Living with osteoarthritis: Patient expenditures, health status, and social impact

Objective. To determine out-of-pocket expenditures related to osteoarthritis (OA) and to explore whether demographic details, health status scores (Medical Outcomes Study 36-item Short Form [SF-36] and Western Ontario and McMaster Universities Osteoarthritis Index [WOMAC]), or perception of social effect were expenditure determinants. Methods. A prospective cohort study of community-dwelling subjects with OA completed 4 consecutive 3-month cost diaries. In addition, subjects completed the SF-36 and WOMAC at baseline and at 12 months. Social impact at baseline was collected. Four groups categorized by age and sex were compared. Patients undergoing joint replacement were excluded. Results. Differences in health status were defined more by age than by sex, especially for physical function. The costs to the patients were high, particularly for women, who spent more on medications and special equipment. Women also reported receiving more assistance from family and friends. Higher disease-related expenditures were associated with greater pain levels, poorer social function and mental health, and longer duration of disease. Significant independent predictors of total patient expenditures related to OA were being female and having joint stiffness. Conclusion. Despite having heavily subsidized health care and access to the Pharmaceutical Benefits Scheme, out-of-pocket costs for patients with OA in Australia are considerable. Higher expenditures for patients with OA are related to more advanced disease, especially for women.

Diversity of Phytophthora clandestina isolated from subterranean clover in southern australia: Analysis of virulence and RAPD profiles

The virulence spectrum of 112 isolates of Phytophthora clandestina collected from 56 sites in four subterranean clover-growing states in southern Australia was determined using differential cultivars of subterranean clover. Five races were detected, with race 0 in all states except New South Wales, race 1 in all states, race 2 only in Victoria, race 3 only in New South Wales, and race 4 in Victoria and Western Australia. The level of genotypic diversity among the different P. clandestina populations was investigated using five RAPD primers. Among 30 bands amplified, only two were polymorphic. This enabled identification of four multilocus RAPD genotypes. Three of the four genotypes occurred in all four states. Races 2 and 3 occurred with RAPD genotypes 1 and 2 only whereas races 0 and 1 occurred in all four multilocus RAPD genotypes. These results indicate that the pathogenicity spectrum of P. clandestina can change rapidly.

Cloning and gastrointestinal expression of rat hephaestin: relationship to other iron transport proteins

The membrane-bound ceruloplasmin homolog hephaestin plays a critical role in intestinal iron absorption. The aims of this study were to clone the rat hephaestin gene and to examine its expression in the gastrointestinal tract in relation to other genes encoding iron transport proteins. The rat hephaestin gene was isolated from intestinal mRNA and was found to encode a protein 96% identical to mouse hephaestin. Analysis by ribonuclease protection assay and Western blotting showed that hephaestin was expressed at high levels throughout the small intestine and colon. Immunofluorescence localized the hephaestin protein to the mature villus enterocytes with little or no expression in the crypts. Variations in iron status had a small but nonsignificant effect on hephaestin expression in the duodenum. The high sequence conservation between rat and mouse hephaestin is consistent with this protein playing a central role in intestinal iron absorption, although its precise function remains to be determined.

A soluble form of CD83 is released from activated dendritic cells and B lymphocytes, and is detectable in normal human sera

CD83 is an inducible glycoprotein expressed predominantly by dendritic cells (DC) and B lymphocytes. Expression of membrane CD83 (mCD83) is widely used as a marker of differentiated/ activated DC but its function and ligand(s) are presently unknown. We report the existence of a soluble form of CD83 (sCD83). Using both a sCD83-specific ELISA and Western blotting, we could demonstrate the release of sCD83 by mCD83(+) B cell and Hodgkin's disease-derived cell lines, but not mCD83(-) cells. Inhibition of de novo protein synthesis did not affect the release of sCD83 during short-term (2 h) culture of cell lines although mCD83 expression was significantly reduced, suggesting sCD83 is generated by the release of mCD83. Isolated tonsillar B lymphocytes and monocyte-derived DC, which are mCD83(low), released only low levels of sCD83 during culture. However, the differentiation/activation of these populations both up-regulated mCD83 and increased sCD83 release significantly. Analysis of sera from normal donors demonstrated the presence of low levels (121 +/- 3.6 pg/ml) of circulating sCD83. Further studies utilizing purified sCD83 and the analysis of sCD83 levels in disease may provide clues to the function and ligand(s) of CD83.

Genetic differentiation between Australian and North American populations of the monarch butterfly Danaus plexippus (L.) (Lepidoptera : Nymphalidae): an exploration using allozyme electrophoresis

Allozyme analysis was used to address the question of the source of the Australian populations of the monarch butterfly Danaus plexippus (L.). The study had three major aims: (1) To compare the levels of diversity of Australian and Hawaiian populations with potential source populations. (2) To determine whether eastern and western North American populations were sufficiently divergent for the Australian populations to be aligned to a source population. (3) To compare the differentiation among regions in Australia and North America to test the prediction of greater genetic structure in Australia, as a consequence of reduced migratory behaviour. The reverse was found, with F-ST values an order of magnitude lower in Australia than in North America. Predictably, Australian and Hawaiian populations had lower allelic diversity, but unexpected higher heterozygosity values than North American populations. It was not possible to assign the Australian populations to a definitive source, although the high levels of similarity of Australian populations to each other suggest a single colonization event. The possibility that the Australian populations have not been here long enough to reach equilibrium is discussed. (C) 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 75, 437-452.

