Redox Switches Controlling Nitric Oxide Signaling in the Resistance Vasculature and Implications for Blood Pressure Regulation: Mid-Career Award for Research Excellence 2020.

The arterial resistance vasculature modulates blood pressure and flow to match oxygen delivery to tissue metabolic demand. As such, resistance arteries and arterioles have evolved a series of highly orchestrated cell-cell communication mechanisms between endothelial cells and vascular smooth muscle cells to regulate vascular tone. In response to neurohormonal agonists, release of several intracellular molecules, including nitric oxide, evokes changes in vascular tone. We and others have uncovered novel redox switches in the walls of resistance arteries that govern nitric oxide compartmentalization and diffusion. In this review, we discuss our current understanding of redox switches controlling nitric oxide signaling in endothelial and vascular smooth muscle cells, focusing on new mechanistic insights, physiological and pathophysiological implications, and advances in therapeutic strategies for hypertension and other diseases.

Nitric oxide impacts bovine sperm capacitation in a cGMP-dependent and cGMP-independent manner.

We aimed to elucidate whether NO acts in in vitro sperm capacitation in bovine via cGMP/PKG1 pathway. For this, cryopreserved bovine sperm were capacitated in vitro with 20 µg/ml heparin (Control) plus treatments: 1 mM L-arginine (L-arg, NO precursor), 50 µM Rp-8-Bromo-ß-phenyl-1,N2 -ethenoguanosine-3',5'-cyclic monophosphorothioate (Rp-8-Br-cGMPS, selective inhibitor of the binding site for cGMP in PKG1), 1 mM 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO, NO scavenger), and the combinations of L-arg + RP-8-Br-cGMPS and L-arg + PTIO. Sperm motility and vigour were determined by phase-contrast microscopy, capacitation status by chlortetracycline staining, and the intracellular concentration of cGMP was measured by ELISA. Data were subjected to analysis of variance and means compared with SNK test at 5% probability. Motility and vigour were lower in sperm treated with PTIO when compared to Control and other treatments (p < .05). The L-arg treatment showed the highest percentage of capacitated sperm when compared to the Control and other treatments (Rp-8-Br-cGMPS, L-arg + Rp-8-Br-cGMPS and PTIO) (69.8 ± 3.4%, 51.2 ± 3.0, 51.1 ± 2.1, 51.2 ± 3.0 and 45.5 ± 2.7, respectively) (p < .05). The capacitation ratio (%) was lower in treatments with Rp-8-Br-cGMPS, L-arg + Rp-8-Br-cGMPS and PTIO, respectively (p < .05). Lastly, cGMP concentration (pmol/ml) was lower in PTIO and L-arg + PTIO (1.3 ± 0.3 and 1.6 ± 0.4) and was higher in Rp-8-Br-cGMPS and L-arg + Rp-8-Br-cGMPS (3.7 ± 0.4 and 4.0 ± 0.5) treatments. We showed that during in vitro capacitation of cattle: (a) NO influences sperm motility and vigour; (b) NO is associated with cGMP synthesis through two independent pathways and (c) the cGMP/PKG1 pathway has a partial role in sperm capacitation and does not involve the L-arg/NO.

The granulopoietic cytokine granulocyte colony-stimulating factor (G-CSF) induces pain: analgesia by rutin.

Rutin is a glycone form of the flavonol quercetin and it reduces inflammatory pain in animal models. Therapy with granulocyte colony-stimulating factor (G-CSF) is known by the pain caused as its main side effect. The effect of rutin and its mechanisms of action were evaluated in a model of hyperalgesia induced by G-CSF in mice. The mechanical hyperalgesia induced by G-CSF was reduced by treatment with rutin in a dose-dependent manner. Treatment with both rutin + morphine or rutin + indomethacin, at doses that are ineffectual per se, significantly reduced the pain caused by G-CSF. The nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG)-ATP-sensitive potassium channel (KATP) signaling pathway activation is one of the analgesic mechanisms of rutin. Rutin also reduced the pro-hyperalgesic and increased anti-hyperalgesic cytokine production induced by G-CSF. Furthermore, rutin inhibited the activation of the nuclear factor kappa-light-chain enhancer of activated B cells (NFκB), which might explain the inhibition of the cytokine production. Treatment with rutin upregulated the decreased mRNA expression of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) combined with enhancement of the mRNA expression of the Nrf2 downstream target heme oxygenase (HO-1). Intraperitoneal (i.p.) treatment with rutin did not alter the mobilization of neutrophils induced by G-CSF. The analgesia by rutin can be explained by: NO-cGMP-PKG-KATP channel signaling activation, inhibition of NFκB and triggering the Nrf2/HO-1 pathway. The present study demonstrates rutin as a promising pharmacological approach to treat the pain induced by G-CSF without impairing its primary therapeutic benefit of mobilizing hematopoietic progenitor cells into the blood.

