Dissertations / Theses on the topic 'Cardiovascular receptors'

Trace amines (TAs), including p-phenylethylamine (p-PEA), tyramine and octopamine are structurally and functionally related to biogenic amines such as catecholamines and serotonin and to amphetamines. They are present in trace levels in the nervous system and in chocolate, cheese and wine. TAs are usually regarded as indirectly-acting sympathomimetic amines (ISAs) exerting vasoconstriction via a-adrenoceptors. However, they also stimulate trace amine-associated receptors (TAARs), of which only TAAR1 and TAAR4 are sensitive to TAs. The aim of the thesis was to examine whether vasoconstriction by TAs in blood vessels is via ISA or TA mechanisms. TAs caused concentration-related and endothelium-independent contractions in rat isolated aortic rings in the presence of prazosin (ai-adrenoceptor antagonist), cocaine (catecholamine uptake inhibitor), ICI-118,551-adrenoceptor antagonist) and pargyline (MAO A and B inhibitor). The persistent and inhibitor-independent contractions suggest that mechanisms other than ISA and a- and p- adrenoceptor stimulation are involved, possibly TAARs. Differences in the profile of vasoconstrictor activities to a range of TAs were identified in rat and guinea-pig aorta, suggesting species variations in receptor distribution. Tyramine was identified as a partial agonist in isolated rat aorta and an antagonist of other TAs in this tissue. Finally, the presence of TAAR1 mRNA and protein was demonstrated for the first time in rat aorta by RT-PCR and Western blotting, respectively. Most information about TAs relates to studies which have been done on the brain, or cloned receptors expressed in transfected cells. This study of different TAs and structurally related derivatives in aortic tissues has expanded the knowledge of the vasoconstrictor effects of TAs in isolated tissues. The molecular biological confirmation of the presence of TAAR1 and the pharmacological findings regarding the effects of TAs in rat aortic rings might explain their hypertensive effects and their role in coronary heart disease and migraine headache.

Les maladies cardiovasculaires restent la première cause de mortalité et deviennent vite la préoccupation de santé numéro un dans le monde. L'identification de nouveaux facteurs responsables de la régulation du système cardiovasculaire et la génération des modèles animaux des maladies cardiovasculaires sont des étapes importantes pour mieux comprendre les pathologies de l'insuffisance cardiaque incluant les maladies cardiaques congénitales et pour développer des nouvelles thérapies. Les prokinéticines sont des facteurs angiogènic potentiels qui se liant aux récepteurs couplés aux protéines G (PKR1 et PKR2) pour initier leurs effets biologiques. D'abord, nous émettons l’hypothèse que la voie de signalisation du récepteur 1 de la prokinéticine (PKR1) peut contribuer à la survie des cardiomyocytes ou à la protection du coeur après un infarctus du myocarde. Puisque nous avons montré que la prokinéticine-2 et PKR1 sont exprimés dans le coeur des souris adultes et dans les cellules cardiaques, nous avons étudié le rôle de la prokinéticine-2 et PKR1 sur la fonction des cardiomyocytes. Dans les cardiomyocytes et les cellules H9c2, la prokinéticine-2 ou la surexpression de PKR1 active Akt pour protéger les cardiomyocytes contre le stress oxydatif. Puis nous avons déterminé si le transfert de gène, ADN codant PKR1, intracardiaque peut améliorer les fonctions du myocarde après l'infarctus du myocarde dans le modèle de souris. Le transfert transitoire du gène PKR1 après ligation de l’artère coronaire réduit la mortalité et préserve la fonction du ventricule gauche en favorisant la néovascularisation et en réduisant l’apoptose des cardiomyocytes. Nos résultats suggèrent que PKR1 peut représenter une cible thérapeutique originale pour limiter les lésions du myocarde suite à une ischémie. Ensuite, nous avons déterminé les conséquences pathologiques suite à la surexpression de PKR1 dans les cardiomyocytes in vivo. Nous avons généré des souris transgéniques surexprimant PKR1 dans les cardiomyocytes (TG-PKR1) en utilisant le promoteur α-MHC. Les coeurs TG-PKR1 ne montrent aucune anomalie spontanée dans les cardiomyocytes, mais on observe une augmentation de la densité capillaire et des artérioles. De plus, en surexprimant PKR1 dans les cardiomyoblasts H9c2 ou dans les coeurs transgéniques l'expression de la prokinéticine-2 est augmentée. La surexpression de PKR1 dans les cardiomyocytes augmente la libération son propre ligand la prokinéticine-2 qui agit comme un facteur paracrine, en déclenchant la prolifération/différentiation des cellules progénitrices de l’épicarde (EPDCs) pour induire la néovascularisation. Cette étude offre un nouvel aperçu des stratégies thérapeutiques possibles ayant pour but la restauration de la pluripotence des cellules adultes EPDCs pour promouvoir la neovascularisation en induisant la voie de signalisation de PKR1 dans les cardiomyocytes. Comme PKR1 et PKR2 sont identiques à 85 % et sont exprimés dans les tissus cardiovasculaires, nous avons ensuite