No description available.

[Truncated abstract] Cells need to be able to detect changes in their surrounding environment and transduce these signals into the appropriate cellular compartments. One of the major ways that the cell achieves this signal transduction is through the process of phosphorylation. Protein kinases are the enzymes responsible for catalysing this transfer of phosphate groups from ATP to amino acid residues of their specific substrates. A subfamily of serine/threonine kinases known as the Mitogen-Activated Protein Kinases (MAPKs) is essential in a diverse range of cell processes including growth, metabolism, differentiation and death. The first identified MAPKs, the Extracellular Signal-Regulated Kinases (ERKs), were found to be activated in response to mitogenic stimuli such as growth factors. However, since the discovery of the ERKs, other pathways leading to the activation of related kinases have been recognised. These kinases are preferentially activated in response to stress, and are thus termed “Stress-Activated Protein Kinases” or SAPKs. They consist of the c-Jun N-terminal kinase isoforms 1, 2 and 3 (also called SAPK1γ, SAPK1α and SAPKβ respectively) and the p38 MAPKs, p38α, p38β, p38γ and p38δ (also called SAPK2a, SAPK2b, SAPK3 and SAPK4 respectively). A major challenge in this field has been to identify the substrates and functions of the SAPKs. This has been partly achieved by the development of inhibitors for the JNK MAPKs and SAPK2a/b. However, no inhibitors currently exist that specifically inhibit SAPK3 and SAPK4. Therefore, elucidating the function of these SAPKs has proved more difficult. Recent studies suggest that SAPK3 may play a unique role in the cell compared to other members of the p38 MAPK family. For example, several signalling proteins appear to specifically activate SAPK3 in certain circumstances while not activating other members of the p38 MAPK family. In addition, SAPK3 contains a unique sequence motif that allows it to bind to specialised domains known as PDZ domains. The interaction of SAPK3 with proteins containing these domains may regulate its subcellular localisation and interactions with other proteins in the cell. This project was undertaken to expand the knowledge on the expression, localisation, substrate specificity and binding partners of SAPK3. In Chapter 3 of this thesis, a SAPK3 monoclonal antibody was evaluated for its ability to specifically recognise endogenous SAPK3 protein. SAPK3 was found to be expressed in immortalised cell lines and primary cultures of neonatal rat myocytes, and to be colocalised with the mitochondria of these cells. This co-localisation remained unaltered in response to treatment with the nuclear export inhibitor Leptomycin B, and with exposure to osmotic shock, suggesting that SAPK3 substrates may be localised at the mitochondria

No description available.

Parathyroid hormone (PTH) secretion maintains free-ionised extracellular calcium (Ca2+o) homeostasis under the control of the calcium-sensing receptor (CaR). In humans and dogs, blood acidosis and alkalosis is associated with increased or suppressed PTH secretion respectively. Furthermore, large (1.0 pH unit) changes in extracellular pH (pHo) alter Ca2+o sensitivity of the CaR in CaR-transfected HEK-293 cells (CaR-HEK). Indeed, it has been found in this laboratory that even pathophysiological acidosis (pH 7.2) renders CaR less sensitive to Ca2+o while pathophysiological alkalosis (pH 7.6) increases its Ca2+o sensitivity, both in CaR-HEK and parathyroid cells. If true in vivo, then CaR’s pHo sensitivity might represent a mechanistic link between metabolic acidosis and hyperparathyroidism in ageing and renal disease. However, in acidosis one might speculate that the additional H+ could displace Ca2+ bound to plasma albumin, thus increasing free-Ca2+ concentration and so compensating for the decreased CaR responsiveness. Therefore, I first demonstrated that a physiologically-relevant concentration of albumin (5% w/v) failed to overcome the inhibitory effect of pH 7.2 or stimulatory effect of pH 7.6 on CaR-induced intracellular Ca2+ (Ca2+i) mobilisation. Determining the molecular basis of CaR pHo sensitivity would help explain cationic activation of CaR and permit the generation of experimental CaR models that specifically lack pHo sensitivity. With extracellular histidine and free cysteine residues the most likely candidates for pHo sensing (given their sidechains’ pK values), all 17 such CaR residues were mutated to non-ionisable residues. However, none of the resulting CaR mutants exhibited significantly decreased CaR pHo sensitivity. Even co-mutation of the two residues whose individual mutation appeared to elicit modest reductions (CaRH429V and CaRH495V) failed to exhibit any change in CaR pHo sensitivity. I conclude therefore, that neither extracellular histidine nor free cysteine residues account for CaR pHo sensitivity. Next, it is known that cytosolic cAMP drives PTH secretion in vivo and that cAMP potentiates Ca2+o-induced Ca2+i mobilisation in CaR-HEK cells. Given the physiological importance of tightly controlled PTH secretion and Ca2+o homeostasis, here I investigated the influence of cAMP on CaR signalling in CaR-HEK cells. Agents that increase cytosolic cAMP levels such as forskolin and isoproterenol potentiated Ca2+o-induced Ca2+i mobilisation and lowered the Ca2+o threshold for Ca2+i mobilisation. Indeed, forskolin lowered the EC50 for Ca2+o on CaR (2.3 ± 0.1 vs. 3.0 ± 0.1 mM control, P<0.001). Forskolin also potentiated CaR-induced ERK phosphorylation; however protein kinase A activation appeared uninvolved in any of these effects. Pertussis toxin, used to block CaR-induced suppression of cAMP accumulation, also lowered the Ca2+o threshold for Ca2+i mobilisation though appeared to do so by increasing efficacy (Emax). Furthermore, mutation of the CaR’s two putative PKA consensus sequences (CaRS899 and CaRS900) to a non-phosphorylatable residue (alanine) failed to alter the potency of Ca2+o for CaR or attenuate the forskolin response. In contrast, phosphomimetic mutation of CaRS899 (to aspartate) did increase CaR sensitivity to Ca2+o. Together this suggests that PKA-mediated CaRS899 phosphorylation could potentiate CaR activity but that this does not occur following Ca2+o treatment in CaR-HEK cells. Together, these data show that cAMP regulates the Ca2+o threshold for Ca2+i mobilisation, thus helping to explain differential efficacy between CaR downstream signals. If true in vivo, this could help explain how multiple physiological signal inputs may be integrated in parathyroid cells.