Fibroblast growth factor receptor 4 (FGFR4) expression in newborn murine calvaria and primary osteoblast cultures

Fibroblast growth factor receptor (FGFR) signalling is important in the initiation and regulation of osteogenesis. Although mutations in FGFR1, 2 and 3 genes are known to cause skeletal deformities, the expression of FGFR4 in bony tissue remains unclear. We have investigated the expression pattern of FGFR4 in the neonatal mouse calvaria and compared it to the expression pattern in cultures of primary osteoblasts. Immunohistochemistry demonstrated that FGFR4 was highly expressed in rudimentary membranous bone and strictly localised to the cellular components (osteoblasts) between the periosteal and endosteal layers. Cells in close proximity to the newly formed osteoid (preosteoblasts) also expressed FGFR4 on both the endosteal and periosteal surfaces. Immunocytochemical analysis of primary osteoblast cultures taken from the same cranial region also revealed high levels of FGFR4 expression, suggesting a similar pattern of cellular expression in vivo and in vitro. RT-PCR and Western blotting for FGFR4 confirmed its presence in primary osteoblast cultures. These results suggest that FGFR4 may be an important regulator of osteogenesis with involvement in preosteoblast proliferation and differentiation as well as osteoblast functioning during intramembranous ossification. The consistent expression of FGFR4 in vivo and in vitro supports the use of primary osteoblast cultures for elucidating the role of FGFR4 during osteogenesis.

Conceptions of health and illness held by Australian Aboriginal, Torres Strait Islander and Papua New Guinea Health Science students

Health is considered to be a fundamental human right and developing a better understanding of health is assumed to be a global social goal (Bloom, 1987). Yet many third-world countries and some subpopulations within developed countries do not enjoy a healthy existence. The research reported in this paper examined the conceptions of health and conceptions of illness for a group of Aboriginal, Torres Strait Islander, and Papua New Guinea university students studying health science courses. Results found three conceptions of health and three conceptions of illness that indicated these students held a mix of traditional cultural and Western beliefs. These findings may contribute to overcoming the dissonance between traditional and Western beliefs about health and the development of health care courses that are more specific to how these students understand health. This may also serve to improve the educational status of Aboriginal and Torres Strait Islander people and potentially improve the health status within these communities.

Oceanic interchange and nonequilibrium population structure in the estuarine dependent Indo-Pacific tasselfish, Polynemus sheridani

We assayed mtDNA haplotype [300 base pairs (bp) control region] geography and genealogy in the Indo-Pacific tasselfish, Polynemus sheridani from its contiguous estuarine distribution across northern Australia (n = 169). Eight estuaries were sampled from three oceanographic regions (Timor Sea, Gulf of Carpentaria and the Coral Sea) to assess the impact of Pleistocene sea level changes on the historical connectivity among P. sheridani populations. Specifically, we investigated the genetic consequences of disruption to Indian-Pacific Ocean connectivity brought about by the closure of the Torres Strait. Overall there was significant population subdivision among estuaries (F-ST = 0.161, (Phi(ST) = 0.187). Despite a linear distribution, P. sheridani did not show isolation by distance over the entire sampled range because of genetic similarity of estuaries greater than 3000 km apart. However, significant isolation by distance was detected between estuaries separated by less than 3000 km of coastline. Unlike many genetic studies of Indo-Pacific marine species, there was no evidence for an historical division between eastern and western populations. Instead, phylogeographical patterns were dominated by a starlike intraspecific phylogeny coupled with evidence for population expansion in both the Gulf of Carpentaria and the Coral Sea but not the Timor Sea. This was interpreted as evidence for recent west to east recolonization across of northern Australia following the last postglacial marine advance. We argue that although sufficient time has elapsed postcolonization for populations to approach gene flow-drift equilibrium over smaller spatial scales (< 3000 km), the signal of historical colonization persists to obscure the expected equilibrium pattern of isolation by distance over large spatial scales (> 3000 km).

Species of Stephanostomum Looss, 1899 (Digenea : Acanthocolpidae) from fishes of Australian and South Pacific waters, including five new species

Nine species of Stephanostomum are described from Australian and Southern Pacific marine fishes: Stephanostomum madhaviae n. sp. [syn. S. orientalis of Madhavi ( 1976)] from Caranx ignobilis, off Hope Island, Queensland, with 30-34 circum-oral spines and vitelline fields almost reaching to the posterior extremity of the cirrus-sac; S. bicoronatum (Stossich, 1883) from Argyrosomus hololepidotus, off Southport Broadwater, Queensland; S. votonimoli n. sp. from Scomberoides lysan, off Moorea, French Polynesia ( type-locality) and Western Samoa, with 33-38 circum-oral spines, a uroproct and the vitelline fields not reaching the cirrus-sac; S. nyoomwa n. sp. from Caranx sexfasciatus, off Heron Island, Queensland, with 33-38 circum-oral spines, a uroproct and the vitelline fields reaching the cirrus-sac; S. cobia n. sp. from Rachycentron canadum, off Heron Island, with 36 circum-oral spines, a uroproct and the vitelline fields reaching the cirrus-sac; S. petimba Yamaguti, 1970 from Seriola hippos, off Rottnest Island, Western Australia; S. pacificum ( Yamaguti, 1951) from Pseudocaranx wrighti, off Fremantle, Western Australia; S. aaravi n. sp. from Lethrinus miniatus, off Heron Island, with 36-39 circumoral spines, probably a uroproct and the vitelline fields reaching the ventral sucker; S. pagrosomi ( Yamaguti, 1939) from L. nebulosus, L. miniatus and L. atkinsoni off Heron Island, Pagrus auratus, off Rottnest Island, Western Australia and Gymnocranius audleyi, off Heron Island. A digest of described species of Stephanostomum is included as an appendix.

