Genotype × region and genotype × production level interactions in Holstein cows.

The identification of the presence of genotype by environment interaction effects on important traits in Holstein cattle allows for the use of international genetic evaluations and a more efficient design of regional genetic evaluation programmes. The aim of this study was to determine the genotype × environment interaction effects in Chilean Holstein dairy cattle through the analysis of records corresponding to calvings between 1998 and 2015. Herds were classified in the central and southern regions of Chile based on herd location as well as by high and low levels of production environments based on the fat plus protein yield averages per herd within each region. The central region has a Mediterranean climate and a confined production system while the southern region has a humid temperate climate and a production system based on grazing with supplementation. Traits studied were milk yield (MY), fat yield (FY), protein yield (PY), fat content (FC) and protein content (PC) by lactation, age at first calving (AFC) and calving interval (CI). Several four-trait mixed animal models were applied to environmental category data as different traits, which included herd-year-calving season (herd-year-birth season for AFC) and lactation number as fixed effects, and animal additive genetic, sire-herd, permanent environment and residual effects as random effects. Genetic correlations (rg) for MY, FY, FC, PC and CI were found to decrease as differences between environmental categories increased. The rg between the most extreme environmental categories considered in this study for AFC (0.26) was the only one found statistically lower than 0.60. Genetic correlation values statistically lower than 0.80 (P < 0.05) were observed for AFC, CI, MY, FY and PY between some environmental categories. If separate genetic evaluations are adopted as practical criteria when the value of rg is lower than 0.60, the consequence of improving a multi-trait economic breeding objective in this population is likely to be small unless extreme environmental categories are considered. However, a moderate decrease in selection response and re-ranking of selection candidates is expected for AFC, CI and yield traits when selection is performed in different environmental conditions. Genotype × environment interaction effects involving production systems in a Mediterranean climate and confinement vs. Temperate Oceanic climate and grazing with supplementation, and between two fat plus protein yield level categories within each environment, were at most moderate for the studied traits.

DIMINUIÇÃO DO ÍNDICE DE NECROSE EM ENXERTOS CUTÂNEOS EM COELHOS MEDIANTE A APLICAÇÃO DE CÉLULAS TRONCO MESENQUIMAIS XENÓGENAS

O presente estudo teve como objetivo avaliar a inflamação em auto-enxertos cutâneos obtidos no terceiro, sétimo e décimo quarto dia de pós-operatório, além disso, buscou-se determinar diferenças no processo de cicatrização no grupo tratado com células tronco mesenquimais xenógenas em relação ao grupo controle utilizando a avaliação microscópica e imuno-histoquímico. A avaliação microscópica foi realizada utilizando cortes histológicos corados pela técnica de histoquímica com hematoxilina-eosina (HE), e a imuno-histoquímica, com cortes submetidos a anticorpos específicos. As variáveis analisadas foram quantidade de vasos, células inflamatórias, COX-2, Macrófagos e presença de necrose. Os dados foram analisados estatisticamente pelo software R. A quantidade de vasos foi maior (p<0,0001) no grupo tratamento (GT) durante o dia 3, enquanto no grupo controle (GC) foi maior no dia 7. No dia 3 houve menor porcentagem de necrose no grupo tratamento (GT) (p = 0,038). Nos demais dias avaliados não houve diferença entre a porcentagem de necrose observada nos dois tratamentos (p = 0,98), sendo de 53% para o grupo controle (GC) e 47% para o grupo tratamento (GT). Em relação ao número de macrófagos não houve diferença entre os grupos (p = 0,5637). Entretanto, entre os dias houve diferença significativa (p = 0,0223), sendo menor número de macrófagos no terceiro dia. A imunomarcação de C

Diminuição do índice de necrose em enxertos cutâneos em coelhos mediante a aplicação de células tronco mesenquimais xenógenas / Reducing of the necrosis index in skin grafts in rabbits through application of xenogenous mesenchymal stem cells

