Angela M. Finch

Pharmacological characterization of antagonists of the C5a receptor

Potent and highly selective small molecule antagonists have recently been developed by us for C5a receptors (C5aR) on human polymorphonuclear leukocytes (PMN). In this study we compared a new cyclic antagonist, F‐[OPdChaWR], with an acyclic derivative, MeFKPdChaWr, for their capacities to bind to C5aR on human PMN and human umbilical artery membranes. We also compared their inhibition of myeloperoxidase (MPO) secretion from human PMNs and their inhibition of human umbilical artery contraction induced by human recombinant C5a. In both PMNs and umbilical artery, the cyclic and acyclic C5a antagonists displayed insurmountable antagonism against C5a. There were differences in selectivities for the C5aR with F‐[OPdChaWR] (pKb 8.64±0.21) being 30 times more potent than MeFKPdChaWr (pKb 7.16±0.11, P<0.05) in PMNs, but of similar potency (pKb 8.19±0.38 vs pKb 8.28±0.29, respectively) in umbilical artery. This trend was also reflected in their relative binding affinities, both antagonists having similar affinities (−logIC50 values) for C5aR in umbilical artery membranes (F‐[OPdChaWR], 7.00±0.46; MeFKPdChaWr, 7.23±0.17), whereas in PMN membranes the C5aR affinity of the cycle F‐[OPdChaWR] (7.05±0.06) was four times higher than that of acyclic MeFKPdChaWr (6.43±0.24, P<0.05). In summary, the results reveal that these antagonists are insurmountable in nature against C5a for C5aR on at least two human cell types, and the differences in relative receptor binding affinities and antagonistic potencies against C5a are consistent with differences in receptors within these cell types. The nature of these differences is yet to be elucidated.

The effect of feeding salmon oil to sows throughout pregnancy on pre-weaning mortality of piglets

Salmon oil (16·5 kg /t), a source of long-chain polyunsaturated n-3 fatty acids, was included in diets offered to multiparous sows during pregnancy and lactation to measure responses in pre-weaning mortality and performance of piglets in two studies. The first study, carried out under commercial conditions, included 196 sows which were offered salmon oil and control diets from immediately post service until weaning. The same diets were also offered to 10 sows per treatment from day 58 of pregnancy in a controlled nutritional study which measured the effects of salmon oil on piglet tissue fatty acid composition. Offering salmon oil to the sow significantly increased gestation length and decreased individual piglet birth weight but had no effect on litter size at birth. Overall, salmon oil reduced pre-weaning mortality from 11·7% to 10·2% mainly by reducing the incidence of deaths from crushing by the sow. More detailed analysis of mortality using a general linear mixed model and 2294 piglet records, demonstrated that the incidence of pre-weaning mortality was significantly decreased with increasing individual piglet birth weight and by inclusion of salmon oil in the diet; the incidence of mortality increased with average piglet birth weight in a litter. Salmon oil inclusion had no effect on weight of litter weaned, sow lactation food intake or subsequent reproductive performance. In both studies, dietary salmon oil increased the proportions of long-chain n-3 polyunsaturated fatty acids in colostrum to a similar extent. In the nutritional study, inclusion of salmon oil reduced the proportions of 20: 4 n-6 in piglet liver and brain at birth and increased the proportions of long-chain n-3 polyunsaturated fatty acids. Therefore, despite reducing piglet birth weight, offering sows salmon oil reduced pre-weaning mortality of piglets. The nutritional study showed that the amount and type of marine oil used may not have been optimal.

Effects of a new C5a receptor antagonist on C5a- and endotoxin-induced neutropenia in the rat.

A new C5a receptor antagonist, the cyclic peptide Phe‐[Orn‐Pro‐D‐cyclohexylalanine‐Trp‐Arg], (F‐[OPdChaWR]), was tested for its ability to antagonize the neutropenic effects of both C5a and endotoxin in rats. Human recombinant C5a (2 μg kg−1 i.v.) caused rapid neutropenia, characterized by an 83% decrease in circulating polymorphonuclear leukocytes (PMNs) at 5 min. Administration of F‐[OPdChaWR] (0.3–3 mg kg−1 i.v.), did not affect the levels of circulating PMNs but, when given 10 min prior to C5a, it inhibited the C5a‐induced neutropenia by up to 70%. Administration of E. Coli lipopolysaccharide (LPS, 1 mg kg−1 i.v.) also caused neutropenia with an 88% decrease in circulating PMNs after 30 min. When rats were pretreated with F‐[OPdChaWR] (0.3–10 mg kg−1 i.v.) 10 min prior to LPS, there was a dose‐dependent antagonism of the neutropenia caused by LPS, with up to 69% reversal of neutropenia observed 30 min after LPS administration. These findings suggest that C5a receptor antagonists may have therapeutic potential in the many diseases known to involve either endotoxin or C5a.