Mosquito host-feeding patterns and implications for Japanese encephalitis virus transmission in northern Australia and Papua New Guinea

Japanese encephalitis (JE) virus spread to northern Australia during the 1990s, transmitted by Culex annulirostris Skuse and other mosquitoes (Diptera: Culicidae). To determine the relative importance of various hosts for potential vectors of JE virus, we investigated the host-feeding patterns of mosquitoes in northern Australia and Western Province of Papua New Guinea, with particular attention to pigs, Sus scrofa L. - the main amplifying host of JE virus in South-east Asia. Mosquitoes were collected by CDC light traps baited with dry ice and 1-octen-3-ol, run 16.00-08.00 hours, mostly set away from human habitations, if possible in places frequented by feral pigs. Bloodmeals of 2569 mosquitoes, representing 15 species, were identified by gel diffusion assay. All species had fed mostly on mammals: only 30%) were trapped where domestic pigs were kept close to human habitation. From seven of eight locations on the Australian mainland, the majority of Cx. annulirostris had obtained their bloodmeals from marsupials, probably the Agile wallaby Macropus agilis (Gould). Overall proportions of mosquito bloodmeals identified as marsupial were 60% from the Gulf Plains region of Australia, 78% from the Cape York Peninsula and 64% from the Daru area of Papua New Guinea. Thus, despite the abundance of feral pigs in northern Australia, our findings suggest that marsupials divert host-seeking Cx. annulirostris away from pigs. As marsupials are poor JE virus hosts, the prevalence of marsupials may impede the establishment of JE virus in Australia.

HTLV-I/II and blood donors: determinants associated with seropositivity in a low risk population

OBJECTIVE: Blood donors in Brazil have been routinely screened for HTLV-I/II since 1993. A study was performed to estimate the prevalence of HTLV-I/II infection in a low risk population and to better understand determinants associated with seropositivity. METHODS: HTLV-I/II seropositive (n=135), indeterminate (n=167) and seronegative blood donors (n=116) were enrolled in an open prevalence prospective cohort study. A cross-sectional epidemiological study of positive, indeterminate and seronegative HTLV-I/II subjects was conducted to assess behavioral and environmental risk factors for seropositivity. HTLV-I/II serological status was confirmed using enzyme-linked immunosorbent assay (EIA) and Western blot (WB). RESULTS: The three groups were not homogeneous. HTLV-I/II seropositivity was associated to past blood transfusion and years of schooling, a marker of socioeconomic status, and use of non-intravenous illegal drugs. CONCLUSIONS: The study results reinforce the importance of continuous monitoring and improvement of blood donor selection process.

Global material analysis on phosphorus flows, supply horizon and impact assessment on phosphorus depletion

Different problems are daily discuss on environmental aspects such acid rain, eutrophication, global warming and an others problems. Rarely do we find some discussions about phosphorus problematic. Through the years the phosphorus as been a real problem and must be more discussed. On this thesis was done a global material flow analysis of phosphorus, based on data from the year 2004, the production of phosphate rock in that year was 18.9 million tones, almost this amount it was used as fertilizer on the soil and the plants only can uptake, on average, 20% of the input of fertilizer to grow up, the remainder is lost for the phosphorus soil. In the phosphorus soil there is equilibrium between the phosphorus available to uptake from the plants and the phosphorus associate with other compounds, this equilibrium depends of the kind of soil and is related with the soil pH. A reserve inventory was done and we have 15,000 million tones as reserve, the amount that is economical available. The reserve base is estimated in 47,000 million tones. The major reserves can be found in Morocco and Western Sahara, United Sates, China and South Africa. The reserve estimated in 2009 was 15,000 million tone of phosphate rock or 1,963 million tone of P. If every year the mined phosphate rock is around 22 Mt/yr (phosphorus production on 2008 USGS 2009), and each year the consumption of phosphorus increases because of the food demand, the reserves of phosphate rock will be finished in about 90 years, or maybe even less. About the value/impact assessment was done a qualitative analysis, if on the future we don’t have more phosphate rock to produce fertilizers, it is expected a drop on the crops yields, each depends of the kind of the soil and the impact on the humans feed and animal production will not be a relevant problem. We can recovery phosphorus from different waste streams such as ploughing crop residues back into the soil, Food processing plants and food retailers, Human and animal excreta, Meat and bone meal, Manure fibre, Sewage sludge and wastewater. Some of these examples are developed in the paper.

