Methylene Blue Inhibits Cromakalim-Activated K+ Currents in Follicle-Enclosed Oocytes.

The effects of methylene blue (MB) on cromakalim-induced K+ currents were investigated in follicle-enclosed Xenopus oocytes. In concentrations ranging from 3-300 µM, MB inhibited K+ currents (IC50: 22.4 µM) activated by cromakalim, which activates KATP channels. MB inhibited cromakalim-activated K+ currents in a noncompetitive and voltage-independent manner. The respective EC50 and slope values for cromakalim-activation of K+ currents were 194 ± 21 µM and 0.91 for controls, and 206 ± 24 µM and 0.87 in the presence of 30 µM MB. The inhibition of cromakalim-induced K+ currents by MB was not altered by pretreatment with the Ca2+ chelator BAPTA, which suggests that MB does not influence Ca2+-activated second messenger pathways. K+ currents mediated through a C-terminally deleted form of Kir6.2 (KirΔC26), which does not contain the sulfonylurea receptor, were still inhibited by MB, indicating direct interaction of MB with the channel-forming Kir6.2 subunit. The binding characteristics of the KATP ligand [3H]glibenclamide are not altered by MB in a concentration range between 1 µM-1 mM, as suggested by radioligand binding assay. The presence of a membrane permeable cGMP analogue (8-Br-cGMP, 100 µM) and a guanylate cyclase activator (BAY 58-2667, 3 µM) did not affect the inhibitory effects of MB, suggesting that MB does not inhibit cromakalim-activated K+ currents through guanylate cyclase. Collectively, these results suggest that MB directly inhibits cromakalim-activated K+ currents in follicular cells of Xenopus oocytes.

Multifunctional angiotensin converting enzyme 2, the SARS-CoV-2 entry receptor, and critical appraisal of its role in acute lung injury.

The recent emergence of coronavirus disease-2019 (COVID-19) as a pandemic affecting millions of individuals has raised great concern throughout the world, and the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was identified as the causative agent for COVID-19. The multifunctional protein angiotensin converting enzyme 2 (ACE2) is accepted as its primary target for entry into host cells. In its enzymatic function, ACE2, like its homologue ACE, regulates the renin-angiotensin system (RAS) critical for cardiovascular and renal homeostasis in mammals. Unlike ACE, however, ACE2 drives an alternative RAS pathway by degrading Ang-II and thus operates to balance RAS homeostasis in the context of hypertension, heart failure, and cardiovascular as well as renal complications of diabetes. Outside the RAS, ACE2 hydrolyzes key peptides, such as amyloid-ß, apelin, and [des-Arg9]-bradykinin. In addition to its enzymatic functions, ACE2 is found to regulate intestinal amino acid homeostasis and the gut microbiome. Although the non-enzymatic function of ACE2 as the entry receptor for SARS-CoV-2 has been well established, the contribution of enzymatic functions of ACE2 to the pathogenesis of COVID-19-related lung injury has been a matter of debate. A complete understanding of this central enzyme may begin to explain the various symptoms and pathologies seen in SARS-CoV-2 infected individuals, and may aid in the development of novel treatments for COVID-19.

A comprehensive guide to the pharmacologic regulation of angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 entry receptor.

The recent emergence of coronavirus disease-2019 (COVID-19) as a global pandemic has prompted scientists to address an urgent need for defining mechanisms of disease pathology and treatment. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for COVID-19, employs angiotensin converting enzyme 2 (ACE2) as its primary target for cell surface attachment and likely entry into the host cell. Thus, understanding factors that may regulate the expression and function of ACE2 in the healthy and diseased body is critical for clinical intervention. Over 66% of all adults in the United States are currently using a prescription drug and while earlier findings have focused on possible upregulation of ACE2 expression through the use of renin angiotensin system (RAS) inhibitors, mounting evidence suggests that various other widely administered drugs used in the treatment of hypertension, heart failure, diabetes mellitus, hyperlipidemias, coagulation disorders, and pulmonary disease may also present a varied risk for COVID-19. Specifically, we summarize mechanisms on how heparin, statins, steroids and phytochemicals, besides their established therapeutic effects, may also interfere with SARS-CoV-2 viral entry into cells. We also describe evidence on the effect of several vitamins, phytochemicals, and naturally occurring compounds on ACE2 expression and activity in various tissues and disease models. This comprehensive review aims to provide a timely compendium on the potential impact of commonly prescribed drugs and pharmacologically active compounds on COVID-19 pathology and risk through regulation of ACE2 and RAS signaling.

