Functional serotonin 5-HT4 receptors in porcine and human ventricular myocardium with increased 5-HT4 mRNA in heart failure

Serotonin (5-hydroxytryptamine, 5-HT) increases contractile force and elicits arrhythmias through 5-HT4 receptors in porcine and human atrium, but its ventricular effects are unknown. We now report functional 5-HT4 receptors in porcine and human ventricle. 5-HT4 mRNA levels were determined in porcine and human ventricles and contractility studied in ventricular trabeculae. Cyclic AMP-dependent protein kinase (PKA) activity was measured in porcine ventricle. Porcine and human ventricles expressed 5-HT4 receptor mRNA. Ventricular 5-HT4(b) mRNA was increased by four times in 20 failing human hearts compared with five donor hearts. 5-HT increased contractile force maximally by 16% (EC50=890 nM) and PKA activity by 20% of the effects of (-)-isoproterenol (200 muM) in ventricular trabeculae from new-born piglets in the presence of the phosphodiesterase-inhibitor 3-isobutyl-1-methylxanthine. In ventricular trabeculae from adult pigs (3-isobutyl-1-methylxanthine present) 5-HT increased force by 32% (EC50=60 nM) and PKA activity by 39% of (-)-iso-proterenol. In right and left ventricular trabeculae from failing hearts, exposed to modified Krebs solution, 5-HT produced variable increases in contractile force in right ventricular trabeculae from 4 out of 6 hearts and in left ventricular trabeculae from 3 out of 3 hearts- range 1-39% of (-)-isoproterenol, average 8%. In 11 left ventricular trabeculae from the failing hearts of four beta-blocker-treated patients, pre-exposed to a relaxant solution with 0.5 mM Ca2+ and 1.2 mM Mg2+ followed by a switch to 2.5 mM Ca2+ and 1 mM Mg2+, 5-HT (1-100 muM, 3-isobutyl-1-melhylxanthine present) consistently increased contractile force and hastened relaxation by 46% and 25% of (-)-isoproterenol respectively. 5-HT caused arrhythmias in three trabeculae from 3 out of I I patients. In the absence of phosphodiesterase inhibitor, 5-HT increased force in two trabeculae, but not in another six trabeculae from 4 patients. All 5-HT responses were blocked by 5-HT4 receptor antagonists. We conclude that phosphodiesterase inhibition uncovers functional ventricular 5-HT4 receptors, coupled to a PKA pathway, through which 5-HT enhances contractility, hastens relaxation and can potentially cause arrhythmias.

Polymorphic CAG repeat length in the androgen receptor gene and association with neurodegeneration in a heterozygous female carrier of Kennedy's disease

Kennedy's disease (spinobulbar muscular atrophy) is an X-linked form of motor neuron disease affecting adult males carrying a CAG trinucleotide repeat expansion within the androgen receptor gene. While expression of Kennedy's disease is thought to be confined to males carrying the causative mutation, subclinical manifestations have been reported in a few female carriers of the disease. The reasons that females are protected from the disease are not clear, especially given that all other diseases caused by CAG expansions display dominant expression. In the current study, we report the identification of a heterozygote female carrying the Kennedy's disease mutation who was clinically diagnosed with motor neuron disease. We describe analysis of CAG repeat number in this individual as well as 33 relatives within the pedigree, including two male carriers of the Kennedy's mutation. The female heterozygote carried one expanded allele of the androgen receptor gene with CAG repeats numbering in the Kennedy's disease range (44 CAGs), with the normal allele numbering in the upper-normal range (28 CAGs). The subject has two sons, one of whom carries the mutant allele of the gene and has been clinically diagnosed with Kennedy's disease, whilst the other son carries the second allele of the gene with CAGs numbering in the upper normal range and displays a normal phenotype. This coexistence of motor neuron disease and the presence of one expanded allele and one allele at the upper limit of the normal range may be a coincidence. However, we hypothesize that the expression of the Kennedy's disease mutation combined with a second allele with a large but normal CAG repeat sequence may have contributed to the motor neuron degeneration displayed in the heterozygote female and discuss the possible reasons for phenotypic expression in particular individuals.

Fundamental considerations of beta-adrenoceptor subtypes in human heart failure

beta-Adrenoceptor antagonists have revolutionized the management of heart failure in humans. However, fundamental questions remain concerning their use. Currently, there is considerable debate about the role of beta(2)-adrenoceptors in heart failure and whether incremental clinical benefit can be obtained by blockade of beta(2)-adrenoceptors in addition to beta(1)-adrenoceptors. Polymorphic forms of beta(1)- and beta(2)-adrenoceptors exist, which might contribute to the variable clinical outcomes that are observed with P-adrenoceptor antagonists. There is evidence for a low-affinity state of beta(1)-adrenoceptors and ventricular beta(3)-adrenoceptors, and these are discussed in the context of heart failure. Finally, there is seemingly paradoxical evidence that restoration and normalization of the beta-adrenoceptor system is beneficial in animal models of heart failure. We reconcile this view with the current clinical use and proven benefit of beta-adrenoceptor antagonists.

