RESUMO
BACKGROUND: Simulation models are increasingly important for skill acquisition during microsurgery training. Prosthetics, living and non-living biological models have been proposed in the literature in the optics of recreating real-life scenarios in a controlled environment. This study aims to validate and prove the reusability of a novel non-living biological model: the porcine placenta. METHODS: A prospective comparative study was carried out to assess face and content validities of the proposed model, as well as the reusability and quality of the Thiel-embalming method. Participants were asked answer a questionnaire for each anastomosis they performed on porcine placental vessels of ≤2mm (small) and 2-4mm (large). Scores were classified according to different subgroups, either small or large vessels and first or second sessions. Reliability analysis of the questionnaire was carried out using Cronbach's α, to ensure an α>0.7. Median scores for each question were analyzed using boxplots and compared amongst each subgroup using a non-parametric independent Mann-Whitney U test. RESULTS: With nine participants, the Cronbach's α for each category of question was 0.867, 0.778, 0.720 and 0.593. Statistical differences were found between responses of small and large vessels on 5/10 questions, where large vessels reported higher validity. No statistical differences were found between scores of the first and second sessions. CONCLUSION: By evaluating face and content validity, the Thiel-embalmed porcine placenta has proven its suitability as a microsurgery model, especially for vessels of larger caliber. Qualities that distinguish this model is its reliable reusability, its low cost-effectiveness, and its ethical acceptability.
Assuntos
Embalsamamento , Placenta , Animais , Cadáver , Feminino , Humanos , Gravidez , Estudos Prospectivos , Reprodutibilidade dos Testes , SuínosRESUMO
OBJECTIVE: 12/15-Lipoxygenase (12/15-LOX) catalyzes the generation of various anti-inflammatory lipid mediators, and has been implicated in several inflammatory and degenerative diseases. However, there is currently no evidence that 12/15-LOX has a role in osteoarthritis (OA). The aim of this study was to investigate the role of 12/15-LOX in the pathogenesis of OA. METHODS: The development of aging-associated and destabilization of the medial meniscus (DMM)-induced OA were compared in 12/15-LOX-deficient (12/15-LOX-/-) and wild-type (WT) mice. The extent of cartilage damage was evaluated by histology. The expression of OA markers was evaluated by immunohistochemistry and RT-PCR. Cartilage explants were stimulated with IL-1α in the absence or presence of the 12/15-LOX metabolites, 15-hydroxyeicosatetraenoic acids (15-HETE), 13-hydroxyoctadecadienoic acid (13-HODE) or lipoxin A4 (LXA4), and the levels of matrix metalloproteinases-13 (MMP-13), Nitric oxide (NO) and prostaglandin E2 (PGE2) were determined. The effect of LXA4 on the progression of OA was evaluated in wild type (WT) mice. RESULTS: The expression of 12/15-LOX in cartilage increased during the progression of DMM-induced OA and with aging in WT mice. Cartilage degeneration was more severe in 12/15-LOX-/- mice compared to WT mice in both models of OA, and this was associated with increased expression of MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs, aggrecanases (ADAMTS5), inducible NO synthases (iNOS), and mPGES-1. Treatment of cartilage explants with 12/15-LOX metabolites, suppressed IL-1α-induced production of MMP-13, NO and PGE2, with LXA4 being the most potent. Intra-peritoneal injection of LXA4 reduced the severity of DMM-induced cartilage degradation. CONCLUSIONS: These data suggest an important role of 12/15-LOX in the pathogenesis of OA. They also suggest that activation of this pathway may provide a novel strategy for prevention and treatment of OA.
