G-protein Complex Research Articles

Porcine hepatic response to sepsis and its amplification by an adrenergic receptor α1 agonist and a β2 antagonist

1. We investigated the effect of adrenergic receptor stimulation or inhibition on the hepatic ultrastructural changes in a porcine faecal peritonitis model of multi-organ failure. We infused either the alpha1 adrenergic receptor agonist methoxamine or the beta2 adrenergic receptor antagonist ICI 118551 during 8 h of the study.2. Anaesthetized pigs (25-30 kg) were divided into four non-septic groups (control, non-septic, non-septic methoxamine and non-septic ICI 118551) and three septic groups (septic, septic methoxamine and septic ICI 118551).3. Changes in hepatic ultrastructure were measured by morphometric analysis. The septic group was significantly worse than all the non-septic groups. Septic methoxamine and septic ICI 118551 were significantly worse than the septic group.4. Septic methoxamine and septic ICI 118551 had a significantly increased perisinusoidal space; septic methoxamine had significant hepatocyte vacuolation.5. Hepatic ultrastructural changes were independent of hepatic blood flow.6. Septic methoxamine had significant myocardial depression.7. The alpha1 adrenergic receptor agonist methoxamine or the beta2 antagonist ICI 118551 both amplified the hepatic injury normally found during sepsis in our porcine model.8. These findings suggest that during sepsis a protective endogenous beta2 adrenergic receptor-mediated anti-inflammatory response is activated via cell membrane transduction to stimulate the trimeric G-protein complex Gs and activate the second cell messenger cAMP.9. In addition, it is likely that alpha1 adrenergic receptor agonists amplify the inflammatory response by stimulating the cell-surface receptor-linked trimeric G-protein complex to activate Gq and the second cell messenger phospholipase C.

The Calculus of Rod Phototransduction

In this issue, two articles present major advances in the quantitative analysis of the molecular mechanisms underlying rod phototransduction. One is by Nikonov et al. (1998), the other by Calvert et al. (1998). These two papers are complimentary, but with substantial areas of intersection. At the present time, the activation cascade in rod phototransduction that leads to the hydrolysis of the internal transmitter, cyclic GMP (cGMP) and to the closure of light-sensitive channels is fairly well understood. The inactivation steps responsible for the termination of the photoresponse and the feedback mechanisms, which modulate sensitivity and kinetics and also contribute to response termination, are not understood nearly as well. The field of phototransduction has always been fraught with controversy: for every point, there has been a counterpoint. However, one can argue, with little fear of inciting controversy, that a complete understanding of phototransduction must include an understanding of the steps by which the photoresponse is initiated and the steps by which it is terminated. For this reason, the Nikonov et al. paper, “The Kinetics of Recovery of the Dark-adapted Salamander Rod Photoresponse” is especially significant. This paper moves us closer to a definitive answer to an old, controversial question: What is the rate-limiting biochemical reaction that determines the time course of recovery of the photocurrent from a flash bright enough to temporarily shut off all light-sensitive current? Two main contenders have been the inactivation of rhodopsin and the inactivation of the activated phosphodiesterase–G-protein complex. The authors present a case for the latter. A highlight of the paper is a new approach to quantify the extent of guanylyl cyclase activation in a feedback pathway mediated by calcium. The role of cyclase in determining the time at which photocurrent recovery begins and its role in sculpting the waveform of recovery are quantified. This analysis supports the existence of at least one more significant target for calcium feedback. A notable feature of the paper is that the authors make stunning progress largely through powerful new theoretical analysis applied to data gathered with stateof-the-art techniques. A number of important observations and conclusions are stated in a formal manner in mathematical language, which includes theorems, lemmas, and proofs. The rigorous approach in this paper has the advantages of clarity and completeness. The aspects of phototransduction that can now be well understood are highlighted by a mathematical model, and the gaps in our knowledge are set off in stark contrast. Fortunately for the reader with less mathematical background, sufficient explanatory discussion surrounds the mathematical statements, such that a reader may even choose to skip the theorems entirely without major loss of information.

