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INTRODUCTION: No pharmacy program, however well-resourced, has sufficient time or resources to teach students all current, practice-relevant knowledge. And while the volume of potential pharmacy education curriculum content increases exponentially each year, available time for direct instruction continues to decline. Given these constraints, pharmacy curricula must focus on promoting deep learning of the most critical, fundamental, broadly applicable, and lasting knowledge. Yet, in terms of didactic knowledge, pharmacy education currently has no agreed upon, evidence-based criteria for determining which foundational concepts are most important to teach nor any research-based assessment tools to demonstrate how well students have learned those core concepts. PERSPECTIVE: This lack of consensus regarding core conceptual knowledge makes disparities in learning outcomes both more likely to occur and less likely to be detected or addressed. Over the past 30 years, several scientific disciplines undergirding pharmacy have developed research-based lists of core concepts and related concept inventories, demonstrating their transformative educational potential. Core concepts are big, fundamental ideas that experts agree are critical for all students in their discipline to learn, remember, understand, and apply. Concept inventories are research-based, psychometrically validated, multiple-choice tests designed to uncover learners' prior knowledge and potential misconceptions and determine their depth of understanding of disciplinary core concepts. IMPLICATIONS: This commentary proposes adapting and applying this evidence-based core concepts approach to enhance pharmacy education's overall effectiveness and efficiency and outlines an ongoing, multinational research initiative to identify and define essential pharmacy concepts to be taught, learned, and assessed.
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Educación en Farmacia , Curriculum , Humanos , Conocimiento , EstudiantesRESUMEN
This study investigated the in vitro inhibitory potential of different solvent extracts of leaves of Barbeya oleoides on key enzymes related to type 2 diabetes mellitus (α-glucosidase and α-amylase) in combination with an aggregation assay (using 0.01% Triton X-100 detergent) to assess the specificity of action. The methanol extract was the most active in inhibiting α-glucosidase and α-amylase, with IC50 values of 6.67 ± 0.30 and 25.62 ± 4.12 µg/mL, respectively. However, these activities were significantly attenuated in the presence of 0.01% Triton X-100. The chemical analysis of the methanol extract was conducted utilizing a dereplication approach combing LC-ESI-MS/MS and database searching. The chemical analysis detected 27 major peaks in the negative ion mode, and 24 phenolic compounds, predominantly tannins and flavonol glycosides derivatives, were tentatively identified. Our data indicate that the enzyme inhibitory activity was probably due to aggregation-based inhibition, perhaps linked to polyphenols.
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Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores de Glicósido Hidrolasas/química , Inhibidores de Glicósido Hidrolasas/farmacología , Rosales/química , Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/aislamiento & purificación , Flavonoides/química , Flavonoides/aislamiento & purificación , Flavonoides/farmacología , Inhibidores de Glicósido Hidrolasas/aislamiento & purificación , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/aislamiento & purificación , Hipoglucemiantes/farmacología , Técnicas In Vitro , Fitoquímicos/química , Fitoquímicos/farmacología , Fitoterapia , Extractos Vegetales/química , Extractos Vegetales/farmacología , Hojas de la Planta/química , Plantas Medicinales/química , Polifenoles/química , Polifenoles/aislamiento & purificación , Polifenoles/farmacología , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , alfa-Amilasas/antagonistas & inhibidoresRESUMEN
Currently, type 2 diabetes mellitus (T2DM) and obesity are major global public health issues, and their prevalence in the United Arab Emirates (UAE) are among the highest in the world. In 2019, The UAE diabetes national prevalence was 15.4%. In recent years there has been a considerable investigation of predictive biomarkers associated with these conditions. This study analysed fasting (8 h) blood samples from an obese, normoglycemic cohort and an obese, T2DM cohort of UAE nationals, employing clinical chemistry analysis, 1D 1H NMR and mass spectroscopy (FIA-MS/MS and LC-MS/MS) techniques. The novel findings reported for the first time in a UAE population revealed significant differences in a number of metabolites in the T2DM cohort. Metabolic fingerprints identified by NMR included BCAAs, trimethylamine N-oxide, ß-hydroxybutyrate, trimethyl uric acid, and alanine. A targeted MS approach showed significant differences in lysophosphatidylcholines, phosphatidylcholines, acylcarnitine, amino acids and sphingomyelins; Lyso.PC.a.C18.0, PC.ae.C34.2, C3.DC..C4.OH, glutamine and SM.C16.1, being the most significant metabolites. Pearson's correlation studies showed associations between these metabolites and the clinical chemistry parameters across both cohorts. This report identified differences in metabolites in response to T2DM in agreement with many published population studies. This contributes to the global search for a bank of metabolite biomarkers that can predict the advent of T2DM and give insight to its pathogenic mechanisms.