Assuntos
Araquidonato 12-Lipoxigenase/fisiologia , Araquidonato 15-Lipoxigenase/fisiologia , Artrite Experimental/enzimologia , Osteoartrite/enzimologia , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Araquidonato 12-Lipoxigenase/deficiência , Araquidonato 12-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/deficiência , Araquidonato 15-Lipoxigenase/genética , Artrite Experimental/etiologia , Artrite Experimental/prevenção & controle , Cartilagem Articular/metabolismo , Progressão da Doença , Mediadores da Inflamação/metabolismo , Instabilidade Articular/complicações , Lipoxinas/uso terapêutico , Masculino , Camundongos Knockout , Osteoartrite/etiologia , Osteoartrite/prevenção & controle , Lesões do Menisco Tibial/complicações , Técnicas de Cultura de Tecidos , Regulação para CimaRESUMO
The aim of this randomized, placebo-controlled and double-blinded trial was to compare the effect of a veterinary therapeutic diet (VTD) rich in omega-3 fatty acids (omega-3) from fish origin to a regular diet used as control (CTR) over a period of 13 weeks in dogs afflicted by naturally occurring osteoarthritis (OA). Thirty privately owned dogs were selected. Dogs had lameness confirmed by an orthopaedic examination, had stifle/hip OA and had locomotor disability based on the peak of the vertically oriented ground reaction force (PVF) measured using a force platform. At Baseline, all owners were asked to determine 2-5 activities of daily living that were the most impaired. Activities were scores (0-4) in accordance with severity using case-specific outcome measures (CSOM). The PVF was also measured. Dogs (15/group) were then randomly assigned to receive either the CTR or the VTD. The CSOM was completed twice weekly. The recording of PVF was repeated at Week 7 and 13. The VTD-fed dogs showed a significantly higher PVF at Week 7 (p < 0.001) and at Week 13 (p < 0.001) when compared to Baseline. From Baseline to Week 13, VTD-fed dogs had a mean (± SD) change in PVF recording of 3.5 ± 6.8% of body weight (%BW) compared with 0.5 ± 6.1%BW (p = 0.211) in CTR-fed dogs. This change in primary outcome was consistent with an effect size of 0.5. Conversely, dogs fed the CTR did not show significant change in PVF measurements. At the end of the study, the CSOM was significantly decreased (p = 0.047) only in VTD fed dogs. In lame OA dogs, a VTD that contains high level of omega-3 from fish origin improved the locomotor disability and the performance in activities of daily living. Such nutritional approach appears interesting for the management of OA.
Assuntos
Gorduras na Dieta/farmacologia , Doenças do Cão/dietoterapia , Ácidos Graxos Ômega-3/farmacologia , Osteoartrite/veterinária , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Dieta/veterinária , Gorduras na Dieta/administração & dosagem , Cães , Relação Dose-Resposta a Droga , Ácidos Graxos Ômega-3/administração & dosagem , Osteoartrite/dietoterapiaRESUMO
OBJECTIVE: To evaluate the effects of moderate exercise on kinetic gait analysis using a force platform in dogs with hindlimb lameness due to osteoarthritis (OA). METHODS: Ten control dogs (Control) and 10 dogs presented with chronic and stable hindlimb lameness (OA) were recruited. Dogs were subjected to force platform gait analysis to determine baseline data. They were thereafter trotted for a distance of 1.2 km on a short leash, lead by the same handler at a gait convenient for each of them (ranging from slow to fast trot), after which the gait analysis was immediately repeated to determine post-exercise values. Peak and impulse of the vertical and braking / propulsion forces were analysed using a linear model for repeated measures and Bonferroni sequential correction. RESULTS: In the Control group, the differences between baseline and post-exercise data were not significant. Conversely, post-exercise peak (p = 0.020) and impulse (p = 0.009) values of the vertical force, as well as the peak of the propulsion force (p = 0.009) values were significantly lower than baseline in the OA group. CLINICAL RELEVANCE: This study demonstrates the significant effect of a moderate amount of exercise in exacerbating hindlimb lameness in dogs clinically afflicted by OA. It is suggested that: 1) exercise should be considered as a potential factor of variation in future force platform gait analyses and an effort should be made to limit bias in data recording; and 2) an exercise-based protocol could be added to the standard force platform gait analysis to potentially increase its sensitivity in the detection of lame dogs.