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Obesity has reached epidemic proportions worldwide and is currently a serious health problem. It is associated with metabolic abnormalities, oxidative stress, hypertension, insulin resistance and an increased disposition for the development of cardiovascular disease. Elucidation of the pathophysiological mechanisms underlying obesity and its relationship with metabolic and cardiovascular diseases is essential for prevention and management of these disorders. Melatonin, the pineal gland hormone, is a powerful antioxidant and has been shown to protect the myocardium against ischaemia/reperfusion (I/R) injury. Long- as well as shortterm melatonin treatment also reversed several of the harmful effects of obesity in an animal model of hyperphagia-induced obesity (DIO). However, its effects on myocardial I/R injury and intracellular signalling in obesity induced by a high fat diet (HFD) are still unknown. Aims of study: (i) To evaluate the ability of a high fat diet (HFD) to induce obesity in rats. Apart from evaluating its effects on the biometric parameters and resistance to ischaemia/reperfusion injury (as indicated by infarct size in regional ischaemia and functional recovery after global ischaemia), special attention will be given on the interplay between adiponectin, AMPK, leptin, and FFA in this model. (ii) To evaluate the effect of daily oral administration of melatonin to rats on the HFD as well as their littermate controls, on the parameters listed above as well as on the development of obesity. In this study melatonin will be administered from the onset of the feeding of the high fat diet. Methods: Male Wistar rats were divided into 4 groups: (i) control rats (receiving normal rat chow) (C); (ii) control rats receiving melatonin (CM); (iii) obese rats (receiving HFD) (HFD); (iv) obese rats receiving melatonin (HM). Animals were kept on the diet for 16 weeks and melatonin treatment (10mg/kg/day, added to the drinking water) started at the onset of the feeding. Following feeding and treatment, the animals were grouped into fasted/ non-fasted of which biometric parameters were recorded and blood collected at the time of sacrifice for metabolic and biochemical assays. Hearts were perfused in the working mode for evaluation of myocardial function and infarct size determination after exposure to 35min regional ischaemia/60min reperfusion. For study of intracellular signaling, hearts were perfused in the working mode, subjected to 20min global ischaemia/10min reperfusion and freeze-clamped for Western blotting. Plasma leptin, adiponectin, free fatty acid, triglycerides, total cholesterol, phospholipids, conjugated dienes and thiobarbituric reactive substances (TBARS) levels were determined. Several kinases were investigated including, the RISK (reperfusion injury salvage kinase) (PKB/Akt and ERK p44/42) and SAFE (survivor activating factor enhancement) (STAT-3) pathways, AMPK and JNK under baseline conditions or following 10 min reperfusion. In addition, expression of UCP-3 and PGC1-α was determined. Results: Significant increases in body weight, visceral fat, blood glucose, insulin, HOMA index and leptin and a reduction in adiponectin levels were observed in the fasted high fat diet (HFD) group when compared with controls (C). Significant increases in free fatty acid and triglyceride levels were also noted the HFD group while other serum lipid parameters, including TBARS, remained unchanged. No differences in functional recovery during reperfusion or infarct size after exposure to 35 min regional ischaemia, as well as functional recovery during reperfusion after 20 min global ischaemia were observed between the control and HFD groups. Baseline and 10 min reperfusion data were similar for the RISK and SAFE pathway kinases for the control vs HFD groups. The HFD also had no effect on the expression and phosphorylation of myocardial AMPK and JNK, as well as on the expression of UCP-3 and PGC1-α, when compared to the controls. Treatment with melatonin significantly reduced body weight, visceral fat, blood glucose, HOMA index and serum leptin levels in HFD treated groups, while having no effect on the lipid profile. Although melatonin significantly reduced infarct size in both control [% of area at risk: 20.59 ± 2.29 (CM) vs 38.08 ± 2.77 (C)] and high-fat diet groups [% of area at risk: 11.43 ± 2.94 (HM) vs 38.06 ± 3.59 (H)], it was without effect on myocardial functional recovery during reperfusion. Melatonin had no effect on the intracellular signaling pathways studied. Conclusions: The HFD proved to be a useful model of diet-induced obesity with a more pronounced impact on biometric and metabolic changes compared to the DIO model. Long-term melatonin treatment successfully prevented the development of metabolic abnormalities associated with the high fat diet and obesity as well as significantly reduced myocardial infarct size. The mechanisms involved in melatonin-induced cardioprotection in obesity have not been fully elucidated in this study and require further investigation. However, the anti-obesogenic and cardioprotective properties of melatonin were very significant indeed and support the suggestion of this hormone as a potential tool in the treatment of obesity and associated cardiovascular