Raman spectroscopy study of 7,8-dihydrofolate inhibition on the Wuhan strain SARS-CoV-2 binding to human ACE2 receptor.

Emerging evidence suggests that elevated levels of folic acid in the bloodstream may confer protection against Wuhan-SARS-CoV-2 infection and mitigate its associated symptoms. Notably, two comprehensive studies of COVID-19 patients in Israel and UK uncovered a remarkable trend, wherein individuals with heightened folic acid levels exhibited only mild symptoms and necessitated no ventilatory support. In parallel, research has underscored the potential connection between decreased folic acid levels and the severity of Covid-19 among hospitalized patients. Yet, the underlying mechanisms governing this intriguing inhibition remain elusive. In a quest to elucidate these mechanisms, we conducted a molecular dynamics simulation approach followed by a Raman spectroscopy study to delve into the intricate interplay between the folic acid metabolite, 7,8-dihydrofolate (DHF), and the angiotensin-converting enzyme ACE2 receptor, coupled with its interaction with the receptor-binding domain (RBD) of the Wuhan strain of SARS-CoV-2. Through a meticulous exploration, we scrutinized the transformation of the ACE2 + RBD complex, allowing these reactants to form bonds. This was juxtaposed with a similar investigation where ACE2 was initially permitted to react with DHF, followed by the exposure of the ACE2 + DHF complex to RBD. We find that DHF, when bonded to ACE2, functions as a physical barrier, effectively inhibiting the binding of the Wuhan strain RBD. This physicochemical process offers a cogent explanation for the observed inhibition of host cell infection in subjects receiving supplementary folic acid doses, as epidemiologically substantiated in multiple studies. This study not only sheds light on a potential avenue for mitigating SARS-CoV-2 infection but also underscores the crucial role of folic acid metabolites in host-virus interactions. This research paves the way for novel therapeutic strategies in the battle against COVID-19 and reinforces the significance of investigating the molecular mechanisms underlying the protective effects of folic acid in the context of viral infections.

Fatty Acid-Binding Proteins Identification during the Evolution of Metabolic Syndrome: A Raman Spectroscopy-Based Approach.

Excess fat in abdominal deposits is a risk factor for multiple conditions, including metabolic syndrome (MetS); lipid metabolism plays an essential role in these pathologies; fatty acid-binding proteins (FABPs) are dedicated to the cytosolic transport of fat. FABP4, whose primary source is adipose tissue, is released into the circulation, acting as an adipokine, while FABP5 also accompanies the adverse effects of MetS. FABP4 and 5 are potential biomarkers of MetS, but their behavior during syndrome evolution has not been determined. Raman spectroscopy has been applied as an alternative method to disease biomarker detection. In this work, we detected spectral changes related to FABP4 and 5 in the serum at different points of time, using an animal model of a high-fat diet-induced MetS. FABP4 and 5 spectral changes show a contribution during the evolution of MetS, which indicates alteration to a molecular level that predisposes to established MetS. These findings place FABPs as potential biomarkers of MetS and Raman spectroscopy as an alternative method for MetS assessment.

Feeding during the resting phase causes gastrointestinal tract dysfunction and desynchronization of metabolic and neuronal rhythms in rats.