Role of protein kinase G on the post-shock mesenteric lymph blockage ameliorating vascular calcium sensitivity.

PURPOSE: To investigate the role of protein kinase G (PKG) in blocking post-shock mesenteric lymph (PSML) return ameliorating the calcium sensitivity in hemorrhagic shock rats. METHODS: Male Wistar rats were randomly divided into sham, shock, shock+ligation (shock plus mesenteric lymph duct ligation (MLDL)), shock+drainage (shock plus PSML drainage) groups. After shock (hypotension 40 mm Hg) for three hours or corresponding times, the superior mesenteric artery (SMA) was taken out for detecting the PKG and phospho PKG (p-PKG) contents, and the vascular rings of SMA were prepared for assaying the calcium sensitivity using an isolated organ perfusion system. RESULTS: The PKG and p-PKG contents of SMA in shock group were significantly increased than that of sham group, and MLDL or PSML drainage reducing the levels of PKG and p-PKG. Meanwhile, the vascular calcium sensitivity in shock group was significantly lower than that of sham group, MLDL or PSML drainage enhanced the calcium sensitivity. After incubating with PKG regulators in shock+ligation and shock+drainage groups, the PKG agonist 8Br-cGMP reduced the contractility of vascular rings to gradient calcium ions and Emax and the PKG inhibitor agonist KT5823 elevated the calcium sensitivity significantly. CONCLUSION: Protein kinase G plays an important role in post-shock mesenteric lymph blockage improving vascular calcium sensitivity.

Role of protein kinase G on the post-shock mesenteric lymph blockage ameliorating vascular calcium sensitivity

PURPOSE: To investigate the role of protein kinase G (PKG) in blocking post-shock mesenteric lymph (PSML) return ameliorating the calcium sensitivity in hemorrhagic shock rats. METHODS: Male Wistar rats were randomly divided into sham, shock, shock+ligation (shock plus mesenteric lymph duct ligation (MLDL)), shock+drainage (shock plus PSML drainage) groups. After shock (hypotension 40mmHg) for three hours or corresponding times, the superior mesenteric artery (SMA) was taken out for detecting the PKG and phospho PKG (p-PKG) contents, and the vascular rings of SMA were prepared for assaying the calcium sensitivity using an isolated organ perfusion system. RESULTS: The PKG and p-PKG contents of SMA in shock group were significantly increased than that of sham group, and MLDL or PSML drainage reducing the levels of PKG and p-PKG. Meanwhile, the vascular calcium sensitivity in shock group was significantly lower than that of sham group, MLDL or PSML drainage enhanced the calcium sensitivity. After incubating with PKG regulators in shock+ligation and shock+drainage groups, the PKG agonist 8Br-cGMP reduced the contractility of vascular rings to gradient calcium ions and Emax and the PKG inhibitor agonist KT5823 elevated the calcium sensitivity significantly. CONCLUSION: Protein kinase G plays an important role in post-shock mesenteric lymph blockage improving vascular calcium sensitivity.

Vasorelaxant action of the total alkaloid fraction obtained from Solanum paludosum Moric. (Solanaceae) involves NO/cGMP/PKG pathway and potassium channels.