complications. / AFRIKAANSE OPSOMMING: Inleiding: Vetsug (obesiteit) het wêreldwyd epidemiese afmetings aangeneem en word tans as ‘n ‘n ernstige gesondheidsprobleem beskou. Vetsug word geassosieer met metaboliese afwykings, oksidatiewe stres, hipertensie, insulienweerstandigheid en is‘n belangrike risikofaktor vir die ontwikkeling van kardiovaskulêre siekte. Ten spyte hiervan, het onlangse studies ‘n gunstige effek van vetsug op die uitkomste van miokardiale infarksie in pasiënte gerapporteer, die sg obesiteitsparadoks. Kennis van die patofisiologiese meganismes onderliggend aan vetsug en die ontstaan van metaboliese afwykinge en hartsiekte is noodsaaklik vir die voorkoming en behandeling van hierdie toestande. Melatonien, die hormoon afgeskei deur die pineaalklier, is ‘n kragtige antioksidant en vry radikaal opruimer. Dit is voorheen aangetoon dat dit die hart teen iskemie/herperfusie (I/H) besering kan beskerm en sommige van die skadelike gevolge van vetsug in diermodelle kan omkeer. Die effek van melatonien op miokardiale I/H besering en intrasellulêre seintransduksie prosesse in vetsug geïduseer deur ‘n hoë vet dieet is egter nog onbekend. Doelstellings: (i) Die ontwikkeling en karakterisering van ‘n nuwe model van vetsug en insulienweerstandigheid geïnduseer deur 'n hoë vet dieet (HVD) en die evaluering van die effek daarvan op miokardiale I/H besering en die gepaardgaande intrasellulêre seintransduksieprosesse; (ii) Bepaling van die effek van daaglikse toediening van melatonien aan rotte op die HVD sowel as aan kontroles op ‘n standard dieet, op die ontwikkeling van dieet-geïnduseerde metaboliese veranderinge, miokardiale infarktgrootte en funksionele herstel na koronêre arterie afbinding, sowel as intrasellulêre seintransduksie. Metodiek: Vier groepe van manlike Wistar rotte is bestudeer: (i) kontrole rotte (op‘n standaard dieet) (K); (ii) kontrole rotte op ‘n standard dieet plus melatonien (KM); (iii) dieetrotte (op‘n HVD); (iv) HVD rotte wat melatonien ontvang (HM). Die HVD en melatonien (10mg/kg/dag in die drinkwater) is vir 16 weke toegedien. Na die periode van behandeling, is die diere in vastende en nie-vastende groepe verdeel, die biometriese parameters genoteer en bloedmonsters vir metaboliese en biochemiese bepalings versamel, tydens verwydering van die harte. Harte is geperfuseer volgens die werkhartmodel vir bepaling van miokardiale funksie en infarktgrootte na blootstelling aan 35min streeksiskemie. Vir evaluering van intrasellulêre seintransduksie, is geperfuseerde werkende rotharte blootgestel aan 15min globale iskemie/10 min herperfusie en gevriesklamp vir latere analises volgens die Western kladtegniek.hart. Serum leptien, adiponektien, vryvetsure, trigliseried, totale cholesterol, fosfolipiede, gekonjugeerde diene en tiobarbituursuur reaktiewe stowwe (TBARS) is bepaal. Met gebruik van Western kladtegniek, is die aktivering en/of uitdrukking van die RISK (PKB/ Akt en ERK p44/42) en SAFE (STAT-3) seintransduksiepaaie, AMPK, JNK, UCP-3 en PGC1-α, onder basislyn toestande of na 10 min herperfusie bestudeer. Resultate:‘n Beduidende toename in liggaamsgewig, visserale vet, die HOMA indeks, insulien en leptien vlakke is in die HVD groep waargeneem vergeleke met die kontrole (K) rotte. Adiponektien vlakke was laer in die HVD groep. Die HVD groep is ook gekenmerk deur ‘n beduidende styging in serum vryvetsuur en trigliseried vlakke, terwyl die ander lipied parameters, insluitende die TBARS vlakke, onveranderd was. Infarktgrootte en funksionele herstel tydens herperfusie na blootstelling aan 35 min streeksiskemie, asook funksionele herstel tydens herperfusie na 20 min globale iskemie het nie verskil tussen harte van die kontrole en HVD rotte nie. Aktivering van PKB/Akt, ERK p44/p42, STAT3, AMPK en JNK by basislyn en na 10 min herperfusie was soortgelyk in die kontrole en HFD groepe. Die HVD het ook geen effek op die uitdrukking van UCP-3 en PGC1-α in vergelyking met die kontrole gehad nie. Behandeling met melatonien het die liggaamsgewig, visserale vet, bloedglukose, HOMA indeks en serum leptien vlakke in die HVD groepe statisties beduidend verlaag, terwyl dit geen invloed op die lipiedprofiel gehad het nie. Melatonien behandeling het die miokardiale infarktgrootte beduidend en tot dieselfde mate verminder in beide kontrole [20.59 ± 2.29 (KM) vs 38.08 ± 2.77% (K)] en HVD groepe [11.43 ± 2.94 (HM) vs 38.06 ± 3.59% (HVD)]. Geen verskille is egter tussen die funksionele herstel gedurende herperfusie van die behandelde en onbehandelde kontrole en HVD groepe waargeneem nie. Melatonien het ook geen uitwerking op die intrasellulêre seintransduksiepaaie gehad nie. Gevolgtrekkings: Die resultate het getoon dat die HFD 'n goeie model van dieetgeïnduseerde vetsug en insulien weerstandigheid ontlok, met 'n meer uitgesproke impak op biometriese en metaboliese veranderinge as die voorheen gebruikte hoë-sukrose dieet. Langtermyn melatonien- behandeling het die ontwikkeling van metaboliese abnormaliteite geassosieer met die HVD, voorkom, asook miokardiale infarktgrootte na koronêre afbinding beduidend verminder. Die meganismes betrokke in melatonien-geïnduseerde miokardiale beskerming moet egter in meer detail ondersoek word. Die resultate verkry steun die voorstel dat melatonientoediening voordelig sal wees in die behandeling van vetsug en sy kardiovaskulêre komplikasies.

