We compared the occurrence of calcium-dependent electrophysiological alterations in left ventricular myocytes isolated from the heart of old spontaneously hypertensive (SHR) and age-matched ...normotensive Wistar Kyoto rats (WKY). Electrical and mechanical activity were measured in patch-clamped myocytes in which intracellular Ca2+was allowed to change freely. In parallel experiments, intracellular Ca2+concentration was monitored in fura-2-AM preloaded cells using an image analysis system. Left ventricular myocytes from SHR showed a significantly greater membrane capacitance (an index of cell size), a prolonged duration of the action potential and a higher incidence of delayed afterdepolarizations (DADs) and associated aftercontractions, and of spontaneous activity. DADs were likely generated by intracellular Ca2+waves due to spontaneous and local Ca2+release from intracellular stores. These results demonstate that DADs, possibly related to spontaneous intracellular Ca2+waves, occur frequently in hypertrophied rat cardiomyocytes and may be a relevant arrhythmogenic mechanism.
Previous studies have shown that (R)-(+)-hyoscyamine has analgesic activity as a consequence of increased ACh release following antagonism of central muscarinic autoreceptors. Since the enhancement ...of central cholinergic transmission could be beneficial for cognitive disorders, we manipulated (R)-(+)-hyoscyamine, synthesizing several derivatives of tropic and 2-phenylpropionic acids, with the aim of obtaining drugs which are able to increase ACh release and consequently to show analgesic and nootropic activities. The results showed that several new compounds are indeed potent analgesics (with an analgesic efficacy comparable to that of morphine) and that the most potent one ((+/-)-19, PG9) also has remarkable cognition-enhancing properties. Our study confirmed that the mechanism of action involves ACh release even if it is still unclear whether only muscarinic autoreceptors or, also, heteroreceptors are involved.
The effect of the NSAIDs indomethacin, indoprofen, diclofenac and acetylsalicylic acid on the increase in guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) induced by nitric oxide‐donor agents was ...tested in human whole platelets and in platelet crude homogenate.
In whole platelets, indomethacin reduced the increase in cyclic GMP induced by the nitric oxide‐donors (NO‐donors) sodium nitroprusside (NaNP) and S‐nitroso‐N‐acetylpenicillamine (SNAP) in a dose‐dependent way, its IC50 being 13.7 μm and 15.8 μm, respectively.
Of the other cyclooxygenase inhibitors tested, only indoprofen reduced the increase in cyclic GMP induced by both NO‐donors in a dose‐dependent way (IC50=32.7 μm, NaNP and 25.0 μm, SNAP), while acetylsalicylic acid (up to 1000 μm) and diclofenac (up to 100 μm) were ineffective.
However, in platelet crude homogenate neither indomethacin nor indoprofen reduced the cyclic GMP production.
Indomethacin (10 μm), indoprofen (30 μm), diclofenac (100 μm) and acetylsalicylic acid (1000 μm) showed a comparable efficacy in inhibiting platelet thromboxane B2 (TXB2) production, suggesting that the inhibitory effect of indomethacin and indoprofen on the increase in cyclic GMP induced by both NO‐donors was not mediated by inhibition of cyclooxygenase.
In vitro, the NSAIDs analysed did not interfere with nitrite production of SNAP.
The unhomogeneous behaviour of NSAIDs on the increase in cyclic GMP induced by NO‐donors in whole platelets may contribute to the different pharmacological and toxicological characteristics of the drugs, providing new knowledge on the effect of indomethacin and indoprofen.
British Journal of Pharmacology (1998) 123, 1457–1463; doi:10.1038/sj.bjp.0701745
1
In Fura‐2 preloaded human platelets, the increase in cytosolic calcium induced by α‐thrombin was reduced by some l‐ and d‐arginine ester compounds the IC50 (μm) values of which were 7.4 for TAEE, ...56.9 for BAEE, 77.6 for TAME, 560 for T(d)AME, 656.3 for l‐ArgOMe and 2206.7 for d‐ArgOMe. α‐tosyl‐l‐Arginine, l‐ and d‐arginine were inactive.
2
The inhibitory activity of the l‐arginine esters was not modified when platelets were pretreated with 100 μm Nω‐monomethyl‐l‐arginine.
3
The l‐arginine esters did not increase cyclic GMP content in platelets either in the presence or absence of indomethacin and apyrase at rest and after α‐thrombin stimulation.
4
The kinetic parameters of platelet Na+/H+ antiporter (amiloride‐inhibitable, evaluated after cytosolic nigericin‐induced acidification) were modified by l‐ and d‐arginine esters, while the native amino acids were ineffective.
