Molecular Handoffs in Nitrergic Neurotransmission

Postsynaptic density (PSD) proteins in excitatory synapses are relatively immobile components, while there is a structured organization of mobile scaffolding proteins lying beneath the PSDs. For example, shank proteins are located further away from the membrane in the cytosolic faces of the PSDs, facing the actin cytoskeleton. The rationale of this organization may be related to important roles of these proteins as “exchange hubs” for the signaling proteins for their migration from the subcortical cytosol to the membrane. Notably, PSD95 have also been demonstrated in prejunctional nerve terminals of nitrergic neuronal varicosities traversing the gastrointestinal smooth muscles. It has been recently reported that motor proteins like myosin Va play important role in transcytosis of nNOS. In this review, the hypothesis is forwarded that nNOS delivered to subcortical cytoskeleton requires interactions with scaffolding proteins prior to docking at the membrane. This may involve significant role of “shank,” named for SRC-homology (SH3) and multiple ankyrin repeat domains, in nitric oxide synthesis. Dynein light chain LC8–nNOS from acto-myosin Va is possibly exchanged with shank, which thereafter facilitates transposition of nNOS for binding with palmitoyl-PSD95 at the nerve terminal membrane. Shank knockout mice, which present with features of autism spectrum disorders, may help delineate the role of shank in enteric nitrergic neuromuscular transmission. Deletion of shank3 in humans is a monogenic cause of autism called Phelan–McDermid syndrome. One fourth of these patients present with cyclical vomiting, which may be explained by junctionopathy resulting from shank deficit in enteric nitrergic nerve terminals

Myosin Va Plays a Role in Nitrergic Smooth Muscle Relaxation in Gastric Fundus and Corpora Cavernosa of Penis

The intracellular motor protein myosin Va is involved in nitrergic neurotransmission possibly by trafficking of neuronal nitric oxide synthase (nNOS) within the nerve terminals. In this study, we examined the role of myosin Va in the stomach and penis, proto-typical smooth muscle organs in which nitric oxide (NO) mediated relaxation is critical for function. We used confocal microscopy and co-immunoprecipitation of tissue from the gastric fundus (GF) and penile corpus cavernosum (CCP) to localize myosin Va with nNOS and demonstrate their molecular interaction. We utilized in vitro mechanical studies to test whether smooth muscle relaxations during nitrergic neuromuscular neurotransmission is altered in DBA (dilute, brown, non-agouti) mice which lack functional myosin Va. Myosin Va was localized in nNOS-positive nerve terminals and was co-immunoprecipitated with nNOS in both GF and CCP. In comparison to C57BL/6J wild type (WT) mice, electrical field stimulation (EFS) of precontracted smooth muscles of GF and CCP from DBA animals showed significant impairment of nitrergic relaxation. An NO donor, Sodium nitroprusside (SNP), caused comparable levels of relaxation in smooth muscles of WT and DBA mice. These normal postjunctional responses to SNP in DBA tissues suggest that impairment of smooth muscle relaxation resulted from inhibition of NO synthesis in prejunctional nerve terminals. Our results suggest that normal physiological processes of relaxation of gastric and cavernosal smooth muscles that facilitate food accommodation and penile erection, respectively, may be disrupted under conditions of myosin Va deficiency, resulting in complications like gastroparesis and erectile dysfunction

Zaštitna uloga amlodipina, blokatora kalcijevih kanala, protiv oštećenja mitohondrija kod ishemičnih i reperfuzijskih ozljeda jetre štakora

Ca2+ accumulation and Ca2+ overloading into mitochondria are responsible for the cell abnormality associated with ischemia and reperfusion injury. The present study was aimed to evaluate the efficacy of Ca2+ channel blocker- amlodipine on the mitochondrial Ca2+ accumulation, mitochondrial antioxidant status and mitochondrial respiratory enzymes in ischemic - reperfusion (I/R) induced liver injury. I/R injury induced mitochondrial damage in rats was assessed in terms of decrease in activities (p< 0.05) of respiratory marker enzymes (malate dehydrogenase, succinate dehydrogenase and NADH –dehydrogenase), mitochondrial antioxidant enzymes (glutathione, superoxide dismutase, catalase), level of lipid peroxidation.(LPO), and Ca2+ accumulation were assessed as marker of mitochondrial damage. Mitochondrial damage was confirmed by transmission electron microscopic (TEM) examination. Pretreatment with amlodipine effectively counteracted the alternation in mitochondrial enzymes induced by ischemia-reperfusion liver damage. TEM study confirms the restoration of cellular normalcy and accredits the cytoprotective role of amlodipine against I/R induced hepatic injury. On the basis of our findings it may be concluded that amlodipine not only possesses Ca2+ channel antagonist properties but it may also reduce the extent of mitochondrial damage by its antioxidant activity.Akumulacija Ca2+ iona i njihovo nakupljanje u mitohondrijima uzrok je abnormalnosti u stanicama nakon ishemičnih i reperfuzijskih ozljeda. Cilj ovog rada bio je ispitati učinak amlodipina, blokatora kalcijevih kanala, na nakupljanje iona kalcija u mitohondrijima, antioksidativni status mitohondrija i na aktivnost enzima u dišnom lancu kod ishemičnih i reperfuzijskih (I/R) ozljeda jetre. Kod tih ozljeda smanjeno je djelovanje (p < 0,05) enzima dišnog lanca (malat dehidrogenaze, sukcinat dehidrogenaze i NADH-dehidrogenaze), antioksidativnih enzima mitohondrija (glutation, superoksid dismutaze, katalaze), stupanj lipidne peroksidacije (LPO) i nakupljanje iona Ca2+. Oštećenje mitohondrija potvrđeno je transmisijskom elektronskom mikroskopijom (TEM). Prethodna obrada s amlodipinom učinkovito sprječava promjenu aktivnosti enzima mitohondrija uzrokovanih I/R oštećenjima jetre. TEM potvrđuje uspostavljanje normalnih uvjeta u stanici i citoprotektivno djelovanje amlodipina. Na temelju rezultata naših istraživanja može se zaključiti da amlodipin zbog antioksidativnog djelovanja reducira oštećenje mitohondrija