déterminé les conséquences pathologiques de la surexpression du récepteur 2 de la prokinéticine (PKR2) dans les cardiomyocytes in vivo. Nous avons généré des souris transgéniques surexprimant PKR2 dans les cardiomyocytes (TG-PKR2) en utilisant le promoteur α-MHC. Nous émettons une hypothèse que PKR2 peut aussi contribuer à la croissance des cardiomyocytes et à la vascularisation. Les coeurs TG-PKR2 ont montré une augmentation de l'expression des gènes de l’hypertrophie et de l'hypertrophie excentrique montrant l'augmentation des diamètres du ventricule gauche et l’augmentation de la longueur des cardiomyocytes. L'analyse morphologique quantitative a montré que les coeurs TG-PKR2 ont une densité des microvaisseaux et un nombre de points de branchement normaux, cependant les coeurs TG-PKR2 ont montré une augmentation des formes et des ultrastructures anormales des cellules endothéliales ce qui indiquent la fenestration des vaisseaux sanguins. L'application de milieu conditionné par les cardioblasts H9c2 surexprimant PKR2 de façon significative induit une diminution de la localisation de jonction serrée ZO-1 dans les cellules endothéliales cardiaques, mimant le modèle TG-PKR2. Ces conclusions fournissent la première évidence génétique que la voie de la signalisation de PKR2 dans les cardiomyocytes cause l'hypertrophie excentrique par régulation autocrine et a diminué l'intégrité des cellules endothéliales par régulation paracrine sans induire l’angiogenèse. Ces souris TG-PKR2 peuvent fournir un nouveau modèle génétique des maladies du coeur. Ensuite nous avons déterminé si PKR2 peut directement induire la fenestration dans les cellules endothéliales cardiaques. Les cellules endothéliales cardiaques surexprimant PKR2 ont montré une augmentation de l'expression de caveolin-1 et une diminution de l'expression de la protéine de jonction serrée ZO-1. De plus, ces cellules ont montré une perturbation de la localisation ZO-1. Ces données indiquent que PKR2 peut induire le dysfonctionnement de la barrière des cellules ayant pour effet directe la fenestration dans les cellules endothéliales cardiaques. Après la stimulation des cellules endothéliales surexprimant PKR2 par la prokinéticine-2, nous avons observé l'internalisation et réduction de la protéin VE-cadhérine. Ces données indiquent la possibilité que la protéine Gα12 couplée avec PKR2 induit un dysfonctionnement de la barrière des cellules endothéliales cardiaques. Pour conclure, pour la première fois nous avons montré que la balance entre l'activation de la voie de signalisation de PKR1 et de PKR2 pouvait être très importante pour protéger les cardiomyocytes des lésions causées par l'ischémie et/ou d’induire la neovascularisation dans le coeur, puisque les rôles des récepteurs de la prokinéticine dans le coeur sont impliqués dans la survie des cellules et l’angiogenèse via PKR1 et dans l'hypertrophie cardiaque et la fenestration via PKR2
Cardiovascular disease remains the number one cause of mortality and is fast becoming the number one health concern worldwide. Identification of new factors responsible for regulation of the cardiovascular system and generation of animal models of cardiovascular disease are important steps to better understand the pathogenesis of the heart failure including congenital heart disease and to develop new therapies. Prokineticins are potent angiogenic factors that bind to two G protein-coupled receptors (PKR1 and PKR2) to initiate their biological effects. First, we hypothesize that prokineticin receptor-1 (PKR1) signaling may contribute to cardiomyocyte survival or repair in myocardial infarction. Since we showed that prokineticin-2 and PKR1 are expressed in adult mouse heart and cardiac cells, we investigated the role of prokineticin-2 and PKR1 on cardiomyocytes function. In cardiomyocytes and H9c2 cells, prokineticin-2 or overexpressing PKR1 activates Akt to protect cardiomyocytes against oxidative stress. We thus, further investigated whether intramyocardial gene transfer of DNA encoding PKR1 may rescue the myocardium against myocardial infarction in mouse model. Transient PKR1 gene transfer after coronary ligation reduces mortality and preserves left ventricular function by promoting neovascularization and protecting cardiomyocytes. Our results suggest that PKR1 may represent a novel therapeutic target to limit myocardial injury following ischemic events. Next, we investigated the pathological consequences of overexpressing PKR1 in cardiomyocytes in vivo. We have generated transgenic mice overexpressing PKR1 in cardiomyocytes (TG-PKR1) using α-MHC promoter. TG-PKR1 hearts displayed no spontaneous abnormalities in cardiomyocytes but showed an increased number of capillary density and arterioles. Moreover, overexpressing PKR1 in H9c2 cardiomyoblasts or in TG-PKR1 hearts upregulated prokineticin-2 expression. Cardiomyocyte-PKR1 signaling upregulates its own ligand prokineticin-2 that acts as a paracrine factor, triggering Epicardial-derived Progenitor cells (EPDCs) proliferation/differentiation to induce neovascularization. This study provides a novel insight for possible therapeutic