Assuntos
Doenças do Cão/fisiopatologia , Marcha/fisiologia , Osteoartrite/veterinária , Condicionamento Físico Animal , Animais , Tornozelo/fisiopatologia , Peso Corporal , Progressão da Doença , Doenças do Cão/terapia , Cães , Feminino , Articulação do Quadril/fisiopatologia , Coxeadura Animal/terapia , Masculino , Osteoartrite/fisiopatologia , Osteoartrite/terapia , Joelho de Quadrúpedes/fisiopatologiaRESUMO
To evaluate the effect of licofelone, an arachidonic acid substrate with combined inhibitory activity against 5-lipoxygenase and cyclooxygenases 1 and 2, a double-blind, randomised and placebo-controlled study was conducted in 33 client-owned dogs that were lame owing to hindlimb osteoarthritis. Seventeen of the dogs received a placebo and 16 were treated with 2.5 mg/kg licofelone twice a day for 28 days. The dogs' lameness was assessed on a visual analogue scale (vas), and by force plate analyses at baseline and 14 and 28 days after starting the treatment. After 14 days the mean (se) change in peak vertical force in the licofelone-treated dogs (1.7 [0.8] per cent bodyweight) was significantly greater (P<0.05) than in the placebo-treated dogs (-0.3 [0.6] per cent bodyweight), and after 28 days the difference had increased. In contrast, the dogs' lameness, as assessed by the vas values, had decreased significantly over baseline in both the treated and control groups.
Assuntos
Acetatos/uso terapêutico , Anti-Inflamatórios não Esteroides/uso terapêutico , Doenças do Cão/tratamento farmacológico , Osteoartrite/veterinária , Pirróis/uso terapêutico , Animais , Cães , Método Duplo-Cego , Feminino , Marcha/efeitos dos fármacos , Coxeadura Animal/tratamento farmacológico , Masculino , Osteoartrite/tratamento farmacológicoRESUMO
GH-releasing factor (GRF)-stimulated GH release is dependent on a biphasic increase in free intracellular Ca2+ concentration [( Ca2+]i), resulting from an influx of Ca2+ into somatotrophs, while the inhibitory action of somatostatin (SRIF) on basal and GRF-induced GH release results from its ability to lower [Ca2+]i by inhibiting Ca2+ influx. This study was carried out to investigate the mechanism by which GRF and SRIF regulate [Ca2+]i to control GH release. The roles of ion channels, cAMP-dependent processes, and protein kinase-C (PKC) were investigated by measuring changes in [Ca2+]i, 45Ca influx, and GH release when purified rat somatotrophs were exposed to high K+, cAMP analogs, prostaglandin E2, as well as the PKC activators 1,2-dioctanoyl-glycerol and phorbol 12-myristate 13-acetate. High K+ depolarization produced a rapid and transient increase in [Ca2+]i, while cAMP and prostaglandin E2 led to a sustained elevated [Ca2+]i. PKC activators produced a transient increase in [Ca2+]i, followed by a decrease to below baseline. All secretagogues tested raised [Ca2+]i by stimulating Ca2+ influx through L-type voltage-sensitive Ca2+ channels (VSCC), since the increases in [Ca2+]i were blocked by incubation in Ca2(+)-free medium and by the dihydropyridine Ca2+ antagonist nifedipine. SRIF lowered [Ca2+]i by blocking the Ca2+ influx stimulated by all of these GH secretagogues except high K+. These results are consistent with the model in which GRF initiates its action by increasing Na+ conductance to depolarize the somatotroph via cAMP. This depolarization would stimulate Ca2+ influx through VSCC, which would result in the first phase of the GRF-dependent increase in [Ca2+]i. This increase in [Ca2+]i would stimulate Ca2+ removal from the cytosol by activating Ca-ATPase via Ca-calmodulin and/or PKC. This would result in the lowering of [Ca2+]i to the plateau level of the second phase of the GRF response. SRIF prevents the GRF-induced increase in [Ca2+]i by increasing K+ conductance and, thus, hyperpolarizing the cell. Hyperpolarization would close VSCC, leading to a decrease in Ca2+ influx, with a subsequent drop in [Ca2+]i.