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Biomarcadores/sangre , Diabetes Mellitus Tipo 2/diagnóstico , Obesidad/complicaciones , Ácido 3-Hidroxibutírico/sangre , Adulto , Alanina/sangre , Aminoácidos de Cadena Ramificada/sangre , Cromatografía Liquida , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/etiología , Femenino , Humanos , Masculino , Metabolómica , Metilaminas/sangre , Persona de Mediana Edad , Obesidad/sangre , Espectrometría de Masas en Tándem , Emiratos Árabes Unidos , Ácido Úrico/sangre , Adulto JovenRESUMEN
Steatosis is the accumulation of triglycerides in hepatic cells wherein fats exceed 5% of the entire liver weight. Although steatotic liver damage is reversible due to the liver's regenerative capability, protracted damage often and typically leads to irreversible conditions such as cirrhosis and hepatocellular carcinoma (HCC). Therefore, early steatotic detection is critical for preventing progression to advanced liver diseases. This also becomes particularly important given the higher prevalence of drug usage, as drugs are a frequent cause of liver damage. Currently, the recommendation to diagnose steatosis is using liver enzymes and performing a liver biopsy. Liver biopsy remains the gold standard method of detection, but the procedure is invasive and an unreliable diagnostic tool. Non-invasive, specific and sensitive diagnostic solutions such as biomarkers are therefore needed for the early detection of steatosis. Our aim is to identify changes in urinary metabolites in tetracycline-induced hepatic steatotic rats at different stages of the diseases using metabolomic-based techniques. Sprague Dawley male rats are treated by intraperitoneal injection (I.P.) with either 62.5 mg kg-1 or 125 mg kg-1 tetracycline, an antibiotic previously known to induce steatosis. We analyse the metabolic profile of the urinary tetracycline induced hepatic steatotic rats using 1H nuclear magnetic resonance (NMR), 2D 1H-1H TOCSY (total correlation spectroscopy) and electrospray liquid chromatography-mass spectrometry (ESI-LC-MS/MS) based metabolomics. The combined analysis of haematoxylin & eosin (H&E), oil red O (ORO) and direct measurement of triglyceride content in the liver tissues of the control samples against 125 mg kg-1 and 62.5 mg kg-1 treated samples, reveals that 125 mg kg-1 tetracycline exposure potentially induces steatosis. The combination of 1H NMR, 2D 1H-1H TOCSY and ESI-LC-MS/MS alongside multivariate statistical analysis, detected a total of 6 urinary metabolites changes, across 6 metabolic pathways. Furthermore, lysine concentration correlates with liver damage as tetracycline dose concentration increases, whilst both H&E and ORO fail to detect hepatocellular damage at the lowest dose concentration. We conclude that the combination of 1H NMR and ESI-LC-MS/MS suggests that these are suitable platforms for studying the pathogenesis of steatosis development, prior to morphological alterations observed in staining techniques and offer a more detailed description of the severity of the steatotic disease.