O presente estudo teve como objetivo avaliar a inflamação em auto-enxertos cutâneos obtidos no terceiro, sétimo e décimo quarto dia de pós-operatório, além disso, buscou-se determinar diferenças no processo de cicatrização no grupo tratado com células tronco mesenquimais xenógenas em relação ao grupo controle utilizando a avaliação microscópica e imuno-histoquímico. A avaliação microscópica foi realizada utilizando cortes histológicos corados pela técnica de histoquímica com hematoxilina-eosina (HE), e a imuno-histoquímica, com cortes submetidos a anticorpos específicos. As variáveis analisadas foram quantidade de vasos, células inflamatórias, COX-2, Macrófagos e presença de necrose. Os dados foram analisados estatisticamente pelo software R. A quantidade de vasos foi maior (p<0,0001) no grupo tratamento (GT) durante o dia 3, enquanto no grupo controle (GC) foi maior no dia 7. No dia 3 houve menor porcentagem de necrose no grupo tratamento (GT) (p = 0,038). Nos demais dias avaliados não houve diferença entre a porcentagem de necrose observada nos dois tratamentos (p = 0,98), sendo de 53% para o grupo controle (GC) e 47% para o grupo tratamento (GT). Em relação ao número de macrófagos não houve diferença entre os grupos (p = 0,5637). Entretanto, entre os dias houve diferença significativa (p = 0,0223), sendo menor número de macrófagos no terceiro dia. A imunomarcação de COX-2 foi similar entre os grupos (p = 0,5637) e entre os dias (p = 0,9843). Portanto, o emprego das células tronco mesenquimais xenógenas em enxertos cutâneos promoveu menor ocorrência de necrose, favorecendo sua cicatrização, e não induziu o processo inflamatório, sendo assim factível seu uso em cirurgias reconstrutivas.(AU)

The present study aimed to assess inflammation in skin autografts obtained on the third, seventh and fourteenth postoperative day, in addition, it sought to determine differences in the healing process in the group treated with xenogenous mesenchymal stem cells in relation to to the control group using microscopic and immunohistochemical evaluation. Microscopic evaluation was performed using histological sections, stained by the hematoxylin-eosin (HE) histochemistry technique, and immunohistochemistry with sections were subjected to specific antibodies. The variables analyzed were the number of vessels, inflammatory cells (COX-2 and Macrophages) and the presence of necrosis. The data were analyzed statistically by software R. The number of vessels was higher (p< 0.0001) ) in the treatment group (GT) during day 3, while in the control group (CG) it was higher on day 7. On day 3 there was a lower percentage of necrosis in the treatment group (GT) (p = 0.038). On the other evaluated days, there was no difference between the percentage of necrosis observed in the two treatments (p = 0.98), being 53% for the control group (CG) and 47% for the treatment group (GT). Regarding the number of macrophages, there was no difference between groups (p = 0.5637). However, between days there was a significant difference (p = 0.0223), with a lower number of macrophages on the third day. The immunostaining of COX-2 was similar between groups (p = 0.5637) and between days (p = 0.9843). Therefore, the use of xenogenous mesenchymal stem cells in skin grafts promoted a lower occurrence of necrosis, favoring its healing, and did not induce the inflammatory process, thus making its use in reconstructive surgery feasible.(AU)

Diminuição do índice de necrose em enxertos cutâneos em coelhos mediante a aplicação de células tronco mesenquimais xenógenas / Reducing of the necrosis index in skin grafts in rabbits through application of xenogenous mesenchymal stem cells

O presente estudo teve como objetivo avaliar a inflamação em auto-enxertos cutâneos obtidos no terceiro, sétimo e décimo quarto dia de pós-operatório, além disso, buscou-se determinar diferenças no processo de cicatrização no grupo tratado com células tronco mesenquimais xenógenas em relação ao grupo controle utilizando a avaliação microscópica e imuno-histoquímico. A avaliação microscópica foi realizada utilizando cortes histológicos corados pela técnica de histoquímica com hematoxilina-eosina (HE), e a imuno-histoquímica, com cortes submetidos a anticorpos específicos. As variáveis analisadas foram quantidade de vasos, células inflamatórias, COX-2, Macrófagos e presença de necrose. Os dados foram analisados estatisticamente pelo software R. A quantidade de vasos foi maior (p<0,0001) no grupo tratamento (GT) durante o dia 3, enquanto no grupo controle (GC) foi maior no dia 7. No dia 3 houve menor porcentagem de necrose no grupo tratamento (GT) (p = 0,038). Nos demais dias avaliados não houve diferença entre a porcentagem de necrose observada nos dois tratamentos (p = 0,98), sendo de 53% para o grupo controle (GC) e 47% para o grupo tratamento (GT). Em relação ao número de macrófagos não houve diferença entre os grupos (p = 0,5637). Entretanto, entre os dias houve diferença significativa (p = 0,0223), sendo menor número de macrófagos no terceiro dia. A imunomarcação de COX-2 foi similar entre os grupos (p = 0,5637) e entre os dias (p = 0,9843). Portanto, o emprego das células tronco mesenquimais xenógenas em enxertos cutâneos promoveu menor ocorrência de necrose, favorecendo sua cicatrização, e não induziu o processo inflamatório, sendo assim factível seu uso em cirurgias reconstrutivas.