BACKGROUND: Disrupted circadian rhythms may result from a misalignment between the environmental cycles (due to shift work, sleep restriction, feeding at an unusual time of day) and endogenous rhythms or by physiological aging. Among the numerous adverse effects, disrupted rhythms affect the brain-gut axis, contributing to the pathogenesis of several diseases in the gastrointestinal tract, for example, abdominal pain, constipation, gastric dyspepsia, inflammatory bowel disease, irritable bowel syndrome, and others. METHODS: This study evaluated the rat gastric emptying, gastrointestinal motility, a clock gene, gut hormones, and the neuron activity on the nucleus of tractus solitarius (NTS), area postrema (AP), and the dorsal motor nucleus of the vagus (DMV) in rats with restricted food access to the rest phase for 4 weeks. KEY RESULTS: Our results show that food restricted to the rest light period disturbed the expression pattern of a series of transcripts, including metabolic and circadian regulation. Also, the secretion of gastrointestinal hormones, gastric emptying, intestinal motility, and NTS, AP, and DMV activity were altered. CONCLUSIONS & INFERENCES: These data indicate the importance of the time of the day food is ingested on the regulation of energy balance and the endocrine activity of the stomach and small intestine, emphasizing the importance of food as a powerful circadian synchronizer and an essential factor for the triggering of gastrointestinal diseases and metabolic problems. These findings offer a novel clue regarding the obesity-promoting effect attributed to feeding time and open the possibility of treating this and other intestinal disorders.

Raman Spectroscopy for Adipose Tissue Assessment in Rat Models of Obesity and Type 1 Diabetes.

Adipose tissue presents structural and functional changes in obesity and type 1 diabetes mellitus (T1DM). In obesity, the size and number of adipocytes and adipokine secretion increases. In T1DM, a loss of adipose tissue suggests changes in the metabolic activity of this tissue. A significant challenge is to find alternative noninvasive methods to evaluate molecular changes in adipose tissue related to obesity and T1DM. Recently, Raman spectroscopy and chemometrics techniques have emerged as a tool for biological tissue analysis. In this work, we propose the use of Raman spectroscopy to characterize spectral differences in adipose tissue from different rat groups (control, obese, and T1DM). The Raman spectra were analyzed using direct band analysis, ratiometric analysis, and chemometric methods (principal component analysis (PCA) and support vector machines (SVMs)). We found that the Raman spectra of obese rats showed significant spectral differences compared to control and diabetic groups related to fatty acids Raman bands. Also, the obese group has a significant decrease in the degree of unsaturation of lipids. The PCA-SVM models showed classification performance ranging from 71.43% to 71.79% accuracy for brown and white adipose tissue samples, respectively. In conclusion, the results demonstrate that Raman spectroscopy can be used as a nondestructive method to assess adipose tissue according to a metabolic condition.

Early prognostic capacity of serum lactate for severe postpartum hemorrhage.

OBJECTIVE: To evaluate whether the concentration of serum lactate during the diagnosis of postpartum hemorrhage (bleeding ≥500 mL during labor or ≥1000 mL during cesarean delivery) predicts severe hemorrhage (SPPH; blood loss ≥1500 mL at end of labor or in the following 24 h). METHODS: A prospective cohort pilot study was conducted of women with a vaginal or cesarean delivery from February 2018 to March 2019 who presented with bleeding ≥500 mL measured by the gravimetric method in a reference hospital in San Luis Potosi, Mexico. Venous blood samples were taken for analysis of serum lactate. A receiver operating characteristic curve determined the serum lactate threshold value for SPPH and χ2 test assessed the difference in serum lactate elevation between SPPH and non-SPPH groups. Lastly, the prognostic capacity between the thresholds was compared. RESULTS: SPPH developed in 43.33% of the 30 women in the study group. The best prognostic threshold was 2.68 mmol/L of serum lactate (odds ratio [OR] 17.88, 95% confidence interval [CI] 2.7-16.8, P < 0.001); sensitivity was 0.85 (95% CI 0.55-0.98); specificity was 0.76 (95% CI 0.50-0.93). CONCLUSION: Serum lactate may be a useful prognostic marker for SPPH, more studies are needed to validate these findings.

The Folate Concentration and/or Folic Acid Metabolites in Plasma as Factor for COVID-19 Infection.