The invisible factors that can make the difference. Language management and knowledge transfer in multinational sites

The knowledge-based society we live in has stressed the importance of human capital and brought talent to the top of most wanted skills, especially to companies who want to succeed in turbulent environments worldwide. In fact, streams, sequences of decisions and resource commitments characterize the day-to-day of multinational companies (MNCs). Such decision-making activities encompass major strategic moves like internationalization and new market entries or diversification and acquisitions. In most companies, these strategic decisions are extensively discussed and debated and are generally framed, formulated, and articulated in specialized language often developed by the best minds in the company. Yet the language used in such deliberations, in detailing and enacting the implementation strategy is usually taken for granted and receives little if any explicit attention (Brannen & Doz, 2012) an can still be a “forgotten factor” (Marschan et al. 1997). Literature on language management and international business refers to lack of awareness of business managers of the impact that language can have not only in communication effectiveness but especially in knowledge transfer and knowledge management in business environments. In the context of MNCs, management is, for many different reasons, more complex and demanding than that of a national company, mainly because of diversity factors inherent to internationalization, namely geographical and cultural spaces, i.e, varied mindsets. Moreover, the way of functioning, and managing language, of the MNC depends on its vision, its values and its internationalization model, i.e on in the way the MNE adapts to and controls the new markets, which can vary essentially from a more ethnocentric to a more pluricentric focus. Regardless of the internationalization model followed by the MNC, communication between different business units is essential to achieve unity in diversity and business sustainability. For the business flow and prosperity, inter-subsidiary, intra-company and company-client (customers, suppliers, governments, municipalities, etc..) communication must work in various directions and levels of the organization. If not well managed, this diversity can be a barrier to global coordination and create turbulent environments, even if a good technological support is available (Feely et al., 2002: 4). According to Marchan-Piekkari (1999) the tongue can be both (i) a barrier, (ii) a facilitator and (iii) a source of power. Moreover, the lack of preparation for the barriers of linguistic diversity can lead to various costs, including negotiations’ failure and failure on internationalization.. On the other hand, communication and language fluency is not just a message transfer procedure, but above all a knowledge transfer process, which requires extra-linguistic skills (persuasion, assertiveness …) in order to promote credibility of both parties. For this reason, MNCs need a common code to communicate and trade information inside and outside the company, which will require one or more strategies, in order to overcome possible barriers and organization distortions.

Histamine induces ATP release from human subcutaneous fibroblasts via pannexin-1 hemichannels leading to Ca2+ mobilization and cell proliferation

Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca2+ ([Ca2+]i) and ATP release from human subcutaneous fibroblasts via H1 receptor activation. Histamine-induced [Ca2+]i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca2+]i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with 10Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca2+]i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H1 receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y1 receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADPsensitive P2Y1 receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca2+]i mobilization and cell growth through the cooperation of H1 and P2 (probably P2Y1) receptors.

Functional significance of adenosine in carotid body chemosensory activity in control and chronically hypoxic animals

