SummaryGram-negative bacterial peptidoglycan is specifically recognized by the host intracellular sensor NOD1, resulting in the generation of innate immune responses. Although epithelial cells are normally refractory to external stimulation with peptidoglycan, these cells have been shown to respond in a NOD1-dependent manner to Gramnegative pathogens that can either invade or secrete factors into host cells. In the present work, we report that Gram-negative bacteria can deliver peptidoglycan to cytosolic NOD1 in host cells via a novel mechanism involving outer membrane vesicles (OMVs). We purified OMVs from the Gramnegative mucosal pathogens: Helicobacter pylori, Pseudomonas aeruginosa and Neisseria gonorrhoea and demonstrated that these peptidoglycan containing OMVs upregulated NF-kB and NOD1-dependent responses in vitro. These OMVs entered epithelial cells through lipid rafts thereby inducing NOD1-dependent responses in vitro. Moreover, OMVs delivered intragastrically to miceinduced innate and adaptive immune responses via a NOD1-dependent but TLR-independent mechanism. Collectively, our findings identify OMVs as a generalized mechanism whereby Gramnegative bacteria deliver peptidoglycan to cytosolic NOD1. We propose that OMVs released by bacteria in vivo may promote inflammation and pathology in infected hosts.

We identified a gene in the ovine hypothalamus encoding for RFamide-related peptide-3 (RFRP-3), and tested the hypothesis that this system produces a hypophysiotropic hormone that inhibits the function of pituitary gonadotropes. The RFRP-3 gene encodes for a peptide that appears identical to human RFRP-3 homolog. Using an antiserum raised against RFRP-3, cells were localized to the dorsomedial hypothalamic nucleus/paraventricular nucleus of the ovine brain and shown to project to the neurosecretory zone of the ovine median eminence, predicating a role for this peptide in the regulation of anterior pituitary gland function. Ovine RFRP-3 peptide was tested for biological activity in vitro and in vivo, and was shown to reduce LH and FSH secretion in a specific manner. RFRP-3 potently inhibited GnRH-stimulated mobilization of intracellular calcium in gonadotropes. These data indicate that RFRP-3 is a specific and potent mammalian gonadotropin-inhibiting hormone, and that it acts upon pituitary gonadotropes to reduce GnRH-stimulated gonadotropin secretion.

Abstract-The nature of the vasodilator endothelium-derived hyperpolarizing factor (EDHF) is controversial, putatively involving diffusible factors and/or electrotonic spread of hyperpolarization generated in the endothelium via myoendothelial gap junctions (MEGJs). In this study, we investigated the relationship between the existence of MEGJs, endothelial cell (EC) hyperpolarization, and EDHF-attributed smooth muscle cell (SMC) hyperpolarization in two different arteries: the rat mesenteric artery, where EDHF-mediated vasodilation is prominent, and the femoral artery, where there is no EDHF-dependent relaxation. In the rat mesenteric artery, stimulation of the endothelium with acetylcholine (ACh) evoked hyperpolarization of both ECs and SMCs, and characteristic pentalaminar MEGJs were found connecting the two cell layers. Key Words: endothelium-derived hyperpolarizing factor Ⅲ myoendothelial gap junctions Ⅲ endothelium Ⅲ smooth muscle Ⅲ electrical coupling T he endothelium plays a central role in the regulation of vascular tone. 1 The endothelium is capable of exerting a profound relaxing influence on the underlying smooth muscle, mediated by at least three different factors, depending on the vascular bed. These include nitric oxide (NO) and prostacyclin, both diffusible factors. [2][3][4][5][6] In addition, after blockade of NO and prostacyclin synthesis, stimulation of the endothelium is capable of evoking vascular smooth muscle relaxation that has been attributed to a third factor, endothelium-derived hyperpolarizing factor (EDHF). [7][8][9][10][11][12][13][14][15] The hallmark of the vasorelaxation attributed to EDHF is that it is accompanied by membrane hyperpolarization. Consensus regarding the nature of EDHF is lacking, with evidence suggesting the involvement of a diffusible factor(s) released from the endothelium in some vascular beds. 11,12,16,17 Others propose that the hyperpolarization generated in endothelial cells (ECs) is capable of spreading electrotonically to the underlying smooth muscle cells (SMCs), 18,19,21,23,25 most likely via myoendothelial gap junctions (MEGJs). 9,18,19,21,[23][24][25][26] Evidence to support the transfer of a small molecule (eg, cAMP) via MEGJs has also been presented. 20,22 Support for the MEGJ hypothesis has come from studies in which gap junctions have been pharmacologically blocked, either with peptide mimetics of connexin 43 (Cx43) [27][28][29][30] or with glycyrrhetinic acid derivatives. 13,26,[31][32][33] Although the former have been demonstrated to indeed reduce dye coupling between Cx-transfected cells, 34 the latter seem to have some nonspecific actions. [31][32][33]35 An alternative and complementary approach would be to study an artery that lacked an EDHF-dependent relaxation and to test for the existence of agonist-induced hyperpolarization in the ECs and for the presence of MEGJs. The femoral artery represents such a tissue because EDHF does not contribute to endothelium-dependent SMC relaxation in this vessel. 36,37 In the present study, this artery was ...