G-protein-mediated signaling in cholesterol-enriched arterial smooth muscle cells. 1. Reduced membrane-associated G-protein content due to diminished isoprenylation of G-gamma subunits and p21ras.

Mechanisms contributing to altered heterotrimeric G-protein expression and subsequent signaling events during cholesterol accretion have been unexplored. The influence of cholesterol enrichment on G-protein expression was examined in cultured smooth muscle cells that resemble human atherosclerotic cells by exposure to cationized LDL (cLDL). cLDL, which increases cellular free and esterified cholesterol 2-fold and 10-fold, respectively, reduced the cell membrane content of Galphai-1, Galphai-2, Galphai-3, Gq/11, and Galphas. The following evidence supports the premise that the mechanism by which this occurs is due to reduced isoprenylation of the Ggamma-subunit. First, the inhibitory effect of cholesterol enrichment on the membrane content of Galphai subunits was found to be post-transcriptional, since the mRNA steady-state levels of Galphai(1-3) were unchanged following cholesterol enrichment. Second, the membrane expression of alpha and beta subunits was mimicked by cholesterol and 17-ketocholesterol, both of which inhibit HMG-CoA reductase. Third, inhibition of Galphai and Gbeta expression in cholesterol-enriched cells was overcome by mevalonate, the immediate product of HMG-CoA reductase. Fourth, pulse-chase experiments revealed that cholesterol enrichment did not reduce the degradation rate of membrane-associated Galphai subunits. Fifth, cholesterol enrichment also reduced membrane expression of Ggamma-5, Ggamma-7upper; these gamma subunits are responsible for trafficking of the heterotrimeric G-protein complex to the cell membrane as a result of HMG-CoA reductase-dependent post-translational lipid modification (geranylgeranylation) and subsequent membrane association. Cholesterol enrichment did not alter expression of G-gamma-5 mRNA, as assessed by reverse transcriptase polymerase chain reaction, supporting a post-transcriptional defect in Ggamma subunit expression. Fifth, cholesterol enrichment also reduced the membrane content of p21ras (a low molecular weight G-protein requiring farnesylation for membrane targeting) but did not alter the membrane content of the two proteins that do not require isoprenylation for membrane association&sbd;PDGF-receptor or p60-src. Reduced G-protein content in cholesterol-laden cells was reflected by reduced G-protein-mediated signaling events, including ATP-induced GTPase activity, thrombin-induced inhibition of cyclic AMP accumulation, and MAP kinase activity. Collectively, these results demonstrate that cholesterol enrichment reduces G-protein expression and signaling by inhibiting isoprenylation and subsequent membrane targeting. These results provide a molecular basis for altered G-protein-mediated cell signaling processes in cholesterol-enriched cells.

G-protein function is reduced in hypertension.

A functional impairment in vasodilator tone may be important in the pathogenesis and/or maintenance of elevated peripheral vascular resistance in hypertension. Previous studies of hypertensive subjects have demonstrated impaired beta-adrenergic-mediated vasodilation paralleling a reduction in lymphocyte beta-adrenergic-stimulated adenylyl cyclase activity. We have suggested that this impairment is related to a defect in G-protein function. To determine whether this defect alters the coupling between the G-protein complex and adenylyl cyclase, we performed [3H]forskolin binding studies in lymphocytes from hypertensive subjects, older normotensive subjects, and younger normotensive control subjects. Maximal specific [3H]forskolin binding was used as an index of adenylyl cyclase binding sites. Gpp(NH)p-, NaF/AlCl3-, and isoproterenol-stimulated binding were used as indices of G-protein/adenylyl cyclase coupling. In the absence of other stimulators, maximal [3H]forskolin binding was not significantly different among groups. However, both Gpp(NH)p- and isoproterenol-stimulated [3H]forskolin binding were significantly decreased in lymphocytes from hypertensive subjects. Overall, Gpp(NH)p- and isoproterenol-stimulated [3H]forskolin binding were significantly inversely correlated with blood pressure. No differences in NaF/AlCl3-stimulated [3H]forskolin binding were detected between groups. These studies indicate that G-protein/adenylyl cyclase coupling is impaired in lymphocytes from younger hypertensive subjects and may contribute to the blood pressure-related defect in beta-adrenoceptor-stimulated adenylyl cyclase activity.

