Effect of depolarizing concentrations of potassium on calcium uptake and metabolism in rat liver

AbstractExposure of perfused livers of fed rats to 60 mM K+ induces rapid responses in the Ca2+-sensitive metabolic events, glycogenolysis, cytoplasmic and mitochondrial NADH/NAD ratios and octanoate oxidation. All increase within 45 s of K+ addition. Metabolic responses were not observed following K+ addition to livers perfused in the absence of added Ca2+. Movements of Ca2+ into the liver were suggested from experiments in which 45Ca2+ uptake was measured. The Ca2+ antagonists verapamil, diltiazem and Ni2+ essentially abolished changes to tissue metabolism and Ca2+ fluxes induced by K+ addition. K+-induced changes were consistent with Ca2+ channel activiation.LiverPotassium depolarizationGlycogenolysisCalcium antagonis

Heterogeneous Responses to Antioxidants in Noradrenergic Neurons of the Locus Coeruleus Indicate Differing Susceptibility to Free Radical Content

The present study investigated the effects of the antioxidants trolox and dithiothreitol (DTT) on mouse Locus coeruleus (LC) neurons. Electrophysiological measurement of action potential discharge and whole cell current responses in the presence of each antioxidant suggested that there are three neuronal subpopulations within the LC. In current clamp experiments, most neurons (55%; 6/11) did not respond to the antioxidants. The remaining neurons exhibited either hyperpolarization and decreased firing rate (27%; 3/11) or depolarization and increased firing rate (18%; 2/11). Calcium and JC-1 imaging demonstrated that these effects did not change intracellular Ca2+ concentration but may influence mitochondrial function as both antioxidant treatments modulated mitochondrial membrane potential. These suggest that the antioxidant-sensitive subpopulations of LC neurons may be more susceptible to oxidative stress (e.g., due to ATP depletion and/or overactivation of Ca2+-dependent pathways). Indeed it may be that this subpopulation of LC neurons is preferentially destroyed in neurological pathologies such as Parkinson's disease. If this is the case, there may be a protective role for antioxidant therapies

Heterogeneous Responses to Antioxidants in Noradrenergic Neurons of the Locus Coeruleus Indicate Differing Susceptibility to Free Radical Content

The present study investigated the effects of the antioxidants trolox and dithiothreitol (DTT) on mouse Locus coeruleus (LC) neurons. Electrophysiological measurement of action potential discharge and whole cell current responses in the presence of each antioxidant suggested that there are three neuronal subpopulations within the LC. In current clamp experiments, most neurons (55%; 6/11) did not respond to the antioxidants. The remaining neurons exhibited either hyperpolarization and decreased firing rate (27%; 3/11) or depolarization and increased firing rate (18%; 2/11). Calcium and JC-1 imaging demonstrated that these effects did not change intracellular Ca2+ concentration but may influence mitochondrial function as both antioxidant treatments modulated mitochondrial membrane potential. These suggest that the antioxidant-sensitive subpopulations of LC neurons may be more susceptible to oxidative stress (e.g., due to ATP depletion and/or overactivation of Ca2+-dependent pathways). Indeed it may be that this subpopulation of LC neurons is preferentially destroyed in neurological pathologies such as Parkinson's disease. If this is the case, there may be a protective role for antioxidant therapies

Acetilkolinesteraza u eritrocitima i butirilkolinesteraza u plazmi - Važni pokazatelji za liječenje osoba otrovanih organofosfornim spojevima