The present study aimed to assess inflammation in skin autografts obtained on the third, seventh and fourteenth postoperative day, in addition, it sought to determine differences in the healing process in the group treated with xenogenous mesenchymal stem cells in relation to to the control group using microscopic and immunohistochemical evaluation. Microscopic evaluation was performed using histological sections, stained by the hematoxylin-eosin (HE) histochemistry technique, and immunohistochemistry with sections were subjected to specific antibodies. The variables analyzed were the number of vessels, inflammatory cells (COX-2 and Macrophages) and the presence of necrosis. The data were analyzed statistically by software R. The number of vessels was higher (p< 0.0001) ) in the treatment group (GT) during day 3, while in the control group (CG) it was higher on day 7. On day 3 there was a lower percentage of necrosis in the treatment group (GT) (p = 0.038). On the other evaluated days, there was no difference between the percentage of necrosis observed in the two treatments (p = 0.98), being 53% for the control group (CG) and 47% for the treatment group (GT). Regarding the number of macrophages, there was no difference between groups (p = 0.5637). However, between days there was a significant difference (p = 0.0223), with a lower number of macrophages on the third day. The immunostaining of COX-2 was similar between groups (p = 0.5637) and between days (p = 0.9843). Therefore, the use of xenogenous mesenchymal stem cells in skin grafts promoted a lower occurrence of necrosis, favoring its healing, and did not induce the inflammatory process, thus making its use in reconstructive surgery feasible.

Metástase hepática de tumor gastrointestinal estromal (gist) em cão: relato de caso / Hepatic metastasis of gastrointestinal stromal tumors (gist) in dog: case report

Os tumores mesenquimais originados a partir de células intersticiais de Cajal, denominados tumores gastrointestinais estromais (GIST) são raros em humanos e com incidência ainda desconhecida em cães. A diferenciação de GIST, leiomiossarcoma e leiomioma com base apenas em exames de imagem, morfológicos e colorações simples por meio da histologia é muitas vezes difícil, sendo indicada realização de imuno-histoquímica. Foi atendida uma cadela, sem padrão racial definido, oito anos, 17kg de peso corporal, com discreto aumento de volume abdominal, sendo evidenciada por ultrassom abdominal neoformação intra-abdominal em região mesogástrica, sem demais alterações clínicas. Durante celiotomia exploratória, identificou-se que a neoformação intestinal localizava-se em jejuno, optando pela realização de enterectomia, sendo posteriormente diagnosticada como leiomiossarcoma pela análise histopatológica. Preconizou-se a instituição de quimioterapia adjuvante com o uso de doxorrubicina (30 mg/m²), a cada 21 dias, totalizando seis sessões. Após dez meses do tratamento cirúrgico, a paciente foi submetida a novo estadiamento clínico, sendo visibilizado, mediante ultrassom abdominal neoformações sólidas em fígado. A partir de biópsia hepática guiada por ultrassom com agulha “tru-cut” e análise histológica e imuno-histoquímica, definiu-se que as neoformações hepáticas eram metástases de GIST, instituindo o tratamento com fosfato de toceranib (Palladia®). Desde o momento da enterectomia ao óbito da paciente, contabilizou-se 20,4 meses de sobrevida global.(AU)

Mesenchymal tumors originating from interstitial cells of Cajal, called stromal gastrointestinal tumors (GIST) are rare in humans and their incidence is still unknown in dogs. The differentiation of GIST, leiomyosarcoma and leiomyoma based only on imaging, morphological and simple staining through histology is often difficult, and immunohistochemistry is indicated. A female dog, mixed breed, eight years old, 17kg of body weight, with a slight increase in abdominal volume, and ultrasound evidence of an intra-abdominal neoformation in the mesogastric region, without other clinical changes was seen. During exploratory celiotomy, it was identified that the intestinal neoformation in jejunum, opting for enterectomy, being identified as leiomyosarcoma according to histopathological analysis. Adjuvant chemotherapy was instituted using doxorubicin (30mg / m²) every 21 days, totaling six sessions. The patient underwent a new clinical staging, 10 months after enterectomy, where were visualized, by abdominal ultrasound, neoformations in the liver. Based on ultrasound guided liver biopsy with “tru-cut” needle and histological and immunohistochemical analysis, the neoformation was defined as GIST liver metastasis, being instituted treatment with toceranib phosphate (Palladia®). From the time of enterectomy to the death of the patient, 20,4 months of overall survival were counted.(AU)

Metástase hepática de tumor gastrointestinal estromal (gist) em cão: relato de caso / Hepatic metastasis of gastrointestinal stromal tumors (gist) in dog: case report

