The effect of estrogen on brown adipose tissue activity in male rats.

OBJECTIVE: Centrally administered estrogen can increase sympathetic nerve activity to brown adipose tissue, resulting in thermogenesis. The central thermogenic effects of estrogen have not been investigated in males. Therefore, this study sought to investigate the effects of peripherally and centrally administered estrogen on thermogenesis, heart rate and mean arterial pressure in male rats. Thermogenesis was assessed by monitoring brown adipose tissue temperature. RESULTS: Peripherally administered estrogen elicited no significant effect on brown adipose tissue temperature, heart rate or mean arterial pressure. Centrally administered estrogen elicited a coincident increase in both brown adipose tissue and core temperature. Centrally administered estrogen also resulted in a decrease in mean arterial pressure but had no effect on heart rate. With the present data it is not possible to elucidate whether changes in temperature were the result of thermogenic or thermoregulatory mechanisms.

Stimulatory, but not anxiogenic, doses of caffeine act centrally to activate interscapular brown adipose tissue thermogenesis in anesthetized male rats.

The role of central orexin in the sympathetic control of interscapular brown adipose tissue (iBAT) thermogenesis has been established in rodents. Stimulatory doses of caffeine activate orexin positive neurons in the lateral hypothalamus, a region of the brain implicated in stimulating BAT thermogenesis. This study tests the hypothesis that central administration of caffeine is sufficient to activate BAT. Low doses of caffeine administered either systemically (intravenous [IV]; 10 mg/kg) and centrally (intracerebroventricular [ICV]; 5-10 µg) increases BAT thermogenesis, in anaesthetised (1.5 g/kg urethane, IV) free breathing male rats. Cardiovascular function was monitored via an indwelling intra-arterial cannula and exhibited no response to the caffeine. Core temperature did not significantly differ after administration of caffeine via either route of administration. Caffeine administered both IV and ICV increased neuronal activity, as measured by c-Fos-immunoreactivity within subregions of the hypothalamic area, previously implicated in regulating BAT thermogenesis. Significantly, there appears to be no neural anxiety response to the low dose of caffeine as indicated by no change in activity in the basolateral amygdala. Having measured the physiological correlate of thermogenesis (heat production) we have not measured indirect molecular correlates of BAT activation. Nevertheless, our results demonstrate that caffeine, at stimulatory doses, acting via the central nervous system can increase thermogenesis, without adverse cardio-dynamic impact.

IRAK3 modulates downstream innate immune signalling through its guanylate cyclase activity.

Interleukin-1 receptor associated kinase 3 (IRAK3) is a cytoplasmic homeostatic mediator of inflammatory responses and is potentially useful as a prognostic marker in inflammation. IRAK3 inhibits signalling cascades downstream of myddosome complexes associated with toll like receptors. IRAK3 contains a death domain that interacts with other IRAK family members, a pseudokinase domain and a C-terminus domain involved with tumour necrosis factor receptor associated factor 6 (TRAF6). Previous bioinformatic studies revealed that IRAK3 contained a guanylate cyclase centre in its pseudokinase domain but its role in IRAK3 action is unresolved. We demonstrate that wildtype IRAK3 is capable of producing cGMP. Furthermore, we show that a specific point mutation in the guanylate cyclase centre reduced cGMP production. Cells containing toll like receptor 4 and a nuclear factor kappa-light-chain-enhancer of activated B cells (NFĸB) reporter system were transfected with IRAK3 or mutant IRAK3 proteins. Cell-permeable cGMP treatment of untransfected control cells suppresses downstream signalling through modulation of the NFĸB in the presence of lipopolysaccharides. Cells transfected with wildtype IRAK3 also suppress lipopolysaccharide induced NFĸB activity in the absence of exogenous cGMP. Lipopolysaccharide induced NFĸB activity was not suppressed in cells transfected with the IRAK3 mutant with reduced cGMP-generating capacity. Whereas in the presence of exogenously applied cell-permeable cGMP the IRAK3 mutant was able to retain its function by suppressing lipopolysaccharide induced NFĸB activity. Furthermore, increasing the amount of membrane permeable cGMP did not affect IRAK3's ability to reduce NFĸB activity. These results suggest that cGMP generated by IRAK3 may be involved in regulatory function of the protein where the presence of cGMP may selectively affect downstream signalling pathway(s) by modulating binding and/or activity of nearby proteins that interact in the inflammatory signalling cascade.