Expression of adrenoceptor subtypes in preterm piglet heart is different to term heart.

Preterm delivery increases the risk of inadequate systemic blood flow and hypotension, and many preterm infants fail to respond to conventional inotrope treatments. If the profile of cardiac adrenoceptor subtypes in the preterm neonate is different to that at term this may contribute to these clinical problems. This study measured mRNA expression of β1, β2, α1A, α2A and α2B-adrenoceptor subtypes by real time PCR in term (113d), preterm (91d) and preterm piglets (91d) exposed to maternal glucocorticoid treatment. Abundance of β-adrenoceptor binding sites in the left ventricle was measured using saturation binding assays. Relative abundance of β1-adrenoceptor mRNA in untreated preterm hearts was ∼50% of term abundance in both left and right ventricles (P<0.001). Trends in receptor binding site density measurements supported this observation (P = 0.07). Glucocorticoid exposure increased β1-adrenoceptor mRNA levels in the right ventricle of preterm hearts (P = 0.008) but did not alter expression in the left ventricle (P>0.1). Relative abundance of α1A-adrenoceptor mRNA was the same in preterm and term piglet hearts (P = >0.1) but was reduced by maternal glucocorticoid treatment (P<0.01); α2A-adrenoceptor mRNA abundance was higher in untreated and glucocorticoid exposed preterm piglet hearts than in term piglets (P<0.001). There was no difference between male and female piglets in mRNA abundance of any of the genes studied. In conclusion, there is reduced mRNA abundance of β1-adrenoceptors in the preterm pig heart. If this lower expression of β-adrenoceptors occurs in human preterm infants, it could explain their poor cardiovascular function and their frequent failure to respond to commonly used inotropes.

Reversibility of tachyphylaxis to C5a in guinea pig tissues, perfused human placental lobule, and umbilical artery

The spasmogenic effect of C5a is mediated by histamine and/or eicosanoids. Tachyphylaxis to this effect of C5a occurs rapidly, but the spasmogenic effects of C5a on a guinea pig lung parenchymal strips, field-stimulated ventricular papillary muscle, and human umbilical artery were completely restored by a 1-h period of drug-free rest, whereas that of guinea pig ileum was not. Perfusion of the isolated human placental lobule with C5a caused a transient pressor response that was largely abolished by indomethacin (5 μM), indicating mediation by cyclooxygenase metabolites. This pressor response to C5a was also completely restored following a 1-h rest period. The results show that tissue rest reverses tachyphylaxis to the spasmogenic effects of C5a in tissues where the response is mediated by cyclooxygenase metabolites. Where the response is mediated by histamine released by mast cells, restoration does not occur, presumably because of the catastrophic nature of mast cell degranulation. Histamine released in guinea pig papillary muscle by C5a may be from non-mast-cell sources.

The α1D-Adrenergic Receptor: Cinderella or Ugly Stepsister

This Perspective focuses on the α1D-adrenergic receptor (AR), the often neglected sibling of the α1-AR family. This neglect is due in part to its poor cell-surface expression. However, it has recently been shown that dimerization of the α1D-AR with either the α1B-AR or the β2-AR increases α1D-AR cell-surface expression, and in this issue of Molecular Pharmacology, Hague et al. (p. 45) demonstrate that dimerization of the α1D-AR with the α1B-AR not only leads to increased cell-surface expression but also results in the formation of a novel functional entity.