ETHNOPHARMACOLOGICAL RELEVANCE: Solanum paludosum Moric. (jurubeba-roxa) is commonly used to treat hypertension as a substitute for Solanum paniculatum L. (jurubeba verdadeira). The total ethanolic extract from the root bark of Solanum paludosum have been found to cause hypotension in rats. AIM OF THE STUDY: To investigate the mechanism by which the total alkaloid fraction obtained from the root bark of Solanum paludosum (FAT-SP) acts as a vasorelaxant agent on rat thoracic aorta. MATERIALS AND METHODS: Rings of rat aorta were suspended in organ bath containing Krebs solution at 37°C, bubbled with carbogen mixture (95% O(2) and 5% CO(2)) under a resting tension of 1 g. Isometric contractions were measured using a force transducer coupled to an amplifier and a microcomputer. RESULTS: FAT-SP has been found cause relaxation of the aortic rings pre-contracted with phenylephrine (Phe) in a concentration-dependent manner, in the presence and absence of endothelium. This effect was more potent on the endothelium-intact aorta. In the presence of endothelium, neither indomethacin (non-selective cyclooxygenase inhibitor) nor atropine (non-selective muscarinic receptor antagonist), produced significant changes on the relaxation response. On the other hand, in the presence of calmidazolium (a calmodulin inhibitor), N-nitro-L-arginine methyl ester (L-NAME, nitric oxide synthase inhibitor), hydroxocobalamin (HDX) (scavenger of free-radical nitric oxide), 1-H-[1,2,4]-oxadiazolo-[4,3a]-quinoxalin-1-one (ODQ, selective blocker of soluble guanylate cyclase), Rp-8-bromo-ß-phenyl-1,N(2)-ethenoguanosine 3':5'-cyclic monophosphorothioate sodium salt hydrate (Rp-8-Br-PET-cGMPS, competitive inhibitor of cGMP-dependent protein kinase G) or TEA(+) (tetraethylammonium, nonselective potassium channel blocker), the vasorelaxant effect was significantly reduced, suggesting the involvement of NO/sCG/PKG pathway and potassium channel opening in vasorelaxant action of the FAT-SP. CONCLUSION: The mechanism of vasorelaxant activity of the FAT-SP on rat aorta involves both NO/sCG/PKG pathway and potassium channels.

Biotin increases glucokinase expression via soluble guanylate cyclase/protein kinase G, adenosine triphosphate production and autocrine action of insulin in pancreatic rat islets.

Besides its role as a carboxylase prosthetic group, biotin has important effects on gene expression. However, the molecular mechanisms through which biotin exerts these effects are largely unknown. We previously found that biotin increases pancreatic glucokinase expression. We have now explored the mechanisms underlying this effect. Pancreatic islets from Wistar rats were treated with biotin, in the presence or absence of different types of inhibitors. Glucokinase mRNA and 18s rRNA abundance were determined by real-time PCR. Adenosine triphosphate (ATP) content was analyzed by fluorometry. Biotin treatment increased glucokinase mRNA abundance approximately one fold after 2 h; the effect was sustained up to 24 h. Inhibition of soluble guanylate cyclase or protein kinase G (PKG) signalling suppressed biotin-induced glucokinase expression. The cascade of events downstream of PKG in biotin-mediated gene transcription is not known. We found that inhibition of insulin secretion with diazoxide or nifedipine prevented biotin-stimulated glucokinase mRNA increase. Biotin treatment increased islet ATP content (control: 4.68+/-0.28; biotin treated: 6.62+/-0.26 pmol/islet) at 30 min. Inhibition of PKG activity suppressed the effects of biotin on ATP content. Insulin antibodies or inhibitors of phosphoinositol-3-kinase/Akt insulin signalling pathway prevented biotin-induced glucokinase expression. The nucleotide 8-Br-cGMP mimicked the biotin effects. We propose that the induction of pancreatic glucokinase mRNA by biotin involves guanylate cyclase and PKG activation, which leads to an increase in ATP content. This induces insulin secretion via ATP-sensitive potassium channels. Autocrine insulin, in turn, activates phosphoinositol-3-kinase/Akt signalling. Our results offer new insights into the pathways that participate in biotin-mediated gene expression.

Possible participation of the nitric oxide-cyclic GMP-protein kinase G-K+ channels pathway in the peripheral antinociception of melatonin.

The possible participation of the nitric oxide (NO)-cyclic GMP-protein kinase G (PKG)-K(+) channel pathway on melatonin-induced local antinociception was assessed during the second phase of the formalin test. The local peripheral ipsilateral, but not contralateral, administration of melatonin (150-600 microg/paw) produced a dose-related antinociception during both phases of the formalin test in rats. Moreover, local pretreatment with N(G)-L-nitro-arginine methyl ester (L-NAME, NO synthesis inhibitor, 10-100 microg/paw), 1H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one (ODQ, guanylyl cyclase inhibitor, 5-50 microg/paw), (9S, 10R, 12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo [1,2,3-fg:3',2',1'-kl]pyrrolo [3,4-i][1,6] benzodiazocine-10-carboxylic acid methyl ester (KT-5823, specific PKG inhibitor, 50-500 ng/paw), glibenclamide (ATP-sensitive K(+) channel blocker, 5-50 microg/paw), apamin (small-conductance Ca(2+)-activated K(+) channel blocker, 0.1-1 microg/paw) or charybdotoxin (large- and intermediate-conductance Ca(2+)-activated K(+) channel blocker, 0.03-0.3 microg/paw), but not N(G)-D-nitro-arginine methyl ester (D-NAME, inactive isomer of L-NAME, 100 microg/paw) or vehicle, significantly prevented melatonin (300 microg/paw)-induced antinociception. Data suggest that melatonin-induced local peripheral antinociception during the second phase of the test could be due to activation of the NO-cyclic GMP-PKG-ATP-sensitive and Ca(2+)-activated K(+) channels pathway.

