 400-1500-fold compared to that of normal kidneys, and were approximately 60-fold greater than those in the livers of the hemorrhaged sheep.

β3-Adrenoceptor regulation and relaxation responses in mouse ileum

This study examines the relationship between β3a‐ and β3b‐adrenoceptor (AR) mRNA levels, β3‐AR binding and changes in ileum responses in mice treated with the β3‐AR agonist (R,R)‐5‐[2[[2‐(3‐chlorophenyl)‐2‐hydroxyethyl]‐amino]‐propyl]1,3‐benzodioxole‐2,2‐dicarboxylate (CL316243), or the β3‐AR antagonist 3‐(2‐ethylphenoxy)‐1‐[(1S)‐1,2,3,4‐tetrahydronapth‐1‐ylamino]‐2S‐2‐propanol oxalate (SR59230A), or dexamethasone or forskolin. Levels of β3a‐ and β3b‐AR mRNA and the maximum number of binding sites (Bmax) in ileum were unaffected following CL316243 treatment, although responses to CL316243 were reduced by 50% following 4 and 24u2003h treatment, indicating another desensitization mechanism not involving changes in receptor expression or number. β3a‐AR mRNA levels were reduced in both brown (BAT) and white adipose tissue (WAT) but β3b‐AR mRNA levels were significantly reduced only in WAT. Levels of β3a‐ and β3b‐mRNA returned towards normal with continued treatment. SR59230A treatment markedly increased β3‐AR mRNA levels in ileum and BAT but not in WAT. The increase in β3‐AR mRNA levels in ileum was associated with increased Bmax levels in binding analysis and increased responses to CL316243, suggesting these as the cause of sensitization. Treatment with forskolin (4u2003h) or dexamethasone (4u2003h) significantly reduced β3a‐AR mRNA levels in BAT and WAT but did not alter levels in ileum. Responses to CL316243 in ileum were unaffected by either treatment. In summary, the β3‐AR is differently regulated in adipose tissue and ileum: Treatment with SR59230A increased β3‐AR number, mRNA and responsiveness in ileum, whereas treatment with CL316243 reduced responses without affecting β3‐AR number or mRNA levels.