ABSTRACT: Carotid bodies (CB) are peripheral chemoreceptor organs sensing changes in arterial blood O2, CO2 and pH levels. Hypoxia and acidosis or hypercapnia activates CB chemoreceptor cells, which respond by releasing neurotransmitters in order to increase the action potential frequency in their sensory nerve, the carotid sinus nerve (CSN). CSN activity is integrated in the brainstem to induce a fan of cardiorespiratory reflex responses, aimed at normalising the altered blood gases. Exogenously applied adenosine (Ado) increases CSN chemosensory activity inducing hyperventilation through activation of A2 receptors. The importance of the effects of adenosine in chemoreception was reinforced by data obtained in humans, in which the intravenous infusion of Ado causes hyperventilation and dyspnoea, an effect that has been attributed to the activation of CB because Ado does not cross blood-brain barrier and because the ventilatory effects are higher the closer to the CB it is injected. The present work was performed in order to establish the functional significance of adenosine in chemoreception at the carotid body in control and chronically hypoxic rats. To achieve this objective we investigated: 1) The release of adenosine from a rat carotid body in vitro preparation in response to moderate hypoxia and the specificity of this release. We also investigated the metabolic pathways of adenosine production and release in the organ in normoxia and hypoxia; 2) The modulation of adenosine/ATP release from rat carotid body chemoreceptor cells by nicotinic ACh receptors; 3) The effects of caffeine on peripheral control of breathing and the identity of the adenosine receptors involved in adenosine and caffeine effects on carotid body chemoreceptors; 4) The interactions between dopamine D2 receptors and adenosine A2B receptors that modulate the release of catecholamines (CA) from the rat carotid body; 5) The effect of chronic caffeine intake i.e. the continuous blockage of adenosine receptors thereby simulating a caffeine dependence, on the carotid body function in control and chronically hypoxic rats. The methodologies used in this work included: molecular biology techniques (e.g. immunocytochemistry and western-blot), biochemical techniques (e.g. neurotransmitter quantification by HPLC, bioluminescence and radioisotopic methods), electrophysiological techniques (e.g. action potential recordings) and ventilatory recordings using whole-body plethysmography. It was observed that: 1) CB chemoreceptor sensitivity to hypoxia could be related to its low threshold for the release of adenosine because moderate acute hypoxia (10% O2) increased adenosine concentrations released from the CB by 44% but was not a strong enough stimulus to evoke adenosine release from superior cervical ganglia and arterial tissue; 2) Acetylcholine (ACh) modulates the release of adenosine/5’-adenosine triphosphate (ATP) from CB in moderate hypoxia through the activation of nicotinic receptors with α4 and ß2 receptor subunits, suggesting that the excitatory role of ACh in chemosensory activity includes indirect activation of purinergic receptors by adenosine and ATP, which strongly supports the hypothesis that ATP/adenosine are important mediators in chemotransduction; 3) adenosine increases the release of CA from rat CB chemoreceptor cells via A2B receptors; 4) the inhibitory effects of caffeine on CB chemoreceptors are mediated by antagonism of postsynaptic A2A and presynaptic A2B adenosine receptors indicating that chemosensory activity elicited by hypoxia is controlled by adenosine; 5) The release of CA from rat CB chemoreceptor cells is modulated by adenosine through an antagonistic interaction between A2B and D2 receptors, for the first time herein described; 6) chronic caffeine treatment did not significantly alter the basal function of CB in normoxic rats assessed as the dynamics of their neurotransmitters, dopamine, ATP and adenosine, and the CSN chemosensory activity. In contrast, the responses to hypoxia in these animals were facilitated by chronic caffeine intake because it increased the ventilatory response, slightly increased CSN chemosensory activity and increased dopamine (DA) and ATP release; 7) In comparison with normoxic rats, chronically hypoxic rats exhibited an increase in several parameters: ventilatory hypoxic response; basal and hypoxic CSN activity; tyrosine hydroxylase expression, CA content, synthesis and release; basal and hypoxic adenosine release; and in contrast a normal basal release and diminished hypoxia-induced ATP release; 8) Finally, in contrast to chronically hypoxic rats, chronic caffeine treatment did not alter the basal CSN chemosensory activity. Nevertheless, the responses to mild and intense hypoxia, and hypercapnia, were diminished. This inhibitory effect of chronic caffeine in CB output is compensated by central mechanisms, as the minute ventilation parameter in basal conditions and in response to acute hypoxic challenges remained unaltered in rats exposed to chronic hypoxia. We can conclude that adenosine both in acute and chronically hypoxic conditions have an excitatory role in the CB chemosensory activity, acting directly on adenosine A2A receptors present postsynaptically in CSN, and acting presynaptically via A2B receptors controlling the release of dopamine in chemoreceptor cells. We suggest that A2B -D2 adenosine / dopamine interactions at the CB could explain the increase in CA metabolism caused by chronic ingestion of caffeine during chronic hypoxia. It was also concluded that adenosine facilitates CB sensitisation to chronic hypoxia although this effect is further compensated at the central nervous system.