[Signal transduction, pillar of the neurobiological integration of memory. An alternative view to the cholinergic hypothesis]. / Transducción de la señal, pivote de la integración neurobiológica de la memoria. Una propuesta diferente a la tradicional hipótesis del sistema colinérgico.

Neurophysiological, biochemical and molecular processes described in the integration of memory are closely related with neurotransmitters such as glutamate and serotonin (SHT) and with the function of calcium and potassium ion channels more than with cholinergic activity. Infact, glutamate and 5-HT receptors are closely related with Long-Term potentiation (L TP) processes, the mechanism by which memory is preserved throughout time. That is, the activation of the 5-HTI receptor triggers a transduction signal that after influencing nuclear cell activity, provokes several presynaptic changes, which leads to the displacement of magnesium from the postsynaptic area depolarizing the neuron and leading to the activation of N-methyl-D-aspartate receptors (NMDA). As a whole, this process contributes to the support and perpetuation of LTP, which consists of the following processes: LTPI that depends on protein kinase activity; LTP2 linked to translation of genes; and LTP3 closely related to genes transcription. On the opposite side but in perfect balance, we find the mechanism of Long-Term depression (LTD), which is triggered instead when the Ca+ +flow decreases in the presynaptic neuron activating the inhibitor-1 enzyme that promotes the dephosphorylation of a calmodulin-dependent protein kinasell and as a result, the inhibition of autophosphorylation and consequently of LTP too. Despite the widespread dissemination of the cholinergic hypothesis in Alzheimer's disease, memory build up rather than involving acetylcholine essentially depends on the participation of other neurotransmitters such as 5-HT and glutamate, which have not been adequately considered in the treatment of this disease. However, beyond neurotransmission, it is the cellular mechanism of autophosphorylation of several protein kinases, the process susceptible of being activated or controlled by the action of distinct substances. In such a case, it would be possible to exert some influence on gene expression improving perhaps, some of the physiopathological deficits that characterize memory disruption.

Transducción de la señal, pivote de la integración neurobiológica de la memoria: Una propuesta diferente a la tradicional hipótesis del sistema colinérgico / Signal transduction, pillar of the neurobiological integration of memory: An alternative view to the cholinergic hypothesis

Los procesos neurofisiológicos, bioquímicos y moleculares descritos en la integración de la memoria, más que estar relacionados con la actividad colinérgica involucran fundamentalmente a neurotransmisores como la serotonina y el glutamato, así como a diversos canales iónicos como los del calcio y los del potasio. De hecho, los receptores de estos neurotransmisores están ligados directamente con la activación de la potenciación a largo plazo (LTP), mecanismo que contribuye a la preservación de la memoria. De esta forma que la activación del receptor 5HT desencadena una señal de transducción que al influenciar bioquímicamente al núcleo produce diversos cambios presinápticos con los que se expulsa al magnesio del área postsináptica, despolarizando a la neurona y activando simultáneamente a los receptores N metilD Aspartato dependientes (NMDAR), contribuyendo en esta forma a perpetuar el mecanismo de LTP en sus distintas fases: LTP1 que depende de la activación de proteincinasas; LTP2 ligada con la traslación genética; y LTP3 relacionada con la transcripción. A este poderoso mecanismo de activación neuronal, se contrapone el fenómeno de depresión a largo plazo (LTD), que se inicia cuando la neurona pre sináptica activa al inhibidor 1 en el momento en que detecta una reducción en el influjo de calcio, promoviendo en esta forma la defosforilación de una proteincinasa tipo II calcio calmodulin dependiente, lo que detiene el desarrollo del proceso de autofosforilación y con ello, el mecanismo de LTP. No obstante lo difundido de la hipótesis colinérgica en la enfermedad de Alzheimer, la integración de la memoria depende fundamentalmente de la intervención de otros sistemas de neurotransmisión como lo son el serotonérgico y el glutamatérgico, los que no han sido debidamente considerados en el tratamiento de esta enfermedad; sin embargo más allá de estos sistemas, se encuentran los mecanismos de autofosforilación de distintas proteincinasas cuyo control, además de repercutir sobre la expresión genética, podría restituir algunos de los trastornos que afectan la función cognoscitiva.

