Cicletanine reverses vasoconstriction induced by the endogenous sodium pump ligand, marinobufagenin, via a protein kinase C dependent mechanism

RATIONALE: Cicletanine (CIC), an anti-hypertensive compound with direct vascular and natriuretic actions, is especially effective in salt-sensitive hypertension, in which dysregulation of the sodium pump plays an important pathogenic role, and digitalis-like cardiotonic steroids contribute to increased vascular tone. The purpose of the present study was to investigate whether, and by what mechanisms, cicletanine antagonizes the vasoconstrictor effects of cardiotonic steroids in isolated human arteries. METHODS: The effects of cicletanine on vascular tone were studied in isolated, endothelium-denuded rings of 2nd-3rd-order branches of human mesenteric arteries pre-contracted with bufodienolide marinobufagenin (MBG), an Na/K-ATPase inhibitor, or endothelin-1 (ET-1). Na/K-ATPase activity was measured in sarcolemmal membranes from the mesenteric artery. Activity of rat brain protein kinase C (PKC) was measured using the PepTag phosphorylation assay. RESULTS: MBG and ET-1 both induced sustained vasoconstriction in human mesenteric artery rings, and cicletanine relaxed rings pre-contracted with either MBG (EC50 = 11 +/- 2 micromol/l) or ET-1 (EC50 = 6.4 +/- 1.1 micromol/l). Although 8-Br-cGMP (100 micromol/l) caused complete vasorelaxation of arterial rings pre-contracted with ET-1, it did not affect the MBG-induced vasoconstriction. An activator of PKC, phorbol diacetate (PDA) (50 nmol/l), attenuated CIC-induced vasorelaxation of mesenteric artery rings pre-contracted with MBG (EC50 > 100 micromol/l), but not rings pre-contracted with ET-1 (EC50 = 6.5 +/- 1.2 micromol/l). In mesenteric artery sarcolemma, 100 nmol/l MBG inhibited the Na/K-ATPase by 68 +/- 5% and cicletanine (100 micromol/l) attenuated this Na/K-ATPase inhibition by 85 +/- 6%. In the PepTag PKC assay, cicletanine produced a concentration-dependent inhibition of rat brain PKC activity (IC50 45 +/- 11 micromol/l). In the presence of 50 nmol/l PDA, 100 micromol/l cicletanine did not antagonize the Na/K-ATPase inhibition by MBG, and did not inhibit the PKC from rat brain. CONCLUSIONS: Cicletanine antagonizes vasoconstriction induced by Na/K-ATPase inhibition via a PKC-dependent mechanism that does not involve inhibition of cyclic GMP phosphodiesterase (cGMP-PDE). This mechanism of action may be relevant to the greater potency of cicletanine in salt-sensitive hypertension in which plasma levels of endogenous digitalis-like cardiotonic steroids are elevated. Our findings also suggest that PKC is an important factor for cardiotonic steroid-Na/K-ATPase interactions on the vascular tone, and is therefore a potential target for therapeutic intervention in hypertension.     

GABAB receptor deficiency causes failure of neuronal homeostasis in hippocampal networks

Stabilization of neuronal activity by homeostatic control systems is fundamental for proper functioning of neural circuits. Failure in neuronal homeostasis has been hypothesized to underlie common pathophysiological mechanisms in a variety of brain disorders. However, the key molecules regulating homeostasis in central mammalian neural circuits remain obscure. Here, we show that selective inactivation of GABA_B, but not GABA_A, receptors impairs firing rate homeostasis by disrupting synaptic homeostatic plasticity in hippocampal networks. Pharmacological GABA_B receptor (GABA_BR) blockade or genetic deletion of the GB_1a receptor subunit disrupts homeostatic regulation of synaptic vesicle release. GABA_BRs mediate adaptive presynaptic enhancement to neuronal inactivity by two principle mechanisms: First, neuronal silencing promotes syntaxin-1 switch from a closed to an open conformation to accelerate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly, and second, it boosts spike-evoked presynaptic calcium flux. In both cases, neuronal inactivity removes tonic block imposed by the presynaptic, GB_1a-containing receptors on syntaxin-1 opening and calcium entry to enhance probability of vesicle fusion. We identified the GB_1a intracellular domain essential for the presynaptic homeostatic response by tuning intermolecular interactions among the receptor, syntaxin-1, and the Ca_V2.2 channel. The presynaptic adaptations were accompanied by scaling of excitatory quantal amplitude via the postsynaptic, GB_1b-containing receptors. Thus, GABA_BRs sense chronic perturbations in GABA levels and transduce it to homeostatic changes in synaptic strength. Our results reveal a novel role for GABA_BR as a key regulator of population firing stability and propose that disruption of homeostatic synaptic plasticity may underlie seizure''s persistence in the absence of functional GABA_BRs.Neural circuits achieve an ongoing balance between plasticity and stability to enable adaptations to constantly changing environments while maintaining neuronal activity within a stable regime. Hebbian-like plasticity, reflected by persistent changes in synaptic and intrinsic properties, is crucial for refinement of neural circuits and information storage; however, alone it is unlikely to account for the stable functioning of neural networks (1). In the last 2 decades, major progress has been made toward understanding the homeostatic negative feedback systems underlying restoration of a baseline neuronal function after prolonged activity perturbations (2–4). Homeostatic processes may counteract the instability by adjusting intrinsic neuronal excitability, inhibition-to-excitation balance, and synaptic strength via postsynaptic or presynaptic modifications (5, 6) through a profound molecular reorganization of synaptic proteins (7, 8). These stabilizing mechanisms have been collectively termed homeostatic plasticity. Homeostatic mechanisms enable invariant firing rates and patterns of neural networks composed from intrinsically unstable activity patterns of individual neurons (9).However, nervous systems are not always capable of maintaining constant output. Although some mutations, genetic knockouts, or pharmacologic perturbations induce a compensatory response that restores network firing properties around a predefined “set point” (10), the others remain uncompensated, or their compensation leads to pathological function (11). The inability of neural networks to compensate for a perturbation may result in epilepsy and various types of psychiatric disorders (12). Therefore, determining under which conditions activity-dependent regulation fails to compensate for a perturbation and identifying the key regulatory molecules of neuronal homeostasis is critical for understanding the function and malfunction of central neural circuits.In this work, we explored the mechanisms underlying the failure in stabilizing hippocampal network activity by combining long-term extracellular spike recordings by multielectrode arrays (MEAs), intracellular patch-clamp recordings of synaptic responses, imaging of synaptic vesicle exocytosis, and calcium dynamics, together with FRET-based analysis of intermolecular interactions at individual synapses. Our results demonstrate that metabotropic, G protein-coupled receptors for GABA, GABA_BRs, are essential for firing rate homeostasis in hippocampal networks. We explored the mechanisms by which GABA_BRs gate homeostatic synaptic plasticity. Our study raises the possibility that persistence of epileptic seizures in GABA_BR-deficient mice (13–15) is directly linked to impairments in a homeostatic control system.     

