Os estudos foram realizados em cultura de células HEK-293 (human embrionic kidney cells). Por microscopia de fluorescência, avaliou-se a velocidade de recuperação do pHi (dpHi/dt). Por Western blot, avaliou-se a expressão de SGLTs e NHEs e a translocação dos SGLTs foi avaliada por imunofluorescência. Resultados: No controle, a dpHi/dt foi de 0,169 ± 0,020 unid pH/min (n=6). A glicose modula dose e tempo dependentemente a dpHi/dt. O tratamento crônico aumentou esse parâmetro e somente Florizina (inibidor dos SGLTs), H-89 (inibidor da PKA) e BAPTA (quelante de Ca2+intracelular Ca2+i) reduziram esse efeito. O tratamento crônico induziu a internalização do SGLT1, manteve o SGLT2 no citosol e aumentou sua expressão. Conclusões: No tratamento crônico, a internalização do SGLT1 depende da PKA, independe de Ca2+i e a permanência do SGLT2 no citosol depende tanto da PKA quanto do Ca2+i. Assim, a distribuição celular do SGLT2 altera a atividade dos NHEs. / In this work we used human embryonic kidney (HEK-293 cells). The pHi recovery rate (dpHi/dt) was evaluated through fluorescence microscopy. The expression of SGLT´s and NHEs was analysed through Western blot and translocation of SGLTs was evaluated through Imunofluorescence. Results: In the control situation, the dpHi/dt was 0,169 ± 0,020 units pH/min (n=6). This parameter was modulated by glucose in a concentration and time dependent manner. Chronic treatment increased the dpHi/dt and this stimulatory effect was inhibited by Phlorizin (SGLTs inhibitor), H-89 (PKA inhibitor) and BAPTA (intracellular Ca2+ cheleator - Ca2+i). The chronic treatment induced internalization of SGLT1, increased the expression of SGLT2 and kept it in the cytosol. Conclusions: In chronic treatment, the internalization of SGLT1 involves a PKA-dependent and Ca2+i- independent mechanism. The maintenance of SGLT2 in the cytosol depends on PKA and Ca2+i. Thus, the cellular distribution of SGLT2 is associated with NHEs activity.

O objetivo do presente trabalho foi analisar o papel do ANP na ação não genômica da Aldosterona sobre o trocador Na+/H+ no segmento S3 do túbulo proximal de rato, isolado, in vitro. Os resultados indicam que o pHi basal do segmento S3 proximal de ratos é 7.20 + ou - 0.009 (n = 47/209). O valor médio da velocidade de extrusão celular de H+ na condição controle é de 0.195 + ou - 0.012 pHi/min (n = 16/96). Os dados confirmam que a aldosterona apresenta um efeito bifásico sobre o NHE1: em baixas doses (10-12 M) o estimula, enquanto que em altas doses (10-6M), o inibe. O ANP (10-6 M) não possui efeito sobre o NHE1; contudo, o ANP previne ambos os efeitos da aldosterona sobre esse trocador. O valor médio da concentração do cálcio no citosol ([Ca2+]i) na condição controle é 100 ± 1 (n = 5) hM Adicionalmente, nossos estudos mostram que o ANP diminui a [Ca2+]i e inibe o efeito estimulatório de ambas as doses de aldosterona sobre esse parâmetro. / The effects of aldosterone and ANP(2 min preincubation) on the intracellular pH recovery rate (pHirr) after the acid load induced by NH4Cl and on the [Ca2+]i were investigated in isolated rat S3 segment. The basal pHi was 7.20 + ou - 0.009(n=47/209) and the basal pHirr via the Na+/H+ exchanger was 0.195 + ou - 0.012 pHi/min(n=16/96). Aldosterone(10-12M) caused an increase in the pHirr, but aldosterone(10-6M) decreased it. ANP(10-6M) alone or plus aldosterone(10-12 or 10-6 M) had no effect on pHirr. The basal [Ca2+]i was 100 + ou - 1(n=5)hM. After 1 min of Aldosterone pi there was a transient and dose-dependent increase of the [Ca2+]i and after 6 min pi there was a new increase of [Ca2+]i. ANP alone decreased the [Ca2+]i and prevented the stimulatory effects of aldosterone(10-12 or 10-6M) on this parameter. The data indicate a nongenomic action of aldosterone and ANP on the Na+/H+ exchanger and on [Ca2+]i and are compatible with stimulation of the this exchanger by increases in [Ca2+]i in the lower range (at10-12M aldosterone) and inhibition by increases at high levels (at10-6M aldosterone).