Two-pore channels (TPCs, gene name Tpcn) have recently been identified as endolysosomal cation channels modulated by the potent calcium (Ca2+) releasing messenger nicotinic acid adenine dinucleotide phosphate (NAADP). Gene knockout (KO) and RNA knockdown studies have implicated TPCs in fundamental cellular processes, including secretion, of insulin in pancreatic islets, and differentiation, of skeletal myoblasts and osteoclasts. Investigations of Tpcn1 and Tpcn2 mRNA expression have indicated widespread tissue distribution, but a lack of suitable antibodies has impeded study of the endogenous proteins. In this study, an anti-TPC1 antibody was purified from immune sera and used in immunoblotting investigations to demonstrate TPC1 protein expression in a wide range of mouse tissues, with highest expression levels observed in kidney, liver and adipose tissue. Endogenous mouse TPC1 was demonstrated to be glycosylated, with apparent differences in the extent of glycosylation in different tissues based on the indicated molecular weight before and after treatment with a deglycosylating enzyme, which may have implications for the functional regulation of channel activity. Given the increasing prevalence of type 2 diabetes and obesity, an understanding of the molecular basis of glucose homeostasis and adipose tissue formation and function is an important scientific goal. Tpcn KO mice have been developed; in both Tpcn1 KOs and Tpcn2 KOs, impaired pancreatic β-cell Ca2+ signalling and reduced insulin secretion from the whole pancreas were demonstrated. However, the whole-animal phenotype has not been extensively researched. In this study, intraperitoneal glucose tolerance tests were conducted in Tpcn KO mice. These indicated that glucose homeostasis was not significantly affected in Tpcn2 KOs or Tpcn1/2 double KOs (DKOs), and only mildly impaired in Tpcn1 KOs, despite the defects previously observed at the cellular and tissue level. In addition, body composition was investigated in Tpcn1 KO, Tpcn2 KO and Tpcn1/2 DKO animals using magnetic resonance spectroscopy and time domain-nuclear magnetic resonance. Single Tpcn KOs were found to have lower adipose tissue levels as a percentage of body composition, while Tpcn1/2 DKOs were shown to have increased bodyweight but normal body composition. To investigate potential roles for TPCs in adipose tissue formation, Tpcn expression during adipogenesis was investigated using an in vitro multipotent mesenchymal stem cell line model of adipogenic differentiation. Tpcn2 mRNA levels were demonstrated by quantitative PCR to be transiently increased during the early stages of adipogenic differentiation, and cyclic AMP (cAMP) was identified as the factor that induced this upregulation. Lentiviruses were developed to express fluorescently-tagged TPCs, and overexpression of TPC2 was demonstrated to partially overcome the requirement for the cAMP-inducing agent in the medium used for the induction of adipogenesis. Collectively, these data suggest that TPCs may play a role in the formation and/or function of adipose tissue.

