GTP<Subscript>γS</Subscript> increases Na<Subscript>v</Subscript>1.8 current in small-diameter dorsal root ganglia neurons

Tetrodotoxin-resistant (TTX-R) sodium current in small-size dorsal root ganglia (DRG) neurons is upregulated by prostaglandin E₂ and serotonin through a protein kinase A (PKA)/protein kinase (PKC) pathway, suggesting G protein modulation of one or more TTX-R channels in these neurons. Recently, GTP_S, a hydrolysis-resistant analogue of GTP, was shown to increase the persistent current produced by the TTX-R Na_v1.9. In this study, we investigated the modulation of another TTX-R channel, Na_v1.8, by GTP_S in small-diameter DRG neurons from rats using whole-cell voltage clamp recordings. Because it has been suggested that fluoride, often used in intracellular recording solutions, may bind to trace amounts of aluminum and activate G proteins, we recorded Na_v1.8 currents with and without intracellular fluoride, and with the addition of deferoxamine, an aluminum chelator, to prevent fluoride–aluminum binding. Our results show that GTP_S (100 µM) caused a significant increase in Na_v1.8 current (67%) with a chloride-based intracellular solution. Although the inclusion of fluoride instead of chloride in the pipette solution increased the Na_v1.8 current by 177%, GTP_S further increased Na_v1.8 current by 67% under these conditions. While the effect of GTP_S was prevented by pretreatment with H7 (100 µM), a non-selective PKA/PKC inhibitor, the fluoride-induced increase in Na_v1.8 current was not sensitive to H7 (100 µM), or to inclusion of deferoxamine (1 mM) in the intracellular solution. We conclude that G protein activation by GTP_S increases Na_v1.8 current through a PKA/PKC mechanism and that addition of fluoride to the pipette solution further enhances the current, but is not a confounding variable in the study of Na_v1.8 channel modulation by G proteins independent of a PKA/PKC pathway or binding to aluminum.     

The Role of Integrin α<Subscript>4</Subscript>β<Subscript>7</Subscript> in HIV Pathogenesis and Treatment

Purpose of Review

Acute HIV infection is characterized by high-level viral replication throughout the body’s lymphoid system, particularly in gut-associated lymphoid tissues resulting in damage to structural components of gut tissue. This damage is irreversible and believed to contribute to the development of immune deficiencies. Antiretroviral therapy (ART) does not restore gut structure and function. Studies in macaques point to an alternative treatment strategy that may ameliorate gut damage. Integrin α₄β₇ mediates the homing of lymphocytes to gut tissues. Vedolizumab, a monoclonal antibody (mAb) antagonist of α₄β₇, has demonstrated efficacy and has been approved for the treatment of inflammatory bowel disease in humans. Here, we describe our current knowledge, and the gaps in our understanding, of the role of α₄β₇ in HIV pathogenesis and treatment.

Recent Findings

When administered to macaques prior to infection, a nonhuman primate analogue of vedolizumab prevents transmission of SIV. In combination with ART, this mAb facilitates durable virologic control following treatment interruption.

Summary

Targeting α₄β₇ represents a novel therapeutic approach to prevent and treat HIV infection.

     

The differential contribution of dopamine D<Subscript>1</Subscript> and D<Subscript>2</Subscript> receptors to μ-opioidergic immunomodulation

Activation of μ-opioid receptors (μ-OR) by the highly selective agonist DAGO (100 μg/kg) significantly increased the immune response in CBA mice. This effect of the μ agonist was prevented by prior blockade of dopamine D₂ receptors with haloperidol (2 mg/kg). In contrast, the selective D₁ receptor antagonist SCH 23390 (1 mg/kg) had no effect on the nature of the immune reaction in response to antigen (sheep erythrocytes, 5·10⁸ cells). However, blockade of both types of dopamine receptor led to the same effect-immunosuppression. These data lead to the suggestion that D₁ and D₂ receptors make different contributions to modulating immunogenesis on activation of μ-OR. __________ Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 92, No. 5, pp. 546–551, May, 2006.     