Assuntos
Cálcio/metabolismo , Hormônio Liberador de Hormônio do Crescimento/farmacologia , Hormônio do Crescimento/metabolismo , Adeno-Hipófise/metabolismo , Somatostatina/farmacologia , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Bucladesina/farmacologia , Separação Celular/métodos , Centrifugação com Gradiente de Concentração/métodos , AMP Cíclico/metabolismo , Corantes Fluorescentes , Indóis , Cinética , Masculino , Modelos Biológicos , Nifedipino/farmacologia , Adeno-Hipófise/citologia , Adeno-Hipófise/efeitos dos fármacos , Potássio/farmacologia , Ratos , Ratos Endogâmicos , Espectrometria de FluorescênciaRESUMO
GH feeds back at the level of the central nervous system to alter the release of somatostatin and GRF, resulting in altered GH release. The purpose of this study was to see whether the concentration of GH, impinging directly on the somatotrophs of the adenohypophysis, would alter the responsiveness of the somatotrophs to GRF. Using a perifusion system and dispersed pituitary cells, we found that the GH response to a pulse of GRF is unaltered over a wide range of GH concentrations. We conclude that GH does not feed back at the level of the adenohypophysis to alter the responsiveness of the somatotrophs to GRF.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/farmacologia , Hormônio do Crescimento/fisiologia , Adeno-Hipófise/fisiologia , Animais , Masculino , Perfusão , Adeno-Hipófise/citologia , Ratos , Valores de Referência , Fatores de TempoRESUMO
To examine the role of protein kinase-C in the mediation of GH release we used acutely dispersed purified somatotrophs in static incubation and acutely dispersed adenohypophyses in perifusion. In static incubation, activation of protein kinase-C by phorbol 12-myristate 13-acetate (PMA) and 1,2-dioctanoyl-rac-glycerol (diC8) resulted in an increase in GH release and a concurrent concentration-dependent increase in cAMP accumulation. The GH response to diC8 in perifusion was reversible and repeatable. On the other hand, the GH response to PMA was not repeatable. The lack of repeatability is most likely due to the depletion of protein kinase-C by prolonged treatment with PMA. This assumption is strengthened by the observation that 1 h of perifusion with PMA left the somatotrophs refractory to a subsequent application of diC8. When graded pulses of GRF were applied during treatment with PMA, the GH response to GRF was not altered. Somatostatin reduced (in static incubation) or blocked (in perifusion) the release of GH induced by diC8 and PMA, but the accumulation of cAMP was not affected. We conclude that 1) activation of protein kinase-C in normal somatotrophs results in GH release which may not be completely independent of the cAMP pathway; 2) activation of protein kinase-C is not essential for GRF-induced GH release; and 3) SRIF acts at a site distal to or independent of cAMP to inhibit GH release induced by activators of protein kinase-C.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/farmacologia , Hormônio do Crescimento/metabolismo , Adeno-Hipófise/metabolismo , Proteína Quinase C/fisiologia , Animais , AMP Cíclico/metabolismo , Técnicas Citológicas , Ativação Enzimática , Perfusão/métodos , Adeno-Hipófise/citologia , Proteína Quinase C/metabolismo , Ratos , Somatostatina/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The purpose of this study was to characterize the adenylate cyclase system in a purified population of normal somatotrophs derived from rat pituitary and to determine the responses of this system to GRF, somatostatin, guanine nucleotides, and cations. Additionally, experiments were performed to evaluate the interrelationships among changes in adenylate cyclase activity, cellular cAMP levels, and GH release induced by GRF and somatostatin. The results obtained using homogenates and membrane preparations from somatotrophs included the following. 1) GRF caused guanine nucleotide-dependent concentration-related (Ka, approximately 10(-8) M) stimulation of adenylate cyclase activity. 2) Guanine nucleotides were effective in stimulating cyclase in the absence of GRF; the concentration of guanine nucleotide required for half-maximal stimulation was decreased more than 10-fold in the presence of GRF. 3) Adenylate cyclase activity increased with increasing concentrations of free Mg2+ (0.25-20 mM); activation by GRF and guanine nucleotide resulted in an approximately 7-fold increase in the enzyme's affinity for free Mg2+. 4) Somatostatin, up to 10(-6) M, did not alter basal or GRF-stimulated adenylate cyclase activity. 