Bovine serum albumin adsorbed on eremomycin and grafted on silica as new mixed-binary chiral sorbent for improved enantioseparation of drugs

A new silica-based, mixed-binary chiral sorbent grafted with the macrocyclic antibiotic eremomycin and bovine serum albumin (BSA) was obtained. The sorbent-filled high-performance liquid chromatography column was capable of enantioseparation of racemic drugs, such as profens, in reversed-phase-chromatography mode. The mixed-binary eremomycin-BSA-sorbent showed better capability for profens enantioseparation as compared with a sorbent containing eremomycin only. BSA grafted onto the sorbent surface significantly reduced retention times of other proteins from the analyte solution, and free proteins (including BSA) injected as analytes were not retained on the column, and subsequently eluted with a dead volume. The drastic difference observed in the binding of profens and other proteins using the sorbent was tested for determination and enantioseparation of profens in artificial-urine solutions.     

Potential of impression cytology in diagnosis and evaluation of efficacy of pharmacological correction of dry eye syndrome associated with contact lens wearing

Potential of impression cytology in diagnosis and evaluation of efficacy of pharmacological correction of dry eye syndrome (DES) associated with contact lens wearing was studied. When wearing contact lenses for a long time DES with tear film instability and reduction of tear production occurs in more than 50% patients. Morphological changes of epithelium of tarsal and bulbar conjunctiva manifest consequently. Impression cytology reveals structural damage of epithelium with keratinization signs and decrease of goblet cells density down to total absence. After tear substitution therapy tear break-up time increased by 65,3% and total tear production by 11,4%. In control impression cytology of tarsal and bulbar conjunctiva during tear substitution therapy the following changes were revealed: recovery of goblet cells density and differentiation, recovery of epithelial structure and reduction of epithelium keratinization.     

Influenza virus inactivated by artificial ribonucleases as a prospective killed virus vaccine

The inactivation of viral particles with agents causing minimal damage to the structure of surface epitopes is a well-established approach for the production of killed virus vaccines. Here, we describe new agents for the inactivation of influenza virus, artificial ribonucleases (aRNases), which are chemical compounds capable of cleaving RNA molecules. Several aRNases were identified, exhibiting significant virucidal activity against the influenza A virus and causing a minimal effect on the affinity of monoclonal antibodies for the inactivated virus. Using a murine model of the influenza virus infection, a high protective activity of the aRNase-inactivated virus as a vaccine was demonstrated. The results of the experiments demonstrate the efficacy of novel chemical agents in the preparation of vaccines against influenza and, perhaps, against other infections caused by RNA viruses.     

PRE-HOSPITAL EMERGENCY CARE: EVALUATION OF AN AUSTRALIAN SYSTEM

The efficacy of the Sydney ambulance paramedic service in dealing with out-of-hospital cardiac and other emergencies was examined. The outcome of 182 cases (from a total of 1,799 casualty calls) treated by a paramedic service was compared with the outcome of 104 similar cases (from a total of 2,376 calls) treated by a general duties service. There were 33 cases of cardiac arrest in the general duties group; resuscitation was attempted in 12 and none survived. There were 49 cases of cardiac arrest in the paramedic group; resuscitation was attempted in 21 cases and 4 survived. There were 35 cases of suspected myocardial infarction in the general duties group; 7 died compared with 58 cases and 4 deaths, in the paramedic group (mortality 20%, cf. 7%; difference not significant). The increased cost of a paramedic call, less than half of an entire hospital day, appears justified by better results.