Capsaicin Is a Negative Allosteric Modulator of the 5-HT3 Receptor.

In this study, effects of capsaicin, an active ingredient of the capsicum plant, were investigated on human 5-hydroxytryptamine type 3 (5-HT3) receptors. Capsaicin reversibly inhibited serotonin (5-HT)-induced currents recorded by two-electrode voltage clamp method in Xenopus oocytes. The inhibition was time- and concentration-dependent with an IC50 = 62 µM. The effect of capsaicin was not altered in the presence of capsazepine, and by intracellular BAPTA injections or trans-membrane potential changes. In radio-ligand binding studies, capsaicin did not change the specific binding of the 5-HT3 antagonist [3H]GR65630, indicating that it is a noncompetitive inhibitor of 5-HT3 receptor. In HEK-293 cells, capsaicin inhibited 5-HT3 receptor induced aequorin luminescence with an IC50 of 54 µM and inhibition was not reversed by increasing concentrations of 5-HT. In conclusion, the results indicate that capsaicin acts as a negative allosteric modulator of human 5-HT3 receptors.

Extracellular matrix alterations, accelerated leukocyte infiltration and enhanced axonal sprouting after spinal cord hemisection in tenascin-C-deficient mice.

The extracellular matrix glycoprotein tenascin-C has been implicated in wound repair and axonal growth. Its role in mammalian spinal cord injury is largely unknown. In vitro it can be both neurite-outgrowth promoting and repellent. To assess its effects on glial reactions, extracellular matrix formation, and axonal regrowth/sprouting in vivo, 20 tenascin-C-deficient and 20 wild type control mice underwent lumbar spinal cord hemisection. One, three, seven and fourteen days post-surgery, cryostat sections of the spinal cord were examined by conventional histology and by immunohistochemistry using antibodies against F4/80 (microglia/macrophage), GFAP (astroglia), neurofilament, fibronectin, laminin and collagen type IV. Fibronectin immunoreactivity was significantly down-regulated in tenascin-C-deficient mice. Moreover, fourteen days after injury, immunodensity of neurofilament-positive fibers was two orders of magnitude higher along the incision edges of tenascin-C-deficient mice as compared to control mice. In addition, lymphocyte infiltration was seen two days earlier in tenascin-C-deficient mice than in control mice and neutrophil infiltration was increased seven days after injury. The increase in thin neurofilament positive fibers in tenascin-C-deficient mice indicates that lack of tenascin-C alters the inflammatory reaction and extracellular matrix composition in a way that penetration of axonal fibers into spinal cord scar tissue may be facilitated.

Study of the spinal cords of the sturgeon Acipenser schrenckii, gar Lepisosteus oculatus, and goldfish Carassius auratus by morphological, immunohistochemical, and biochemical approaches.

Little is known about the spinal cords of phylogenetically ancient actinopterygeans. The spinal cords of the chondrostean Acipenser schrenckii (Amur sturgeon), holostean Lepisosteus oculatus (spotted gar), and teleost Carassius auratus (goldfish) were, therefore, analyzed by immunohistochemistry, electron microscopy and two-dimensional gel electrophoresis. Morphology showed numerous similarities between sturgeons and gars. In both, a dorsal column between the two dorsal horns was lacking, giving the grey matter an inverted Y-shape. In goldfish, a small dorsal column was seen, the grey matter occupied a larger area, neuronal density was much higher, and a ventral commissure was apparent, which was absent in sturgeons and gars. In the white matter of sturgeons and gars, small caliber axons predominated, whereas larger axons were frequent in goldfish. Choline acetyltransferase immunoreactive neurons were prevalent in the ventral horns of all three fish, mainly in motoneurons, but stained fibers were only found in sturgeons and gars. gamma-aminobutyric acid positive cells were seen in both the ventral and the dorsal horns of all three fish. Distribution of serotonin (5-HT) and tyrosine hydroxylase (TH) immunoreaction was similar in sturgeons and gars, being located in both the ventral and the dorsal horns. In goldfish, 5-HT label was confined to the ventral horn and TH label was mainly observed in a cell group located ventromedially. Two-dimensional gel electrophoresis showed a gradual increase in protein number from sturgeons to gars to goldfish. In conclusion, the spinal cords of sturgeons and gars share many morphological and chemical features, distinguishing them from the goldfish spinal cord.