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INTRODUCTION AND BACKGROUND: In recent decades diabetes mellitus has become a considerable health problem in countries like Sri Lanka and results in an increasing economic burden hampering the social and economic development of these countries. About 60% to 70% of the rural population in Sri Lanka rely on indigenous medicinal systems as their main source for primary health care. Siddha (Tamil) Medicine is one of the four Sri Lankan traditional medicinal systems and it is practised mostly in the eastern and northern provinces of Sri Lanka where the majority of Tamils reside. AIM: The foundation of this study is a documentation of plant species recorded in historical and modern Sri Lankan Siddha Medical documents used to treat diabetes. Based on the systematic documentation and analysis of Siddha concepts about diabetes and its signs and preparations used to treat diabetes in Sri Lankan Siddha Medicine, the plant species included in these preparations (excluding globally or very widely used, very well studied species) were evaluated in terms of the current state-of-the-art about these species' pharmacology and effectiveness in order to lay a foundation for their further development. METHOD: Historic and modern Sri Lankan university texts books in Tamil were used as sources for information on diabetes Siddha concepts and antidiabetic Sri Lankan Siddha Medicine preparations. Information on the known antidiabetic effects of extracts and compounds obtained from these species were used in order to assess the current state of the art of these species. RESULTS AND DISCUSSION: Information of ingredients, preparation methods, amount of ingredients used, and dosages of 60 antidiabetic Sri Lankan Siddha Medicine preparations were obtained. Animal parts including marine organisms, inorganic substances, and plants are the three types of ingredients used. Overall 171 plant species in 73 families were documented. Senna auriculata (L.) Roxb. (Fabaceae) was identified as the most frequently cited species. Globally distributed and very well studied plants were excluded in the pharmacological and clinical literature review which includes 123 plant species. The majority (48%) of the plant species reviewed were studied up to in vivo level as the current maximum level of scientific evidence available. Followed by 41% of species have not been studied for antidiabetic activities or did not show antidiabetic activity. Moreover, 6% and 5% were studied up to in vitro and in clinical levels, respectively. The majority of the species were studied only in the models that represent type 1 diabetes. CONCLUSION: This is the first study systematically assessing the importance of preparations and plants used in antidiabetic Sri Lankan Siddha Medicine preparations. Antidiabetic plants are a crucial health care resource in Sri Lankan Siddha Medicine. This study also identified a wide range of methodological problems in the studies conducted so far. More and better type 2 diabetes models should be employed in future studies. This comprehensive review creates the basis for a more systematic study of these local resources.
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Diabetes Mellitus/tratamiento farmacológico , Preparaciones de Plantas/farmacología , Plantas Medicinales/química , Animales , Etnofarmacología , Humanos , Hipoglucemiantes/aislamiento & purificación , Hipoglucemiantes/farmacología , Medicina Ayurvédica , Fitoterapia/métodos , Preparaciones de Plantas/aislamiento & purificación , Sri LankaRESUMEN
Hexachloro-1:3-butadiene (HCBD) causes segment-specific injury to the proximal renal tubule. A time course study of traditional and more recently proposed urinary biomarkers was performed in male Hanover Wistar rats receiving a single intraperitoneal (ip) injection of 45 mg/kg HCBD. Animals were killed on days 1, 2, 3, 4, 5, 6, 7, 10, 14, and 28 postdosing and the temporal response of renal biomarkers was characterized using kidney histopathology, urinary and serum biochemistry, and gene expression. Histopathologic evidence of tubular degeneration was seen from day 1 until day 3 postdosing and correlated with increased urinary levels of α-glutathione S-transferase (α-GST), albumin, glucose, and kidney injury molecule-1 (KIM-1), and increased gene expression of KIM-1, NAD(P)H dehydrogenase, quinone 1, and heme oxygenase (decycling) 1. Histopathologic evidence of tubular regeneration was seen from day 2 postdosing and correlated with raised levels of urinary KIM-1 and osteopontin and increased gene expression of KIM-1 and annexin A7. Traditional renal biomarkers generally demonstrated low sensitivity. It is concluded that in rat proximal tubular injury, measurement of a range of renal biomarkers, in conjunction with gene expression analysis, provides an understanding of the extent of degenerative changes induced in the kidney and the process of regeneration.