Inhibition of acetylcholinesterase (AChE) is regarded as the primary toxic mechanism of organophosphorus compounds (OP). Therapeutic strategies are directed to antagonise overstimulation of muscarinic receptors with atropine and to reactivate inhibited AChE with oximes. Reactivation is crucial within the neuromuscular synapse, where atropine is ineffective, since peripheral neuromuscular block eventually leads to respiratory failure. Patients with OP intoxication have to be identified as early as possible. During an international NBC-defence exercise anesthetised pigs were poisoned with sarin, followed by treatment with atropine and oxime. Blood samples were drawn and red blood cell (RBC)-AChE activity determined with a fielded test system on-site. Within a few minutes the poisoning was verified. After administration of HI-6, RBC-AChE activity increased rapidly. Blood samples were reanalysed in our laboratory in Munich. Almost identical course of the AChE activities was recorded by both systems. The more comprehensive cholinesterase status was determined in Munich. Oxime administration can be stopped when AChE is aged completely, but has to be continued as long as poison is present in the body and reactivation is possible. To aid the on-site physician in optimising diagnosis and treatment, a fielded test system should be available to allow rapid determination of the complete cholinesterase status.Inhibicija acetilkolinesteraze (AChE) smatra se primarnim mehanizmom toksičnoga djelovanja organofosfornih spojeva (OP). Strategije liječenja idu za zaustavljanjem prekomjerne stimulacije muskarinskih receptora atropinom i reaktiviranjem inhibiranog AChE oksimima. Ključna je reaktivacija u neuromuskularnoj sinapsi, u kojoj atropin nije djelotvoran, budući da neuromuskularna blokada u konačnici vodi do prestanka disanja. Važno je što ranije prepoznati otrovanje organofosfornim spojem. U jednoj međunarodnoj vježbi zaštite od nuklearnog, biološkog i kemijskog napada svinje pod anestezijom otrovane su sarinom te liječene atropinom i oksimom. Uzeti su im uzorci krvi te s pomoću terenskoga testa na licu mjesta određena aktivnost AChE u eritrocitima. Otrovanje je potvrđeno za nekoliko minuta. Nakon primjene HI-6, aktivnost AChE brzo je porasla. Isti su uzorci krvi ponovno analizirani u našem laboratoriju u Münchenu. Oba su testa zabilježila gotovo istovjetan tijek aktivnosti AChE. U Münchenu je međutim napravljen potpuniji nalaz kolinesteraza. Liječenje oksimima može se prekinuti kada AChE potpuno “ostari” (tj. dealkilira), ali ga valja nastaviti dokle god je otrov u tijelu, a reaktivacija moguća. Liječnici na terenu trebali bi raspolagati terenskim testovima radi brzoga i potpunog utvrđivanja statusa kolinesteraza, a time i kvalitetnije dijagnoze

Three independent mechanisms contribute to tetracaine inhibition of cardiac calcium release channels

Tetracaine is a tertiary amine local anaesthetic which inhibits ryanodine receptors (RyRs), the calcium release channels of the sarcoplasmic reticulum (SR). Tetracaine has been extensively used to study the role of the SR Ca2+ fluxes in muscle cells, yet a detailed understanding of tetracaine action on RyR channels is lacking. Here we investigate tetracaine effects in single channel recording of sheep cardiac RyRs in lipid bilayers. Tetracaine decreased channel conductance (block) and open probability (inhibition). The IC50 for inhibition had complex dependencies on membrane voltage and cytoplasmic [ATP], [Ca2+] and pH. We identify three mechanisms underlying these actions. First, a voltage-dependent, slow inhibition in which luminal and cytoplasmic tetracaine compete for a common neutral/cation binding site within the trans-membrane RyR domain to induce long closed events (~ 100 ms). The apparent binding rate is proportional to the RyR closed probability, indicating that it only operates on closed channels. Second, a voltage-independent, pH sensitive fast inhibition in which cytoplasmic and luminal tetracaine compete for a site located on the cytoplasmic domain of the RyR to induce fast closed events (~ 2 ms). Its IC50 is not dependent on the open/closed conformation of RyR. Finally, a voltage-dependent block of the channel by cytoplasmic tetracaine reduced channel conductance. We develop a model for tetracaine inhibition which predicts that under diastolic conditions, i.e. when RyRs are mainly closed, the slow mechanism has the highest potency (IC50 ~ 200 μM) of the three mechanisms and is therefore the dominant form of inhibition. However, during periods of Ca2+ release, i.e. when RyRs are open, the slow mechanism becomes ineffective, leaving the fast inhibition (IC50 ~ 2 mM) as the dominant effect. Because of this closed state inhibition property, tetracaine loses its efficacy when RyRs open. This has the effect of increasing the feedback on SR Ca2+ release generated by cytoplasmic and luminal Ca2+