Heterogeneity amongst 5-HT3 receptor subunits: is this significant?

The serotonin 3 (5-HT3) receptor is a ligand gated ion channel unlike the other 5-HT receptors which are G protein coupled receptors. The functional 5-HT3 receptor forms a pentamer of five symmetrically arranged subunits surrounding a central pore. The 5-HT(3A) subunit was first identified at a molecular level and can form functional homomers or heteromers with the 5-HT(3B) subunit. Recently, three new 5-HT3 subunits have been discovered and these can also form functional heteromers with the 5-HT(3A) subunit. In addition, splice variants of the 5-HT3 subunits have also been reported. These findings have markedly increased the complexity of the 5-HT3 receptor and may form part of the explanation of unresolved differences between studies investigating 5-HT3 receptor function in cell lines compared with native tissues. In this review we discuss the properties of the different subunits and their distribution to determine if they contribute to functional changes in the 5-HT3 receptor. Several recent pharmacogenomic studies have revealed single nucleotide polymorphisms (SNPs) and other variations in the different 5-HT3 receptor subunits that are associated with various clinical conditions. We discuss the implications of these findings with respect to drug design and tailored pharmacogenomic therapies.

Synthesis and preliminary screening of novel tryptamines as 5-HT4 receptor ligands.

For the development of novel 5-HT(4) receptor ligands we have designed and synthesized two series of 5-methoxytryptamine derivatives varying the substitution on the primary amine. Their biological activities were evaluated in a receptor binding assay where a subset of compounds showed comparable potency to the agonists serotonin and 5-methoxytryptamine. Structure-activity analyses have highlighted promising avenues for further synthetic work and binding modes were proposed by docking these compounds into a homology model of the 5-HT(4) receptor.

The use of tripeptides for lead discovery of 5-HT4 receptor ligands.

A series of 30 tripeptides were synthesized and tested as novel 5-HT4 receptor ligands. Receptor binding assays showed that a subset of compounds had reasonable potency relative to the agonists serotonin and 5-methoxytryptamine. Structure-activity analyses and molecular docking have highlighted avenues for further synthetic work.

Distribution of serotonin receptors and interacting proteins in the human sigmoid colon.

This study aimed to examine the distribution of 5-HT receptors in the human colon. 5-HT induces desensitization of the circular muscle and as this is facilitated by G-protein coupled receptor kinases (GRKs) and other proteins, we also examined their distribution. Human sigmoid colon samples were dissected into three separate layers (mucosa, taeniae coli and intertaenial strips) and RNA was amplified by RT-PCR. The 5-HT(2B) receptor and all 5-HT(7) receptor splice variants were expressed in all tissues. 5-HT(4) a,b,c and n splice variants were also expressed in all tissues and 5-HT(4d), 5-HT(4g) and 5-HT(4i) were only detected in some samples. The 5-HT(2A) receptor was seen predominantly in the intertaenial strips of the colon. Only one transcript of the serotonin transporter (SERT) was detected in the muscle layers. Variation was seen in GRK expression with GRK2 and 3 predominantly expressed in the mucosa, while GRK5 and 6 were found more commonly in the taeniae coli. PDZ (named after postsynaptic density protein, Drosophila disc large tumour suppressor and tight junction protein ZO-1) domain containing proteins, which may be involved in 5-HT receptor trafficking, were also detected throughout the sigmoid colon. The 5-HT(3A) subunit was expressed in all tissues, whereas the 5-HT(3E) subunit was mainly found in the mucosa layer while the 5-HT(3B) subunit was more common in the muscle layers. Receptor interacting chaperone (RIC-3), which is involved in transporting 5-HT(3) receptor subunits, is expressed less in mucosa compared to muscle layers. In conclusion, these results show that there is variation in distribution of 5-HT receptors and interacting proteins within the sigmoid colon that may contribute to colonic function.