Para estudar os mecanismos celulares que levam à mudança de cor cromomotora em crustáceos investigamos os cromatóforos ovarianos vermelhos do camarão de água doce Macrobrachium olfersi. A natureza do receptor do hormônio agregador de pigmento vermelho (RPCH) localizado na membrana plasmática é desconhecida. Muitos eventos das cascatas de sinalização induzidas por Ca2+ e GMPc, assim como os tipos de motores moleculares por elas ativados, são ainda obscuros. Avaliamos, farmacologicamente, pela perfusão in vitro dos cromatossomos com pigmentos inicialmente dispersos, possíveis funções do receptor acoplado à proteína G (GPCR), de receptores de glutamato não-NMDA (rGlu), da óxido nítrico sintase (NOS), da proteína cinase G (PKG), da cinase (MLCK) e da fosfatase (MLCP) da cadeia leve da miosina, da protéina cinase Rho (ROCK) e da miosina II não-muscular no mecanismo que induz a translocação pigmentar. Também investigamos a presença de microfilamentos de actina, microtúbulos, miosinas, cinesina e dineína, por microscopia de fluorescência. A inibição do GPCR com GDP--S (10 µM) não tem efeito significativo, mas com AntPG (5 µM) a agregação induzida por RPCH é inibida em 50%, e tem velocidade máxima de 13,3 ± 2,1 m/min (= RPCH-controle, 16,7 ± 1,6 m/min, P=0,85), seguida de dispersão espontânea. A inibição de rGlu com CNQX (50 µM) causa sutil hiperdispersão e inibe 25% da agregação induzida por RPCH, com velocidade máxima de 16 ± 1,5 µm/min (= RPCH-controle, P=0,95). A estimulação de rGlu com AMPA (30 µM) causa forte hiperdispersão (115%) e não afeta a agregação em relação ao RPCH-controle (velocidade máxima de 16,3 ± 1,8 µm/min, P=0,86). Com a inibição da NOS por L-NAME (5 mM), a agregação induzida por RPCH dura 14 min e chega aos 43,5 ± 10% de dispersão, com velocidade máxima de 11,1 ± 1,3 µm/min (= RPCH-controle, P=0,38). Com a PKG inibida por rp-sGMPc-trietilamina (3 µM), a agregação induzida por RPCH chega aos 36,2 ± 5,6% de dispersão em 12 min, com velocidade máxima de 16,9 ± 1,8 µm/min (= RPCH-controle, P=0,626), seguida de dispersão espontânea. A inibição da MLCP com cantaridina (10 µM) acelera a fase rápida da agregação induzida pelo RPCH (25,1 ± 2,6 µm/min, P= 0,017) e inibe sutilmente sua fase final (9,2 ± 5,1% após 30 min). A inibição da MLCK com ML-7 (10 µM) não afeta significativamente a agregação induzida pelo RPCH, que atinge 8,7 ± 3,14% de dispersão com velocidade máxima de 14,1 ± 1,6 µm/min (= RPCH-controle, P= 0,277). As inibições da ROCK com Y-27632 a 3 µM e H-1152 a 50 nM afetam a agregação pigmentar induzida por RPCH em 15,4 ± 4,8% e 32,8 ± 14,3%, e as velocidades máximas são similares ao RPCH-controle, de 18 ± 3,5 m/min (P=0,86) e 13,9 ± 2,3 m/min (P=0,9), respectivamente. Com H-1152 ocorre dispersão espontânea; e com ambos os compostos a dispersão durante a lavagem do RPCH é acelerada. A inibição da miosina II não-muscular com blebistatina reduz a resposta ao RPCH, havendo agregação até os 47 ± 6,2% em 16 min, com velocidade máxima de 9,1 ± 1,5 µm/min, (= RPCH-controle, P= 0,007), seguida de dispersão espontânea; a dispersão com a lavagem do RPCH ocorre normalmente. Por microscopia de fluorescência foram identificados microtúbulos, presentes nas extensões celulares com o pigmento agregado; microfilamentos de actina, aparentemente formandos trilhos aos grânulos pigmentares; miosina II não-muscular, em associação ao citoesqueleto; miosina esquelética e muscular, cinesina e dineína, em associação aos grânulos pigmentares. Evidenciamos que o receptor do RPCH pode ser do tipo GPCR. Os receptores pGlu não parecem ter papel na transdução de sinal deste neuropeptídeo. A NOS, a PKG, a MLCP e a ROCK têm papéis importante na agregação pigmentar, mas a MLCK aparentemente não. Sugerimos que o RPCH se acopla a um receptor associado à proteína G0 na membrana plasmática, e concomitantemente à elevação da concentração intracelular de Ca2+, desencadeia a ativação da NOS, que produz NO, estimulando da GC-S a liberar GMPc. Este segundo mensageiro ativa a PKG, que fosforila um sítio de ativação da miosina. O movimento da miosina é impulsionado por ciclos de fosforilação/defosforilação em um sítio regulatório de suas cadeias leves, catalizados pela MLCP e pela ROCK. Um dos tipos de miosina ativada pela PKG pode ser a miosina II não-muscular, que parece efetuar principalmente a fase lenta da agregação pigmentar. Outras miosinas e a dineína possivelmente também participam da agregação, enquanto que a cinesina parece ter papel na dispersão pigmentar. / To study the cellular mechanisms that lead to cromomotor color changes in crustaceans, we investigated the red ovarian chromatophores of the freshwater shrimp Macrobrachium olfersi. The nature of the receptor for red pigment concentrating hormone (RPCH) in the plasma membrane is unknown. Many events of the induced Ca2+ and GMPc signaling cascades, as well types of molecular motors activated are still obscure. We evaluated, using pharmacological perfusions in vitro of chromatossomes with initially dispersed pigments, putative functions of a G protein coupled receptor (GPCR), non-NMDA glutamate receptors (rGlu), nitric oxide sintase (NOS), protein kinase G (PKG), myosin light chain kinase (MLCK) and phosphatase (MLCP), Rho protein kinase (ROCK) and non-muscular myosin II in the mechanism that induces pigment translocation. We also investigated by fluorescence microscopy the presence of