Requirement of the F‐actin‐binding activity of l‐afadin for enhancing the formation of adherens and tight junctions

The apical junctional complex consists of adherens junctions (AJs) and tight junctions (TJs) in polarized epithelial cells, which are attached to each other to form a sheet. Actin filaments (F‐actin) are associated with AJs and TJs and required for the formation and maintenance of this complex. l‐Afadin is an F‐actin‐binding protein, which is localized at AJs through binding to the cell adhesion molecule nectin, and regulates the formation of AJs and TJs. However, the role of the F‐actin‐binding activity of l‐afadin for the formation of the apical junctional complex remains unknown. We generated here the cultured EpH4 mouse mammary epithelial cells in which afadin was genetically ablated. In the Ca²⁺ switch assay, the formation of both AJs and TJs was markedly impaired in the afadin‐deficient cells. Re‐expression of l‐afadin in the afadin‐deficient cells fully restored the formation of both AJs and TJs, but the re‐expression of the l‐afadin mutant lacking the FAB domain did not completely restore the formation of AJs or TJs. These results indicate that the F‐actin‐binding activity of l‐afadin is required for enhancing the formation of both AJs and TJs.     

Novel role of nectin: implication in the co-localization of JAM-A and claudin-1 at the same cell-cell adhesion membrane domain

Tight junctions (TJs) are formed at the apical side of adherens junctions (AJs) in epithelial cells. Major cell adhesion molecules (CAMs) at TJs are JAM and claudin, whereas major CAMs at AJs are nectin and cadherin. We previously showed that nectin initially forms cell–cell adhesion and then recruits cadherin to the nectin-based cell–cell adhesion sites to form AJs, followed by the recruitment of JAM and claudin to the apical side of AJs to form TJs. We investigated the roles of nectin in the formation of TJs by expressing various combinations of CAMs in L fibroblasts with no TJs or AJs. Co-expression of one of the AJ CAMs and one of the TJ CAMs formed two separate cell–cell adhesion membrane domains (CAMDs). Co-expression of nectin-3 and E-cadherin formed the same CAMD, but co-expression of JAM-A and claudin-1 did not form the same CAMD. Co-expression of JAM-A and claudin-1 with nectin-3, but not E-cadherin, made them form the same CAMD, which was separated from the nectin-based CAMD. Nectin-3 required afadin, a nectin- and F-actin-binding protein, for this ability. In conclusion, nectin plays a novel role in the co-localization of JAM and claudin at the same CAMD.     

Interaction and localization of Necl-5 and PDGF receptor beta at the leading edges of moving NIH3T3 cells: Implications for directional cell movement

It was previously shown that platelet-derived growth factor (PDGF) receptor physically and functionally interacts with integrin α_vβ₃, effectively inducing cell movement. We previously showed that Necl-5, originally identified as a poliovirus receptor, interacts with integrin α_vβ₃ and enhances its clustering and the formation of focal complexes at the leading edges of moving cells, resulting in an enhancement of cell movement. We showed here that Necl-5 additionally interacts with PDGF receptor in NIH3T3 cells and regulates the interaction between PDGF receptor and integrin α_vβ₃, effectively inducing directional cell movement. PDGF receptor co-localized with Necl-5 and integrin α_vβ₃ at peripheral ruffles over lamellipodia, which were formed at the leading edges of moving cells in response to PDGF, but not at the focal complexes under these ruffles, whereas Necl-5 and integrin α_vβ₃ co-localized at these focal complexes. The clustering of these three molecules at peripheral ruffles required the activation of integrin α_vβ₃ by vitronectin and the PDGF-induced activation of the small G protein Rac and subsequent re-organization of the actin cytoskeleton. These results indicate a key role of Necl-5 in directional cell movement by physically and functionally interacting with both integrin α_vβ₃ and PDGF receptor.     