Estimation of<Emphasis Type="Italic"> V̇</Emphasis>O<Subscript>2max</Subscript> from the ratio between HR<Subscript>max </Subscript>and HR<Subscript>rest</Subscript> – the Heart Rate Ratio Method

The effects of ̇raining and/or ageing upon maximal oxygen uptake (O_2max) and heart rate values at rest (HR_rest) and maximal exercise (HR_max), respectively, suggest a relationship between O_2max and the HR_max-to-HR_rest ratio which may be of use for indirect testing of O_2max. Fick principle calculations supplemented by literature data on maximum-to-rest ratios for stroke volume and the arterio-venous O₂ difference suggest that the conversion factor between mass-specific O_2max (ml·min^–1·kg^–1) and HR_max·HR_rest ^–1 is ~15. In the study we experimentally examined this relationship and evaluated its potential for prediction of O_2max. O_2max was measured in 46 well-trained men (age 21–51 years) during a treadmill protocol. A subgroup (n=10) demonstrated that the proportionality factor between HR_max·HR_rest ^–1 and mass-specific O_2max was 15.3 (0.7) ml·min^–1·kg^–1. Using this value, O_2max in the remaining 36 individuals could be estimated with an SEE of 0.21 l·min^–1 or 2.7 ml·min^–1·kg^–1 (~4.5%). This compares favourably with other common indirect tests. When replacing measured HR_max with an age-predicted one, SEE was 0.37 l·min^–1 and 4.7 ml·min^–1·kg^–1 (~7.8%), which is still comparable with other indirect tests. We conclude that the HR_max-to-HR_rest ratio may provide a tool for estimation of O_2max in well-trained men. The applicability of the test principle in relation to other groups will have to await direct validation. O_2max can be estimated indirectly from the measured HR_max-to-HR_rest ratio with an accuracy that compares favourably with that of other common indirect tests. The results also suggest that the test may be of use for O_2max estimation based on resting measurements alone.An erratum to this article can be found at     

Interaction of Afobazole with σ<Subscript>1</Subscript>-Receptors

The capacity of living systems to perceive low-intensity stimuli sometimes inducing protective reactions is still little studied. Incubation of neurons under conditions increasing the content of cAMP and Ca²⁺ increases the amplitude of their responses to lidocaine (10⁻³ M). After cell preconditioning with low concentrations of lidocaine (10⁻¹⁵ M) under these conditions, the protective effects of “ineffective” concentrations were detected, because the response amplitude did not decrease. It was hypothesized that the basic amplitude responses retrieved by lidocaine in a concentration of 10⁻³ M are memory traces about the effects of this compound in subthreshold concentrations. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 147, No. 1, pp. 43-46, January, 2009     

Oxytocin and prostaglandins F<Subscript>2α</Subscript> and E<Subscript>2</Subscript> do not enhance HIV antigen production <Emphasis Type="Italic">in vitro</Emphasis>

Summary.  Oxytocin and prostaglandins (PGs) are hormones involved in labor and are used clinically for its induction. In this study the effect of oxytocin, PGF_2α, and PGE₂ on Humour immunodeficiency virus-1 production in acutely and persistently infected cells was measured. No significant effect on p24 antigen production was found with oxytocin or PGs, except for a transient decrease in persistently infected cells treated with 1 μM PGF_2α. These results showed that oxytocin and PGs could be used clinically for labor induction without any direct enhancement in viral production. Besides, the results with PGF_2α at the highest concentration studied may indicate a pharmacological effect. Received October 10, 2002; accepted October 28, 2002     

Does NAD(P)H oxidase-derived H<Subscript>2</Subscript>O<Subscript>2</Subscript> participate in hypotonicity-induced insulin release by activating VRAC in β-cells?