Morphogenesis of the different types of photoreceptors of the chicken (Gallus domesticus) retina and the effect of amblyopia in neonatal chicken.

Despite the great variety in chicken photoreceptors, existing morphogenetic studies only deal with two types: rods and cones. We have therefore examined by scanning electron microscopy the first appearance and maturation of different retinal photoreceptors in 36 chicken embryos (Gallus domesticus), aged 5-19 days prehatching. On day 5 of incubation, chicken retinae were only composed of proliferating ventricular cells devoid of photoreceptors. On day 8, outer mitotic cells were separated from inner differentiating photoreceptors, by the transient layer of Chievitz. Ball-like protrusions appeared at the ventricular surface, representing the first signs of photoreceptor inner segment formation. From day 10 onward, double cones, single cones, and rods could be clearly distinguished, and occasional cilia were detected at their tip. On day 12, inner segments had increased in length and diameter, and frequently carried a cilium representing the beginning of outer segment formation. On day 14, most photoreceptors displayed a distinct outer segment. On day 19, photoreceptors had essentially assumed adult morphology. Based on the shape of their outer segments, two subtypes of cones and three subtypes of double cones could be distinguished. Throughout development, we observed microvilli close to maturing photoreceptors, either originating from their lateral sides, from their tip, or from Müller cells. Microvillus density peaked between day 12 and 14, indicating an important role in photoreceptor morphogenesis. Unilateral occlusion of the eyes of posthatching chicken reduced the proportion of double cones to single cones in the retina, indicating dependence of retinal morphogenesis upon functional activity of visual cells.

Early postnatal sound exposure induces lasting neuronal changes in the inferior colliculus of senescence accelerated mice (SAMP8): a morphometric study on GABAergic neurons and NMDA expression.

Senescence-acceleration-prone mice (SAMP8) provide a model to study the influence of early postnatal sound exposure upon the aging auditory midbrain. SAMP8 were exposed to a 9-kHz monotone of either 53- or 65-dB sound pressure level during the first 30 postnatal days, the neurons in the auditory midbrain responding selectively to 9 kHz were localized by c-fos immunohistochemistry and the following parameters were compared to control SAMP8 not exposed to sound: mortality after sound exposure, dendritic spine density, and quantitative neurochemical alterations in this 9-kHz isofrequency lamina. For morphometric analysis, animals were examined at 1, 4, and 8 months of age. Serial sections of the inferior colliculus were Golgi impregnated or stained immunohistochemically for the expression of epsilon1 subunit of NMDA receptor or GABA. Mortality after exposure to 53 dB was the same as in controls, but was markedly increased from 7 months of age onward after postnatal exposure to 65 dB. No gross morphological alterations were observed in the auditory midbrain after sound exposure. However, sound exposure to 53 or 65 dB significantly reduced dendritic spine density by 11% at 4 months or by 11-17% both at 1 and 4 months of age, respectively. The effect of sound exposure upon neurons expressing the NMDAepsilon1 subunit was dose-dependent. Increasing with age until 4 months in control mice and remaining essentially stable thereafter, the percentage of NMDAepsilon1-immunoreactive neurons was significantly elevated by 40-66% in 1- and 8-month-old SAMP8 exposed to 53 dB, whereas no significant effect of 65 dB was apparent. The proportion of GABAergic cells declined with age in controls. It was significantly decreased at 1 month after 53 and 65 dB sound exposure. In contrast, it was elevated at later stages, being significantly increased at 4 months after exposure to 53 dB and at 8 months after exposure to 65 dB. The total cell number in the 9-kHz isofrequency lamina of SAMP8 decreased with age, but was not affected by exposure to either 53 or 65 dB. The present results indicate that early postnatal exposure to a monotone of mild intensity has long-term effects upon the aging auditory brain stem. Some of the changes induced by sound exposure, e.g., decline in spine density, are interpreted as accelerations of the normal aging process, whereas other effects, e.g., increased NMDAepsilon1 expression after 53 dB and elevated GABA expression after both 53 and 65 dB, are not merely explicable by accelerated aging.