Role of nectin in organization of tight junctions in epithelial cells

BACKGROUND: In polarized epithelial cells, cell-cell adhesion forms specialized membrane structures comprised of claudin-based tight junctions (TJs) and of E-cadherin-based adherens junctions (AJs). These structures are aligned from the apical to the basal side of the lateral membrane, but the mechanism of this organization remains unknown. Nectin is a Ca2+ independent immunoglobulin-like cell-cell adhesion molecule which localizes at AJs. Nectin is associated with E-cadherin through their respective cytoplasmic tail-binding proteins, afadin and catenins, and involved in the formation of AJs in cooperation with E-cadherin. We show here that nectin is also involved in the formation of TJs. RESULTS: During the formation of the junctional complex consisting of AJs and TJs in Madin-Darby canine kidney (MDCK) cells, claudin and occludin accumulated at the apical sites of the nectin-based cell-cell adhesion sites. This accumulation of claudin and occludin was inhibited by inhibitors acting on the trans interaction of nectin. The barrier function of TJs was also impaired by the nectin inhibitors. It has been shown that a phorbol ester promotes the formation of a TJ-like structure in an E-cadherin-independent manner. This phorbol ester-induced formation of the TJ-like structure was also inhibited by the nectin inhibitors. CONCLUSIONS: These results suggest a role of the nectin-afadin system in the organization of TJs as well as AJs in epithelial cells.     

Involvement of integrin-induced activation of protein kinase C in the formation of adherens junctions

In epithelial cells, tight junctions (TJs) and adherens junctions (AJs) form junctional complexes. At AJs, cadherins and nectins are the major cell-cell adhesion molecules. Nectins first form cell-cell adhesions and then recruit cadherins to the nectin-based cell-cell adhesion sites to form AJs in coordination with the activation of integrin alpha(v)beta(3), followed by the formation of TJs. We previously demonstrated that when MDCK cells precultured at a low Ca(2+) concentration were treated with the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA), incomplete AJs and a TJ-like structure were achieved. However, it remains unknown how PKC is activated and how it regulates the formation of cell-cell junctions. When MDCK cells precultured at a low Ca(2+) concentration were treated with TPA, incomplete AJs were formed without the activation of integrin alpha(v)beta(3). Treatment of cells with TPA also enhanced the phosphorylation of FAK, which transmits the outside-in signal of integrin and plays a role in the nectin-induced formation of AJs. In addition, inhibition of PKC suppressed the formation of AJs. These results indicate that the activation of PKC functions downstream of integrin alpha(v)beta(3) and upstream of FAK, and is important for the nectin-induced formation of AJs.     

Characterization of Monoclonal Antibodies to the αIIbβ3 Integrin on Bovine Platelets

A set of monoclonal antibodies (MoAbs) to leucocyte antigens is an essential tool to identify different cell types and functional membrane molecules involved in immune responses. Since no MoAbs existed to bovine integrins, except against the β₂ subfamily, we generated MoAbs to β₃ integrin after the immunization of mice with bovine platelets. Two MoAbs, IL-A164 (IgG_2a) and IL-A166 (IgG₁), were selected that reacted specifically with bovine platelets and detected the same membrane molecule. The antigen was a heterodimer of two polypeptide chains of 122 kDa and 95 kDa as resolved by SDS-PAGE under reducing conditions. Although the Mr of the smaller subunit is identical to that of β₂ integrin, pre-absorption with an antibody to β₂ (or CD18) did not remove the bovine antigen. Comparing the molecular masses of the two subunits in reduced and non-reduced forms showed a pattern that was similar to that of human GPIIb/IIIa (also called α_IIbβ₃ or CD41a). Reduction of the bovine molecule increased the apparent Mr of the light chain from 76 kDa to 95 kDa, while the heavy subunit changed from 136 kDa to 122 kDa. As with human GPIIb, the decrease in Mr of the α-subunit is probably a result of a small disulphide-linked polypeptide, although no additional evidence for this was detected for the bovine integrin. Sequencing of the N-terminal amino acids of both bovine polypeptides showed identity of the bovine integrin with human GPIIb/IIIa.     

Expression of Integrin αvβ3 in Gliomas Correlates with Tumor Grade and Is not Restricted to Tumor Vasculature

In malignant gliomas, the integrin adhesion receptors seem to play a key role for invasive growth and angiogenesis. However, there is still a controversy about the expression and the distribution of α_vβ₃ integrin caused by malignancy. The aim of our study was to assess the extent and pattern of α_vβ₃ integrin expression within primary glioblastomas (GBMs) compared with low-grade gliomas (LGGs). Tumor samples were immunostained for the detection of α_vβ₃ integrin and quantified by an imaging software. The expression of α_vβ₃ was found to be significantly higher in GBMs than in LGGs, whereby focal strong reactivity was restricted to GBMs only. Subsequent analysis revealed that not only endothelial cells but also, to a large extent, glial tumor cells contribute to the overall amount of α_vβ₃ integrin in the tumors. To further analyze the integrin subunits, Western blots from histologic sections were performed, which demonstrated a significant difference in the expression of the β₃ integrin subunit between GBMs and LGGs. The presented data lead to new insights in the pattern of α_vβ₃ integrin in gliomas and are of relevance for the inhibition of α_vβ₃ integrin with specific RGD peptides and interfering drugs to reduce angiogenesis and tumor growth.     