Pharmacological evidence for activation of phospholipid and small GTP binding protein signalling cascades by Nod factors.

The effects of lipo-chitin oligosaccharide Nod factors (NodNGR[S] from Rhizobium sp. NGR234) on root hair deformation in Vigna unguiculata (L.) Walp. were studied using pharmacological agents to mimic and/or inhibit their action. It was hypothesised that the rearrangement of the cytoskeleton seen during Nod factor induced root hair deformation is modulated by protein kinase C, monomeric G proteins of the Rho superfamily and the location and amount of phosphatidylinositol 3-phosphates (PI3Ps). This hypothesis is supported by the following observations. The protein kinase C activators, 12-deoxyphorbol 13-acetate (DPA) and diacylglycerol kinase inhibitor 1, stimulated root hair deformation to a level similar to that seen with Nod factors or mastoparan, whereas the inhibitor Gö 6976 inhibited root hair deformations induced by NodNGR[S], mastoparan, DPA and diacylglycerol kinase inhibitor 1. The Ras antagonists mevastatin and sulindac sulphide, and the Rho antagonist exoenzyme C3 toxin from Clostridium botulinum all inhibited Nod factor stimulated root hair deformation. Pasteurella multocida toxin activates Rho and stimulated root hair deformation, this stimulation was inhibited by both neomycin and exoenzyme C3 toxin. The PI3 kinase inhibitors, wortmannin and LY-294002 attenuated Nod factor induced root hair deformation. These studies were complemented with actin immunoprecipitations of root hair enriched microsomal membrane preparations from V. unguiculata which pulled down small GTP binding proteins. Root hair deformation is an important early stage in the formation of nitrogen fixing nodules and this study highlights that these processes may depend on signalling cascades involving phospholipids and small GTP binding proteins.

Influences of gender and region on responses to 5-HT in the rat small intestine.

The aim of this study was to obtain more information regarding the 'atypical' 5-HT7 receptor of the rat jejunum. 5-HT7-induced contractions of the jejunum were elicited by 5-HT in the presence of ondansetron. Maximal responses were slightly larger in tissues from male compared to female rats of comparable age, with Emax values of 97.2 +/- 3.3 and 84.25 +/- 4.3% respectively compared to acetylcholine as an internal standard. However, the pEC50 values for 5-HT were not significantly different. The mRNA expression levels of the 5-HT7 receptor were similar in whole jejunum and longitudinal muscle tissues taken from males and females. It was also shown that the maximal response of the jejunum from male rats was larger than the responses from mid intestine and ileum. However, in female tissues, the Emax of the mid intestine was significantly larger than the ileum, but not different from the jejunum. The results provide further insights into the 'atypical' 5-HT7 receptor of the rat jejunum and are also useful in optimising the preparation for further studies.

Natriuretic peptides--a class of heterologous molecules in plants.

Immunological and physiological evidence suggests the presence of biologically active natriuretic peptide hormones (NPs) in plants. Evidence includes specific binding of rat atrial NP, [rANP (99-126)] to plant membranes and the promotion of cyclic guanosine-3',5'-monophosphate (cGMP) mediated stomatal responses. Furthermore, anti-ANP affinity purifies biologically active plant immunoreactants (irPNPs) and a biologically active Arabidopsis thaliana irPNP (AtPNP-A) has been identified. AtPNP-A belongs to a novel class of molecules that share some similarity with the cell wall loosening expansins but do not contain the carbohydrate-binding wall anchor, thus suggesting that irPNPs and ANP are heterologues. We hypothesise that irPNP-like molecules have evolved from primitive glucanase-like molecules that have been recruited to become systemically mobile modulators of homeostasis acting via the plasma membrane. Such a function is compatible with localisation in the conductive tissue and the physiological and cellular modes of action of irPNPs reported to-date.