5) Ca2+ (0.5-11.9 microM) produced concentration-dependent inhibition of basal (up to 28%) and GRF-stimulated (up to 47%) cyclase activities; the inhibitory effect of Ca2+ was accompanied by a decrement (2- to 3-fold) in the apparent affinities of the enzyme for both GRF and guanine nucleotide. In intact somatotrophs, GRF produced concentration-dependent stimulation of GH release (Ka, approximately 6 x 10(-11) M), preceded by a marked elevation of cAMP levels. While somatostatin blocked GRF-induced GH release, the augmented cAMP levels were only slightly reduced.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Adenilil Ciclases/metabolismo , Hormônio Liberador de Hormônio do Crescimento/farmacologia , Nucleotídeos de Guanina/farmacologia , Hipófise/enzimologia , Somatostatina/farmacologia , Regulação Alostérica , Animais , Cálcio/farmacologia , Membrana Celular/enzimologia , AMP Cíclico/biossíntese , Ativação Enzimática/efeitos dos fármacos , Guanosina Trifosfato/farmacologia , Guanilil Imidodifosfato/farmacologia , Magnésio/farmacologia , Masculino , Hipófise/efeitos dos fármacos , RatosRESUMO
This study was carried out to investigate the role of free intracellular Ca2+ ([Ca2+]i) in the action of GH-releasing factor (GRF) by determining whether GRF causes and increase in [Ca2+]i and whether this increase results from changes in Ca2+ influx/efflux and/or mobilization of intracellular Ca2+ stores. We used a purified preparation of normal rat somatotrophs and examined the changes in 45Ca uptake, [Ca2+]i measured with indo-1, intracellular cAMP, and GH release induced by GRF. GRF stimulated a concentration-related biphasic increase in [Ca2+]i. Both the GRF-dependent increase in [Ca2+]i and GH release were blocked by incubation in low Ca2+ medium and by the organic Ca2+ antagonists nifedipine and diltiazem. The measurement of 45Ca uptake, in both steady state and nonsteady state conditions, demonstrated directly that GRF stimulates Ca2+ influx into somatotrophs. These data demonstrate that the GRF-stimulated increase in [Ca2+]i is dependent on Ca2+ influx. Redistribution of intracellularly stored Ca2+ could not be detected, even though intracellular Ca2+ stores were present. Therefore, the increase is due to Ca2+ influx, and the biphasic nature of the increase in [Ca2+]i induced by GRF is due to a difference in the rate of activation of Ca2+ influx and Ca2+ removal from the cytosol.
Assuntos
Cálcio/metabolismo , Hormônio Liberador de Hormônio do Crescimento/farmacologia , Adeno-Hipófise/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Separação Celular/métodos , Células Cultivadas , Centrifugação com Gradiente de Concentração , AMP Cíclico/metabolismo , Diltiazem/farmacologia , Corantes Fluorescentes , Hormônio do Crescimento/metabolismo , Indóis , Cinética , Masculino , Nifedipino/farmacologia , Adeno-Hipófise/citologia , Adeno-Hipófise/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Espectrometria de FluorescênciaRESUMO
This study was carried out to investigate the role of Ca2+ in the somatostatin (SRIF)-induced inhibition of GH release. We examined the effect of SRIF on basal and GH-releasing factor (GRF)-induced increases in Ca2+ influx and free intracellular Ca2+ concentration ([Ca2+]i) in normal somatotrophs and examined the effect of SRIF on 45Ca uptake, [Ca2+]i measured with indo-1, and GH release. SRIF inhibited basal and GRF-induced GH release concurrently with a reduction in steady state 45Ca uptake. In nonsteady state experiments, SRIF also decreased basal 45Ca uptake. SRIF decreased baseline [Ca2+]i in a concentration-dependent manner and inhibited the GRF-induced biphasic increase in [Ca2+]i, but in a differential fashion. Low concentrations of SRIF abolished the peak (first phase) without affecting the plateau (second phase), while at high concentrations, both phases were inhibited. SRIF blocked the GRF-induced increase in [Ca2+]i regardless of whether it was applied before or during GRF stimulation. These data indicate that the SRIF-dependent decrease in 45Ca uptake is due to a decrease in Ca2+ influx. This is further supported by the fact that the GRF-dependent increase in [Ca2+]i, which is dependent on Ca2+ influx, is blocked by SRIF. The reported ability of SRIF to reduce the activation rate of Ca2+ currents, decrease Ca2+ conductance, and hyperpolarize the cell would explain the differential effect of SRIF on the GRF-induced [Ca2+]i increase. The inhibitory effect of SRIF on GH release would then be dependent on the ability of SRIF to decrease, or prevent, an increase in [Ca2+]i.