myosins, kinesin, dinein, actin microfilaments and microtubules. GPCR inhibition with 10 µM GDP--S has no significant effect, but 5 µM PGAnt inhibits 50% of RPCH-triggered aggregation, that has maximum velocity of 13,3 ± 2,1 m/min (= RPCH-control, 16,7 ± 1,6 m/min, P=0,85), followed by spontaneous dispersion. rGlu inhibition with 50 µM CNQX causes subtle hyperdispersion and inhibits 25% RPCH induced aggregation, with a maximum velocity of 16 ± 1,5 µm/min (= RPCH-control, P=0,95). rGlu stimulation with 30 µM AMPA causes strong pigment hyperdispersion (115%) but does not affect aggregation compared to RPCH-control (16,3 ± 1,8 µm/min maximum velocity, P=0,86). NOS inhibition with 5 mM L-NAME affects RPCH-triggered aggregation, that lasts 14 min and reaches 43,5 ± 10% dispersion, with maximum velocity of 11,1 ± 1,3 µm/min (= RPCH-control, P=0,38). PKG inhibition with 3 µM rp-cGMPs-thrietylamine affects RPCH-triggered aggregation, that lasts 2 min and reaches 36,2 ± 5,6% dispersion with maximum velocity of 16,9 ± 1,8 µm/min (= RPCH-control, P=0,626), followed by spontaneous dispersion. MLCP inhibition with 10 µM cantharidin accelerates the RPCH-triggered aggregation fast phase (25,1 ± 2,6 µm/min, P= 0,017) and subtly inhibits final aggregation (9,2 ± 5,1% after 30 min). MLCK inhibition with 10 µM ML-7 does not significantly affect RPCH-induced aggregation, that reaches 8,7 ± 3,14% dispersion with a maximum velocity of 4,1 ± 1,6 µm/min (= RPCH-control, P= 0,277). ROCK inhibition with 3µM Y-27632 or 50 nM H-1152 decreases RPCH-triggered pigment aggregation by 15,4 ± 4,8% and 32,8 ± 14,3%; maximum velocities are similar to RPCH-control, 18 ± 3,5 m/min (P=0,86) and 13,9 ± 2,3 m/min (P=0,9), respectively. H-1152 induces spontaneous dispersion; dispersion during RPCH washout is accelerated by both Y-27632 and H-1152. Non-muscular myosin II inhibited with blebbistatin reduces the response to RPCH, aggregation reaching 47 ± 6,2% in 16 min, with a maximum velocity of 9,1 ± 1,5 µm/min (= RPCH-control, P= 0,007), followed by spontaneous dispersion; RPCH washout leads to normal dispersion. Microtubules are present in the cellular extensions in chromatophores with aggregated pigments; actin microfilaments, apparently form trails to associate with pigment granules; non-muscular myosin II is associated with the cytoskeleton; skeletal and muscular myosin, kinesin and dinein, associated with the granules, were revealed by fluorescence microscopy. We showed that the RPCH receptor may be a GPCR. A pGlu receptor does not seem to be present and play a role in signal transduction. NOS, PKG, MLCP and ROCK play important roles in pigment aggregation, although MLCK apparently does not. We suggest that RPCH binds to a G0 protein coupled receptor in the plasma membrane, and together with cytosolic [Ca2+] increase, triggers NOS activation, producing NO, that stimulates GC-S to release cGMP. This second messenger activates PKG, that phosphorylates an activation site on myosin, whose movements are driven by a phosphorylation/dephosphorylation cycle at a regulatory site on the myosin light chain, catalyzed by MLCP and ROCK. One of the PKG activated myosins may be non-muscular myosin II, which seems to effect mainly the slow phase of pigment aggregation. Other myosins and dinein possibly also participate in pigment aggregation, while kynesin seems to play a role in pigment dispersion.

[Truncated abstract] The calcium-sensing receptor (CaR) is a G protein-coupled receptor that can respond to changes in extracellular calcium and plays an integral role in calcium homeostasis. Later studies revealed that the CaR was stimulated by not just calcium, but a diverse range of stimuli and that activation of the receptor regulated a host of different biological processes. The CaR is linked to these cellular responses via the various signalling pathways initiated by the receptor. Recent yeast two-hybrid studies have identified a number of accessory proteins that, through their interaction with the intracellular tail of the CaR, are able to regulate important functional aspects of the receptor, including its signalling and degradation. We hypothesised that many more proteins that bind to the CaR-tail await identification, especially since most of the previous studies used the yeast two-hybrid system to screen cDNA libraries generated from tissues that are important to whole body calcium homeostasis, such as the parathyroid gland and kidney. In order to identify novel binding partners of the CaR, which may affect its function, particularly in biological processes that might be unrelated to calcium homeostasis, our laboratory performed a yeast two-hybrid screen of an EMLC.1 mouse pluripotent haemopoietic cell line library using the intracellular tail of the human CaR as bait. This screen revealed a large number of