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Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/orina , Butadienos/toxicidad , Lesión Renal Aguda/genética , Lesión Renal Aguda/patología , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Biomarcadores/orina , Expresión Génica , Corteza Renal/química , Corteza Renal/efectos de los fármacos , Corteza Renal/metabolismo , Corteza Renal/patología , Masculino , Estrés Oxidativo/genética , Ratas , Ratas WistarRESUMEN
Hexachloro-1:3-butadiene (HCBD) causes damage specifically to the renal proximal tubule in rats. In the present study, injury to the nephron of male Hanover Wistar rats was characterized at 24 h following dosing with HCBD in the range 5-90 mg kg⻹ to determine the most sensitive biomarkers of damage, that is, the biomarkers demonstrating significant changes at the lowest dose of HCBD, using a range of measurements in serum and urine, renal histopathology, and renal and hepatic gene expression. Histologically, kidney degeneration was noted at doses as low as 10 mg kg⻹ HCBD. Significant changes in the hepatic and renal gene expression categories of xenobiotic metabolism and oxidative stress were observed at 5 mg kg⻹ HCBD, and in the kidney alone, evidence of inflammation at 90 mg kg⻹ HCBD. Increases in the urinary excretion of α-glutathione S-transferase (α-GST) and kidney injury molecule-1 (KIM-1) were seen at 10 mg kg⻹ HCBD, and increases in urinary excretion of albumin and total protein were evident at 15 mg kg⻹ HCBD. The most sensitive, noninvasive biomarkers of HCBD-induced renal toxicity in Hanover Wistar rats were urinary α-GST and KIM-1. Urinary albumin measurement is also recommended as, although it is not the most sensitive biomarker, together with α-GST, albumin showed the largest relative increase of all the biomarkers investigated, and the protein is easily measured.
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Biomarcadores , Butadienos/toxicidad , Fungicidas Industriales/toxicidad , Enfermedades Renales/inducido químicamente , Riñón/efectos de los fármacos , Albuminuria/sangre , Albuminuria/diagnóstico , Albuminuria/orina , Animales , Apoptosis/efectos de los fármacos , Biomarcadores/sangre , Biomarcadores/orina , Moléculas de Adhesión Celular/sangre , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/orina , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Glutatión Transferasa/sangre , Glutatión Transferasa/orina , Inyecciones Intraperitoneales , Isoenzimas/sangre , Isoenzimas/orina , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/ultraestructura , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Tamaño de los Órganos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND: The reproductive ground plan hypothesis of social evolution suggests that reproductive controls of a solitary ancestor have been co-opted during social evolution, facilitating the division of labor among social insect workers. Despite substantial empirical support, the generality of this hypothesis is not universally accepted. Thus, we investigated the prediction of particular genes with pleiotropic effects on ovarian traits and social behavior in worker honey bees as a stringent test of the reproductive ground plan hypothesis. We complemented these tests with a comprehensive genome scan for additional quantitative trait loci (QTL) to gain a better understanding of the genetic architecture of the ovary size of honey bee workers, a morphological trait that is significant for understanding social insect caste evolution and general insect biology. RESULTS: Back-crossing hybrid European x Africanized honey bee queens to the Africanized parent colony generated two study populations with extraordinarily large worker ovaries. Despite the transgressive ovary phenotypes, several previously mapped QTL for social foraging behavior demonstrated ovary size effects, confirming the prediction of pleiotropic genetic effects on reproductive traits and social behavior. One major QTL for ovary size was detected in each backcross, along with several smaller effects and two QTL for ovary asymmetry. One of the main ovary size QTL coincided with a major QTL for ovary activation, explaining 3/4 of the phenotypic variance, although no simple positive correlation between ovary size and activation was observed. CONCLUSIONS: Our results provide strong support for the reproductive ground plan hypothesis of evolution in study populations that are independent of the genetic stocks that originally led to the formulation of this hypothesis. As predicted, worker ovary size is genetically linked to multiple correlated traits of the complex division of labor in worker honey bees, known as the pollen hoarding syndrome. The genetic architecture of worker ovary size presumably consists of a combination of trait-specific loci and general regulators that affect the whole behavioral syndrome and may even play a role in caste determination. Several promising candidate genes in the QTL intervals await further study to clarify their potential role in social insect evolution and the regulation of insect fertility in general.