Pregnant women appear to be more susceptible to infectious diseases than women in reproductive age. According to the California Department of Public Health pregnant women were 9.6-folds more likely to be hospitalized during the 2009 influenza outbreak when compared to non-pregnant women in reproductive age. In contrast, it was reported that of 16,749 COVID-19 patients that were hospitalized in the UK, the probability for pregnant women to require in-patient care due to infection by SARS-CoV-2 was 0.95 versus non-pregnant women. Therefore 9.6/0.95 = 10.10, which brings us to the conclusion that pregnant women are 10.10-folds less likely to be hospitalized for a SARS-CoV-2 infection than for the 2009 H1N1 pandemic. Folic acid supplementation during pregnancy could be the factor that is protecting these patients against SARS-CoV-2 infection. Two independent papers that used informatic simulation proved that folic acid reduced the replication of this virus. One of them showed that folic acid inhibits the furin protease which the virus needs in order to enter its host cell, while the other one explained that folic acid inactivates protease 3CL pro , a protein that the virus needs to replicate. Nonetheless the probability that folic acid blocks two different proteins is very low, therefore the mechanism by which folic acid has apparently protected pregnant women during the COVID-19 pandemic has not been determined.

Purinergic Signaling During Hyperglycemia in Vascular Smooth Muscle Cells.

The activation of purinergic receptors by nucleotides and/or nucleosides plays an important role in the control of vascular function, including modulation of vascular smooth muscle excitability, and vascular reactivity. Accordingly, purinergic receptor actions, acting as either ion channels (P2X) or G protein-coupled receptors (GCPRs) (P1, P2Y), target diverse downstream effectors, and substrates to regulate vascular smooth muscle function and vascular reactivity. Both vasorelaxant and vasoconstrictive effects have been shown to be mediated by different purinergic receptors in a vascular bed- and species-specific manner. Purinergic signaling has been shown to play a key role in altering vascular smooth muscle excitability and vascular reactivity following acute and short-term elevations in extracellular glucose (e.g., hyperglycemia). Moreover, there is evidence that vascular smooth muscle excitability and vascular reactivity is severely impaired during diabetes and that this is mediated, at least in part, by activation of purinergic receptors. Thus, purinergic receptors present themselves as important candidates mediating vascular reactivity in hyperglycemia, with potentially important clinical and therapeutic potential. In this review, we provide a narrative summarizing our current understanding of the expression, function, and signaling of purinergic receptors specifically in vascular smooth muscle cells and discuss their role in vascular complications following hyperglycemia and diabetes.

Evaluation of cardiovascular responses to silver nanoparticles (AgNPs) in spontaneously hypertensive rats.

Silver nanoparticles (AgNPs) are used in the medical, pharmaceutical and food industry. Adverse effects and toxicity induced by AgNPs upon cardiac function related to nitric oxide (NO) and oxidative stress (OS) are described. AgNPs-toxicity may be influenced by cardiovascular pathologies such as hypertension. However, the molecules involved under pathophysiological conditions are not well studied. The aim of this work was to evaluate perfusion pressure (PP) and left ventricle pressure (LVP) as physiological parameters of cardiovascular function in response to AgNPs, using isolated perfused hearts from spontaneously hypertensive rats (SHR), and identify the role of NO and OS. The results suggest that AgNPs reduced NO derived from endothelial/inducible NO-synthase and increased OS, leading to increased and sustained vasoconstriction and myocardial contractility. Additionally, the hypertension condition alters phenylephrine (Phe) and acetylcholine (ACh) classic effects. These data suggest that hypertension intensified AgNPs-cardiotoxicity. Nevertheless, the precise mechanism of action is still under elucidation.

Effect of silver nanoparticles upon the myocardial and coronary vascular function in isolated and perfused diabetic rat hearts.

Silver nanoparticles (AgNPs) are promising antibacterial nanomaterials for diagnostic and treatment of diabetes. However, toxicity and adverse cardiac responses induced by AgNPs related to nitric oxide (NO) and oxidative stress (OS) are described. Moreover, little is known about the diabetes influence upon AgNPs-toxicity. The aim of this work was to evaluate cardiovascular function in response to AgNPs through measuring perfusion pressure (PP) and left ventricle pressure (LVP), using perfused hearts from streptozotocin (STZ)-induced diabetic rats and identify the role of NO and OS. High concentrations but not the lower concentrations of AgNPs, promotes increases in PP and LVP, as well as increased OS. Additionally, diabetes alters the classic effects of phenylephrine (Phe) and acetylcholine (ACh). These data suggest that diabetes may intensify AgNPs-cardiotoxicity. Nevertheless, the precise mechanism of action is still under elucidation.