Os tumores mesenquimais originados a partir de células intersticiais de Cajal, denominados tumores gastrointestinais estromais (GIST) são raros em humanos e com incidência ainda desconhecida em cães. A diferenciação de GIST, leiomiossarcoma e leiomioma com base apenas em exames de imagem, morfológicos e colorações simples por meio da histologia é muitas vezes difícil, sendo indicada realização de imuno-histoquímica. Foi atendida uma cadela, sem padrão racial definido, oito anos, 17kg de peso corporal, com discreto aumento de volume abdominal, sendo evidenciada por ultrassom abdominal neoformação intra-abdominal em região mesogástrica, sem demais alterações clínicas. Durante celiotomia exploratória, identificou-se que a neoformação intestinal localizava-se em jejuno, optando pela realização de enterectomia, sendo posteriormente diagnosticada como leiomiossarcoma pela análise histopatológica. Preconizou-se a instituição de quimioterapia adjuvante com o uso de doxorrubicina (30 mg/m²), a cada 21 dias, totalizando seis sessões. Após dez meses do tratamento cirúrgico, a paciente foi submetida a novo estadiamento clínico, sendo visibilizado, mediante ultrassom abdominal neoformações sólidas em fígado. A partir de biópsia hepática guiada por ultrassom com agulha “tru-cut” e análise histológica e imuno-histoquímica, definiu-se que as neoformações hepáticas eram metástases de GIST, instituindo o tratamento com fosfato de toceranib (Palladia®). Desde o momento da enterectomia ao óbito da paciente, contabilizou-se 20,4 meses de sobrevida global.

Mesenchymal tumors originating from interstitial cells of Cajal, called stromal gastrointestinal tumors (GIST) are rare in humans and their incidence is still unknown in dogs. The differentiation of GIST, leiomyosarcoma and leiomyoma based only on imaging, morphological and simple staining through histology is often difficult, and immunohistochemistry is indicated. A female dog, mixed breed, eight years old, 17kg of body weight, with a slight increase in abdominal volume, and ultrasound evidence of an intra-abdominal neoformation in the mesogastric region, without other clinical changes was seen. During exploratory celiotomy, it was identified that the intestinal neoformation in jejunum, opting for enterectomy, being identified as leiomyosarcoma according to histopathological analysis. Adjuvant chemotherapy was instituted using doxorubicin (30mg / m²) every 21 days, totaling six sessions. The patient underwent a new clinical staging, 10 months after enterectomy, where were visualized, by abdominal ultrasound, neoformations in the liver. Based on ultrasound guided liver biopsy with “tru-cut” needle and histological and immunohistochemical analysis, the neoformation was defined as GIST liver metastasis, being instituted treatment with toceranib phosphate (Palladia®). From the time of enterectomy to the death of the patient, 20,4 months of overall survival were counted.

Zinc modulation of basal and ß-adrenergically stimulated L-type Ca2+ current in rat ventricular cardiomyocytes: consequences in cardiac diseases.

Zinc exists in biological systems as bound and histochemically reactive free Zn(2+) in the nanomolar range. Zinc is required as either structural or catalytic component for a large number of enzymes. It also modulates current passage through many ion channels. Here, we reinvestigated the effects of extracellular and intracellular Zn(2+) on the L-type Ca(2+) current (I (CaL)) and its modulation by ß-adrenergic stimulation in rat ventricular cardiomyocytes. In the absence of Ca(2+) ions, Zn(2+) could permeate through the L-type channel at much lower concentrations and at a more positive voltage range, but with a lower permeability than Ca(2+). In the presence of Ca(2+), extracellular Zn(2+) demonstrated strong bimodal inhibitory effects on the I (CaL), with half-inhibition occurring around 30 nM, i.e., in the range of concentrations found in the plasma. Intracellular Zn(2+) also significantly inhibited the I (CaL) with a half-inhibitory effect at 12.7 nM. Moreover, ß-adrenergic stimulation was markedly reduced by intracellular Zn(2+) at even lower concentrations (<1 nM) as a consequence of Zn(2+)-induced inhibition of the adenylyl cyclase. All these effects appeared independent of redox variations and were not affected by dithiothreitol. Thus, both basal intracellular and extracellular Zn(2+) modulate transmembrane Ca(2+) movements and their regulation by ß-adrenergic stimulation. Considering that, in many pathological situations, including diabetes, the extracellular Zn(2+) concentration is reduced and the intracellular one is increased, our results help to explain both Ca(2+) overload and marked reduction in the ß-adrenergic stimulation in these diseases.