Developmental changes in expression of GABA(A) receptor-channels in rat intrinsic cardiac ganglion neurones

The effects of gamma-aminobutyric acid (GABA) on the electrophysiological properties of intracardiac neurones were investigated in the intracardiac ganglion plexus in situ and in dissociated neurones from neonatal, juvenile and adult rat hearts. Focal application of GABA evoked a depolarizing, excitatory response in both intact and dissociated intracardiac ganglion neurones. Under voltage clamp, both GABA and muscimol elicited inward currents at -60 mV in a concentration-dependent manner. The fast, desensitizing currents were mimicked by the GABA(A) receptor agonists muscimol and taurine, and inhibited by the GABA(A) receptor antagonists, bicuculline and picrotoxin. The GABA(A0) antagonist (1,2,5,6-tetrahydropyridin-4-yl)methyl phosphonic acid (TPMPA), had no effect on GABA-induced currents, suggesting that GABA(A) receptor-channels mediate the response. The GABA-evoked current amplitude recorded from dissociated neurones was age dependent whereby the peak current density measured at -100 mV was similar to 20 times higher for intracardiac neurones obtained from neonatal rats (P2-5) compared with adult rats (P45-49). The decrease in GABA sensitivity occurred during the first two postnatal weeks and coincides with maturation of the sympathetic innervation of the rat heart. Immunohistochemical staining using antibodies against GABA demonstrate the presence of GABA in the intracardiac ganglion plexus of the neonatal rat heart. Taken together, these results suggest that GABA and taurine may act as modulators of neurotransmission and cardiac function in the developing mammalian intrinsic cardiac nervous system.

Dissection of peripheral and central endogenous opioid modulation of systemic interleukin-1 beta responses using c-fos expression in the rat brain

In opiate addicts or patients receiving morphine treatment, it has been reported that the immune system is often compromised. The mechanisms responsible for the adverse effects of opioids on responses to infection are not clear but it is possible that central and/or peripheral opioid receptors may be important. We have utilised an experimental immune challenge model in rats, the systemic administration of the human pro-inflammatory cytokine interleukin-1 beta (IL-1 beta) to study the effects of selectively blocking peripheral opioid receptors only (using naloxone methiodide) or after blocking both central and peripheral opioid receptors (using naloxone). Pre-treatment with naloxone methiodide decreased (15%) IL-1 beta-induced Fos-immunoreactivity (Fos-IR) in medial parvocellular paraventricular nucleus (mPVN) corticotropin-releasing hormone (CRH) neurons but increased responses in the ventrolateral medulla (VLM) C1 (65%) and nucleus tractus solitarius (NTS) A2 (110%) catecholamine cell groups and area postrema (136%). However no effect of blocking peripheral opioid receptors was detected in the central nucleus of the amygdala (CeA) or dorsal bed nucleus of the stria terminalis (BNST). We next determined the effect of blocking both central and peripheral opioid receptors with naloxone and, when compared to the naloxone methiodide pre-treated group, a further 60% decrease in Fos-IR mPVN CRH neurons induced by IL-1 beta was detected, which was attributed to block of central opioid receptors. Similar comparisons also detected decreases in Fos-IR neurons induced by IL-1 beta in the VLM A1, VLM C1 and NTS A2 catecholamine cell groups, area postrema, and parabrachial nucleus. In contrast, pre-treatment with naloxone increased Fos-IR neurons in CeA (98%) and dorsal BNST (72%). These results provide novel evidence that endogenous opioids can influence central neural responses to systemic IL-1 beta and also suggest that the differential patterns of activation may arise because of actions at central and/or peripheral opioid receptors that might be important in regulating behavioural, hypothalamic-pituitary-adrenal axis and sympathetic nervous system responses during an immune challenge. (c) 2005 Elsevier Ltd. All rights reserved.