5
The inhibitory effects of the l‐ and d‐arginine esters on platelet activation appear to be mainly due to their inhibitory effect on Na+/H+ antiporter.
A dor neuropática (DN) é causada por uma lesão primária ou por uma disfunção no sistema nervoso periférico (SNP) ou central (SNC), sendo que os principais sintomas são a alodinia mecânica e a ...hiperalgesia a estímulos térmicos e mecânicos. A DN apresenta resposta analgésica insuficiente com terapeuticas farmacológicas clássicas, sendo um desafio para o tratamento clínico. Técnicas de neuromodulação central, como a estimulação transcraniana por corrente contínua (ETCC), representam um recurso promissor no manejo da dor, uma vez que promovem neuroplasticidade em vias envolvidas com o processo doloroso, sendo um método não-invasivo que pode ser combinado com outras terapias. Sendo assim, o objetivo deste estudo foi investigar os efeitos do tratamento repetido com ETCC na resposta hiperalgésica térmica e mecânica em modelo experimental de DN. Adicionalmente, foram avaliados os níveis de IL-1β, IL-10, TNF-α e NGF em estruturas do SNC destes animais. Todos os procedimentos foram aprovado pela Comissão de Ética no Uso de Animais (CEUA/HCPA:120512). Oitenta e quatro ratos machos Wistar foram divididos em 7 grupos: controle, dor neuropática, dor neuropática+ETCC, dor neuropática+sham ETCC, sham dor neuropática, sham dor neuropática+ETCC e sham dor neuropática+sham ETCC. O modelo de DN foi induzido por meio de ligura parcial do nervo isquiático na pata esquerda. O sham do modelo de DN seguiu o mesmo protocolo, com simulação da ligadura parcial do nervo isquiático e o grupo controle não sofreu nenhuma manipulação. O tratamento com ETCC consistiu em 20minutos/dia/8 dias, com intensidade de 0,5mA. Para o sham do tratamento, os eletrodos foram apenas fixados à cabeça do animal durante 20 minutos/dia/8 dias, sem nenhuma estimulação. A hiperalgesia térmica e mecânica foi avaliada por meio dos testes da Placa Quente e de Von Frey, respectivamente, no tempo basal, 7 e 14 dias após a cirurgia e imediatamente, 24 horas e 7 dias após o final do tratamento. Os níveis de IL-1β, IL-10, TNF-α e NGF no cortex cerebral, medula espinhal e tronco cerebral foram determinados por ELISA 48 horas e 7 dias após o final do tratamento. A análise estatística para os testes nociceptivos foi realizada através da Generalized Estimation Equation (GEE)/Bonferroni e para as análises bioquímicas por ANOVA de uma via (IL-1β, IL-10, TNF-α ) e ANOVA de três vias (NGF). Os dados estão expressos como media+erro padrão da média, sendo considerado significativo p<0.05. Nossos resultados demonstraram que a DN altera os níveis de IL-1β, IL-10, TNF-α e NGF no SNC em curto e longo prazo. Além disso, a ETCC reduz a resposta nociceptiva a curto e longo prazo e na modulação dos níveis de citocinas no sistema nervoso central neste modelo. Evidencia-se a importância do papel do sistema imune central nos processos de continuidade da dor neuropática, que pode estar envolvido com as alterações neuroplásticas maladaptativas características dessa patologia.