strategies aiming at restoring pluripotency of adult EPDCs to promote neovasculogenesis by induction of cardiomyocyte PKR1 signaling. Since PKR1 and PKR2 are 85% identical and expressed in cardiovascular tissues, next we investigated the pathological consequences of overexpressing prokineticin receptor-2 (PKR2) in cardiomyocytes in vivo. We have generated transgenic mice overexpressing PKR2 in cardiomyocytes (TG-PKR2) using α-MHC promoter. We hypothesize that PKR2 may also contribute to cardiomyocyte growth and vascularization. TG-PKR2 hearts showed increased hypertrophic gene expression and eccentric hypertrophy which showed that increased left ventricular diameters and increased the length of cardiomyocytes. Quantitative morphological analysis showed that TG-PKR2 hearts have a normal micro vessel density and number of branch points, however TG-PKR2 hearts showed increased abnormal endothelial shape and ultrastucture which indicate the fenestration of blood vessels. Application of media conditioned by H9c2 cardioblast cells overexpressing PKR2 significantly induced impaired ZO-1 tight junction localization in cardiac endothelial cells, mimicking the TG-PKR2 model. These findings provide the first genetic evidence that cardiomyocyte-PKR2 signaling leads to eccentric hypertrophy in an autocrine regulation, and impaired endothelial integrity in a paracrine regulation without inducing angiogenesis. These TG-PKR2 mice may provide a new genetic model for heart disease. Next we investigated whether PKR2 can directly induce fenestration into cardiac endothelial cells. PKR2 overexpressing cardiac endothelial cells showed increased caveolin-1 expression and decreased ZO-1 tight junction protein expression. Moreover, those cells showed disruption of ZO-1 localization. These data indicate PKR2 can induce cell barrier dysfunction resulting in fenestration into cardiac endothelial cells as a direct effect. After prokineticin-2 stimulation in PKR2 overexpressing cardiac endothelial cells, we observed internalization and downregulation of VE-cadherin. These data indicate the possibility of Gα12 coupling with PKR2 to induce cell barrier dysfunction in cardiac endothelial cells. As a conclusion, for the first time we have shown that the balance between the activation of PKR1 and PKR2 signaling could be very important to prevent cardiomyocytes from ischemic insult and/or to induce neovascularization in heart, since the roles of prokineticin receptors in heart are involved in cell survival and angiogenesis via PKR1 and in cardiac hypertrophy and fenestration via PKR2

The natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are members of a family of hormones that play an important role in mammalian fluid and electrolyte balance. In the periphery, natriuretic peptides reduce blood volume and subsequently blood pressure by increasing renal natriuresis and diuresis and relaxation of vascular smooth muscle. The actions of natriuretic peptides are mediated via two membrane-linked guanylate cyclase receptors (NPR-GC); natriuretic peptide receptor-A (NPR-A) which has a high affinity for ANP and BNP; and natriuretic peptide receptor-B (NPR-B)which has the greatest affinity for CNP. A third receptor not linked to guanylate cyclase, natriuretic peptide receptor-C (NPR-C) also exists, which binds to ANP, BNP and CNP with a relatively equal affinity, and is involved with clearance of the peptides from the circulation and tissues. The natriuretic peptides are present in the brain and are particularly predominant in cardiovascular and fluid and electrolyte regulating areas such as the anteroventral third ventricle (AV3V) region. This distribution has led to the suggestion natriuretic peptides play a neuromodulatory role in the central control of fluid homeostasis. Natriuretic peptides in the brain have been observed to inhibit the release of other fluid and electrolyte regulating hormones such as arginine vasopressin (AVP) and angiotensin II (AII). Natriuretic peptides have also been identified in the non-mammalian vertebrates although information regarding the distribution of the peptides and their receptors in the non-mammalian brain is limited. In amphibians, immunohistochemical studies have shown that natriuretic peptides are highly concentrated in the preoptic region of the brain, an area believed to be analogous to the A\T3\ region in mammals, which suggests that natriuretic peptides may also be involved in central fluid and electrolyte regulation in amphibians. To date, CNP is the only natriuretic peptide that has been isolated and cloned from the lower vertebrate brain, although studies on the distribution of CNP binding sites in the brain have only been performed in one fish species. Studies on the distribution of ANP binding sites in the lower vertebrate brain are similarly limited and have only been performed in one fish and two amphibian species. Moreover, the nature and distribution of the natriuretic peptide receptors has not been characterised. The current study therefore, used several approaches to investigate the distribution of natriuretic peptides and their receptors in the brain