Expression of α5 (CD49e) and α6 (CD49f) Integrin Subunits on T Cells in the Circulation and the Lamina Propria of Normal and Inflammatory Bowel Disease Colonic Mucosa

Intestinal lamina propria T cells are believed to be derived, via the systemic circulation, from gut-associated lymphoid tissue. After migration into the lamina propria, T cells are capable of luminally directed migration following the loss of surface epithelial cells. For adhesion and migration within the extracellular matrix, T cells are likely to utilize the integrin family of adhesion molecules. The aim of this study was to quantitatively and qualitatively investigate the expression of α₅ and α₆ integrin subunits on the surface of human T cells that: (a) migrated out of the lamina propria, (b) remained resident within the matrix and (c) were present in the circulation. In both subpopulations of CD4 and CD8-positive T cells, from both normal and inflamed (inflammatory bowel disease) colonic mucosa, there were significantly fewer α₅ and α₆-positive cells than in the peripheral blood. In addition, there were significantly fewer α₆ integrin molecules on the surface of CD4 and CD8-positive lamina propria T-cell subpopulations, compared with those in the circulation. Our studies suggest that, following migration into the lamina propria, there is down-regulation of α₅ and α₆ integrin-subunit expression on the surface of T cells. Molecules other than members of very late activation antigen-5 (VLA-5) (α₅β₁) and VLA-6 (α₆β₁) families of adhesion molecules are likely to be important in interactions with extracellular components in the lamina propria of normal and inflamed human colonic mucosa.     

Rapid changes in shear stress induce dissociation of a Gαq/11–platelet endothelial cell adhesion molecule-1 complex

It has been recently shown that endothelial platelet endothelial cell adhesion molecule-1 (PECAM-1) expression is pro-atherogenic. PECAM-1 is involved in sensing rapid changes in fluid shear stress but the mechanisms for activating signalling complexes at the endothelial cell junction have yet to be elucidated. Additional studies suggest the activation of membrane-bound G proteins Gα_q/11 also mediate flow-induced responses. Here, we investigated whether PECAM-1 and Gα_q/11 could act in unison to rapidly respond to fluid shear stress. With immunohistochemistry, we observed a co-localization of Gα_q/11 and PECAM-1 at the cell–cell junction in the atheroprotected section of mouse aortae. In contrast, Gα_q/11 was absent from junctions in atheroprone areas as well as in all arterial sections of PECAM-1 knockout mice. In primary human endothelial cells, temporal gradients in shear stress led to a rapid dissociation of the Gα_q/11–PECAM-1 complex within 30 s and a partial relocalization of the Gα_q/11 staining to perinuclear areas within 150 min, whereas transitioning fluid flow devoid of temporal gradients did not disrupt the complex. Inhibition of G protein activation eliminated temporal gradient flow-induced Gα_q/11–PECAM-1 dissociation. These results allow us to conclude that Gα_q/11–PECAM-1 forms a mechanosensitive complex and its localization suggests the Gα_q/11–PECAM-1 complex is a critical mediator of vascular diseases.     

Nectin and junctional adhesion molecule are critical cell adhesion molecules for the apico‐basal alignment of adherens and tight junctions in epithelial cells

Tight junctions (TJs) and adherens junctions (AJs) form an apical junctional complex at the apical side of the lateral membranes of epithelial cells, in which TJs are aligned at the apical side of AJs. Many cell adhesion molecules (CAMs) and cell polarity molecules (CPMs) cooperatively regulate the formation of the apical junctional complex, but the mechanism for the alignment of TJs at the apical side of AJs is not fully understood. We developed a cellular system with which epithelial‐like TJs and AJs were reconstituted in fibroblasts and analyzed the cooperative roles of CAMs and CPMs. We exogenously expressed various combinations of CAMs and CPMs in fibroblasts that express negligible amounts of these molecules endogenously. In these cells, the nectin‐based cell–cell adhesion was formed at the apical side of the junctional adhesion molecule (JAM)‐based cell–cell adhesion, and cadherin and claudin were recruited to the nectin‐3‐ and JAM‐based cell–cell adhesion sites to form AJ‐like and TJ‐like domains, respectively. This inversed alignment of the AJ‐like and TJ‐like domains was reversed by complementary expression of CPMs Par‐3, atypical protein kinase C, Par‐6, Crb3, Pals1 and Patj. We describe the cooperative roles of these CAMs and CPMs in the apico‐basal alignment of TJs and AJs in epithelial cells.     