Assuntos
Cálcio/metabolismo , Hormônio do Crescimento/metabolismo , Adeno-Hipófise/metabolismo , Somatostatina/farmacologia , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Separação Celular/métodos , Células Cultivadas , Centrifugação com Gradiente de Concentração/métodos , Corantes Fluorescentes , Indóis , Cinética , Masculino , Adeno-Hipófise/citologia , Adeno-Hipófise/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Espectrometria de FluorescênciaRESUMO
The secretion of GH, in vivo, is pulsatile. We have proposed that the timing of the episodic bursts of GH secretion is set by somatostatin (SRIF) withdrawal, while the magnitude of the bursts is set by the amount of GH-releasing factor (GRF) impinging on the somatotrophs, before and during SRIF withdrawal. We have now used an in vitro model of perifused rat pars distalis cells to further examine the interaction between GRF and SRIF on the magnitude of the burst of GH release that follows SRIF withdrawal. We first characterized the GH response, with time, to constant perifusion with GRF. The initial burst, followed by a rapid decrease in GH release induced by constant perifusion is due to a loss of GRF bioactivity in the perifusion medium and not to a decreasing responsiveness of the somatotrophs. This was followed by studies on the interaction between GRF and SRIF. The burst of GH release after cessation of perifusion with SRIF (10(-9) M) plus GRF (10(-10) M) can be blocked by the administration of SRIF during the burst. Also, the magnitude of the burst is proportional to the concentration of GRF preceding the withdrawal of SRIF. It is likely that similar relations apply in vivo, where SRIF withdrawal sets the timing and duration of the episodic burst of GH release, while GRF sets the magnitude.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/farmacologia , Hormônio do Crescimento/metabolismo , Hipófise/metabolismo , Proteínas Recombinantes/farmacologia , Somatostatina/farmacologia , Animais , Técnicas In Vitro , Cinética , Masculino , Hipófise/efeitos dos fármacos , RatosRESUMO
The secretion of GH is strikingly episodic. We have suggested that the timing of the episodic bursts of GH secretion is set by somatostatin (SRIF) withdrawal, whereas the magnitude of the bursts is determined by the amount of GH-releasing factor (GRF) impinging on the somatotrophs before and during SRIF withdrawal. We have now used an in vitro model of perifused rat pars distalis cells to examine the interaction of SRIF and GRF on GH release and, in particular, to examine the effect of GRF on the magnitude of the burst of GH release that follows SRIF withdrawal. After 30 min of perifusion with SRIF (10(-9) M), there follows an immediate but small burst of GH release. The burst of GH release following concurrent perifusion with SRIF plus GRF (10(-10) M) is increased, with a 7.5- to 9.5-fold increase in the peak secretion rate. When GRF is maintained after the withdrawal of SRIF, the peak secretion rate is not different from that seen after simple withdrawal of both SRIF and GRF, but the duration of the burst is increased. These data demonstrate that the presence of GRF during SRIF perifusion, while not altering basal release, does strikingly increase the post-SRIF release of GH. We propose that a similar relation applies in vivo, where SRIF withdrawal sets the timing of the episodic bursts of GH release, whereas GRF determines the magnitude.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/farmacologia , Hormônio do Crescimento/metabolismo , Somatostatina/fisiologia , Animais , Técnicas In Vitro , Masculino , Perfusão , Periodicidade , Adeno-Hipófise/efeitos dos fármacos , Adeno-Hipófise/metabolismo , RatosRESUMO