Homeopathy in Cuban epidemic neuropathy: an open clinical trial.

In an outbreak of epidemic neuropathy (EN) in Cuba (1992-1993), most patients were improved by vitamin therapy. In subjects with residual symptoms, alternative treatments including homeopathy were suggested to ameliorate optic and peripheral signs of the disease. An open clinical pilot trial was conducted on 31 patients with long standing symptoms of optic (OPTI group, n=15) or peripheral EN (PERI group, n=16). During the trial, OPTI and PERI patients continued the same treatment that they received before. Carboneum sulphuratum and Tabacum in homeopathic dilutions were administered for 30 days. These medicines are specific to optic EN, but not closely linked with peripheral EN. Clinical status was evaluated by neurological and ophthalmologic tests at diagnosis (Ddiag), 7 days before homeopathic treatment (D0) and 90 days after (D90). From D0 to D90, the percentages of improvement were 73.3% for the OPTI form and 12.5% for the PERI form. The percentage of improved OPTI patients was significantly higher after the homeopathic treatment vs the period between Ddiag and D0 for optical EN (P<0.01), but not for PERI subjects (P>0.05). In the OPTI group, colour vision, visual acuity and visual field improved after homeopathic treatment (P<0.001), these parameters did not change between Ddiag and D90 (P>0.05). Carboneum sulphuratum and Tabacum showed a reasonable effectiveness in optical EN, but were not effective in PERI EN.

Cardiac cellular actions of hydrochlorothiazide.

In long term treatment, thiazide diuretics such as hydrochlorothiazide (HCTZ) lower blood pressure by decreasing peripheral resistance rather than by their diuretic effect. This action has been attributed to the opening of Ca2+-activated K+ channels in vascular smooth muscle cells. However, little is known about their cardiac cellular actions. Here we investigated the possible actions of HCTZ on action potential and contraction of rat ventricular muscle strips and on the ionic currents of isolated rat ventricular cardiomyocytes. HCTZ depressed ventricular contraction with an IC30 of 1.85 microM (60% decrease at 100 microM). Action potential duration at -60 mV and maximal rate of depolarization were, however, only slightly decreased by 12% and 22%, respectively, at 100 microM. At the single cell level, HCTZ (100 microM) depressed the fast Na+ current (INa) and the L-type Ca2+ current (ICaL) by 30% and 20%, respectively. The effects on ICaL were not voltage-or frequency-dependent. In cells intracellularly perfused with 50 microM cyclic adenosine, monophosphate HCTZ reduced ICaL by 33%. The transient (Ito), the delayed rectifier and the inward rectifier potassium currents were decreased by 20% at 100 microM HCTZ. The effects on Ito were voltage-dependent. In conclusion, HCTZ at high concentrations possesses a negative inotropic action that could be in part due to its blocking action on INa and ICaL. The actions of HCTZ on multiple cardiac ionic currents could explain its weak effect on action potential duration.

Late post-myocardial infarction induces a tetrodotoxin-resistant Na(+)Current in rat cardiomyocytes.

Left ventricular remodeling after myocardial infarction is accompanied by electrical abnormalities that might predispose to rhythm disturbances. To get insight into the ionic mechanisms involved, we studied myocytes isolated from four different regions of the rat ventricles, 4-6 months after ligation of the left coronary artery. Using the whole-cell patch-clamp technique, we never observed T-type Ca(2+)current in both diseased and control hearts. In contrast, in 41 out of 78 cells isolated from 16 post-myocardial infarcted rats, analysed in the presence of 30 m m Na(+)ions, we found a tetrodotoxin (TTX)-resistant Na(+)current with quite variable amplitude in every investigated region. Albeit being resistant to 100 microM TTX, this Na(+)-dependent current was highly sensitive to lidocaine since 3 microM lidocaine induced about 65% tonic block. It was also inhibited by 5 microM nifedipine and 2 m m Co(2+), but was insensitive to 100 microM Ni(2+). The TTX-resistant Na(+)channel availability was shifted rightward by 25-30 mV with respect to TTX-sensitive Na(+)current; therefore, a large "window current" might flow in the voltage range from -70 to -20 mV. In conclusion, in late post-myocardial infarction, a Na(+)current with specific kinetics and pharmacology may provide inward charges in a critical range of membrane voltages that are able to alter action potential time course and trigger ventricular arrhythmia. These apparent new characteristics of the Na(+)channel might result in part from environmental changes during heart remodeling.

Modulation of voltage-dependent facilitation of the T-type calcium current by sodium ion in isolated frog atrial cells.