The prolactin family hormones regulate vascular tone through NO and prostacyclin production in isolated rat aortic rings.

AIM: Prolactin family hormones include growth hormone, placental lactogen and prolactin, which are able to regulate angiogenesis via NO and prostaglandins. However, their effects on vascular tone are not fully understood. The aim of this study was to evaluate the effects of prolactin family hormones on rat vascular tone in vitro. METHODS: Aortic rings were prepared from adult male rats and precontracted with phenylephrine, then treated with the hormones and drugs. The tension was measured with isometric force displacement transducer connected to a polygraph. NO production and prostacyclin release in physiological solution was determined. Cultured rat aortic endothelial cells (RAECs) were treated with the hormones and drugs, and the phosphorylation of eNOS at serine 1177 was assessed using Western bolt analysis. RESULTS: Administration of growth hormone or placental lactogen (0.01-100 nmol/L) induced endothelium-dependent vasodilation. Both the hormones significantly increased the phosphorylation of eNOS in RAECs and NO level in physiological solution. Preincubation with L-NAME blocked growth hormone- or placental lactogen-induced vasodilation and NO production. Preincubation with an antibody against growth hormone receptors blocked growth hormone- and placental lactogen-induced vasodilation. Addition of a single dose of prolactin (0.01 nmol/L) induced sustained vessel relaxation, whereas multiple doses of prolactin induced a biphasic contraction-relaxation effect. The vascular effects of prolactin depended on endothelium. Prolactin significantly increased the level of prostacyclin I2 in physiological solution. Preincubation with indomethacin or an antibody against prolactin receptors blocked prolactin-induced vasodilation. CONCLUSION: The prolactin family hormones regulate rat vascular tone, selectively promoting either relaxation or contraction of vascular smooth muscle via activation of either growth hormone receptors or prolactin receptors within the endothelium.

Silver nanoparticles induce anti-proliferative effects on airway smooth muscle cells. Role of nitric oxide and muscarinic receptor signaling pathway.

Silver nanoparticles (AgNPs) are used to manufacture materials with new properties and functions. However, little is known about their toxic or beneficial effects on human health, especially in the respiratory system, where its smooth muscle (ASM) regulates the airway contractility by different mediators, such as acetylcholine (ACh) and nitric oxide (NO). The aim of this study was to evaluate the effects of AgNPs on ASM cells. Exposure to AgNPs induced ACh-independent expression of the inducible nitric oxide synthase (iNOS) at 100 µg/mL, associated with excessive production of NO. AgNPs induced the muscarinic receptor activation, since its blockage with atropine and blockage of its downstream signaling pathway inhibited the NO production. AgNPs at 10 and 100 µg/mL induced ACh-independent prolonged cytotoxicity and decreased cellular proliferation mediated by the muscarinic receptor-iNOS pathway. However, the concentration of 100 µg/mL of AgNPs induced muscarinic receptor-independent apoptosis, suggesting the activation of multiple pathways. These data indicate that AgNPs induce prolonged cytotoxic and anti-proliferative effects on ASM cells, suggesting an activation of the muscarinic receptor-iNOS pathway. Further investigation is required to understand the full mechanisms of action of AgNPs on ASM under specific biological conditions.

Effect of 45 nm silver nanoparticles (AgNPs) upon the smooth muscle of rat trachea: role of nitric oxide.