α1-Adrenoceptor and serotonin 5-HT1A receptor affinity of homobivalent 4-aminoquinoline compounds: An investigation of the effect of linker length

α₁-adrenoceptor (α₁-AR) subtype-selective ligands lacking off-target affinity for the 5-HT(1A) receptor (5-HT(1A)-R) will provide therapeutic benefits in the treatment of urogenital conditions such as benign prostatic hyperplasia. In this study we determined the affinity of 4-aminoquinoline and eleven homobivalent 4-aminoquinoline ligands (diquinolines) with alkane linkers of 2-12 atoms (C2-C12) for α(1A), α(1B) and α(1D)-ARs and the 5-HT(1A)-R. These ligands are α(1A)-AR antagonists with nanomolar affinity for α(1A) and α(1B)-ARs. They display linker-length dependent selectivity for α(1A/B)-ARs over α(1D)-AR and the 5-HT(1A)-R. The C2 diquinoline has the highest affinity for α1A-AR (pKi 7.60±0.26) and greater than 30-fold and 600-fold selectivity for α(1A)-AR over α(1D)-AR and 5-HT(1A)-R respectively. A decrease in affinity for α₁-ARs is observed as the linker length increases, reaching a nadir at 5 (α(1A/1B)-ARs) or 6 (α(1D)-AR) atoms; after which affinity increases as the linker is lengthened, peaking at 9 (α(1A/1B/1D)-ARs) or 8 (5-HT(1A)-R) atoms. Docking studies suggest that 4-aminoquinoline and C2 bind within the orthosteric binding site, while for C9 one end is situated within the orthosteric binding pocket, while the other 4-aminoquinoline moiety interacts with the extracellular surface. The limited α(1D)-AR and 5-HT(1A)-R affinity of these compounds makes them promising leads for future drug development of α(1A)-AR selective ligands without α(1D)-AR and the 5-HT(1A)-R off-target activity.

A Novel Structural Framework for α1A/D-Adrenoceptor Selective Antagonists Identified Using Subtype Selective Pharmacophores

In this study four and five-feature pharmacophores for selective antagonists at each of the three α1-adrenoceptor (AR) subtypes were used to identify novel α1-AR subtype selective compounds in the National Cancer Institute and Tripos LeadQuest databases. 12 compounds were selected, based on diversity of structure, predicted high affinity and selectivity at the α1D- subtype compared to α1A- and α1B-ARs. 9 out of 12 of the tested compounds displayed affinity at the α1A and α1D -AR subtypes and 6 displayed affinity at all three α1-AR subtypes, no α1B-AR selective compounds were identified. 8 of the 9 compounds with α1-AR affinity were antagonists and one compound displayed partial agonist characteristics. This virtual screening has successfully identified an α1A/D-AR selective antagonist, with low µM affinity with a novel structural scaffold of a an isoquinoline fused three-ring system and good lead-like qualities ideal for further drug development.

Ligand Binding, Activation, and Agonist Trafficking

Adrenergic receptors are critical mediators of sympaiMolecular Cardiology Program, Vihysiological responses. Activated by the neurotransmitter and neurohormone, norepinephrine and epinephrine, released from sympathetic nerve endings and the adrenal medulla, respectively, they play a central role in this evolutionarily ancient defense system that regulates many physiological functions, including those involved in circulatory, metabolic, respiratory, and central nervous system homeostasis. In addition, alterations in the regulation and molecular structure of adrenergic receptors have been implicated in a variety of diseases, and drugs targeting these receptors are important and widely used therapeutics. The molecular cloning of the first adrenergic receptor in 1986 revealed structural homology with the functionally related rhodopsin visual transduction system—a finding that led to the realization that these receptors formed a new superfamily of proteins, now known as G protein-coupled receptors. Since that time, a plethora of structure-function studies have provided major insights into the molecular determinants of adrenergic receptor ligand-binding, activation, and signaling, many of which are relevant not only to the adrenergic receptor family but also, more generally, to the broader superfamily of G protein-coupled receptors. These advances in our understanding of adrenergic receptor activation, regulation, and functioning are reviewed in this chapter.

The effect of C5a and U46619 on the isolated, perfused human placental lobule: Development of a method for the online estimation of tissue fluid accumulation

A method for the automatic and simultaneous determination of perfusion pressure and fluid accumulation in the isolated, perfused human placental lobule is described. We demonstrated that the inflammatory mediator, C5a, a C5a agonist analogue peptide, and the thromboxane mimetic U46619 caused increased fetal perfusion pressure and increased tissue weight when administered via the fetal arterial circulation. Occlusion of the fetal venous effluent tubing caused significantly greater increases in tissue weight than the pharmacological agents. Detectable increases in tissue weight occurred within 47 +/- 3 sec (n = 21) following pressure increases caused by the pharmacological agents. In each case, the increase in tissue weight was accompanied by an increased permeability of the materno-fetal barrier, shown by the transfer of Evans blue albumin from the fetal circulation to the maternal compartment.