We studied the role of the phosphatidylinositol system in the action of growth hormone-releasing factor (GRF). We asked whether GRF stimulates the activity of phospholipase C by determining GRF-induced changes in 32P labeling of the individual phosphoinositides and inositol phosphates in purified rat somatotrophs. The somatotrophs were challenged with GRF (10(-7)M) for 0.33, 1, 3, 10, 30, and 90 min. GRF did not significantly or consistently alter 32P incorporation into phosphatidylinositol bisphosphate (PIP2), phosphatidylinositol monophosphate (PIP), or phosphatidylinositol (PI), except for a small reduction in PIP labeling at 90 min. In general the level of 32P incorporation into the inositol phosphates did not increase but instead decreased with GRF. There was a small but significant reduction of labeling of inositol trisphosphate (IP3) at 90 min of GRF incubation. There were also small but significant decreases in 32P incorporation into inositol bisphosphate (IP2) at 0.33, 3, and 30 min. GRF did not significantly alter 32P labeling of inositol monophosphate (IP). These results indicate that GRF does not stimulate phospholipase C activity in somatotrophs. We conclude that the phosphatidylinositol second messenger system does not play an essential role in the action of GRF.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/farmacologia , Fosfatidilinositóis/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Adeno-Hipófise/efeitos dos fármacos , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Animais , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Hidrólise , Fosfatos de Inositol/metabolismo , Masculino , Fosfatidilinositol Diacilglicerol-Liase , Adeno-Hipófise/metabolismo , RatosRESUMO
The purpose of this study was to investigate the involvement of protein kinase C in growth hormone-releasing factor (GRF) action by directly measuring the effect of GRF on protein kinase C activity in purified male rat somatotrophs. Somatotrophs were incubated with GRF (10(-7) M) for 0.33, 1, 3, 10, 30 and 90 min. Protein kinase C present in soluble and particulate fractions was partially purified using DEAE-cellulose chromatography, and protein kinase C activity was assayed. In control experiments, to insure protein kinase C activity could be activated, two known protein kinase C activators, phorbol 12-myristate 13-acetate (PMA) and dioctanoyl-rac-glycerol (diC8) were added for 3 min. Protein kinase C activity is present in somatotrophs. Under basal conditions the majority of the enzyme activity is located in the cytosol (approximately 90%). The protein kinase C activators caused a significant translocation of protein kinase C activity from soluble to particulate fractions at 3 min. GRF did not cause a translocation of protein kinase C activity even though GH release was significantly increased by 3 min. GRF did not significantly alter the specific activity of protein kinase C in the soluble or particulate fractions, except for a small (approximately 10%) increase in soluble activity at 90 min. We conclude that protein kinase C is present in the somatotrophs of the anterior pituitary. Protein kinase C, however, does not mediate the action of GRF and its role in signal transduction in somatotrophs awaits elucidation.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/farmacologia , Adeno-Hipófise/enzimologia , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia DEAE-Celulose , Diglicerídeos/farmacologia , Ativação Enzimática , Cinética , Masculino , Dados de Sequência Molecular , Adeno-Hipófise/efeitos dos fármacos , Proteína Quinase C/isolamento & purificação , Ratos , Ratos Endogâmicos , Especificidade por Substrato , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
In nonanesthetized dogs treated with 3 mg/kg . d methyl-prednisolone (MP) for four days the infusion of phlorizin decreases plasma glucose only transiently. The basal level is restored by an increase in hepatic glucose production. The concentration of plasma glucagon (IRG) is raised only about 26%, compared to the increase of 150% observed previously in untreated dogs. In insulin-induced hypoglycemia, hepatic glucose production increases and both the concentrations of epinephrine and IRG in the plasma are elevated significantly. Recovery from hypoglycemia after the cessation of the infusion is significantly faster than observed previously in normal dogs. The following conclusions were reached: In MP-treated dogs during the infusion of phlorizin (in nonhypoglycemic glucoregulation) normoglycemia is restored faster, and by a much smaller increment in plasma glucagon concentration than previously observed in normal dogs. Regulation in overt hypoglycemia too operates more efficiently. In nonhypoglycemic glucoregulation a small change in plasma glucose concentration appears to be the primary stimulus that releases glucagon to the extent necessary to achieve the appropriate increase in hepatic glucose production in a given endocrine milieu.