AgNPs have been used to manufacture nanomaterials with new biophysical properties and functions. However, few experimental approaches have been used to assess their potential toxic or beneficial effects on human health, in association with the size, concentration, and biological target. The aim of this work was to evaluate the effects of the AgNPs on the smooth muscle of rat trachea. A single administration of AgNPs did not modify the smooth muscle tone, but, when the trachea rings were pre-treated with acetylcholine (ACh), AgNPs produced a contractile effect. Simultaneous administration of AgNPs and ACh resulted in a slight increase of smooth muscle contractility induced by ACh. AgNPs pretreatment followed by ACh administration showed that AgNPs exerted an important contraction effect induced by ACh after which muscle tone did not return to the basal level. This effect was associated with an increase in the production of nitric oxide (NO). The contractile response of the AgNPs induced by ACh was completely blocked when the rings were incubated, after the ACh but before the AgNPs administration, with 1400 W (NO blocker). The contractile effect was also abolished by atropine, which suggests that AgNPs alter ACh muscarinic receptor signaling. These data also show that AgNPs modify the contractile action of ACh through NO production and possibly induce hyper-reactivity of tracheal smooth muscle.

Essential role for calcium waves in migration of human vascular smooth muscle cells.

Vascular smooth muscle cell (SMC) migration is characterized by extension of the lamellipodia at the leading edge, lamellipodial attachment to substrate, and release of the rear (uropod) of the cell, all of which enable forward movement. However, little is known regarding the role of intracellular cytosolic Ca(2+) concentration ([Ca(2+)](i)) in coordinating these distinct activities of migrating SMCs. The objective of our study was to determine whether regional changes of Ca(2+) orchestrate the migratory cycle in human vascular SMCs. We carried out Ca(2+) imaging using digital fluorescence microscopy of fura-2 loaded human smooth muscle cells. We found that motile SMCs exhibited Ca(2+) waves that characteristically swept from the rear of polarized cells toward the leading edge. Ca(2+) waves were less evident in nonpolarized, stationary cells, although acute stimulation of these SMCs with the agonists platelet-derived growth factor-BB or histamine could elicit transient rise of [Ca(2+)](i). To investigate a role for Ca(2+) waves in the migratory cycle, we loaded cells with the Ca(2+) chelator BAPTA, which abolished Ca(2+) waves and significantly reduced retraction, supporting a causal role for Ca(2+) in initiation of retraction. However, lamellipod motility was still evident in BAPTA-loaded cells. The incidence of Ca(2+) oscillations was reduced when Ca(2+) release from intracellular stores was disrupted with the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin or by treatment with the inositol 1,4,5-trisphosphate receptor blocker 2-aminoethoxy-diphenyl borate or xestospongin C, implicating Ca(2+) stores in generation of waves. We conclude that Ca(2+) waves are essential for migration of human vascular SMCs and can encode cell polarity.

The 1.3 isoform of Na+–Ca2+ exchanger expressed in guinea pig tracheal smooth muscle is less sensitive to KB-R7943

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The sodium–calcium exchanger (NCX) plays a major role in the regulation of cytosolic Ca2+ in muscle cells. In this work, we performed force experiments to explore the role of NCX during contraction and relaxation of Cch-stimulated guinea pig tracheal smooth muscle strips. This tissue showed low sensitivity to NCX inhibitor KB-R7943 (IC50, 57 ± 2 µM), although a complete relaxation was obtained by NCX inhibition at 100 µM. Interestingly, relaxation after washing the agonist was prolonged in the absence of external Na+, whereas washing without Na+ and in the presence of KB-R7943 resembled control conditions with physiological solution. Altogether, this suggests the reversal of NCX to a Ca2+ influx mode by the manipulation on the Na+ gradient, which can be inhibited by KB-R7943. In order to understand the low sensitivity to KB-R7943, we studied the molecular aspects of the NCX expressed in this tissue and found that the isoform of NCX expressed is 1.3, similar to that described in human tracheal smooth muscle. Sequencing revealed that amino acid 19 in exon B is phenylalanine, whereas in its human counterpart is leucine, and that the first amino acid after exon D is aspartate instead of glutamate in humans. Results herein presented are discussed in term of their possible functional implications in the exchanger activity and thus in airway physiology (AU)

The 1.3 isoform of Na+-Ca 2+ exchanger expressed in guinea pig tracheal smooth muscle is less sensitive to KB-R7943.