Sodium ions have been reported to alter the permeation properties of L- and N-type Ca2+ channels. Here in frog atrial cardiomyocytes under whole-cell patch-clamp conditions, we have examined the effects of lowering the external Na+ concentration on the amplitude of T-type Ca2+ current, ICaT, and on the relief of its steady-state inactivation by large depolarizing prepulses, ICaT facilitation. A partial reduction in Na+ ion concentration did not significantly alter ICaT amplitude elicited at -50 mV. However, after a large depolarization, low- Na+ solutions enhanced the relief of inactivation and induced ICaT facilitation. This facilitation occurred independently of the divalent charge carrier, high intracellular Ca2+ buffering or the intracellular Na+ content. Its effects were additional to the beta-adrenergic effects mediated by a decrease of Gi/o-protein inhibitory tone. In Ca2+-free solution the very large T-type current, then carried by Na+ ions, showed only a weak relief of inactivation. In conclusion, ICaT facilitation--which, as previously reported, is modulated by the transient voltage-dependent relief of Gi-protein inhibitory tone--is further enhanced in a low-Na+ solution. In Ca2+-free solution, relief of inactivation due to re-openings dependent on the divalent charge carrier is improbable. It thus appears that for a short while after a large depolarization, external Na+ compete with Ca2+ ions on permeation-controlling sites, so as to modulate channel re-openings and thus the amplitude of voltage-facilitated ICaT independently of the control exerted by the inhibitory G-protein.

Characteristics of Ca2+ channel blockade by oxodipine and elgodipine in rat cardiomyocytes.

The two novel dihydropyridines, oxodipine and elgodipine greatly depressed the KCl-induced contraction of rabbit aorta and decreased the cardiac force of contraction of rat ventricular strips with lower potency. Both compounds markedly shortened cardiac action potentials. In rat cultured neonatal ventricular myocytes, oxodipine and elgodipine decreased the L-type Ca2+ current (I(CaL)) with IC50 of 0.24 and 0.33 microM respectively while oxodipine was slightly more potent on the T-type Ca2+ current (I(CaT)) than elgodipine (IC50 = 0.41 vs. 2.18 microM). Both compounds were less potent in inhibiting I(CaL) of adult cardiomyocytes. Oxodipine exhibited mostly a tonic block of both currents while elgodipine induced mainly a use-dependent block. Oxodipine and elgodipine increased by at least one order of magnitude their inhibitory potency on I(CaT) and I(CaL) when the cells were partially depolarized. We conclude that the mechanisms of inhibition of Ca2+ channels by these two dihydropyridines are different and suggest that the underlying mechanism of vascular selectivity is the voltage-dependent block of I(CaL), with the use-dependent inhibition of Ca2+ currents by elgodipine further contributing to this selectivity.

Calcium antagonist properties of the bisbenzylisoquinoline alkaloid cycleanine.

The alkaloid cycleanine ([12aR-(12aR,24aR)]-2,3,12a,13,14,15,24,24a-octa hydro-5,6,17,18- tetramethoxy-1,13-dimethyl-8, 11:20,23-dietheno-1H,12H [1,10]dioxacyclooctadecino[2,3,4-ij:11,12,13-i'j']diisoquinolin e) was extracted from the bulbs of Stephania glabra (Roxb) Miers and its effects on cardiac and smooth muscle preparations were studied and compared to those of nifedipine (1,4-dihydro-2, 6-dimethyl-4-(2-nitrophenyl)-3,5-pyridine dicarboxylic acid dimethylesther). Cycleanine inhibited the KCl-induced contraction of rabbit aortic rings with higher potency than nifedipine. IC50s for cycleanine and nifedipine were 0.8 and 7.10(-9) M respectively. Cycleanine had minor effects on the norepinephrine-induced contraction of rabbit aortic rings. Cycleanine and nifedipine also depressed the contraction of rat ventricular preparations but with lower potency (IC50 = 3 and 0.03.10(-6) M respectively). Action potential duration of rat right ventricular strips was decreased by both compounds. L-type Ca-current (ICaL) of single rat ventricular cardiomyocytes was inhibited by cycleanine in a voltage- and frequency-dependent manner. With a higher potency nifedipine inhibited ICaL in a tonic and almost frequency-independent manner. The results suggest that cycleanine can act as a potent vascular selective Ca-antagonist.

Facilitation of T-type calcium current in bullfrog atrial cells: voltage-dependent relief of a G protein inhibitory tone.