Para estudar os mecanismos celulares que levam à mudança de cor cromomotora em crustáceos investigamos os cromatóforos ovarianos vermelhos do camarão de água doce Macrobrachium olfersi. A natureza do receptor do hormônio agregador de pigmento vermelho (RPCH) localizado na membrana plasmática é desconhecida. Muitos eventos das cascatas de sinalização induzidas por Ca2+ e GMPc, assim como os tipos de motores moleculares por elas ativados, são ainda obscuros. Avaliamos, farmacologicamente, pela perfusão in vitro dos cromatossomos com pigmentos inicialmente dispersos, possíveis funções do receptor acoplado à proteína G (GPCR), de receptores de glutamato não-NMDA (rGlu), da óxido nítrico sintase (NOS), da proteína cinase G (PKG), da cinase (MLCK) e da fosfatase (MLCP) da cadeia leve da miosina, da protéina cinase Rho (ROCK) e da miosina II não-muscular no mecanismo que induz a translocação pigmentar. Também investigamos a presença de microfilamentos de actina, microtúbulos, miosinas, cinesina e dineína, por microscopia de fluorescência. A inibição do GPCR com GDP--S (10 µM) não tem efeito significativo, mas com AntPG (5 µM) a agregação induzida por RPCH é inibida em 50%, e tem velocidade máxima de 13,3 ± 2,1 m/min (= RPCH-controle, 16,7 ± 1,6 m/min, P=0,85), seguida de dispersão espontânea. A inibição de rGlu com CNQX (50 µM) causa sutil hiperdispersão e inibe 25% da agregação induzida por RPCH, com velocidade máxima de 16 ± 1,5 µm/min (= RPCH-controle, P=0,95). A estimulação de rGlu com AMPA (30 µM) causa forte hiperdispersão (115%) e não afeta a agregação em relação ao RPCH-controle (velocidade máxima de 16,3 ± 1,8 µm/min, P=0,86). Com a inibição da NOS por L-NAME (5 mM), a agregação induzida por RPCH dura 14 min e chega aos 43,5 ± 10% de dispersão, com velocidade máxima de 11,1 ± 1,3 µm/min (= RPCH-controle, P=0,38). Com a PKG inibida por rp-sGMPc-trietilamina (3 µM), a agregação induzida por RPCH chega aos 36,2 ± 5,6% de dispersão em 12 min, com velocidade máxima de 16,9 ± 1,8 µm/min (= RPCH-controle, P=0,626), seguida de dispersão espontânea. A inibição da MLCP com cantaridina (10 µM) acelera a fase rápida da agregação induzida pelo RPCH (25,1 ± 2,6 µm/min, P= 0,017) e inibe sutilmente sua fase final (9,2 ± 5,1% após 30 min). A inibição da MLCK com ML-7 (10 µM) não afeta significativamente a agregação induzida pelo RPCH, que atinge 8,7 ± 3,14% de dispersão com velocidade máxima de 14,1 ± 1,6 µm/min (= RPCH-controle, P= 0,277). As inibições da ROCK com Y-27632 a 3 µM e H-1152 a 50 nM afetam a agregação pigmentar induzida por RPCH em 15,4 ± 4,8% e 32,8 ± 14,3%, e as velocidades máximas são similares ao RPCH-controle, de 18 ± 3,5 m/min (P=0,86) e 13,9 ± 2,3 m/min (P=0,9), respectivamente. Com H-1152 ocorre dispersão espontânea; e com ambos os compostos a dispersão durante a lavagem do RPCH é acelerada. A inibição da miosina II não-muscular com blebistatina reduz a resposta ao RPCH, havendo agregação até os 47 ± 6,2% em 16 min, com velocidade máxima de 9,1 ± 1,5 µm/min, (= RPCH-controle, P= 0,007), seguida de dispersão espontânea; a dispersão com a lavagem do RPCH ocorre normalmente. Por microscopia de fluorescência foram identificados microtúbulos, presentes nas extensões celulares com o pigmento agregado; microfilamentos de actina, aparentemente formandos trilhos aos grânulos pigmentares; miosina II não-muscular, em associação ao citoesqueleto; miosina esquelética e muscular, cinesina e dineína, em associação aos grânulos pigmentares. Evidenciamos que o receptor do RPCH pode ser do tipo GPCR. Os receptores pGlu não parecem ter papel na transdução de sinal deste neuropeptídeo. A NOS, a PKG, a MLCP e a ROCK têm papéis importante na agregação pigmentar, mas a MLCK aparentemente não. Sugerimos que o RPCH se acopla a um receptor associado à proteína G0 na membrana plasmática, e concomitantemente à elevação da concentração intracelular de Ca2+, desencadeia a ativação da NOS, que produz NO, estimulando da GC-S a liberar GMPc. Este segundo mensageiro ativa a PKG, que fosforila um sítio de ativação da miosina. O movimento da miosina é impulsionado por ciclos de fosforilação/defosforilação em um sítio regulatório de suas cadeias leves, catalizados pela MLCP e pela ROCK. Um dos tipos de miosina ativada pela PKG pode ser a miosina II não-muscular, que parece efetuar principalmente a fase lenta da agregação pigmentar. Outras miosinas e a dineína possivelmente também participam da agregação, enquanto que a cinesina parece ter papel na dispersão pigmentar. / To study the cellular mechanisms that lead to cromomotor color changes in crustaceans, we investigated the red ovarian chromatophores of the freshwater shrimp Macrobrachium olfersi. The nature of the receptor for red pigment concentrating hormone (RPCH) in the plasma membrane is unknown. Many events of the induced Ca2+ and GMPc signaling cascades, as well types of molecular motors activated are still obscure. We evaluated, using pharmacological perfusions in vitro of chromatossomes with initially dispersed pigments, putative functions of a G protein coupled receptor (GPCR), non-NMDA glutamate receptors (rGlu), nitric oxide sintase (NOS), protein kinase G (PKG), myosin light chain kinase (MLCK) and phosphatase (MLCP), Rho protein kinase (ROCK) and non-muscular myosin II in the mechanism that induces pigment translocation. We also investigated by fluorescence microscopy the presence of myosins, kinesin, dinein, actin microfilaments and microtubules. GPCR inhibition with 10 µM GDP--S has no significant effect, but 5 µM PGAnt inhibits 50% of RPCH-triggered aggregation, that has maximum velocity of 13,3 ± 2,1 m/min (= RPCH-control, 16,7 ± 1,6 m/min, P=0,85), followed by spontaneous dispersion. rGlu inhibition with 50 µM CNQX causes subtle hyperdispersion and inhibits 25% RPCH induced aggregation, with a maximum velocity of 16 ± 1,5 µm/min (= RPCH-control, P=0,95). rGlu stimulation with 30 µM AMPA causes strong pigment hyperdispersion (115%) but does not affect