The sodium-calcium exchanger (NCX) plays a major role in the regulation of cytosolic Ca(2+) in muscle cells. In this work, we performed force experiments to explore the role of NCX during contraction and relaxation of Cch-stimulated guinea pig tracheal smooth muscle strips. This tissue showed low sensitivity to NCX inhibitor KB-R7943 (IC50, 57 +/- 2 microM), although a complete relaxation was obtained by NCX inhibition at 100 microM. Interestingly, relaxation after washing the agonist was prolonged in the absence of external Na(+), whereas washing without Na(+) and in the presence of KB-R7943 resembled control conditions with physiological solution. Altogether, this suggests the reversal of NCX to a Ca(2+) influx mode by the manipulation on the Na(+) gradient, which can be inhibited by KB-R7943. In order to understand the low sensitivity to KB-R7943, we studied the molecular aspects of the NCX expressed in this tissue and found that the isoform of NCX expressed is 1.3, similar to that described in human tracheal smooth muscle. Sequencing revealed that amino acid 19 in exon B is phenylalanine, whereas in its human counterpart is leucine, and that the first amino acid after exon D is aspartate instead of glutamate in humans. Results herein presented are discussed in term of their possible functional implications in the exchanger activity and thus in airway physiology.

Role of extracellular Na+, Ca2+-activated Cl- channels and BK channels in the contraction of Ca2+ store-depleted tracheal smooth muscle.

1. In the present study, we investigated the series of events involved in the contraction of tracheal smooth muscle induced by the re-addition of Ca(2+) in an in vitro experimental model in which Ca(2+) stores had been depleted and their refilling had been blocked by thapsigargin. 2. Mean (+/-SEM) contraction was diminished by: (i) inhibitors of store-operated calcium channels (SOCC), namely 100 micromol/L SKF-96365 and 100 micromol/L 1-(2-trifluoromethylphenyl) imidazole (to 66.3 +/- 4.4 and 41.3 +/- 5.2% of control, respectively); (ii) inhibitors of voltage-gated Ca(2+) channels Ca(V)1.2 channels, namely 1 micromol/L nifedipine and 10 micromol/L verapamil (to 86.2 +/- 3.4 and 76.9 +/- 5.9% of control, respectively); and (iii) 20 micromol/L niflumic acid, a non-selective inhibitor of Ca(2+)-dependent Cl(-) channels (to 41.1 +/- 9.8% of control). In contrast, contraction was increased 2.3-fold by 100 nmol/L iberiotoxin, a blocker of the large-conductance Ca(2+)-activated K(+) (BK) channels. 3. Furthermore, contraction was significantly inhibited when Na(+) in the bathing solution was replaced by N-methyl-D-glucamine (NMDG(+)) to 39.9 +/- 7.2% of control, but not when it was replaced by Li(+) (114.5 +/- 24.4% of control). In addition, when Na(+) had been replaced by NMDG(+), contractions were further inhibited by both nifedipine and niflumic acid (to 3.0 +/- 1.8 and 24.4 +/- 8.1% of control, respectively). Nifedipine also reduced contractions when Na(+) had been replaced by Li(+) (to 10.7 +/- 3.4% to control), the niflumic acid had no effect (116.0 +/- 4.5% of control). 4. In conclusion, the data of the present study demonstrate the roles of SOCC, BK channels and Ca(V)1.2 channels in the contractions induced by the re-addition of Ca(2+) to the solution bathing guinea-pig tracheal rings under conditions of Ca(2+)-depleted sarcoplasmic reticulum and inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase. The contractions were highly dependent on extracellular Na(+), suggesting a role for SOCC in mediating the Na(+) influx.