of the amphibian Bufo marinus. The topographical relationship of natriuretic peptides and the fluid and electrolyte regulating hormone arginine vasotocin was also investigated, in order to gain a greater understanding of the role of the natriuretic peptide system in the lower vertebrate brain. Immunohistochemical studies showed natriuretic peptides were distributed throughout the brain and were highly concentrated in the preoptic region and interpeduncular nucleus. No natriuretic peptide-like immunoreactivity (NP-IR) was observed in the pituitary gland. Arginine vasotocin-like immunoreactivity (AvT-IR) was confined to distinct regions, particularly in the preoptic/hypothalamic region and pituitary gland. Double labelling studies of NP-JR and AvT-IR showed the peptides are not colocalised in the same neural pathways. The distribution of natriuretic peptide binding sites using the ligands 125I-rat ANP (125I-rANP) and 125I-porcine CNP (125I-pCNP) showed different distributions in the brain of B. marinus. The specificity of binding was determined by displacement with unlabelled rat ANP, porcine CNP and C-ANF, an NPR-C specific ligand. 125I-rANP binding sites were broadly distributed throughout the brain with the highest concentration in pituitary gland, habenular, medial pallium and olfactory region. Minimal 125I-rANP binding was observed in the preoptic region. Residual 125I-rANP binding in the presence of C-ANF was observed in the olfactory region, habenular and pituitary gland indicating the presence of both NPR-GC and NPR-C in these regions. 125I-pCNP binding was limited to the olfactory region, pallium and posterior pituitary gland. All 125I-pCNP binding was displaced by C-ANF which suggests that CNP in the brain of B. marinus binds only to NPR-C. Affinity cross-linking and SDS-PAGB demonstrated two binding sites at 136 kDa and 65 kDa under reducing conditions. Guanylate cyclase assays showed 0.1 µM ANP increased cGMP levels 50% above basal whilst a 10-fold higher concentration of CNP was required to produce the same result. Molecular cloning studies revealed a 669 base pair fragment showing 91% homology with human and rat NPR-A and 89% homology with human, rat and eel NPR-B. A 432 base pair fragment showing 67% homology to the mammalian NPR-C and 58% homology with eel NPR-D was also obtained. The results show natriuretic peptides and their receptors are distributed throughout the brain of B. marinus which indicates that natriuretic peptides may participate in a range of regulatory functions throughout the brain. The potential for natriuretic peptides to regulate the release of the fluid and electrolyte regulating hormone AVT also exists due to the high number of natriuretic peptide binding sites in the posterior pituitary gland. At least two populations of natriuretic peptide receptors are present in the brain of B. marinus, one linked to guanylate cyclase and one resembling the mammalian clearance receptor. Furthermore, autoradiography and guanylate cyclase studies suggest ANP may be the major ligand in the brain of B. marinus, even though CNP is the only natriuretic peptide that has been isolated from the lower vertebrate brain to date.

Structural information for the zebrafish P2X4 receptor in both an agonist bound and unbound resting state provided a major advance in understanding agonist action and has given insight into movement that occurs in the receptor upon ATP binding. Despite agonist action now being well characterised, the molecular basis of antagonism is poorly understood. In this thesis the mechanism of antagonist action at the hP2X1 receptor has been investigated through determining properties of chimeras and mutant receptors based on differences between antagonist sensitive and insensitive P2X receptors. The antagonists suramin, NF449 and PPADS potently inhibit the human P2X1 receptor but have little or no action at the rat P2X4 receptor. The extracellular loop of the hP2X1 receptor was shown to determine antagonist sensitivity and was therefore split into four sections, residues of which were swapped with corresponding residues of the antagonist insensitive rP2X4 receptor and vice versa. Sub-chimeras and point mutations were then made to identify particular residues and regions which contribute to antagonist action. These experiments identified two regions important for NF449 binding at the receptor. These are a cluster of four positively charged residues at the base of the cysteine rich head region (136-140) and three residues located just below them (T216, H224 and Q231). An NF449 bound model of the hP2X1 receptor has been generated. The introduction of the four positively charged residues at the base of the cysteine rich head region to the rP2X4 receptor introduced suramin and PPADS sensitivity to this previously insensitive receptor. This mutation is thought to cause a conformational change which allows the antagonist to bind at residues which are already present in the wildtype receptor. In summary this thesis has advanced the understanding of antagonist action at the hP2X1 receptor and the antagonist insensitivity of the rP2X4 receptor.