Opposite effects of androgen receptor CAG repeat length on increased risk of left-handedness in males and females

Prenatal exposure to testosterone has been hypothesised to effect lateralization by influencing cell death in the foetal brain. Testosterone binds to the X chromosome linked androgen receptor, which contains a polymorphic polyglutamine CAG repeat, the length of which is positively correlated with testosterone levels in males, and negatively correlated in females. To determine whether the length of the androgen receptor mediates the effects of testosterone on laterality, we examined the association between the number of CAG repeats in the androgen receptor gene and handedness for writing. Association was tested by adding regression terms for the length of the androgen receptor alleles to a multi-factorial-threshold model of liability to left-handedness. In females we found the risk of left-handedness was greater in those with a greater number of repeats (p=0.04), this finding was replicated in a second independent sample of female twins (p=0.014). The length of the androgen receptor explained 6% of the total variance and 24% of the genetic variance in females. In males the risk of left-handedness was greater in those with fewer repeats (p=0.02), with variation in receptor length explaining 10% of the total variance and 24% of the genetic variance. Thus, consistent with Witelson's theory of testosterone action, in all three samples the likelihood of left handedness increased in those individuals with variants of the androgen receptor associated with lower testosterone levels.

Modular alpha-helical mimetics with antiviral activity against respiratory syncitial virus

A 13-residue peptide sequence from a respiratory syncitial virus fusion protein was constrained in an alpha-helical conformation by fusing two back-to-back cyclic alpha-turn mimetics. The resulting peptide, Ac-(3 -> 7; 8 -> 12)-bicyclo-FP[KDEFD][KSIRD]V-NH2, was highly alpha-helical in water by CD and NMR spectroscopy, correctly positioning crucial binding residues (F488, I491, V493) on one face of the helix and side chain-side chain linkers on a noninteracting face of the helix. This compound displayed potent activity in both a recombinant fusion assay and an RSV antiviral assay (IC50 = 36 nM) and demonstrates for the first time that back-to-back modular alpha-helix mimetics can produce functional antagonists of important protein-protein interactions.

Protease-activated receptor-2 peptides activate neurokinin-1 receptors in the mouse isolated trachea

Protective roles for protease-activated receptor-2 (PAR2) in the airways including activation of epithelial chloride (Cl-) secretion are based on the use of presumably PAR(2)-selective peptide agonists. To determine whether PAR(2) peptide-activated Cl- secretion from mouse tracheal epithelium is dependent on PAR(2), changes in ion conductance across the epithelium [short-circuit current (I-SC)] to PAR(2) peptides were measured in Ussing chambers under voltage clamp. In addition, epithelium and endothelium-dependent relaxations to these peptides were measured in two established PAR(2) bioassays, isolated ring segments of mouse trachea and rat thoracic aorta, respectively. Apical application of the PAR(2) peptide SLIGRL caused increases in I-SC, which were inhibited by three structurally different neurokinin receptor-1 (NK1R) antagonists and inhibitors of Cl- channels but not by capsaicin, the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8-37), or the nonselective cyclooxygenase inhibitor indomethacin. Only high concentrations of trypsin caused an increase in I-SC but did not affect the responses to SLIGRL. Relaxations to SLIGRL in the trachea and aorta were unaffected by the NK1R antagonist nolpitantium (SR 140333) but were abolished by trypsin desensitization. The rank order of potency for a range of peptides in the trachea I-SC assay was 2-furoyl-LIGRL > SLCGRL > SLIGRL > SLIGRT > LSIGRL compared with 2-furoyl-LIGRL > SLIGRL > SLIGRT > SLCGRL (LSIGRL inactive) in the aorta relaxation assay. In the mouse trachea, PAR(2) peptides activate both epithelial NK1R coupled to Cl- secretion and PAR(2) coupled to prostaglandin E-2-mediated smooth muscle relaxation. Such a potential lack of specificity of these commonly used peptides needs to be considered when roles for PAR(2) in airway function in health and disease are determined.

Recent developments in C5/C5a inhibitors

Complement factor 5a (C5a) is formed upon complement system activation in response to infection, injury or disease. Whilst C5a is a potent mediator of immune and inflammatory processes, excessive production or inadequate regulation of C5a has been implicated in the pathogenesis of numerous immuno-inflammatory diseases, predominantly through experimental studies utilising animal models of disease. Both acute and chronic conditions may benefit from C5a inhibition, including rheumatoid arthritis, inflammatory bowel disease, asthma, psoriasis, haemorrhagic shock and neurodegenerative conditions. The potentially broad clinical application for treatments that inhibit the activity of C5a at C5a receptors and the large global market for anti-inflammatory therapeutics have made C5a and the C5a receptor attractive targets for academic and commercial drug development programmes. in the past 5 years, interest in C5a as a drug target has grown substantially, and this activity has resulted in a collection of patents and scientific papers reporting novel C5a and C5a receptor inhibitors and antagonists, and generated a secondary stream of patent applications broadly claiming the use of C5/C5a inhibitors as a method of treating various immune and inflammatory conditions. This paper will review the physiology and pathophysiology of C5a and discuss the development of C5a and C5a receptor inhibitors in light of the recent scientific and patent literature.