Functional coupling between the Na+/Ca2+ exchanger and nonselective cation channels during histamine stimulation in guinea pig tracheal smooth muscle.

Airway smooth muscle (ASM) contracts partly due to an increase in cytosolic Ca(2+). In this work, we found that the contraction caused by histamine depends on external Na(+), possibly involving nonselective cationic channels (NSCC) and the Na(+)/Ca(2+) exchanger (NCX). We performed various protocols using isometric force measurement of guinea pig tracheal rings stimulated by histamine. We observed that force reached 53 +/- 1% of control during external Na(+) substitution by N-methyl-D-glucamine(+), whereas substitution by Li(+) led to no significant change (91 +/- 1%). Preincubation with KB-R7943 decreased the maximal force developed (52.3 +/- 5.6%), whereas preincubation with nifedipine did not (89.7 +/- 1.8%). Also, application of the nonspecific NCX blocker KB-R7943 and nifedipine on histamine-precontracted tracheal rings reduced force to 1 +/- 3%, significantly different from nifedipine alone (49 +/- 6%). Moreover, nonspecific NSCC inhibitors SKF-96365 and 2-aminoethyldiphenyl borate reduced force to 1 +/- 1% and 19 +/- 7%, respectively. Intracellular Ca(2+) measurements in isolated ASM cells showed that KB-R7943 and SKF-96365 reduced the peak and sustained response to histamine (0.20 +/- 0.1 and 0.19 +/- 0.09 for KB-R, 0.43 +/- 0.16 and 0.47 +/- 0.18 for SKF, expressed as mean of differences). Moreover, Na(+)-free solution only inhibited the sustained response (0.54 +/- 0.25). These data support an important role for NSCC and NCX during histamine stimulation. We speculate that histamine induces Na(+) influx through NSCC that promotes the Ca(2+) entry mode of NCX and Ca(V)1.2 channel activation, thereby causing contraction.

Novel outwardly rectifying anion conductance in Xenopus oocytes.

We describe a novel, strongly outwardly rectifying anion current in Xenopus laevis oocytes, that we have named I(Cl,Or)- The properties of I(Cl,Or) are different from those of any other anion conductance previously described in these cells. Typically, I(Cl,Or) amplitude was small when extracellular Cl- (Cle) was the permeant anion. However, when Cle was replaced by lyotropic anions I(Cl,Or) became evident as a time-independent current. (ICl,Or) was voltage dependent and showed a remarkable outwards rectification with little or no inwards tail current. The relative selectivity sequence determined from current amplitudes was: SCN- > or = ClO4- > I- > Br- > or = NO3- > Cl- x I(Cl,Or) was insensitive to Gd3+ but was blocked by micromolar concentrations of niflumic acid, DIDS or Zn2+. Furthermore, I(Cl,Or) was not affected by buffering intracellular Ca2+ with BAPTA. Low extracellular pH inhibited I(Cl,Or) with a pK of 5.8. We propose that I(Cl,Or) might result from activation of endogenous ClC-5-like Cl- channels present in Xenopus oocytes.

8Br-cGMP mediates relaxation of tracheal smooth muscle through PKA.

In this study, guinea pig tracheal smooth muscle pre-contracted with histamine was relaxed by the addition of 100microM 8Br-cGMP, a non-hydrolyzable and cell-permeable analog for cGMP. This effect was not sensitive to cGMP-dependent protein kinase (PKG) inhibitors, whereas it was partially blocked by cAMP-dependent protein kinase (PKA) inhibitors. The relaxation observed was also reverted up to 50+/-8.5% by iberiotoxin, a selective inhibitor of large conductance, calcium-activated potassium channels (BK(Ca)). Our results indicate that there exists a crosstalk mechanism between cAMP and cGMP signaling pathways which lead to relaxation of guinea pig tracheal smooth muscle and also that BK(Ca) channels are involved to a certain extent in this phenomenon.