Noradrenergic neuron-specific overexpression of nNOS in cardiac sympathetic nerves decreases neurotransmission.

Gene transfer of neuronal nitric oxide synthase (nNOS) with nonspecific adenoviral vectors can cause promiscuous transduction. We provide direct evidence that nNOS targeted only to cardiac sympathetic neurons inhibits sympathetic neurotransmission. An adenovirus constructed with a noradrenergic neuron-specific promoter (PRSx8), driving nNOS or enhanced green fluorescence protein (eGFP) gene expression caused exclusive expression in tyrosine hydroxylase (TH) positive rat cardiac sympathetic neurons. There was no detectable leakage of transgene expression in other cell types in the preparation nor did the transgene express in choline acetyltransferase (CHAT)-positive intracardiac cholinergic ganglia. Functionally, Ad.PRS-nNOS gene transfer increased nNOS activity and significantly reduced norephinephrine release evoked by field stimulation of isolated right atria. These effects were reversed by the NOS inhibitor N(omega)-Nitro-L-arginine. Our results demonstrate that noradrenergic cell-specific gene transfer with nNOS can inhibit cardiac sympathetic neurotransmission. This targeted technique may provide a novel method for reducing presynaptic sympathetic hyperactivity.

AChR phosphorylation and indirect inhibition of AChR function in seronegative MG.

BACKGROUND: Approximately 10% to 20% of patients with autoimmune MG do not have antibodies to the acetylcholine receptor (AChR), so-called seronegative MG (SNMG). IgG antibodies from up to 70% of SNMG patients bind to the muscle-specific receptor tyrosine kinase, MuSK. The plasmas and non-IgG fractions from SNMG patients (and some with AChR antibodies) also contain a factor, perhaps an IgM antibody, that inhibits AChR function, but it is not clear how this factor acts and whether it is related to the MuSK IgG antibodies. METHODS: The authors studied 12 unselected SNMG plasmas and their non-IgG fractions; seven were positive for MuSK IgG antibodies. Ion flux assays, electrophysiology, phosphorylation, and kinase assays were used to look at mechanisms of action. RESULTS: Eight of the 12 plasmas and their non-IgG fractions inhibited AChR function, but the inhibitory activity was transient and did not correlate with the presence of MuSK IgG antibodies. Two of three plasmas added outside of a cell-attached patch pipette inhibited AChR function within the patch, and these two plasmas also increased AChR phosphorylation. CONCLUSIONS: The authors propose that a plasma factor(s) in SNMG patients, distinct from MuSK IgG antibodies, binds to a muscle membrane receptor and activates a second messenger pathway leading to AChR phosphorylation and reduced AChR function. Identifying the target for this factor should lead to improved diagnosis of MG in MuSK antibody-negative patients and may provide new insights into the function of the neuromuscular junction and pathophysiological mechanisms in MG.

Corticotrophin releasing hormone: its potential for a role in human myometrium.

Aside from its role as a hypothalamic stress hormone, corticotrophin releasing hormone (CRH) is also a placental hormone, at least in primates. Although the function of placentally derived CRH remains to be fully elucidated, elevated CRH levels have been associated with premature labour, suggesting that the hormone may be involved in regulating the duration of pregnancy. Indeed, pregnant human myometrium expresses functional CRH receptors (CRH R1 and CRH R2 subtypes) thought to signal predominantly via the second messenger cAMP. Thus, like other cAMP-producing hormones in the myometrium such as beta(2) agonists, CRH may play a part in maintaining uterine quiescence. However, several of the CRH receptor isoforms identified to date have a reduced ability to activate adenylate cyclase, raising the question as to whether they are linked to other signal transduction pathways. Here, we discuss critically the evidence for the peptide's role in regulating contractility, both directly at the myometrium and indirectly via the fetal membranes and decidua. The possibility of a role in myometrial growth modulation is also described. Experimental Physiology (2001) 86.2, 273-281.

Desensitisation of the oxytocin receptor and other G-protein coupled receptors in the human myometrium.

G-protein coupled receptors (GPCRs) are key to maintaining uterine quiescence and inducing phasic contractions at term. However, the biochemical mechanisms whereby uterine GPCRs are desensitised and re-sensitised during these physiological conditions are unknown. For example, the number of oxytocin receptors (OTRs) on uterine myocytes decrease significantly after the addition of oxytocin. Therefore, further understanding of the desensitisation/re-sensitisation of the OTR and other uterine GPCRs during pregnancy may provide a target for more efficient tocolytic drugs and more selective ways to modulate uterine activity. Here, we briefly review some of the mechanisms that may be involved during OTR and other GPCR desensitisation. Experimental Physiology (2001) 86.2, 303-312.

Interleukin-1 stimulates diglyceride accumulation in the absence of protein kinase C activation.