-------- RESUMO: Os corpos carotídeos (CB) são pequenos orgãos emparelhados localizados na bifurcação da artéria carótida comum. Estes órgãos são sensíveis a variações na PaO2, PaCO2, pH e temperatura sendo responsáveis pela hiperventilação que ocorre em resposta à hipóxia, contribuindo também para a hiperventilação que acompanha a acidose metabólica e respiratória. As células quimiorreceptoras (tipo I ou glómicas) do corpo carotídeo respondem às variações de gases arteriais libertando neurotransmissores que activam as terminações sensitivas do nervo do seio carotídeo (CSN) conduzindo a informação ao centro respiratório central. Está ainda por esclarecer qual o neurotransmissor (ou os neurotransmissores) responsável pela sinalização hipóxica no corpo carotídeo. A adenosina é um neurotransmissor excitatório no CB que aumenta a actividade eléctrica do CSN induzindo a hiperventilação através da activação de receptores A2. A importância destes efeitos da adenosina na quimiorrecepção, descritos em ratos e gatos, foi reforçada por resultados obtidos em voluntários saudáveis onde a infusão intravenosa de adenosina em induz hiperventilação e dispneia, efeito atribuído a uma activação do CB uma vez que a adenosina não atravessa a barreira hemato-encefálica e o efeito é quanto maior quanto mais perto do CB for a administração de adenosina. O presente trabalho foi realizado com o objectivo de esclarecer qual o significado funcional da adenosina na quimiorrecepção no CB em animais controlo e em animais submetidos a hipoxia crónica mantida. Para alcançar este objectivo investigou-se: 1) o efeito da hipóxia moderada sobre a libertação de adenosina numa preparação in vitro de CB e a especificidade desta mesma libertação comparativamente com outros tecidos não quimiossensitivos, assim como as vias metabólicas de produção e libertação de adenosina no CB em normoxia e hipóxia; 2) a modulação da libertação de adenosina/ATP das células quimiorreceptoras do CB por receptores nicotínicos de ACh; 3) os efeitos da cafeína no controlo periférico da ventilação e a identidade dos receptores de adenosina envolvidos nos efeitos da adenosina e da cafeína nos quimiorreceptores do CB; 4) as interacções entre os receptores D2 de dopamina e os receptores A2B de adenosina que modulam a libertação de catecolaminas (CA) no CB de rato e; 5) o efeito da ingestão crónica de cafeína, isto é, o contínuo bloqueio e dos receptores de adenosina, simulando assim o consumo crónico da cafeína, tal como ocorre na população humana mundial e principalmente no ocidente, na função do corpo carotídeo em ratos controlo e em ratos submetidos a hipoxia crónica. Os métodos utilizados neste trabalho incluíram: técnicas de biologia molecular como imunocitoquímica e western-blot; técnicas bioquímicas, tais como a quantificação de neurotransmissores por HPLC, bioluminescência e métodos radioisotópicos; técnicas electrofisiológicas como o registro de potenciais eléctricos do nervo do seio carotídeo in vitro; e registros ventilatórios in vivo em animais não anestesiados e em livre movimento (pletismografia). Observou-se que: 1) a especificidade dos quimiorreceptores do CB como sensores de O2 está correlacionada com o baixo limiar de libertação de adenosina em resposta à hipóxia dado que a libertação de adenosina do CB aumenta 44% em resposta a uma hipóxia moderada (10% O2), que no entanto não é um estímulo suficientemente intenso para evocar a libertação de adenosina do gânglio cervical superior ou do tecido arterial. Observou-se também que aproximadamente 40% da adenosina libertada pelo CB provém do catabolismo extracelular do ATP quer em normóxia quer em hipóxia moderada, sendo que PO2 reduzidas induzem a libertação de adenosina via activação do sistema de transporte equilibrativo ENT1. 2) a ACh modula a libertação de adenosina /ATP do CB em resposta à hipoxia moderada sugerindo que o papel excitatório da ACh na actividade quimiossensora inclui a activação indirecta de receptores purinérgicos pela adenosina e ATP, indicando que a adenosina e o ATP poderiam actuar como mediadores importantes no processo de quimiotransducção uma vez que: a) a activação dos receptores nicotínicos de ACh no CB em normóxia estimula a libertação de adenosina (max 36%) provindo aparentemente da degradação extracelular do ATP. b) a caracterização farmacológica dos receptores nicotínicos de ACh envolvidos na estimulação da libertação de adenosina do CB revelou que os receptores nicotínicos de ACh envolvidos são constituídos por subunidades α4ß2. 3) a adenosina modula a libertação de catecolaminas das células quimiorreceptoras do CB através de receptores de adenosina A2B dado que: a)a cafeína, um antagonista não selectivo dos receptores de adenosina, inibiu a libertação de CA quer em normóxia quer em resposta a estímulos de baixa intensidade sendo ineficaz na libertação induzida por estímulos de intensidade superior; b) o DPCPX e do MRS1754 mimetizaram os efeitos da cafeína no CB sendo o SCH58621 incapaz de induzir a libertação de CA indicando que os efeitos da cafeína seriam mediados por receptores A2B de adenosina cuja presença nas células quimiorreceptoras do CB demonstramos por imunocitoquímica. 4) a aplicação aguda de cafeína inibiu em 52% a actividade quimiossensora do CSN induzida pela hipóxia sendo este efeito mediado respectivamente por receptores de adenosina A2A pós-sinápticos e A2B pré-sinápticos indicando que a actividade quimiossensora induzida pela hipóxia é controlada pela adenosina. 5) existe uma interacção entre os receptores A2B e D2 que controla a libertação de CA do corpo carotídeo de rato uma vez que: a) os antagonistas dos receptores D2, domperidona e haloperidol, aumentaram a libertação basal e evocada de CA das células quimiorreceptoras confirmando a presença de autorreceptores D2 no CB de rato que controlam a libertação de CA através de um mecanismo de feed-back negativo. b) o sulpiride, um antagonista dos receptores D2, aumentou a libertação de CA das células quimiorreceptoras revertendo o efeito inibitório da cafeína sobre esta mesma libertação; c) a propilnorapomorfina, um agonista D2 inibiu a libertação basal e evocada de CA sendo este efeito revertido pela NECA, um agonista dos receptores A2B. O facto de a NECA potenciar o efeito do haloperidol na libertação de CA sugere que a interacção entre os receptores D2 e A2B poderia também ocorrer ao nível de segundos mensageiros, como o cAMP. 6) a ingestão crónica de cafeína em ratos controlo (normóxicos) não alterou significativamente a função basal do CB medida como a dinâmica dos seus neurotransmissores, dopamina, ATP e adenosina e como actividade quimiossensora do CSN. Contrariamente aos efeitos basais, a ingestão crónica de cafeína facilitou a resposta à hipóxia, dado que aumentou o efeito no volume minuto respiratórioapresentando-se também uma clara tendência para aumentar a actividade