NAD(P)H oxidase (NOX)-derived H₂O₂ was recently proposed to act, in several cells, as the signal mediating the activation of volume-regulated anion channels (VRAC) under a variety of physiological conditions. The present study aims at investigating whether a similar situation prevails in insulin-secreting BRIN-BD11 and rat β-cells. Exogenous H₂O₂ (100 to 200 μM) at basal glucose concentration (1.1 to 2.8 mM) stimulated insulin secretion. The inhibitor of VRAC, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) inhibited the secretory response to exogenous H₂O₂. In patch clamp experiments, exogenous H₂O₂ was observed to stimulate NPPB-sensitive anion channel activity, which induced cell membrane depolarization. Exposure of the BRIN-BD11 cells to a hypotonic medium caused a detectable increase in intracellular level of reactive oxygen species (ROS) that was abolished by diphenyleneiodonium chloride (DPI), a universal NOX inhibitor. NOX inhibitors such as DPI and plumbagin nearly totally inhibited insulin release provoked by exposure of the BRIN-BD11 cells to a hypotonic medium. Preincubation with two other drugs also abolished hypotonicity-induced insulin release and reduced basal insulin output: 1) N-acetyl-L-cysteine (NAC), a glutathione precursor that serves as general antioxidant and 2) betulinic acid a compound that almost totally abolished NOX4 expression. As NPPB, each of these inhibitors (DPI, plumbagin, preincubation with NAC or betulinic acid) strongly reduced the volume regulatory decrease observed following a hypotonic shock, providing an independent proof that VRAC activation is mediated by H₂O₂. Taken together, these data suggest that NOX-derived H₂O₂ plays a key role in the insulin secretory response of BRIN-BD11 and native β-cells to extracellular hypotonicity.     

Role of Intracellular Calcium and Cyclic Nucleotides in Realization of Cardioprotective Effects of δ<Subscript>1</Subscript>- and κ<Subscript>1</Subscript>-Opioid Receptor Agonists

The role of cyclic nucleotides (cAMP, cGMP) and Ca²⁺-ATPase of the sarcoplasmic reticulum in the mechanism of cardioprotective effects of selective δ₁- and κ₁-opioid receptor agonists DPDPE and U-50488 was studied under conditions of global ischemia and reperfusion of isolated and perfused rat heart. Activation of both types of opioid receptors 2-fold reduced the reperfusion release of creatine phosphokinase. The cardioprotective effect of U-50488 was paralleled by a 2-fold decrease in cAMP content in the myocardium, while DPDPE did not modify the content of cAMP throughout the experiment. None of these substances changed the content of cGMP in the myocardium. The cardioprotective effect of DPDPE was not observed after inhibition of sarcoplasmic reticulum Ca²⁺-ATPase with cyclopiazonic acid. The cardioprotective effect of U-50488 was associated with reduction of cAMP level in the myocardium, while the cytoprotective effect of DPDPE was mediated by opioidergic modulation of Ca²⁺ transport at the level of the sarcoplasmic reticulum.     

Reactions between β-Casomorphins-7 and 5-HT<Subscript>2</Subscript>-Serotonin Receptors

Radioreceptor analysis showed that human β-casomorphin-7 displaced ³H-spiperone from 5-HT₂-serotonin receptors of the rat cerebral frontal cortex: EC₅₀ 8±1 μM. Human and bovine β-casomorphin-7 dose-dependently blocked serotonin-induced human platelet aggregation: IC₅₀ 5±1 and 20±4 μM, respectively. It was proved that β-casomorphins-7 act as 5-HT₂-serotonin receptor antagonists; one of the mechanisms of their biological effects is presumably associated with modulation of the serotoninergic system. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 140, No. 11, pp. 595–597, November, 2005     

Opioid Peptide Deltorphin II Simulates the Cardioprotective Effect of Ischemic Preconditioning: Role of δ<Subscript>2</Subscript>-Opioid Receptors,Protein Kinase C,and K<Subscript>ATP</Subscript> Channels

The cardioprotective properties of a δ₂-opioid receptor agonist deltorphin II were studied in rats with coronary occlusion and reperfusion. Opioid receptor ligands and inhibitors (glybenclamide, chelerythrine, and 5-hydroxydecanoate) were injected intravenously before ischemia and reperfusion. A δ₂-opioid receptor agonist deltorphin II signifi cantly decreased the infarction zone/risk zone index. This effect was abolished by naltrexone, naloxone methiodide, and δ₂-opioid receptor antagonist naltriben, but not by a δ₁-opioid receptor antagonist BNTX. The infarct-limiting effect of deltorphin II was not observed after inhibition of protein kinase C or blockade of mitochondrial K_ATP channels.     