Despite advances in the knowledge of the intracellular signalling in response to extracellular messengers, the mechanism of action of interleukin-1 (IL-1) has remained an enigma. In the present study, we have employed human dermal fibroblasts (Detroit 532 cells) to investigate IL-1 beta-induced changes in intracellular signals. Both recombinant human IL-1 beta and a native preparation purified from human placental tissue were employed. Cyclic AMP levels in cell monolayers were unaltered by IL-1 beta. Also, IL-1 beta did not influence significantly the levels of phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate in the membrane, nor the water-soluble inositol phosphates, inositol monophosphate, inositol bisphosphate and inositol trisphosphate, in cells prelabelled with myo-[3H]inositol. In addition, intracellular calcium as measured by Quin2 was unaffected by interleukin-1. However, in cells labelled with [3H]glycerol or [3H]arachidonic acid, IL-1 beta caused an immediate rise in diglyceride (DG) accumulation. As the effects of IL-1 beta have been reported to be mimicked by tumour-promoting phorbol esters, this rise in DG suggested the involvement of protein kinase C (PKC). However, repeated experiments failed to reveal any acute effect of IL-1 beta on the activity of this enzyme. Furthermore, IL-1 beta did not cause the translocation of PKC between the membrane and the cytosol as has been found in response to other extracellular signals. Rather, IL-1 beta appeared to increase the synthesis of PKC in both membrane and cytosol preparations, an effect which could be prevented by coincubation with cycloheximide. These findings suggest that the diglyceride formed in response to IL-1 beta does not activate protein kinase C.

Decreased incorporation of glucose into lipids and increased lactate production by adipose tissue after long-term treatment of rats with D-fenfluramine.

Male rats were treated with ten daily doses of 10 mg of D-fenfluramine/kg. Body weight decreased after days 1 and 2, but thereafter the weight gain paralleled that of the control rats. After the tenth injection there were decreases in the weights of the epididymal fat pads, their fat content, and the average size of the adipocytes after collaginase digestion. The rate of glucose uptake by incubated pieces of adipose tissue was maintained after D-fenfluramine treatment, and the production of lactate increased. The incorporation of glucose into fatty acids by adipose tissue pieces decreased by 65-74% after treatment with D-fenfluramine. This effect was not reversed by adding insulin or phenylisopropyladenosine to the incubations. D-Fenfluramine also decreased the incorporation of glucose into glyceride-glycerol, but this effect was less pronounced than that for fatty acid synthesis. Direct addition of D-fenfluramine to the incubation inhibited lipid synthesis from [14C]glucose but only at drug concentrations above 1 mM. It is concluded that the treatment of rats with D-fenfluramine modifies the metabolic balance of adipose tissue so as to direct glucose metabolism away from lipid synthesis and towards lactate production. This could be a significant mechanism in the overall loss of adipose tissue mass caused by the administration of D-fenfluramine.

Interleukin-1 induces a pertussis toxin-sensitive increase in diacylglycerol accumulation in mouse thymoma cells.

The mechanism of action of the cytokine, interleukin-1 (IL-1), has been investigated. Mouse thymoma (EL4 6.1) cells were preincubated with [3H]-glycerol and then incubated with recombinant IL-1 beta for varying periods. Interleukin-1 caused a rapid increase in diacylglycerol production (approx. 2 fold at 30 secs). This reproducible enhancement of diacylglycerol accumulation was abolished by pretreatment of the cells with pertussis toxin. Interestingly, a similar IL-1 induced increase in diacylglycerol was observed when the cells were preincubated with [3H]-myristic acid. These results appear to suggest a novel mode of action of interleukin-1 which involves a G-protein mediated breakdown of a membrane lipid resulting in the production of diacylglycerol. It is suggested that one possible candidate for this parent lipid may be a phosphatidylinositol glycan.

Interactions of insulin and dexamethasone in the control of pyruvate kinase activity and glucose metabolism in sheep adipose tissue.

1. The Vmax. activity of pyruvate kinase of sheep adipose tissue increased during tissue culture up to 48 h; the increase was blocked by actinomycin D (an inhibitor of transcription) and was promoted by insulin and antagonized by dexamethasone. 2. In contrast with their effects on pyruvate kinase, insulin and dexamethasone acted synergistically to increase the activity of glucose-6-phosphate dehydrogenase of sheep adipose tissue maintained in culture. 3. Insulin stimulated, whereas dexamethasone inhibited, glucose utilization by sheep adipose tissue maintained in culture; the two agents were mutually antagonistic, and their effects were prevented by actinomycin D. 4. Antimycin A (an inhibitor of the electron-transport chain) stimulated glucose uptake and lactate output by sheep adipose tissue in the presence of dexamethasone and insulin, suggesting that the effects of dexamethasone on glucose utilization by sheep adipose tissue were not due to an inhibition of glucose transport. 5. Comparison of these findings with previous studies on the endocrine control of hepatic pyruvate kinase shows that there are major differences in the control of these Vmax. activities in liver and adipose tissue. 6. Although glucocorticoid hormones inhibit glucose utilization themselves and can antagonize the stimulatory effects of insulin on glucose utilization in adipose tissue from both sheep and rats, there appear to be major differences in the sites of action of these hormones in the two species.