quimiossensora do CSN e aumentar a libertação de ATP e dopamina.7) após um período de 15 dias de hipóxia crónica era evidente o fenómeno de aclimatização dado que as respostas ventilatórias à hipóxia se encontram aumentadas, assim como a actividade quimiossensora do CSN basal e induzida pela hipóxia. As alterações observadas no metabolismo da dopamina, assim como na libertação basal de dopamina e de adenosina poderiam contribuir para a aclimatização durante a hipoxia crónica. A libertação aumentada de adenosina em resposta à hipóxia aguda em ratos hipóxicos crónicos sugere um papel da adenosina na manutenção/aumento das respostas ventilatórias à hipóxia aguda durante a hipóxia crónica. Observou-se também que a libertação de ATP induzida pela hipóxia aguda se encontra diminuída em hipóxia crónica, contudo a ingestão crónica de cafeína reverteu este efeito para valores similares aos valores controlo, sugerindo que a adenosina possa modular a libertação de ATP em hipóxia crónica. 8) a ingestão crónica de cafeína em ratos hipóxicos crónicos induziu o aumento do metabolismo de CA no CB, medido como expressão de tirosina hidroxilase, conteúdo, síntese e libertação de CA. 9) a ingestão crónica de cafeína não provocou quaisquer alterações na actividade quimiossensora do CSN em ratos hipóxicos crónicos no entanto, as respostas do CSN à hipóxia aguda intensa e moderada e à hipercapnia encontram-se diminuídas. Este efeito inibitório que provém da ingestão crónica de cafeína parece ser compensado ao nível dos quimiorreceptores centrais dado que os parâmetros ventilatórios em condições basais e em resposta à hipoxia aguda não se encontram modificados em ratos expostos durante 15 dias a uma atmosfera hipóxica. Resumindo podemos assim concluir que a adenosina quer em situações de hipoxia aguda quer em condições de hipoxia crónica tem um papel excitatório na actividade quimiossensora do CB actuando directamente nos receptores A2A presentes pós-sinapticamente no CSN, assim como facilitando a libertação de dopamina pré-sinapticamente via receptores A2B presentes nas células quimiorreceptoras. A interacção negativa entre os receptores A2B e D2 observadas nas células quimiorreceptoras do CB poderia explicar o aumento do metabolismo de CA observado após a ingestão crónica de cafeína em animais hipóxicos. Conclui-se ainda que durante a aclimatização à hipóxia a acção inibitória da cafeína, em termos de resposta ventilatória, mediada pelos quimiorreceptores periféricos é compensada pelos efeitos excitatórios desta xantina ao nível do quimiorreceptores centrais.------- RESUMEN Los cuerpos carotídeos (CB) son órganos emparejados que están localizados en la bifurcación de la arteria carótida común. Estos órganos son sensibles a variaciones en la PaO2, en la PaCO2, pH y temperatura siendo responsables de la hiperventilación que ocurre en respuesta a la hipoxia, contribuyendo también a la hiperventilación que acompaña a la acidosis metabólica y respiratoria. Las células quimiorreceptoras (tipo I o glómicas) del cuerpo carotídeo responden a las variaciones de gases arteriales liberando neurotransmissores que activan las terminaciones sensitivas del nervio del seno carotídeo (CSN) llevando la información al centro respiratorio central. Todavía esta por clarificar cual el neurotransmisor (o neurotransmisores) responsable por la señalización hipóxica en el CB. La adenosina es un neurotransmisor excitatório en el CB ya que aumenta la actividad del CSN e induce la hiperventilación a través de la activación de receptores de adenosina del subtipo A2. La importancia de estos efectos de la adenosina en la quimiorrecepción, descritos en ratas y gatos, ha sido fuertemente reforzada por resultados obtenidos en voluntarios sanos en los que la infusión intravenosa de adenosina induce hiperventilación y dispnea, efectos estés que han sido atribuidos a una activación del CB ya que la adenosina no cruza la barrera hemato-encefalica y el efecto es tanto más grande cuanto más cercana del CB es la administración. Este trabajo ha sido realizado con el objetivo de investigar cual el significado funcional de la adenosina en la quimiorrecepción en el CB en animales controlo y en animales sometidos a hipoxia crónica sostenida. Para alcanzar este objetivo se ha estudiado: 1) el efecto de la hipoxia moderada en la liberación de adenosina en una preparación in vitro de CB y la especificidad de esta liberación en comparación con otros tejidos no-quimiosensitivos, así como las vías metabólicas de producción y liberación de adenosina del órgano en normoxia y hipoxia; 2) la modulación de la liberación de adenosina/ATP de las células quimiorreceptoras del CB por receptores nicotínicos de ACh; 3) los efectos de la cafeína en el controlo periférico de la ventilación y la identidad de los receptores de adenosina involucrados en los efectos de la adenosina y cafeína en los quimiorreceptores del CB; 4) las interacciones entre los receptores D2 de dopamina y los receptores A2B de adenosina que modulan la liberación de catecolaminas (CA) en el CB de rata y; 5) el efecto de la ingestión crónica de cafeína, es decir, el bloqueo sostenido de los receptores de adenosina, simulando la dependencia de cafeína observada en la populación mundial del occidente, en la función del CB en ratas controlo y sometidas a hipoxia crónica sostenida. Los métodos utilizados en este trabajo incluirán: técnicas de biología molecular como imunocitoquímica y western-blot; técnicas bioquímicas, tales como la cuantificación de neurotransmissores por HPLC, bioluminescencia y métodos radioisotópicos; técnicas electrofisiológicas como el registro de potenciales eléctricos del nervio do seno carotídeo in vitro; y registros ventilatórios in vivo en animales no anestesiados y en libre movimiento (pletismografia). Se observó que: 1) la sensibilidad de los quimiorreceptores de CB esta correlacionada con un bajo umbral de liberación de adenosina en respuesta a la hipoxia ya que en respuesta a una hipoxia moderada (10% O2) la liberación de adenosina en el CB aumenta un 44%, sin embargo esta PaO2 no es un estimulo suficientemente fuerte para inducir la liberación de adenosina del ganglio cervical superior o del tejido arterial; se observó también que aproximadamente 40% de la adenosina liberada del CB proviene del catabolismo extracelular del ATP en normoxia y en hipoxia moderada, y que bajas PO2 inducen la liberación de adenosina vía activación del sistema de transporte equilibrativo ENT1. 