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Abejas/genética , Evolución Biológica , Sitios de Carácter Cuantitativo , Animales , Abejas/anatomía & histología , Abejas/fisiología , Conducta Animal , Femenino , Tamaño de los Órganos , Ovario/anatomía & histología , Ovario/fisiología , ReproducciónRESUMEN
Hexachloro-1:3-butadiene (HCBD) causes kidney injury specific to the pars recta of the proximal tubule. In the present studies, injury to the nephron was characterized at 24 h following a single dose of HCBD, using a range of quantitative urinary measurements, renal histopathology and gene expression. Multiplexed renal biomarker measurements were performed using both the Meso Scale Discovery (MSD) and Rules Based Medicine platforms. In a second study, rats were treated with a single nephrotoxic dose of HCBD and the time course release of a range of traditional and newer urinary biomarkers was followed over a 25 day period. Urinary albumin (a marker of both proximal tubular function and glomerular integrity) and α-glutathione S-transferase (α-GST, a proximal tubular cell marker of cytoplasmic leakage) showed the largest fold change at 24 h (day 1) after dosing. Most other markers measured on either the MSD or RBM platforms peaked on day 1 or 2 post-dosing, whereas levels of kidney injury molecule-1 (KIM-1), a marker of tubular regeneration, peaked on day 3/4. Therefore, in rat proximal tubular nephrotoxicity, the measurement of urinary albumin, α-GST and KIM-1 is recommended as they potentially provide useful information about the function, degree of damage and repair of the proximal tubule. Gene expression data provided useful confirmatory information regarding exposure of the kidney and liver to HCBD, and the response of these tissues to HCBD in terms of metabolism, oxidative stress, inflammation, and regeneration and repair.
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Lesión Renal Aguda/orina , Albuminuria/inducido químicamente , Butadienos/toxicidad , Moléculas de Adhesión Celular/orina , Expresión Génica/efectos de los fármacos , Riñón/efectos de los fármacos , Lesión Renal Aguda/genética , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Albuminuria/genética , Albuminuria/patología , Albuminuria/orina , Animales , Biomarcadores/orina , Femenino , Perfilación de la Expresión Génica , Inmunoensayo , Riñón/metabolismo , Riñón/patología , Pruebas de Función Renal , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Necrosis , Ratas , Ratas Endogámicas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Previous studies have shown that the enzyme copper/zinc superoxide dismutase (SOD-1) is increased in the urine of rats with carbon tetrachloride (CCl(4))-induced hepatotoxicity. The present experiments aimed to investigate further the usefulness of urinary SOD-1 as a non-invasive biomarker of liver injury. Two investigations were carried out, a dose response study and a time course study. In the dose response study, rats were given a single dose of CCl(4) at 0 (control), 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40 and 0.80 ml/kg and urine samples collected from 12 to 36 h postdosing. In the time course study, rats were dosed at 0.80 ml/kg CCl(4) and urine sampled at 4, 12, 24 and 36 h postdosing. In both studies, the presence of SOD-1 in the urine was confirmed by Western blotting with an SOD-1 antibody. In the dose response study, serum SOD activity was elevated in all CCl(4)-treated animals and urinary SOD-1 activity was increased 2.2 times at the lowest dose (0.10 ml/kg) and 60.4 times at the highest CCl(4) dose level (0.80 ml/kg). In the time course study, urinary SOD-1 was first detected in samples collected from 4 to 12 h postdosing. We conclude that urinary SOD-1 has potential as a sensitive non-invasive biomarker of CCl(4)-induced hepatocellular injury.
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Tetracloruro de Carbono/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Superóxido Dismutasa/orina , Animales , Biomarcadores/sangre , Biomarcadores/orina , Western Blotting/métodos , Tetracloruro de Carbono/administración & dosificación , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Pruebas Enzimáticas Clínicas/métodos , Relación Dosis-Respuesta a Droga , Femenino , Riñón/efectos de los fármacos , Riñón/patología , Hígado/efectos de los fármacos , Hígado/patología , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Proteomic techniques such as two-dimensional gel electrophoresis (2-DGE) and mass spectrometry have become important tools for the identification of novel biomarkers of toxicity and disease. Ideally, such biomarkers need to be sensitive and organ specific, but, recently, it has become apparent that it would be an additional benefit to be able to measure biomarkers in samples obtained using non-invasive methods. The present study is concerned with the identification of novel urinary markers of hepatic fibrosis. In a carbon-tetrachloride-induced liver fibrosis rat model, analysis of urine by 2-DGE revealed an increase in the concentration of a number of proteins in animals with hepatic fibrosis. Using in-gel trypsin digest and nano-scale liquid chromatography combined with electrospray ionisation tandem mass spectrometry, protein spots were identified as copper/zinc superoxide dismutase, D: -dopachrome tautomerase, beta-2-microglobulin and neutrophil gelatinase associated lipocalin. These proteins are known to have important roles in the inflammatory response.