Advances in the molecular understanding of gonadotropins-receptors interactions

The extracellular domain (ECD) of gonadotropin receptors belong to the leucine-rich repeat (LRR) protein superfamily and their transmembrane domain (TMD) is characteristic of the seven α-helices G-protein-coupled receptors (GPCR). The availability of the X-ray strutures of porcine ribonuclease inhibitor (RI), a LRR protein, and bacteriorhodopsin (bR) allows the construction of 3D models of the ECD and the TMD of gonadotropin receptors, respectively. The predicted models are to a large extent consistent with currently available biochemical and mutational data. The models provide a reliable basis for understanding how the hormone binds and activates its receptor. The ECD, in particular the LRR region, serves as a baseball glove which efficiently catches the large hormone and optimally orient the appropriate parts of it for interaction with the seven-transmembrane-helix domain of the receptor. This in turn is expected to lead to a conformational change to be sensed by the appropriate G-protein complex leading to the stimulation of cAMP synthesis and steroids production.

Taste receptor-like cells in the rat gut identified by expression of alpha-gustducin.

The alpha-subunit of the trimeric G-protein complex specific for taste receptor cells of the tongue, alpha-gustducin, is described here to be also expressed in the stomach and intestine. The alpha-gustducin-containing cells were identified as brush cells that are scattered throughout the surface epithelium of the gut and share structural features of taste receptor cells of the tongue. These findings provide clues to the long-sought molecular and cellular basis for chemoreception in the gut.

Gpp(NH)p stimulates [ 3H]raclopride binding to homogenates from human putamen and accumbens

The effects of the stable GTP analogue Gpp(NH)p (5′-guanylyl imido diphosphate) were examined on in vitro [ 3H]raclopride binding to dopamine D 2 receptors in preparations from post mortem human brains. The estimated number of receptors in the brain was 29% and 38% higher in putamen and accumbens, respectively, when determined in the presence of Gpp(NH)p as compared to its absence. The interaction of agonists was biphasic confirming the two affinity state model of the receptor - G-protein complex. The addition of Gpp(NH)p to the assay abolished the two site competition of apomorphine with [ 3H]raclopride binding in both regions studied. The non-specific binding at high concentrations of apomorphine was not significantly affected by the addition of Gpp(NH)p, indicating that only the specific binding of [ 3H]raclopride to the dopamine D 2 receptor is increased.

Isolation of schistosomin, a neuropeptide which antagonizes gonadotropic hormones in a freshwater snail

The molecular mechanisms underlying parasite-induced inhibitory effects on host reproduction were studied in the freshwater snail, Lymnaea stagnalis, infected with the schistosome parasite Trichobilharzia ocellata. This combination is used as a model system for host-parasite interactions involved in schistosomiasis transmission. The female gonadotropic snail neuropeptide, calfluxin, was labelled with fluorescein isothiocyanate (FITC) and used as a ligand in receptor-binding studies on membranes of its target organ, the albumen gland. The binding of calfluxin to its receptor-guanyl-nucleotide-binding-protein (G-protein) complex was inhibited in vitro in the presence of haemolymph of schistosome-infected snails. This inhibition appeared to be established by a peptidergic factor called schistosomin. The receptor assay was used to identify schistosomin from haemolymph during subsequent purification and characterization steps. The peptide could also be purified from the central nervous systems of non-infected snails, indicating that it is produced by the snail itself and released into the haemolymph as a result of infection. Analysis by plasma-desorption mass spectrometry revealed that purified schistosomin has a molecular mass of 8780 Da.