Ca2+ phase waves: a basis for cellular pacemaking and long-range synchronicity in the guinea-pig gastric pylorus

Ca2+ imaging and multiple microelectrode recording procedures were used to investigate a slow wave-like electrical rhythmicity in single bundle strips from the circular muscle layer of the guinea-pig gastric pylorus. The ‘slow waves’ (SWs) consisted of a pacemaker and regenerative component, with both potentials composed of more elementary events variously termed spontaneous transient depolarizations (STDs) or unitary potentials. STDs and SW pacemaker and regenerative potentials exhibited associated local and distributed Ca2+ transients, respectively. Ca2+ transients were often larger in cellular regions that exhibited higher basal Ca2+ indicator-associated fluorescence, typical of regions likely to contain intramuscular interstitial cells of Cajal (ICCIM). The emergence of rhythmicity arose through entrainment of STDs resulting in pacemaker Ca2+ transients and potentials, events that exhibited considerable spatial synchronicity. Application of ACh to strips exhibiting weak rhythmicity caused marked enhancement of SW synchronicity. SWs and underlying Ca2+ increases exhibited very high ‘apparent conduction velocities’ (‘CVs’) orders of magnitude greater than for sequentially conducting Ca2+ waves. Central interruption of either intercellular connectivity or inositol 1,4,5-trisphosphate receptor (IP3R)-mediated store Ca2+ release in strips caused SWs at the two ends to run independently of each other, consistent with a coupled oscillator-based mechanism. Central inhibition of stores required much wider regions of blockade than inhibition of connectivity indicating that stores were voltage-coupled. Simulations, made using a conventional store array model but now including depolarization coupled to IP3R-mediated Ca2+ release, predicted the experimental findings. The linkage between membrane voltage and Ca2+ release provides a means for stores to interact as strongly coupled oscillators, resulting in the emergence of Ca2+ phase waves and associated pacemaker potentials. This distributed pacemaker triggers regenerative Ca2+ release and resultant SWs

Epidermal growth factor induces tyrosine phosphorylation, membrane insertion, and activation of transient receptor potential channel 4

Various members of the canonical family of transient receptor potential channels (TRPCs) exhibit increased cation influx following receptor stimulation or Ca2+ store depletion. Tyrosine phosphorylation of TRP family members also results in increased channel activity; however, the link between the two events is unclear. We report that two tyrosine residues in the C terminus of human TRPC4 (hTRPC4), Tyr-959 and Tyr-972, are phosphorylated following epidermal growth factor (EGF) receptor stimulation of COS-7 cells. This phosphorylation was mediated by Src family tyrosine kinases (STKs), with Fyn appearing to be the dominant kinase. In addition, EGF receptor stimulation induced the exocytotic insertion of hTRPC4 into the plasma membrane dependent on the activity of STKs and was accompanied by a phosphorylation-dependent increase in the association of hTRPC4 with Na+/H+ exchanger regulatory factor. Furthermore, this translocation and association was defective upon mutation of Tyr-959 and Tyr-972 to phenylalanine. Significantly, inhibition of STKs was concomitant with a reduction in Ca2+ influx in both native COS-7 cells and hTRPC4-expressing HEK293 cells, with cells expressing the Y959F/Y972F mutant exhibiting a reduced EGF response. These findings represent the first demonstration of a mechanism for phosphorylation to modulate TRPC channel function