2) la ACh modula la liberación de adenosina /ATP del CB en respuesta a la hipóxia moderada lo que sugiere que el papel excitatório de la ACh en la actividad quimiosensora incluye la activación indirecta de receptores purinérgicos por la adenosina y el ATP, indicando que la adenosina y el ATP pueden actuar como mediadores importantes en el proceso de quimiotransducción ya que: a) la activación de los receptores nicotínicos de ACh en el CB en normoxia estimula la liberación de adenosina (max 36%) que aparentemente proviene de la degradación extracelular del ATP. Se observó también que este aumento de adenosina en el CB en hipoxia ha sido antagonizado parcialmente por antagonistas de estos mismos receptores; b) la caracterización farmacológica de los receptores nicotínicos de ACh involucrados en la estimulación de la liberación de adenosina del CB ha revelado que los receptores nicotínicos de ACh involucrados son constituidos por sub-unidades α4ß2. 3) la adenosina modula la liberación de CA de las células quimiorreceptoras del CB a través de receptores de adenosina A2B ya que: a) la cafeína, un antagonista no selectivo de los receptores de adenosina, ha inhibido la liberación de CA en normoxia y en respuesta a estímulos de baja intensidad siendo ineficaz en la liberación inducida por estímulos de intensidad superior; b) el DPCPX y el MRS1754 ha mimetizado los efectos de la cafeína en el CB y el SCH58621 ha sido incapaz de inducir la liberación de CA lo que sugiere que los efectos de la cafeína son mediados por receptores A2B de adenosina que están localizados pré-sinapticamente en las células quimiorreceptoras del CB. 4) la aplicación aguda de cafeína ha inhibido en 52% la actividad quimiosensora del CSN inducida por la hipoxia siendo este efecto mediado respectivamente por receptores de adenosina A2A pós-sinápticos y A2B pré-sinápticos lo que indica que la actividad quimiosensora inducida por la hipoxia es controlada por la adenosina. 5) existe una interacción entre los receptores A2B y D2 que controla la liberación de CA del CB de rata ya que: a) el sulpiride, un antagonista de los receptores D2, ha aumentado la liberación de CA de las células quimiorreceptoras revertiendo el efecto inhibitorio de la cafeína sobre esta misma liberación; b) los antagonistas de los receptores D2, domperidona y haloperidol, han aumentado la liberación basal e evocada de CA de las células quimiorreceptoras confirmando la presencia de autorreceptores D2 en el CB de rata que controlan la liberación de CA a través de un mecanismo de feed-back negativo; c) la propilnorapomorfina, un agonista D2, ha inhibido la liberación basal e evocada de CA sendo este efecto revertido por la NECA, un agonista de los receptores A2B. Ya que la NECA potencia el efecto del haloperidol en la liberación de CA la interacción entre los D2 y A2B puede también ocurrir al nivel de segundos mensajeros, como el cAMP. 6) la ingestión crónica de cafeína en ratas controlo (normóxicas) no ha cambiado significativamente la función basal del CB medida como la dinámica de sus neurotransmisores, dopamina, ATP y adenosina y como actividad quimiosensora del CSN. Al revés de lo que pasa con los efectos básales, la ingestión crónica de cafeína facilitó la respuesta a la hipóxia, ya que ha aumentado la respuesta ventilatória medida como volumen minuto presentando también una clara tendencia para aumentar la actividad quimiosensora del CSN y aumentar la liberación de ATP y dopamina. 7. Después de un período de 15 días de hipoxia crónica se puede observar el fenómeno de climatización ya que las respuestas ventilatórias a la hipoxia están aumentadas, así como la actividad quimiosensora del CSN basal e inducida por la hipoxia. Los cambios observados en el metabolismo de la dopamina, así como en la liberación basal de dopamina y de adenosina podrían contribuir para la climatización en hipoxia crónica. El aumento en la liberación de adenosina en respuesta a la hipoxia aguda en ratas sometidas a hipoxia crónica sugiere un papel para la adenosina en el mantenimiento/aumento de las respuestas ventilatórias a la hipoxia aguda en hipoxia crónica sostenida. Se ha observado también que la liberación de ATP inducida por la hipoxia aguda está disminuida en hipoxia crónica y que la ingestión crónica de cafeína reverte este efecto para valores similares a los valores controlo, sugiriendo que la adenosina podría modular la liberación de ATP en hipoxia crónica. 8. la ingestión crónica de cafeína ha inducido el aumento del metabolismo de CA en el CB en ratas hipóxicas crónicas, medido como expresión de la tirosina hidroxilase, contenido, síntesis y liberación de CA. 9. la ingestión crónica de cafeína no ha inducido cambios en la actividad quimiosensora del CSN en ratas hipóxicas crónicas sin embargo las respuestas do CSN a una hipoxia intensa y moderada y a la hipercapnia están disminuidas. Este efecto inhibitorio que es debido a la ingestión crónica de cafeína es compensado al nivel de los quimiorreceptores centrales ya que los parámetros ventilatórios en condiciones básales y en respuesta a la hipoxia aguda no están modificados en ratas expuestas durante 15 días a una atmósfera hipóxica. Resumiendo se puede concluir que la adenosina en situaciones de hipoxia aguda así como en hipoxia crónica tiene un papel excitatório en la actividad quimiosensora del CB actuando directamente en los receptores A2A localizados pós-sinapticamente en el CSN, así como controlando la liberación de dopamina pré-sinaptica vía receptores A2B localizados en las células quimiorreceptoras. Las interacciones entre los receptores A2B y D2 observadas en las células quimiorreceptoras del CB podrían explicar el aumento del metabolismo de CA observado después de la ingestión crónica de cafeína en animales hipóxicos. Por fin, pero no menos importante se puede concluir que durante la climatización a la hipoxia la acción inhibitoria de la cafeína, medida como respuesta ventilatória, mediada por los quimiorreceptores periféricos es compensada por los efectos excitatórios de esta xantina al nivel de los quimiorreceptores centrales.