Pathogenic hantaviruses selectively inhibit β<Subscript>3</Subscript> integrin directed endothelial cell migration

Summary.  Hantaviruses cause two diseases of man, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Pathogenic and non-pathogenic hantaviruses use β₃ and β₁ integrins, respectively, to enter endothelial cells. β₃ integrins were recently reported to bind receptors that regulate vascular permeability suggesting that hantavirus β₃ integrin interactions may regulate endothelial cell function and contribute to viral pathogenesis. In this study we investigated the ability of pathogenic and non-pathogenic hantaviruses to regulate β₃ and β₁ integrin directed endothelial cell functions. We found that pathogenic NY-1, SNV, HTN, SEO and PUU viruses blocked endothelial cell migration on β₃, but not β₁, integrin ligands. Migration is similarly inhibited by antibodies to β₃ integrins which selectively block vitronectin directed endothelial cell migration. As a result, the ability of endothelial cells to migrate on integrin ligands was selectively inhibited by only pathogenic hantaviruses. Infection by NY-1 virus inhibited endothelial cell migration as early as 24–48 h post-infection. In contrast, non-pathogenic PH and TUL viruses had no effect on the ability of endothelial cells to migrate on either β₃ or β₁ integrin ligands from 1 to 5 days post-infection. These findings indicate that only hantaviruses which use β₃ integrins, and are associated with HPS and HFRS diseases, functionally dysregulate endothelial cell migration. These findings further demonstrate that hantaviruses regulate only β₃ integrin directed endothelial cell functions and have no effect on β₁ integrin functions. Since β₃ integrins are linked to changes in vascular permeability and the maintenance of vascular integrity, these findings suggest a means by which hantavirus usage and regulation of β₃ integrins may contribute to hantavirus pathogenesis. Received January 23, 2002; accepted May 20, 2002     

Effects of corticoliberin CRF<Subscript>4–6</Subscript> fragment on pain sensitivity in rats

The effects of the tripeptide fragment corticoliberin CRF_4–6 (Pro-Pro-Ile) on pain sensitivity were studied in rats using the hotplate method. CRF_4–6 given centrally (6, 30, and 150 nmol/rat) had dose-dependent antinociceptive actions: the latent period of the paw-licking response increased by 7.4 ± 1.4, 10.1 ± 1.5, and 16.7 ± 4.2 sec from the control level of 10.2 ± 0.9 sec. The durations of the effect were 30 min for CRF_4–6 at a dose of 6 nmol and 60 min for doses of 30 and 150 nmol of tripeptide. Administration of the corticoliberin antagonist ηhCRF_9–41 (centrally, 6.5 nmol) 60 min before tripeptide completely blocked the antinociceptive effects of CRF_4–6 (6 nmol). Thus, corticoliberin receptors are involved in mediating the antinociceptive influence of CRF_4–6. It can be suggested that the tripeptide either directly interacts with corticoliberin receptors or modulates the activity of CRFergic neurons. __________ Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 91, No. 9, pp. 1060–1065, September, 2005.     

Effects of α<Subscript>1</Subscript>-acid glycoprotein on hemostasis in experimental septic peritonitis

Pathogenesis of hemostasis disorders in septic peritonitis and the possibility of their correction with acute phase protein (α₁-acid glycoprotein; two doses of 150 mg/kg) were experimentally studied on outbred albino rats. Platelets count in the peripheral blood and their adhesion to endothelium did not change during peritonitis, while aggregation activity increased due to increased rate and shorter time of aggregation, which was associated with the development of hypercoagulation involving the intrinsic and common coagulation pathways and reduction of antithrombin activity. α₁-Acid glycoprotein increased platelet count above the normal level, normalized aggregation rate, some blood clotting parameters, and antithrombin activity. Hence, α₁-acid glycoprotein is a polyfunctional protein modulating all pathogenetic components in the development of blood clotting disorders during septic peritonitis. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 8, pp. 143–145, August, 2007     

G<Subscript>i2</Subscript>α protein deficiency: A model for inflammatory bowel disease