In situ localization associates biologically active plant natriuretic peptide immuno-analogues with conductive tissue and stomata.

Plant natriuretic peptide immuno-analogues (irPNP) have previously been shown to affect a number of biological processes including stomatal guard cell movements, ion fluxes and osmoticum-dependent water transport. Tissue printing and immunofluorescent labelling techniques have been used here to study the tissue and cellular localization of irPNP in ivy (Hedera helix L.) and potato (Solanum tuberosum L.). Polyclonal antibodies active against human atrial natriuretic peptide (anti-hANP) and antibodies against irPNP from potato (anti-StPNP) were used for immunolabelling. Tissue prints revealed that immunoreactants are concentrated in vascular tissues of leaves, petioles and stems. Phloem-associated cells, xylem cells and parenchymatic xylem cells showed the strongest immunoreaction. Immunofluorescent microscopy with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG supported this finding and, furthermore, revealed strong labelling to stomatal guard cells and the adjacent apoplastic space as well. Biologically active immunoreactants were also detected in xylem exudates of a soft South African perennial forest sage (Plectranthus ciliatus E. Mey ex Benth.) thus strengthening the evidence for a systemic role of the protein. In summary, in situ cellular localization is consistent with physiological responses elicited by irPNPs reported previously and is indicative of a systemic role in plant homeostasis.

Expansin-like molecules: novel functions derived from common domains.

An Arabidopsis thaliana transcript ( AtPNP-A) encoding an immunoreactant plant natriuretic peptide (irPNP) analog was identified and isolated. The encoded protein shows similarity to CjBAp12, a functionally undefined protein from citrus that is induced in response to blight infection. CjBAp12 shows significant sequence identity to domains found in the cell wall loosening expansins but has tested negative for cell wall loosening activity. We have thus undertaken to establish the evolutionary and functional relationships of irPNP-like molecules within the superfamily of expansins, pollen allergens, and distantly related molecules such as endoglucanases. We show that irPNP-like molecules are related to expansins and fall in two groups; one includes CjBAp12 and the other AtPNP-A. Members of both groups share distinct sequence motifs (K[VI]VD and [LM]SxxAFxxI) but do not contain the tryptophan and tyrosine rich C-terminal putative polysaccharide-binding domain typical of expansins or bacterial cellulases and hemicellulases. We argue that both irPNP-like molecules and expansin have evolved from primitive/ancestral glucanase-like molecules that hydrolysed the cell wall. Importantly, we have previously demonstrated that irPNPs act on protoplasts, that is plant cells without cell walls as well as microsomes, indicating that these novel proteins specifically interact with the plasma membrane. It follows that the cell wall cannot be an obligatory substrate for irPNPs. Thus, both irPNP function and domain structure point to these molecules having a systemic role in H2O and solute homeostasis.

Expression patterns of 5-HT7 receptor isoforms in the rat digestive tract.

In this study, we determined the expression patterns of the 5-HT7 receptor isoforms at the mRNA level in the rat jejunum and other regions of the rat digestive tract using reverse transcriptase-PCR. In control studies using the rat thalamus, we detected all three known isoforms of the rat 5-HT7 receptor, 5-HT7(a), 5-HT7(b) and 5-HT7(c). In addition, we also detected in the thalamus a new PCR product that we provisionally name 5-HT7(e). All four isoforms of the 5-HT7 receptor were present throughout the rat digestive tract. However, the spatial distribution and levels of expression of the different isoforms varied between regions of the rat digestive tract. All three known isoforms and the provisional isoform were found in the jejunum, ileum, stomach fundus, oesophagus and colon. The level of expression of the isoforms varied with all four being most consistently found in the ileum. The 5-HT7(a) receptor isoform was predominantly expressed in the rat digestive tissue and 5-HT7(b,, 5-HT7(c) and 5-HT7(e) were only sometimes expressed but always accompanying the receptor isoform 5-HT7(a).