There is an expanding family of cyclic nucleotide-gated cation channels (CNGCs) with expression of family members reported in rod and cone photoreceptor cells, olfactory epithelium, heart, kidney, sperm and aorta. Although functions have been assigned to CNGCs in sensory cells, the function of such channels in non-sensory cells is unknown. A PCR-based screen showed that a CNGC is expressed throughout bovine heart tissue and also in bovine aorta, a bovine aorta endothelial cell line and a human umbilical vein endothelial cell line. Sequence data from these amplified products showed that the CNGC expressed in bovine heart and vasculature is highly related to the bovine rod photoreceptor channel. This was also the case for PCR-generated clones spanning the entire coding sequence of the CNGC from porcine coronary artery smooth muscle tissue. Cyclic GMP is an important messenger in vascular smooth muscle relaxation and therefore this CNGC may play a key role in this process. The second messenger pathways which may be involved in the gating of cardiovascular CNGCs have also been studied by attempting to heterologously co-express a cGMP-generating receptor, ANP-RA, with a CNGC in HEK293 cells. The inward rectifier K+ channel subunits Kir 3.1 and Kir 3.4 have been heterologously co-expressed in MEL cells using a mammalian expression vector which incorporates the ?-globin LCR and promoter allowing high levels of gene expression in a 'position independent' manner. Electrophysiological analysis of these co-expressing cell lines shows that Kir 3.1 and Kir 3.4 form a heteromultimeric ion channel complex which displays the features of the native G-protein activated atrial muscarinic K+ channel, KACh, and it is probable that these two inward rectifier channel subunits are major components of KACh.

The central effect of nicotine on cardiovascular regulation has been extensively studied. However, due to its unselective nature for nicotinic acetylcholine receptors (nAChR) the involvement of specific nAChRs at sites in the brain, in central nervous cardiovascular regulation remains unclear. The effects of intracerebroventricular (i.c.v.) and intracisternal (i.e.) injections of the a7 selective agonist, PSAB-OFP, and the a4p2 selective agonist, TC-2559, were investigated on blood pressure (BP), heart rate (HR) and renal sympathetic nerve activity (RSNA) compared with nicotine, in the anaesthetised rats. PSAB-OFP and TC-2559 i.c.v. caused a delayed dose-related increase in BP and RSNA. When given i.e. the action was similar except the rise in BP was more immediate. The possibility that the pressor response was partly due to the agonists causing the release of the vasoconstrictor vasopressin into the circulation was tested by repeating the i.c.v. and i.e. injections of the agonists in the presence of a selective vasopressin Via antagonist. In the presence of Vi antagonist (i.v.), PSAB-OFP and TC-2559 (i.c.v.) now induced no change in BP or RSNA however i.e., the increase in BP and RSNA was delayed with TC-2559, while PSAB-OFP caused a decrease in BP and no change in RSNA. The cardiovascular effects of i.c.v. PSAB-OFP and TC-2559 in the presence of Vi receptor antagonist (i.c.v.) were also completely blocked. PSAB-OFP and TC-2559 (i.e.) in rats pre-treated with Vi antagonist (i.e.), no longer produced an increase in BP and RSNA. However, the delayed fall in BP caused by PSAB-OFP was potentiated. Nicotine i.c.v. caused a dose-related increase in BP and renal sympathoinhibition while i.e. the rise in BP was larger and now associated with a bradycardia. In the presence of Vj antagonist (i.v.), nicotine's (i.c.v.) cardiovascular effects were blocked however nicotine i.e. caused a decrease in BP, RSNA and HR. In the presence of Vi antagonist (i.c.v.), nicotine caused no change in RSNA, but BP still increased. In the presence of Vi antagonist (i.e.), nicotine i.e. induced a decrease in RSNA and HR, with no change in BP. This study indicates that activation of a4p2 and