Requirement of the actin cytoskeleton for the association of nectins with other cell adhesion molecules at adherens and tight junctions in MDCK cells

Nectins, Ca(2+)-independent immunoglobulin-like cell adhesion molecules (CAMs), first form cell-cell adhesion where cadherins are recruited, forming adherens junctions (AJs) in epithelial cells and fibroblasts. In addition, nectins recruit claudins, occludin, and junctional adhesion molecules (JAMs) to the apical side of AJs, forming tight junctions (TJs) in epithelial cells. Nectins are associated with these CAMs through peripheral membrane proteins (PMPs), many of which are actin filament-binding proteins. We examined here the roles of the actin cytoskeleton in the association of nectins with other CAMs in MDCK cells stably expressing exogenous nectin-1. The nectin-1-based cell-cell adhesion was formed and maintained irrespective of the presence and absence of the actin filament-disrupting agents, such as cytochalasin D and latrunculin A. In the presence of these agents, only afadin remained at the nectin-1-based cell-cell adhesion sites, whereas E-cadherin and other PMPs at AJs, alpha-catenin, beta-catenin, vinculin, alpha-actinin, ADIP, and LMO7, were not concentrated there. The CAMs at TJs, claudin-1, occludin and JAM-1, or the PMPs at TJs, ZO-1 and MAGI-1, were not concentrated there, either. These results indicate that the actin cytoskeleton is required for the association of the nectin-afadin unit with other CAMs and PMPs at AJs and TJs.     

The molecular basis and biological significance of VH replacement

Summary:  First observed in mouse pre-B-cell lines and then in knock-in mice carrying self-reactive IgH transgenes, V _H replacement has now been shown to contribute to the primary B-cell repertoire in humans. Through recombination-activating gene (RAG)-mediated recombination between a cryptic recombination signal sequence (RSS) present in almost all V _H genes and the flanking 23 base pair RSS of an upstream V _H gene, V _H replacement renews the entire V _H -coding region, while leaving behind a short stretch of nucleotides as a V _H replacement footprint. In addition to extending the CDR3 region, the V _H replacement footprints preferentially contribute charged amino acids. V _H replacement rearrangement in immature B cells may either eliminate a self-reactive B-cell receptor or contribute to the generation of self-reactive antibodies. V _H replacement may also rescue non-productive or dysfunctional V _H DJ _H rearrangement in pro-B and pre-B cells. Conversely, V _H replacement of a productive immunoglobulin H gene may generate non-productive V _H replacement to disrupt or temporarily reverse the B-cell differentiation process. V _H replacement can thus play a complex role in the generation of the primary B-cell repertoire.     

Migration of human melanoma cells depends on extracellular pH and Na+/H+ exchange

Their glycolytic metabolism imposes an increased acid load upon tumour cells. The surplus protons are extruded by the Na⁺/H⁺ exchanger (NHE) which causes an extracellular acidification. It is not yet known by what mechanism extracellular pH (pH_e) and NHE activity affect tumour cell migration and thus metastasis. We studied the impact of pH_e and NHE activity on the motility of human melanoma (MV3) cells. Cells were seeded on/in collagen I matrices. Migration was monitored employing time lapse video microscopy and then quantified as the movement of the cell centre. Intracellular pH (pH_i) was measured fluorometrically. Cell–matrix interactions were tested in cell adhesion assays and by the displacement of microbeads inside a collagen matrix. Migration depended on the integrin α2β1. Cells reached their maximum motility at pH_e∼7.0. They hardly migrated at pH_e 6.6 or 7.5, when NHE was inhibited, or when NHE activity was stimulated by loading cells with propionic acid. These procedures also caused characteristic changes in cell morphology and pH_i. The changes in pH_i, however, did not account for the changes in morphology and migratory behaviour. Migration and morphology more likely correlate with the strength of cell–matrix interactions. Adhesion was the strongest at pH_e 6.6. It weakened at basic pH_e, upon NHE inhibition, or upon blockage of the integrin α2β1. We propose that pH_e and NHE activity affect migration of human melanoma cells by modulating cell–matrix interactions. Migration is hindered when the interaction is too strong (acidic pH_e) or too weak (alkaline pH_e or NHE inhibition).     