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Biomarcadores/orina , Tetracloruro de Carbono/toxicidad , Cirrosis Hepática/inducido químicamente , Proteómica , Animales , Cromatografía Liquida , Electroforesis en Gel Bidimensional , Femenino , Cirrosis Hepática/orina , Ratas , Ratas Wistar , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
Carbon tetrachloride (CCl(4)) was used to induce liver fibrosis in the rat. Using this model, we have identified changes in serum and urinary clinical chemistry parameters, and characterized histopathological lesions in the liver. Two experiments were conducted. In Experiment 1, rats were dosed at six levels of CCl(4) (0.06-0.36 ml/kg) twice weekly for 6 weeks, followed by a 6-week non-dosing recovery period (week 12). Livers were removed for histology at 6 and 12 weeks and serum parameters analysed. In Experiment 2, rats were given seven dose levels of CCl(4) (0.4-1.0 ml/kg) twice weekly for 6 weeks, followed by a 6-week recovery period (week 12); urine samples were analysed at 3, 6, 9 and 12 weeks using one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Liver fibrosis was evident at 6 weeks in Experiments 1 and 2, and the activity of serum enzymes (including alanine aminotransferase, aspartate aminotransferase and glutamate dehydrogenase) was increased. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis (Experiment 2) revealed a protein band at 18.4 kDa in urine from rats treated with CCl(4), not present in control urine, which was identified as copper/zinc superoxide dismutase (Cu/Zn SOD). Western blotting revealed that SOD was increased in urine from rats treated with CCl(4) at 3 and 6 weeks, but not at 9 and 12 weeks. We conclude that Cu/Zn SOD is a urinary marker of hepatic necrosis, but not hepatic fibrosis.
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Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Glutamato Deshidrogenasa/sangre , Superóxido Dismutasa/orina , Transaminasas/sangre , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Biomarcadores/sangre , Biomarcadores/orina , Tetracloruro de Carbono , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Enfermedad Hepática Inducida por Sustancias y Drogas/orina , Electroforesis en Gel de Poliacrilamida , Femenino , Fibrosis , Hígado/patología , Modelos Animales , Necrosis , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Noradrenaline-stimulated phosphoinositide breakdown in cultured glia was found to be mediated by alpha(1A)-adrenoceptors. The alpha(1A)-selective agonist A61603 was as effective as noradrenaline in eliciting 3H-inositol phosphate (IP) accumulation but was approximately 50-fold more potent. In addition, the use of selective antagonists revealed a clear rank order of potency in the ability of these drugs to reverse the effect of noradrenaline on phosphoinositide breakdown: RS17053 (alpha(1A)-selective) >>AH11110A (alpha(1B)-selective)>BMY7378 (alpha(1D)-selective). Pre-treatment of cultured glia with the protein phosphatase inhibitor okadaic acid resulted in a concentration- and time-dependent reduction in noradrenaline-evoked 3H-IP accumulation. This effect was mimicked by, but was not additive with, a phorbol ester, was reversed by protein kinase C (PKC) inhibitors and was not evident in cells which had been PKC depleted. The ability of cell extracts to dephosphorylate radiolabelled glycogen phosphorylase revealed the presence of the phosphatases PP1 and PP2A in almost equal abundance. Okadaic acid pre-treatment of intact cultures elicited a marked reduction in total phosphatase activity, particularly that mediated by PP2A. We also determined the effect of okadaic acid pre-treatment on PKC and cyclic AMP-dependent protein kinase (PKA) activities in these cells. PKC and PKA activities in cell extracts were assessed by determining the incorporation of 32P into histone and kemptide, respectively. Okadaic acid elicited increases in both Ca(2+)-dependent and Ca(2+)-independent PKC activity; in addition, increases in both initial and total PKA activities were also recorded. The effect of okadaic acid on noradrenaline-stimulated 3H-IP accumulation were not, however, mimicked by either forskolin or 8-bromo-cyclic AMP, suggesting that this event is not regulated by PKA. Our data point to roles for both PKC and PP2A in the regulation of alpha(1A)-adrenoceptor-linked phosphoinositide metabolism in cultured cortical glia.