Characterisation of muscarinic receptor subtypes in avian smooth muscle.

The identity of the muscarinic receptor subtype in the chick ileum was investigated in functional and binding studies. Preliminary studies [Choo, L.-K., Mitchelson, F., Napier, P. 1988. J. Auton. Pharmacol. 8, 259-266] suggested apparent avian and mammalian family differences in the muscarinic receptor profile of ileal smooth muscle. In the current study, further characterisation was undertaken using a greater range of antagonists exhibiting high affinity for specific muscarinic receptor subtypes. Dissociation constants from functional and binding experiments were compared with published values for antagonists at each of the five muscarinic receptor subtypes. Linear regression and correlation analyses revealed the receptor initiating the contractile response was most likely of the muscarinic M(3) receptor subtype as the slope of the linear regression was 1.01+/-0.14 and the corresponding correlation coefficient (r) was 0.95. The mammalian muscarinic M(5) receptor subtype also showed a high correlation with the data giving a slope of 0.89+/-0.27 and r value of 0.76. These findings were in direct contrast to those from binding experiments in which the single binding site detected was of the muscarinic M(2) receptor subtype. The slope of the linear regression was 1.14+/-0.24 with an r value of 0.87. Thus, these results suggest that there exists a high proportion of the muscarinic M(2) receptor subtype within the tissue that does not contribute to the functional response.

Changes in cytosolic pH and calcium of guard cells precede stomatal movements.

Stomatal opening is induced by indoleacetic acid (IAA), cytokinins, and fusicoccin (FC), whereas stomatal closure is induced by abscisic acid (ABA). To test the effect of these growth regulators on guard cell cytosolic Ca2+ ([Ca2+]cyt) and pH (pHcyt), epidermal strips were taken from the lower side of leaves of the orchid Paphiopedilum tonsum and were loaded with acetomethoxy-esterified forms of the Ca2+ indicator fluo-3 or the pH indicator 2',7'-bis(2-carboxyethyl)-5(6)carboxyfluorescein. Basal [Ca2+]cyt ranged from 0.05 to 0.3 M and was 0.22 +/- 0.015 (n = 21). Increases in both [Ca2+]cyt and pHcyt were observed in guard cells after application of 10-100 M ABA to open stomata, and these preceded stomatal closure. The increase in [Ca2+]cyt ranged from 1.5- to 3-fold and was seen in 7 of 10 experiments. Guard cell alkalinization began within 2 min of ABA treatment and continued for the next 8 min. The increase ranged from 0.04 to 0.3 pH unit and was seen in 13 of 14 experiments. Guard cell [Ca2+]cyt increased, whereas pHcyt decreased after treatment of closed stomata with IAA, kinetin, or FC. In response to 50-100 M IAA, [Ca2+]cyt increased 1.5- to 2-fold in all cases, and pHcyt decreased 0.2-0.4 unit within 5 min in 7 experiments. Within 12 min, 10-100 M kinetin caused [Ca2+]cyt to increase in 28 of 34 experiments (1.3- to 2.5-fold) and pHcyt fell 0.1-0.4 unit in 15 of 17 treatments. The response to 10-50 M FC was similar in both time and magnitude. These results show that stomatal opening is accompanied by an increase in [Ca2+]cyt and cytosolic acidification in the guard cells, whereas stomatal closure is preceded by an increase in [Ca2+]cyt and cytosolic alkalinization in the guard cells. The order of these events is still uncertain, but changes in pHcyt are correlated with stomatal movement, and these changes may be an important factor in the regulation of guard cell movement.

Effects of auxin and abscisic acid on cytosolic calcium and pH in plant cells.