Molecular mechanisms regulating dissociation of cell–cell junction of epithelial cells by oxidative stress

Oxidative stress is regarded as a causative factor in aging and various degenerative diseases. Here, we show the mechanism by which oxidative stress induces disruption of cell–cell junctions using retinal pigment epithelial cells. We demonstrated that reactive oxygen species (ROS)-mediated activation of Src kinase increases the tyrosine phosphorylation state of p120-catenin and rapidly triggers translocation of p120-catenin and internalization of N-cadherin from the cell–cell adhesion sites to an early endosomal compartment. Endosomal accumulation of p120-catenin resulted in stress fiber formation and cell–cell dissociation through the activation of Rho/Rho kinase pathway. However, these cytoskeletal remodeling and cell–cell dissociation induced by oxidative stress were transient, due to the activation of nuclear factor-κB (NF-κB) and the expression of manganese superoxide dismutase (Mn-SOD). Using the NF-κB specific inhibitor DHMEQ, we found that NF-κB is part of a negative feedback loop to control intracellular ROS levels. Finally, we demonstrated that H₂O₂ treatment alone does not induce the epithelial mesenchymal transition (EMT) in retinal pigment epithelial cells, which can be induced by TNF-α treatment. These findings suggest that oxidative stress is a crucial factor to induce the cell–cell dissociation, an initial step of EMT, but does not provide sufficient signals to establish and to maintain the EMT.     

Similar and differential behaviour between the nectin-afadin-ponsin and cadherin-catenin systems during the formation and disruption of the polarized junctional alignment in epithelial cells

BACKGROUND: We have recently identified a novel cell-cell adhesion system, named NAP system, which is localized at cadherin-based cell-cell adherens junctions (AJs). The NAP system is composed of at least nectin, afadin and ponsin. Nectin is an immunoglobulin-like cell adhesion molecule. Afadin is an actin filament-binding protein which associates nectin with the actin cytoskeleton. Ponsin is an afadin-binding protein which furthermore binds to vinculin and provides a possible linkage of nectin-afadin to cadherin-catenin through vinculin. We compared here the behaviour of the NAP and cadherin-catenin systems during the formation and disruption of the polarized junctional alignment in epithelial cells. RESULTS: At the early stage of the formation of the polarized junctional alignment in MTD-1 A cells, primordial spot-like junctions were formed at the tips of thin cellular protrusions radiating from adjacent cells. Nectin, afadin, ponsin, cadherin and catenin were simultaneously recruited to these junctions. As the cell polarization proceeded, the spot-like junctions were gradually fused to form belt-like AJs where all these proteins were concentrated. The disruption of cell-cell AJs in MDCK cells by culturing at a low Ca2+ concentration caused rapid endocytosis of cadherin, but not that of nectin or afadin. Addition of 12-O-tetradecanoylphorbol-13-acetate to the cells formed a tight junction-like structure where nectin and afadin, but not cadherin, accumulated. CONCLUSION: These results indicate that the NAP and cadherin-catenin systems show similar and differential behaviour during the formation and disruption of the polarized junctional alignment in epithelial cells.     

Glycosylation in immune cell trafficking

Summary:  Leukocyte recruitment encompasses cell adhesion and activation steps that enable circulating leukocytes to roll, arrest, and firmly adhere on the endothelial surface before they extravasate into distinct tissue locations. This complex sequence of events relies on adhesive interactions between surface structures on leukocytes and endothelial cells and also on signals generated during the cell–cell contacts. Cell surface glycans play a crucial role in leukocyte recruitment. Several glycosyltransferases such as α1,3 fucosyltransferases, α2,3 sialyltransferases, core 2 N -acetylglucosaminlytransferases, β1,4 galactosyltransferases, and polypeptide N -acetylgalactosaminyltransferases have been implicated in the generation of functional selectin ligands that mediate leukocyte rolling via binding to selectins. Recent evidence also suggests a role of α2,3 sialylated carbohydrate determinants in triggering chemokine-mediated leukocyte arrest and influencing β₁ integrin function. The recent discovery of galectin- and siglec-dependent processes further emphasizes the significant role of glycans for the successful recruitment of leukocytes into tissues. Advancing the knowledge on glycan function into appropriate pathology models is likely to suggest interesting new therapeutic strategies in the treatment of immune- and inflammation-mediated diseases.     