aggregation compared to RPCH-control (16,3 ± 1,8 µm/min maximum velocity, P=0,86). NOS inhibition with 5 mM L-NAME affects RPCH-triggered aggregation, that lasts 14 min and reaches 43,5 ± 10% dispersion, with maximum velocity of 11,1 ± 1,3 µm/min (= RPCH-control, P=0,38). PKG inhibition with 3 µM rp-cGMPs-thrietylamine affects RPCH-triggered aggregation, that lasts 2 min and reaches 36,2 ± 5,6% dispersion with maximum velocity of 16,9 ± 1,8 µm/min (= RPCH-control, P=0,626), followed by spontaneous dispersion. MLCP inhibition with 10 µM cantharidin accelerates the RPCH-triggered aggregation fast phase (25,1 ± 2,6 µm/min, P= 0,017) and subtly inhibits final aggregation (9,2 ± 5,1% after 30 min). MLCK inhibition with 10 µM ML-7 does not significantly affect RPCH-induced aggregation, that reaches 8,7 ± 3,14% dispersion with a maximum velocity of 4,1 ± 1,6 µm/min (= RPCH-control, P= 0,277). ROCK inhibition with 3µM Y-27632 or 50 nM H-1152 decreases RPCH-triggered pigment aggregation by 15,4 ± 4,8% and 32,8 ± 14,3%; maximum velocities are similar to RPCH-control, 18 ± 3,5 m/min (P=0,86) and 13,9 ± 2,3 m/min (P=0,9), respectively. H-1152 induces spontaneous dispersion; dispersion during RPCH washout is accelerated by both Y-27632 and H-1152. Non-muscular myosin II inhibited with blebbistatin reduces the response to RPCH, aggregation reaching 47 ± 6,2% in 16 min, with a maximum velocity of 9,1 ± 1,5 µm/min (= RPCH-control, P= 0,007), followed by spontaneous dispersion; RPCH washout leads to normal dispersion. Microtubules are present in the cellular extensions in chromatophores with aggregated pigments; actin microfilaments, apparently form trails to associate with pigment granules; non-muscular myosin II is associated with the cytoskeleton; skeletal and muscular myosin, kinesin and dinein, associated with the granules, were revealed by fluorescence microscopy. We showed that the RPCH receptor may be a GPCR. A pGlu receptor does not seem to be present and play a role in signal transduction. NOS, PKG, MLCP and ROCK play important roles in pigment aggregation, although MLCK apparently does not. We suggest that RPCH binds to a G0 protein coupled receptor in the plasma membrane, and together with cytosolic [Ca2+] increase, triggers NOS activation, producing NO, that stimulates GC-S to release cGMP. This second messenger activates PKG, that phosphorylates an activation site on myosin, whose movements are driven by a phosphorylation/dephosphorylation cycle at a regulatory site on the myosin light chain, catalyzed by MLCP and ROCK. One of the PKG activated myosins may be non-muscular myosin II, which seems to effect mainly the slow phase of pigment aggregation. Other myosins and dinein possibly also participate in pigment aggregation, while kynesin seems to play a role in pigment dispersion.

Recent research suggests that adding protein (PRO) to a carbohydrate (CHO) supplement can have substantial benefits for endurance exercise performance and recovery beyond that of CHO alone. CHO+PRO supplements are often commercially available formulations consisting of carbohydrates (dextrose, maltodextrin) and whey protein. The effects of a supplement containing moderate protein and a low-CHO mixture on endurance performance has not been investigated. Also, the effects of CHO+PRO supplementation in the form of a natural food, flavored milk, on measures of recovery from acute endurance exercise, as well as on chronic aerobic exercise training adaptations, have not been characterized. Therefore, in this series of four studies, the effects of CHO+PRO supplementation on the following areas of endurance exercise performance, recovery, and adaptation are investigated: acute endurance exercise performance, inflammatory and muscle damage markers, muscle glycogen resynthesis, activation of signaling proteins involved in the initiation of protein synthesis and degradation, subsequent endurance exercise performance, and chronic aerobic training adaptations (maximal oxygen consumption, oxidative enzyme activity, body composition, immune cell levels, and inflammatory markers). Study 1 demonstrated that a supplement containing a low-CHO mixture plus moderate protein significantly improved aerobic endurance when cycling at or below the ventilatory threshold, despite containing 50% less CHO and 30% fewer calories relative to a higher CHO beverage. Study 2 demonstrated that CHO+PRO supplementation in the form of chocolate milk (CM) is an effective post-exercise supplement that can improve subsequent performance and provide a greater intracellular signaling stimulus for protein synthesis compared to CHO and placebo. Study 3 found that post-exercise CM supplementation during 4.5 wks of aerobic exercise training improves the magnitude of cardiovascular adaptations more effectively than isocaloric CHO or placebo, while the fourth study demonstrated that post-exercise CM supplementation during 4.5 wks of aerobic training improves body composition more effectively than isocaloric CHO or placebo. The fourth study also demonstrated that 4.5 wks of training does not appear to perturb resting immune cell concentrations or markers of inflammation and muscle damage. Taken together, the results of this research series suggest that CHO+PRO supplementation extends endurance performance, improves recovery, and increases training adaptations more effectively than CHO or placebo. / text