Dark-grown corn coleoptiles and parsley hypocotyls and their roots were loaded with acetoxymethyl esterified forms of the Ca2+ indicator fluo-3, and the pH indicator 2',7'-bis (2-carboxyethyl)-5(and-6)-carboxyfluorescein. These tissues were treated with the plant growth regulator 2,4-dichlorophenoxyacetic acid (2,4-D), an auxin analogue, or abscisic acid (ABA), and the cytosolic pH (pHcyt) and cytosolic Ca2+ ([Ca2+]cyt) changes were monitored by confocal scanning optical microscopy. Over a period of 4 min pHcyt decreased 0.1-0.2 pH unit and [Ca2+]cyt increased from 280 to 380 nM in response to 2,4-D. ABS, on the other hand, induced cytosolic alkalinization of 0.05-0.1 pH unit with a concomitant increase in [Ca2+]cyt from 240 to 320 nM over a 4-min period. Responses similar to these were observed in all the tissues tested. We suggest that pHcyt profoundly influences signaling by[Ca2+]cyt, possibly by regulating Ca2+-protein binding, and that the divergent effects of auxin and ABA on pHcyt underlie their mutual antagonism.

Phosphatidylcholine breakdown in rat liver plasma membranes. Roles of guanine nucleotides and P2-purinergic agonists.

Release of P-choline and choline from purified rat plasma membrane preparations was increased by GTP and its less hydrolyzable analogues, whereas other nucleotide triphosphates had little or no effect. Stimulation by guanosine 5'-(3-O-thiol)triphosphate (GTP gamma S) was dependent upon magnesium, inhibited by guanosine 5'-(2-O-thiol)diphosphate, and independent of calcium. ATP and ADP (1-100 microM) markedly enhanced the GTP gamma S stimulation of P-choline plus choline release but had no effect alone. ADP was as effective as ATP and nonhydrolyzable ATP analogues produced a similar or greater stimulation, whereas AMP and adenosine were much less effective. Vasopressin (0.1 microM) also produced a small stimulation. Under conditions in which protein kinase C was activated, PMA also stimulated the response to GTP gamma S but was ineffective in its absence. P-choline was the initial product which was hydrolyzed to choline. Guanine nucleotide and purinergic effects were also apparent on phosphatidylcholine degradation. EGTA, at 0.5 mM, completely removed purinergic stimulation but did not affect P-choline plus choline released in response to GTP gamma S alone. Prior treatment of plasma membranes with cholera toxin or prior injection of animals with islet-activating protein did not affect the stimulation of P-choline plus choline release either by GTP gamma S alone or by GTP gamma S plus ATP. These results indicate that a phosphatidylcholine phospholipase C is coupled to purinergic receptors in rat liver plasma membranes by a GTP-binding protein. Hydrolysis of phosphatidylcholine could contribute to hepatic diacylglycerol levels and thus influence protein kinase C activity.

Calcium efflux associated with encystment of Phytophthora palmivora zoospores.

Zoospores of the fungus Phytophthora palmivora, pre-labeled with 45Ca, excreted up to 30% of their total 45Ca when stimulated to encyst. Excretion was essentially completed within 90 sec of the application of the stimulus. Encystment of the population was completed within 5 min. Four different stimuli were used: pectin addition (420 micrograms ml-1), Sr2+ addition (5 mM), cyclic AMP addition (6.7 mM) and mechanical agitation. The kinetics and amount of Ca excretion were essentially the same in each case. The calcium ionophore A23187 increased the rate of 45Ca uptake by motile zoospores, incubated in 100 microM CaCl2, but did not induce encystment under these conditions. The ionophore did not induce 45Ca efflux from pre-labeled zoospores. Incubation in EGTA and in K+ failed to induce either encystment or 45Ca excretion. We conclude that rapid excretion of a significant proportion of the zoospore calcium is linked to the early stage of stimulus-induced encystment, and that this comes from an intracellularly located, non-cytoplasmic source, such as the peripheral vesicles, but that changes in cellular Ca2+ are not necessarily the single controlling factor in the induction of encystment.