Mice deficient for the G protein subunit G_i2α were obtained by gene targeting. They displayed a growth retardation that was apparent at 6 weeks of age. They subsequently developed diffuse colitis with clinical and histopathological features closely resembling those of ulcerative colitis in humans. Seven of 20 G_i2α-deficient mice with colitis also developed adenocarcinomas of the colon. G_i2α-deficient thymocytes displayed two-to fourfold increases in mature CD4⁺8^? and CD4^?8⁺ phenotypes, an approximately threefold increase in highintensity CD3 staining and enhanced proliferative responses to T-cell receptor stimuli. Stimulation of G_i2α-deficient peripheral T cells induced a hyperresponsive profile of interleukin-2, tumor necrosis factor, and interferon-γ production, which may reflect a heightened response of primed cells or a defective negative regulation. We suggest that G_i2α-deficient mice may represent a useful animal model for dissecting the pathomechanisms of inflammatory bowel disease and also for the development of novel therapeutic strategies.     

Endotoxin-induced liver damage in rats is minimized by β<Subscript>2</Subscript>-adrenoceptor stimulation

Objective and Design: To investigate the effects of ₂-adrenoceptor (₂-AR) stimulation on endotoxin-induced liver damage and systemic cytokine levels in rats.Subjects: Standard male Wistar rats.Treatment: A disease-model of lipolysaccharide (LPS)-induced acute systemic inflammation was used. The ₂-selective AR agonist clenbuterol was administered before, during, and after LPS-challenge to investigate its effects on the acute inflammatory response and associated liver-failure.Methods: The following parameters have been measured in plasma: TNF, IL-1, IL-6, IL-10, AST, ALT, and Bilirubin. Liver histological examination was performed to look for changes in tissue morphology.Results: Administration of clenbuterol (p.o.) one hour before, or intravenous at the same time as LPS-challenge resulted in a marked reduction of plasma levels of TNF, IL-1, and IL-6. A change both in plasma-level and in time-concentration profile of the anti-inflammatory cytokine IL-10 was found. Clenbuterol minimized LPS-induced liver damage, as represented by significantly lowered concentrations of several parameters for liver-failure (AST, ALT, Bilirubin), and improved hepatic tissue morphology. Clenbuterol administration after LPS challenge failed to inhibit TNF-release but reduced liver-damage. Simultaneous use of the ₂-AR antagonist propranolol augmented LPS-induced liver failure, suggesting a role of endogenous adrenoceptor-agonists in prevention of organ-failure during systemic inflammation.Conclusions: The results indicate that a selective ₂-AR agonist might be used as an additional therapeutic agent in the clinic for the treatment of (acute) systemic inflammatory disorders in order to reduce or prevent subsequent liver failure.Received 3 March 2003; returned for revision 4 April 2003; returned for final revision 3 October 2003; accepted by M.Parnham 29 October 2003     

A Singular Role of I<Subscript>K1</Subscript> Promoting the Development of Cardiac Automaticity during Cardiomyocyte Differentiation by I<Subscript>K1</Subscript><Emphasis Type="Bold">–</Emphasis>Induced Activation of Pacemaker Current

The inward rectifier potassium current (I_K1) is generally thought to suppress cardiac automaticity by hyperpolarizing membrane potential (MP). We recently observed that I_K1 could promote the spontaneously-firing automaticity induced by upregulation of pacemaker funny current (I_f) in adult ventricular cardiomyocytes (CMs). However, the intriguing ability of I_K1 to activate I_f and thereby promote automaticity has not been explored. In this study, we combined mathematical and experimental assays and found that only I_K1 and I_f, at a proper-ratio of densities, were sufficient to generate rhythmic MP-oscillations even in unexcitable cells (i.e. HEK293T cells and undifferentiated mouse embryonic stem cells [ESCs]). We termed this effect I_K1-induced I_f activation. Consistent with previous findings, our electrophysiological recordings observed that around 50% of mouse (m) and human (h) ESC-differentiated CMs could spontaneously fire action potentials (APs). We found that spontaneously-firing ESC-CMs displayed more hyperpolarized maximum diastolic potential and more outward I_K1 current than quiescent-yet-excitable m/hESC-CMs. Rather than classical depolarization pacing, quiescent mESC-CMs were able to fire APs spontaneously with an electrode-injected small outward-current that hyperpolarizes MP. The automaticity to spontaneously fire APs was also promoted in quiescent hESC-CMs by an I_K1-specific agonist zacopride. In addition, we found that the number of spontaneously-firing m/hESC-CMs was significantly decreased when I_f was acutely upregulated by Ad-CGI-HCN infection. Our study reveals a novel role of I_K1 promoting the development of cardiac automaticity in m/hESC-CMs through a mechanism of I_K1-induced I_f activation and demonstrates a synergistic interaction between I_K1 and I_f that regulates cardiac automaticity.     