Transforming growth factor-β production during the development of renal fibrosis in rats with subtotal renal ablation

The histologic changes observed in the remnant kidney model include progressive mesangial expansion with collapse of capillary lumina, interstitial fibrosis and mononuclear cellular infiltration. Transforming growth factor-beta (TGF-β₁) is an important regulator of extracellular matrix formation. The purpose of this study was to investigate the production and distribution of TGF-β₁ in the kidney during the development of glomerulosclerosis and renal fibrosis in rats with subtotal renal ablation. Eighty-two female Wistar rats weighing 180–220 g were divided into two groups: 49 rats were subjected to 5/6 renal ablation and 33 to sham operation. Urinary albumin excretion, blood pressure and glomerular filtration rate (GFR) were evaluated after the surgical procedure. We also performed histology and immunohistochemistry and determined mRNA for TGF-β₁ in the kidneys of these rats 8, 15, 30 and 90 days after operation. The results showed progressively higher immunohistochemical TGF-β₁ staining in rats with subtotal renal ablation. Cortical renal content of TGF-β₁ mRNA was also higher in these animals and peaked at day 15.
The existence of a temporal association between glomerulosclerosis, interstitial fibrosis and intense mononuclear cellular infiltration on the one hand and higher immunohistochemical TGF-β₁ staining in the renal cortex on the other show that this polypeptide may contribute to the development of renal fibrosis in this model.     

The Interaction between Beta 2-Microglobulin (ßm) and Purified Class-I Major Histocompatibility (MHC) Antigen

The function of MHC class-I molecules is to sample peptides from the intracellular environment and present them to CD8⁺ cytotoxic T lymphocytes. To understand the molecular details of the assembly (and disassembly) of peptide-ß₂m-class-I complexes a biochemical peptidc-class-I binding assay has been generated recently and this paper reports on a similar assay for the interaction between ß₂m and class I. As a model system human ß₂m binding to mouse class I was used. The assay is strictly biochemical using purified reagents which interact in solution and complex formation is determined by size separation. It is specific and highly sensitive. The observed affinity of the interaction, K_D, is close to 0.4 nw. The rate of association at 37 C is very fasi (the k_a is around 5 × 10⁴/M/s) whereas the dissociation is slow (the k_d is around 8 × 10⁻⁶/s); the ratio of dissociation to association yields a calculated K_D close to the observed value. At 37° C almost all of the purified class I participates in binding of the exogenously offered ß₂m showing that a considerable exchange of the endogenous ß₂m occurs. Finally, it was demonstrated that exogenous ß₂m enhances binding to MHC class-I of short perfectly-matching peplides as well as longer peptides.     

Gastric motility in soluble guanylate cyclase α1 knock-out mice

The principal target of the relaxant neurotransmitter nitric oxide (NO) is soluble guanylate cyclase (sGC). As the α₁β₁-isoform of sGC is the predominant one in the gastrointestinal tract, the aim of this study was to investigate the role of sGC in nitrergic regulation of gastric motility in male and female sGCα₁ knock-out (KO) mice. In circular gastric fundus muscle strips, functional responses and cGMP levels were determined in response to nitrergic and non-nitrergic stimuli. sGC subunit mRNA expression in fundus was measured by real-time RT-PCR; in vivo gastric emptying of a phenol red meal was determined. No changes were observed in sGC subunit mRNA levels between wild-type (WT) and KO tissues. Nitrergic relaxations induced by short trains of electrical field stimulation (EFS) were abolished, while those by long trains of EFS were reduced in KO strips; the latter responses were abolished by 1 H [1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The relaxations evoked by exogenous NO and the NO-independent sGC activator BAY 41-2272 were reduced in KO strips but still sensitive to ODQ. Relaxations induced by vasoactive intestinal peptide (VIP) and 8-bromo-cGMP were not influenced. Basal cGMP levels were decreased in KO strips but NO, long train EFS and BAY 41-2272 still induced a moderate ODQ-sensitive increase in cGMP levels. Gastric emptying, measured at 15 and 60 min, was increased at 15 min in male KO mice. sGCα₁β₁ plays an important role in gastric nitrergic relaxation in vitro , but some degree of nitrergic relaxation can occur via sGCα₂β₁ activation in sGCα₁ KO mice, which contributes to the moderate in vivo consequence on gastric emptying.