1. The properties of the low-threshold calcium current, ICa,T, were investigated in bullfrog isolated atrial cardiomyocytes using the whole-cell, patch-clamp technique under control conditions and during beta-adrenergic stimulation. 2. The intracellular application of GTP gamma S or adenosine-5'-O-3-thiotriphosphate (ATP gamma S), poorly hydrolysable analogues of GTP and ATP, respectively, barely affected ICa,T amplitude in control conditions. beta-Adrenergic stimulation effects were more marked in the presence of ATP gamma S. 3. The intracellular application of GDP beta S and ADP reduced ICa,T amplitude. In cells pretreated with pertussis toxin, ICa,T amplitude was significantly increased. In both conditions, the addition of isoprenaline was without effect. 4. Under both control and beta-adrenergic-stimulated conditions, a conditioning prepulse to +70 mV did not fully inactivate ICa,T; rather ICa,T facilitation often occurred after beta-adrenergic stimulation. 5. In GTP gamma S- and ATP gamma S-dialysed cells, ICa,T facilitation was generally observed after a prepulse; it was larger in the ATP gamma S dialysis. Facilitation was sustained but ended immediately upon cessation of conditioning prepulses. After beta-adrenergic stimulation, facilitation was more marked in GTP gamma S- than in ATP gamma S-dialysed cells. 6. ICa,T facilitation was prevented by the intracellular application of GDP beta S and by pertussis toxin pretreatment. 7. ICa,T facilitation developed markedly in the presence of intracellular cyclic AMP. This effect was prevented by pertussis toxin pretreatment of the cells. 8. It is thus proposed that ICa,T is under a double antagonistic control by both a Gs and a Gi protein. Furthermore, the double-pulse-induced facilitation of ICa,T results from a voltage-dependent relief of the Gi protein inhibitory tone. Such an effect is increased by protein kinase A-dependent phosphorylation, presumably of the Gi protein.

Effects of tetrandrine on Ca(2+)- and Na(+)-currents of single bullfrog cardiomyocytes.

Tetrandrine is a natural alkaloid classified as a calcium antagonist. However, its precise actions on Ca(2+)-currents in cardiac cells have not been fully characterized. In the present study, we have investigated the mechanism of action of tetrandrine on the Ca(2+)-currents of single bullfrog cardiac cells, using the patch-clamp technique. Tetrandrine slightly increased ICaL from negative holding potentials (-100 mV) at low concentrations (10 nM-1 microM) and inhibited it at higher concentrations. At depolarized holding potentials (-50 mV) only an enhanced inhibition was seen. Tetrandrine blockade of the L-type Ca(2+)-current (ICaL) was mostly tonic. This is similar to ICaL blockade by nifedipine but not by verapamil, the latter being mostly use-dependent. Use-dependent effects of tetrandrine and nifedipine were evident at high rates. Availability curves were shifted leftwards (10-12 mV) by tetrandrine (10 microM) and nifedipine (1 microM). The T-type Ca(2+)-current (ICaT), although less sensitive, was decreased by both agents in a voltage-independent way. Tetrandrine (10-30 microM) but not nifedipine (1-10 microM), depressed the Na(+)-current (INa) in tonic, use- and voltage-dependent manners. We conclude that tetrandrine and nifedipine share some common actions on cardiac Ca(2+)-channels, while showing differences in their actions on Na(+)-channels. The depression of INa by tetrandrine suggests it could be effective on supraventricular tachycardias.

Properties of the low threshold Ca current in single frog atrial cardiomyocytes. A comparison with the high threshold Ca current.

The properties of the low threshold Ca current (ICaT) in bullfrog (Rana catesbeiana) isolated atrial cardiomyocytes were studied using the whole-cell recording patch-clamp technique and compared with those of the high threshold Ca current (ICaL). In 91% of atrial cells we observed both ICaT and ICaL when collagenase and trypsin were used to dissociate the cells. But when pronase was used, only 30% of the cells exhibited ICaT. ICaT was never found in ventricular cells. ICaT could be investigated more easily when ICaL was inhibited by Cd ions (50 microM). Its kinetics were unchanged by substituting Ba for Ca, or in the presence of high concentrations of Ba. Both ICaT and ICaL exhibited reduced inactivation after high depolarizing prepulses. ICaT was found to be sensitive to dihydropyridines: 1 microM nifedipine decreased this current while 1 microM BAY K 8644 increased it; this occurred without significant variations in the steady-state inactivation curve. ICaT was more sensitive than ICaL to alpha 1-adrenergic and P2-purinergic stimulations, while ICaL was more sensitive to beta-adrenergic stimulation. Isoproterenol was still able to increase ICaT in the presence of high intracellular cAMP. Both currents were increased by 1 microM ouabain (although ICaL only transiently) and decreased by 10 microM ouabain. It is concluded that the two types of Ca channels can be observed in bullfrog atrial cells and that they are specifically altered by pharmacological agents and neuromediators. This may have implications for cardiac behavior.