Assuntos
Glicemia/metabolismo , Glucagon/sangue , Insulina/sangue , Metilprednisolona/farmacologia , Animais , Cães , Epinefrina/sangue , Ácidos Graxos não Esterificados/sangue , Retroalimentação , Glucose/biossíntese , Hipoglicemia/sangue , Infusões Parenterais , Fígado/metabolismo , Florizina/farmacologiaRESUMO
In order to establish whether a prolonged subnormal secretion of insulin may affect glucoregulation against hypoglycemic stimuli, the level of plasma glucose was decreased in alloxan-diabetic dogs by the infusion of either 50 micrograms/kg . min phlorizin (PHL), ie, reducing the concentration of plasma glucose without hyperinsulinemia; or with 7 mU/kg . min insulin (combined hyperinsulinemia and hypoglycemia). The concentration of glucose, immunoreactive glucagon (IRG), and insulin (IRI) and catecholamines were followed in the plasma. Hepatic glucose production (Ra) and the overall rate of glucose removal from the circulation were calculated by a tracer method. During a 200-minute infusion of PHL plasma glucose fell from 328 +/- 29 to 114 +/- 16 mg/dl, while IRG rose from a mean of 470 +/- 123 to 623 +/- 200 pg/mL, however this increase was significant only in 3 out of 6 dogs. There was no change in the plasma level of epinephrine. Plasma IRI decreased significantly, the IRI/IRG ratio remained low, and Ra did not increase. When the animals were treated with insulin for one week, plasma glucose was restored to normal, while plasma IRI and the IRI/IRG ratio were raised above the normal level. Under these circumstances the infusion of PHL increased plasma IRG significantly from 59 +/- 5 to 110 +/- 32 pg/mL, decreased IRI slightly, and increased Ra by an average of 50 +/- 16%. No measurable change in plasma glucose was observed indicating the restoration of nonhypoglycemic glucoregulation. In diabetic dogs during a 95-minute infusion of insulin, plasma glucose dropped from a mean of 338 +/- 5 to 74 +/- 24 mg/dL.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Glicemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Animais , Catecolaminas/sangue , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Tipo 1/metabolismo , Cães , Glucagon/sangue , Humanos , Insulina/sangue , Insulina/fisiologia , Fígado/metabolismo , Florizina/farmacologiaRESUMO
Biocompatible osteoconductive polymer (BOP) shelf arthroplasty was performed on ten nondysplastic dogs, divided into five groups. Each group was evaluated at 6, 13, 17, 26 or 39 weeks postsurgery. Evaluation consisted of clinical, radiological and histological studies. The dogs were injected with three fluorochrome markers, 28 days, 14 days and 6 hours before euthanasia. Transverse sections of undecalcified arthroplasty site were examined by microradiography and fluorescence microscopy; surface-stained sections were evaluated by light microscopy. The BOP shelf arthroplasty was not technically difficult. Minimal mineralization of the shelf was noted by radiography, 26 and 39 weeks postop. A moderate to large amount of fibrous mature connective tissue was observed around the BOP fibers throughout the study. Bone ingrowth occurred around the BOP fibers, but was minimal within them. This osseous proliferation of the arthroplasty was very slow to take place; it was first noted microscopically 17 weeks postsurgery and was still minimal 39 weeks after surgery. These findings suggest that there may be interference to the osteoconductive properties of BOP by fibrous tissue. Ossification of the shelf arthroplasty was too unsatisfactory to recommend its use for the treatment of canine hip dysplasia.
Assuntos
Displasia Pélvica Canina/cirurgia , Prótese de Quadril/veterinária , Teste de Materiais/veterinária , Polímeros , Animais , Cães , Feminino , Displasia Pélvica Canina/diagnóstico por imagem , Masculino , Osseointegração/fisiologia , Complicações Pós-Operatórias/veterinária , Radiografia , Fatores de TempoRESUMO
In order to limit the hemodilution effect during cardiopulmonary bypass (CPB) in low weight animal patients, blood is often used as a component of the prime solution. This study was undertaken to evaluate the effects of the addition of blood to the prime solution on the hemodynamic and respiratory parameters during and following mitral valve replacement in dogs. Ten dogs were randomly assigned to receive either a hemic (HP), 75% blood component, or a nonhemic prime (NP) solution. The hemodilution was 5 +/- 4% and 25 +/- 10% for the HP and NP groups, respectively. Cardiopulmonary measurements were taken 20 minutes before initiating CPB, during CPB, and 20 min after termination. The hematocrit level, the hemoglobin concentration, and the arterial oxygen content were significantly lower in the NP group during and following CPB. However, the systemic oxygen transport index was not significantly different between the NP group (355 +/- 87 mL/min/m2) and the HP group (546 +/- 155 mL/min/m2) following CPB. Our study indicates that, in normal dogs undergoing hemodilution from a nonhemic prime solution, the cardiovascular function is able to maintain the systemic oxygen transport in the period immediately following mitral valve replacement.