HSV1/2 Genital Infection in Mice Cause Reversible Delayed Gastrointestinal Transit: A Model for Enteric Myopathy

In an interesting investigation by Khoury-Hanold et al. (1), genital infection of mice with herpes simplex virus 1 (HSV1) were reported to cause multiple pelvic organ involvement and obstruction. A small subset of mice succumbed after the first week of HSV1 infection. The authors inferred that the mice died due to toxic megacolon. In a severe form of mechanical and/or functional obstruction involving gross dilation of the colon and profound toxemia, the presentation is called “toxic megacolon.” The representative observations by Khoury-Hanold likely do not resemble toxic megacolon. The colon was only slightly dilated and benign appearing. Importantly, HSV1 infection affected the postjunctional mechanisms of smooth muscle relaxation like the sildenafil-response proteins, which may have been responsible for defective nitrergic neurotransmission and the delayed transit. Orally administered polyethylene glycol reversed the gastrointestinal “obstruction,” suggesting a mild functional type of slowed luminal transit, resembling constipation, rather than toxic megacolon, which cannot be reversed by an osmotic laxative without perforating the gut. The authors suggest that the mice did not develop HSV1 encephalitis, the commonly known cause of mortality. The premature death of some of the mice could be related to the bladder outlet obstruction, whose backflow effects may alter renal function, electrolyte abnormalities and death. Muscle strip recordings of mechanical relaxation after electrical field stimulation of gastrointestinal, urinary bladder or cavernosal tissues shall help obtain objective quantitative evidence of whether HSV infection indeed cause pelvic multi-organ dysfunction and impairment of autonomic neurotransmission and postjunctional electromechanical relaxation mechanisms of these organs

Structure-based prediction of Wnt binding affinities for Frizzled-type cysteine-rich domain

Wnt signaling pathways are of significant interest in development and oncogenesis. The first step in these pathways typically involves the binding of a Wnt protein to the cysteine-rich domain (CRD) of a Frizzled receptor; Wnt-Frizzled interactions can be antagonized by secreted Frizzled-related proteins (sFRPs), which also contain a Frizzled-like CRD. The large number of Wnts, Frizzleds and sFRPs, as well as the hydrophobic nature of Wnt, pose challenges to laboratory-based investigations of interactions involving Wnt. Here, utilizing structural knowledge of a representative Wnt-Frizzled CRD interaction, as well as experimentally-determined binding affinities for a selection of Wnt-Frizzled CRD interactions, we generate homology models of Wnt-Frizzled CRD interactions and develop a quantitative structure-activity relationship for predicting their binding affinities. The derived model incorporates a small selection of terms derived from scoring functions used in protein-protein docking, as well as an energetic term considering the contribution made by the lipid of Wnt to the Wnt-Frizzled binding affinity. Validation with an external test set suggests that the model can accurately predict binding affinity for 75% of cases, and that the error associated with the predictions is comparable to the experimental error. The model was applied to predict the binding affinities of the full range of mouse and human Wnt-Frizzled and Wnt-sFRP interactions, indicating trends in Wnt binding affinity for Frizzled and sFRP CRDs. The comprehensive predictions made in this study provide the basis for laboratory-based studies of previously unexplored Wnt-Frizzled and Wnt-sFRP interactions, which in turn, may reveal further Wnt signaling pathways

A Hypothesis for Examining Skeletal Muscle Biopsy-Derived Sarcolemmal nNOSμ as Surrogate for Enteric nNOSα Function

The pathophysiology of gastrointestinal motility disorders is controversial and largely unresolved. This provokes empiric approaches to patient management of these so-called functional gastrointestinal disorders (FGIDs). Preliminary evidence demonstrate that defects in nNOS expression and function, the enzyme that synthesizes nitric oxide (NO), the key inhibitory neurotransmitter mediating mechano-electrical smooth muscle relaxation, is the major pathophysiological basis for sluggishness of oro-aboral transit of luminal contents. This opinion is an ansatz of the potential of skeletal muscle biopsy and examining sarcolemmal nNOSµ to provide complementary insights regarding nNOSα expression, localization and function within enteric nerve terminals, the site of stimulated de novo NO synthesis. The main basis of this thesis is two-folds: (a) the molecular similarity of the structures of nNOS α and µ, similar mechanisms of localizations to active zones of nitrergic synthesis and same mechanisms of electron transfers during NO synthesis (b) pragmatic difficulty to routinely obtain full-thickness biopsies of gastrointestinal tract, even in patients presenting with the most recalcitrant manifestations of stasis and delayed transit of l