Prajmalium, an antiarrhythmic with positive inotropic effect: mechanism of action in rabbit single cardiomyocytes.

The propyl derivative of ajmaline, N-n-propylajamaline (prajmalium), is an antiarrhythmic compound that lacks the commonly reported negative inotropic effects of all others under clinical use. The present study was undertaken to establish and understand its effects at the cellular level in mammalian preparations. Electrical and mechanical activities were recorded from right ventricular strips and Na and L-type Ca currents (INa and ICaL, respectively) were recorded with the whole-cell patch-clamp technique in right ventricular myocytes freshly dissociated from rabbit hearts. Prajmalium decreased the maximal rate of depolarization of the action potential in a dose-dependent manner with an EC50 of 3 microM. This effect was use and frequency dependent. Action potential duration was increased by 1 microM prajamalium but decreased on applying higher concentrations. The force of contraction was slightly (15%) increased at 0.1 microM, not affected at all at 1 microM and depressed by 30% at 20 microM. In single cardiomyocytes maintained at negative holding potentials, INa was slightly depressed by prajmalium at 10 nM and reduced by 75% at 10 microM. ICaL was increased by 30 and 20% on applying prajmalium at 1 and 10 microM, respectively; on the other hand, ICaL was reduced by these two concentrations of prajmalium at less negative holding potentials. A higher prajmalium concentration (100 microM) decreased ICaL at all holding potentials studies and this effect was enhanced with depolarization. The increase in ICaL induced by prajmalium (1 microM) was also observed after ICaL had been fully beta-adrenergic and P2-purinergic stimulated by isoproterenol (1 microM) in the presence of IBMX (100 microM) and ATP (10 microM). It is concluded that prajmalium is able to increase ICaL in rabbit ventricular cells in a voltage-dependent manner, an effect that could account in part for the observed lack of negative inotropism of this antiarrhythmic in clinics.

Dual action of prajmalium on the Ca currents in frog isolated cardiomyocytes.

The effects of N-n-propylajmaline (prajmalium) on the Na and Ca currents of single frog atrial and ventricular cells were studied by means of the whole-cell patch-clamp technique. Prajmalium (10(-9) to 10(-6) M) depressed the Na current (INa) in a dose- and use-dependent manner. In the same range of concentrations, prajmalium induced a dual effect on the high (ICaL) and low (ICaT) threshold Ca currents (the latter being only present in atrial cells). At a low concentration (10(-9) M), prajmalium increased both Ca currents while at high concentrations (10(-6) M) it depressed them. Prajmalium appeared very potent on ICaT although this current is generally reported to be barely sensitive to agonists and drugs. The action of the drug was also accompanied by a shortening in the half-time of inactivation of the Ca currents and a slight hyperpolarizing shift of their availability curves. The increase in ICaL by prajmalium was not prevented by prazosin (10(-7) M) nor by propranolol (10(-6) M), and it was also observed after ICaL had been fully stimulated by isoproterenol (10(-7)M). Nifedipine (10(-6) M), however, was able to prevent or block the prajmalium-induced increase in ICaL. Some similarities between the actions of prajmalium and dihydropyridine agonists on Ca currents are discussed.

[Acute hepatic lesion caused by Giardia lamblia]. / Lesión hepática aguda por Giardia lamblia.

A study was made of 20 rats infested by Giardia muris in which a histologic study was made of the liver, as well as of 25 patients with giardiasis and elevated alanine-aminotransferase levels. Patients with positive A or B hepatitis markers, cholelithiasis or history of drug or alcohol use were excluded. Tests of liver function and liver biopsy were performed and antiparasite therapy was given during three months of follow-up, after which the liver biopsy was repeated. Humoral alterations were compared to those of 30 patients with acute viral hepatitis (15 type A and 15 type B) over the same periods of time. In 20% of the rats, nonspecific liver lesions were found. In the patients liver enzymes and the thymol test normalized a month after treatment and serum bile acids became normal in the third month. The liver biopsy demonstrated hepatic damage in 94% of the patients (in 20 cases cell lesions and in 12 cases inflammatory lesions) which regressed in the third month, the follow-up biopsy being normal after eradication of the parasite was confirmed. The comparative study with viral hepatitis showed highly significant differences in all the variables studied during the follow-up stage. Emphasis is placed on the importance of this lesion and its differential diagnosis to prevent its progression to chronic liver disease.