Of the endothelium-derived relaxing and hyperpolarizing factors, the nature of endothelium-derived hyperpolarizing factor (EDHF) and the ionic mechanisms underlying its actions are least understood (Mombouli & Vanhoutte, 1997;Hecker, 2000). The nature of EDHF as a chemical factor has been explored in a number of studies (Campbell et al. 1996;Popp et al. 1996;Randall et al. 1996;Plane et al. 1997;Chataigneau et al. 1998;Fisslthaler et al. 1999) with a lack of consensus. Some studies suggest that the EDHF-induced hyperpolarization of smooth muscle cells results from electrotonic spread of activity originating in the endothelial cells (Little et al. 1995; B eny, 1997;Chaytor et al. 1998;Yamamoto et al. 1998Yamamoto et al. , 1999 Edwards et al. 1999), while other studies provide evidence against coupling (B eny et al. 1997;Welsh & Segal, 1998). Identification of the nature of EDHF, whether as a diffusible factor or as electrotonic current spread, and the Journal of Physiology (2001) 1. Membrane currents attributed to endothelium-derived hyperpolarizing factor (EDHF) were recorded in short segments of submucosal arterioles of guinea-pigs using single microelectrode voltage clamp. The functional responses of arterioles and human subcutaneous, rat hepatic and guinea-pig coronary arteries were also assessed as changes in membrane potential recorded simultaneously with contractile activity. 2. The current-voltage (I-V) relationship for the conductance due to EDHF displayed outward rectification with little voltage dependence. Components of the current were blocked by charybdotoxin (30-60 nÒ) and apamin (0·25-0·50 ìÒ), which also blocked hyperpolarization and prevented EDHF-induced relaxation. 3. The EDHF-induced current was insensitive to Ba¥ (20-100 ìÒ) andÏor ouabain (1 ìÒ to 1 mÒ). 4. In human subcutaneous arteries and guinea-pig coronary arteries and submucosal arterioles, the EDHF-induced responses were insensitive to Ba¥ andÏor ouabain. Increasing [K¤]ï to 11-21 mÒ evoked depolarization under conditions in which EDHF evoked hyperpolarization. 5. Responses to ACh, sympathetic nerve stimulation and action potentials were indistinguishable between dye-labelled smooth muscle and endothelial cells in arterioles. Action potentials in identified endothelial cells were always associated with constriction of the arterioles. 6. 18â-Glycyrrhetinic acid (30 ìÒ) and carbenoxolone (100 ìÒ) depolarized endothelial cells by 31 ± 6 mV (n = 7 animals) and 33 ± 4 mV (n = 5), respectively, inhibited action potentials in smooth muscle and endothelial cells and reduced the ACh-induced hyperpolarization of endothelial cells by 56 and 58%, respectively. 7. Thus, activation of outwardly rectifying K¤ channels underlies the hyperpolarization and relaxation due to EDHF. These channels have properties similar to those of intermediate conductance (IKCa) and small conductance (SKCa) Ca¥-activated K¤ channels. Strong electrical coupling between endothelial and smooth muscle cells implies that these two layers function as a single electrical syn...