Neurophysiological, biochemical and molecular processes described in the integration of memory are closely related with neurotransmitters such as glutamate and serotonin (5HT) and with the function of calcium and potassium ion channels more than with cholinergic activity. In fact, glutamate and 5 HT receptors are closely related with Long-Term Potentiation (LTP) processes, the mechanism by which memory is preserved throughout time. That is, the activation of the 5 HT4 receptor triggers a transduction signal that after influencing nuclear cell activity, provokes several presynaptic changes, which leads to the displacement of magnesium from the postsynaptic area depolarizing the neuron and leading to the activation of N methyl -D-aspartate receptors (NMDA). As a whole, this process contributes to the support and perpetuation of LTP, which consists of the following processes: LTP1 that depends on protein kinase activity; LTP2 linked to translation of genes; and LTP3 closely related to genes transcription. On the opposite side but in perfect balance, we find the mechanism of Long Term depression (LTD), which is triggered instead when the Ca++ flow decreases in the presynaptic neuron activating the inhibitor 1 enzyme that promotes the dephosphorylation of a calmodulin dependent protein kinase II and as a result, the inhibition of autophosphorylation and consequently of LTP too. Despite the widespread dissemination of the cholinergic hypothesis in Alzheimer's disease, memory build up rather than involving acetylcholine essentially depends on the participation of other neurotransmitters such as 5 HT and glutamate, which have not been adequately considered in the treatment of this disease. However, beyond neurotransmission, it is the cellular mechanism of autophosphorylation of several protein kinases, the process susceptible of being activated or controlled by the action of distinct substances. In such a case, it would be possible to exert some influence on gene expression improving perhaps, some of the physiopathological deficits that characterize memory disruption.

Angiotensin-(1-7) inhibitory mechanism of norepinephrine release in hypertensive rats.

Release of norepinephrine (NE) by the hypothalamic nuclei may contribute to regulation of sympathetic nervous system (SNS) activity. Angiotensin-(1-7) [Ang-(1-7)] has an antihypertensive effect and may decrease SNS activity. We tested the hypothesis that Ang-(1-7) inhibits the release of NE in hypothalami, via the Ang-(1-7) and angiotensin II type 2 (AT2) receptors, acting through a bradykinin (BK)/NO-dependent mechanism. Hypothalami from normotensive controls and spontaneously hypertensive rats (SHR) were isolated and endogenous NE stores labeled by incubating the tissues with [3H]NE. [3H]NE release from the hypothalami was stimulated by KCl in the presence or absence of Ang-(1-7) alone or combined with various antagonists and inhibitors. Ang-(1-7) significantly attenuated K+-induced NE release by hypothalami from normotensive rats but was more potent in SHR. The Ang-(1-7) receptor antagonist [D-Ala7]Ang-(1-7), the AT2 receptor antagonist PD 123319, and the BK B2) receptor antagonist icatibant all blocked the inhibitory effect of Ang-(1-7) on K+-stimulated NE release in SHR. The inhibitory effect of Ang-(1-7) disappeared in the presence of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester and was restored by the precursor of NO, l-arginine. The diminished NE release caused by Ang-(1-7) was blocked by a soluble guanylyl cyclase inhibitor as well as by a cGMP-dependent protein kinase (PKG). We concluded that Ang-(1-7) decreases NE release from the hypothalamus via the Ang-(1-7) or AT2 receptors, acting through a BK/NO-mediated mechanism that stimulates cGMP/PKG signaling. In this way, Ang-(1-7) may decrease SNS activity and exert an antihypertensive effect.

Cyclic 3'-5' guanosine monophosphate-dependent activity in Leishmania amazonensis.

Although there are some data concerning the nitric oxide and the cyclic 3'-5'guanosine monophosphate (cGMP) signaling pathway in trypanosomatids, there is no report about the cGMP-dependent enzymatic activity identification. In this sense, a cGMP dependent activity was detected on soluble fraction from Leishmania amazonensis promastigotes with a high metacyclic level. This information is valuable in order to explore the metabolic pathway of G kinase protein in this parasite.