Neuropathic pain (NP) is caused by a primary insult or dysfunction in the central or peripheral nervous system and its prevalence depends on the type of trauma and related dysfunction. The main symptoms are mechanical allodynia and hyperalgesia to both mechanical and thermal stimuli. NP often shows insufficient response to classic analgesics and remains a challenge to medical treatment and scientific research; and the search for new therapies for this pathology is of fundamental important. Central neuromodulation techniques, such as transcranial direct current stimulation (tDCS), represent a promising resource to pain management since they promote neuroplasticity in the central system of pain. Moreover, tDCS has the advantages of being a noninvasive technique and can be combined with other interventions. The aim of this study was investigated the effects of tDCS in the thermal and mechanical hyperalgesia induced by chronic constriction injury (CCI) of sciatic nerve and measured its effect on the neurochemical markers (IL-1β, IL-10, TNF-α, and NGF levels) on central nervous system structures. All experiments and procedures were approved by the Institutional Animal Care and Use Committee (GPPG-HCPA No.120512) and performed in accordance with the Guide for the Care and Use of Laboratory Animals 8th ed. The CCI of sciatic nerve was used for the induction of NP. For sham surgery, the sciatic nerve was exposed similarly, but it was not ligated. The control group did not undergo surgical procedure. After the establishment of NP, the rats of treated groups were subjected to a 20 minutes session of anodal tDCS, every afternoon for eight days, under a direct constant current of 0.5 mA intensity. The thermal and mechanical hyperalgesia was assessed by Hot plate and Von Frey test, respectively, and evaluated on baseline, 7 and 14 days after surgery; immediately, 24 hours and 7 days after treatment. The IL-1β, IL-10, TNF-α and NGF levels on cortex, spinal cord and brainstem were determined by sandwich-ELISA at 48 hours and 7 days after the end of treatment. Data were expressed as the mean±standard error of the mean (S.E.M). Generalized Estimating Equation (GEE) followed by Bonferroni was performed to compare all groups in different times of nociceptive tests and to biochemical data the one-way ANOVA was used to compare the IL-1β, IL-10, TNF-α and three-way ANOVA was used to compare the NGF levels. P-values less than 0.05 were considered significant. SPSS 19.0 for Windows was used for statistical analysis. In summary, we showed that anodal tDCS is effective to relieve NP and modulate cytokine in CCI rat model, and its effect is observed at long-term. In addition, the CCI model induced increased NGF levels in cerebral cortex and spinal cord at long-lasting time, evidencing the important feature of this neurotrophin in neuropathic pain condition. Additionally, we observed an important role of the central immune system in the neuropathic process, which can be involved with the maladaptative neuroplastic changes.
We studied the interaction between the synthetic prostacyclin analog iloprost and the aggregating agent alpha-thrombin by measuring the internal calcium ion concentration (Ca(2+)i) of human ...fura-2-loaded platelets. Iloprost (0.003-100 micrograms/l) did not modify the resting calcium level; when added 2 minutes before exposure of the platelets to a submaximally active concentration of alpha-thrombin (10 U/l), iloprost dose-dependently antagonized the increase in Ca(2+)i. To evaluate if iloprost retained this antagonistic effect even after a prolonged contact, which is well known to cause a "desensitization" phenomenon, platelets were preincubated with iloprost (35 micrograms/l) for 3 hours. After washout, the effect of newly added iloprost (0.01-100 micrograms/l) on the alpha-thrombin-induced increase in Ca(2+)i was tested. Iloprost was still able to antagonize the increase in Ca(2+)i induced by alpha-thrombin in "desensitized" platelets; however, the dose-inhibitory response curve was significantly shifted to the right when compared with that obtained in control platelets (i.e., platelets preincubated for 3 hours with iloprost's solvent), and the resulting IC50 was significantly higher: 1.78 versus 0.2 micrograms/l (p < 0.001). Since the maximal inhibitory effect of iloprost could also be reached under these experimental conditions, we conclude that iloprost retains its ability to antagonize the increase in Ca(2+)i induced by alpha-thrombin in desensitized platelets.
1
The antiarrhythmic potency of mexiletine was evaluated on three groups of guinea‐pig isolated hearts. Arrhythmias were induced (a) with digitalis intoxication, (b) with hypoxia followed by ...reoxygenation and (c) with ischaemia followed by reperfusion.
2
Mexiletine 10 μM was found to be very effective against all three types of arrhythmias in all three groups.
3
The electrophysiological effects of mexiletine were then studied on sheep cardiac Purkinje fibres manifesting oscillatory afterpotentials and triggered automaticity induced by barium or strophanthidin.
4
Mexiletine 10 μM consistently decreased the amplitude of oscillatory afterpotentials and blocked subsequent triggered activity in sheep Purkinje fibres.
5
In contrast, mexiletine 10 μM had no significant effect on in normal, barium‐ and strophanthidin‐treated preparations.
6
The results are discussed in relation to the mechanisms of antiarrhythmic action of mexiletine.
Further modifications of the leads ((R)-(+)-hyoscyamine and (p-chlorophenyl)propionic acid alpha-tropanyl ester), which show analgesic and nootropic activities as a consequence of increased central ...presynaptic ACh release, are reported. 2-Phenoxy- and 2-(phenylthio)alkanoic acid esters showed the best results. Several members of these classes possess analgesic properties which are comparable to that of morphine and at the same time are able to reverse dicyclomine-induced amnesia. Confirmation was found that the mechanism of action is due to an increase in ACh release at central muscarinic synapses and that both auto- and heteroreceptors controlling ACh release are very likely involved. According to the results obtained with (R)-(+)-hyoscyamine, analgesic activity is stereochemistry dependent, since the R-(+)-enantiomers are always more efficacious than the corresponding S-(-)-ones. On the basis of their potency and acute toxicity, compounds (+/-)-28 (SM21) and (+/-)-42 (SM32) were selected for further study.
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