Prostaglandin I<Subscript>2</Subscript> Analogues Enhance Growth-Related Oncogene-α Expression in Human Monocyte-Derived Dendritic Cells

Chemokines for neutrophils such as growth-related oncogene-α (GRO-α) are important in patients with refractory or severe asthma. Prostaglandin I₂ (PGI₂) analogues were regarded as potential treatments for asthma. Dendritic cells (DCs) are the professional antigen-presenting cells and play a critical role in regulating immune response. However, it is unknown whether PGI₂ analogues have regulatory effects on GRO-α expression in human monocyte-derived DCs (MDDCs). The human MDDCs were pretreated with iloprost and treprostinil (two PGI₂ analogues) or forskolin, a cyclic adenosine monophosphate (cAMP) activator, before stimulation with lipopolysaccharide (LPS). In some cases, I prostanoid (IP) receptor and E prostanoid (EP) antagonists were pretreated before PGI₂ analogue treatment. To investigate the intracellular signaling, nuclear factor (NF)-κB inhibitor and the mitogen-activated protein kinase (MAPK) inhibitors were pretreated before PGI₂ analogue treatment. GRO-α was measured by enzyme-linked immunosorbent assay. Intracellular signaling was also investigated by Western blot. Iloprost and treprostinil enhanced LPS-induced GRO-α expression in MDDCs. This effect could be reversed by an I prostanoid receptor antagonist, CAY10449, but not EP receptor antagonists. Forskolin conferred a similar modulating effect as that noted in iloprost- and treprostinil-treated MDDCs. PGI₂ analogue-enhanced LPS-induced GRO-α expression was reduced by MAPK-p38 inhibitor, SB203580. PGI₂ analogues enhanced LPS-induced phospho-p38 expression. PGI₂ analogues enhanced LPS-induced GRO-α expression via the IP receptor–cAMP and p38-MAPK pathways in human MDDCs, which may further recruit neutrophil accumulation and adversely affect patients with refractory or severe asthma because of airway neutrophilia. These effects should be considered for PGI₂ analogues as candidates for the treatment of asthma.     

Effect of an α<Subscript>1</Subscript>-adrenergic blocker on plasticity elicited by motor training

Recovery of motor function elicited by motor training after cortical lesions in rats is enhanced by norepinephrine (neurotransmitter mediating α₁-adrenergic function) and downregulated by α₁-adrenergic antagonists. In spite of this, α₁-adrenergic antagonists are used to treat elderly patients with hypertension and prostate hyperplasia in stroke settings. The purpose of this study was to determine the effects of a single oral dose of the α₁-adrenergic antagonist prazosin on training-dependent plasticity in intact humans, a function thought to contribute to recovery of motor function after cortical lesions. We report that prazosin decreased the ability of motor training to elicit training-dependent plasticity relative to a drug-free condition. These data suggest caution when using α₁-adrenergic blockers in rehabilitative clinical settings following brain lesions. Electronic Publication     

Comparative study of analgesic potency of ACTH<Subscript>4–10</Subscript> fragment and its analog semax

The effects of ACTH_4–10 fragment and its analog semax on nociception were examined on various animal models. ACTH_4–10 in a dose of 0.5 mg/kg decreased nociception in rats during hindpaw compression test and in mice subjected to acetic acid writhing test. Lower doses of ACTH_4–10 produced no analgesic effect. Semax (0.015–0.500 mg/kg) decreased pain sensitivity in all experimental models. Hence, the substitution of three C-terminal amino acid residues in ACTH_4–10 for Pro-Gly-Pro sequence augmented the analgesic potency of the peptide after its peripheral injection. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 1, pp. 8–12, January, 2007