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH. In the myocardium, NHE1 has been implicated in ischemia/reperfusion (I/R) and cardiac hypertrophy (CH). Hormonal, autocrine and paracrine stimuli, acidosis, cardiotoxic metabolites released during I/R and CH increases NHE1 protein expression and activity. The involvement of NHE1 in CH and I/R has been further supported with the use of NHE1 inhibitors, which have been beneficial in the prevention/regression of several models of CH and I/R injury. Despite the fact that elevation of NHE1 expression and activity have been demonstrated in several models of heart disease, it was unclear whether elevation of NHE1 protein expression was sufficient to induce a specific cardiac pathology, or whether activation of the protein was required. To understand the direct role of NHE1 in CH and I/R, an in vivo and in vitro gain-of-function model, expressing varying levels and activities of NHE1 were examined. In vivo, our N-line mice expressed wild type NHE1 and our K-line mice expressed constitutively active NHE1. In vitro, neonatal rat ventricular cardiomyocytes were infected with the IRM adenovirus containing wild type NHE1 or the K-IRM adenovirus containing active NHE1. We demonstrated that expression of constitutively active NHE1 promotes CH to a much greater degree than expression of wild type NHE1 alone, both in vivo and in vitro. This NHE1-dependent hypertrophic response occurred independent of signaling pathways involved in CH including, mitogen activated protein kinases, p90 ribosomal S6 kinase, calcineurin and glycogen synthase kinase. The NHE1-dependent hypertrophic effect also occurred independent of gender. In addition, the expression of active NHE1 increased the susceptibility of intact mice to neurohormonal stimulation and progressed the hypertrophic response. When these hearts expressing active NHE1 were subjected to I/R using the ex vivo working heart perfusion model, fatty acid (FA) oxidation and glycolysis rates increased, thus generating greater ATP production rates. This was associated with cardioprotective effects in the myocardium, as well as a more energetically efficient myocardium. Expression of the endoplasmic reticulum (ER) stress response proteins, calreticulin and PDI were also shown to be increased relative to controls, and may contribute to the cardioprotection observed. We demonstrate that active NHE1 induces cardioprotection and alters cardiac metabolism in working hearts subjected to I/R. Overall, our results suggest that expression of active NHE1 has a double edged sword effect, on one side it induces CH while on the other side, it protects the heart against I/R injury.

No description available.

Os estudos foram realizados em cultura de células HEK-293 (human embrionic kidney cells). Por microscopia de fluorescência, avaliou-se a velocidade de recuperação do pHi (dpHi/dt). Por Western blot, avaliou-se a expressão de SGLTs e NHEs e a translocação dos SGLTs foi avaliada por imunofluorescência. Resultados: No controle, a dpHi/dt foi de 0,169 ± 0,020 unid pH/min (n=6). A glicose modula dose e tempo dependentemente a dpHi/dt. O tratamento crônico aumentou esse parâmetro e somente Florizina (inibidor dos SGLTs), H-89 (inibidor da PKA) e BAPTA (quelante de Ca2+intracelular Ca2+i) reduziram esse efeito. O tratamento crônico induziu a internalização do SGLT1, manteve o SGLT2 no citosol e aumentou sua expressão. Conclusões: No tratamento crônico, a internalização do SGLT1 depende da PKA, independe de Ca2+i e a permanência do SGLT2 no citosol depende tanto da PKA quanto do Ca2+i. Assim, a distribuição celular do SGLT2 altera a atividade dos NHEs. / In this work we used human embryonic kidney (HEK-293 cells). The pHi recovery rate (dpHi/dt) was evaluated through fluorescence microscopy. The expression of SGLT´s and NHEs was analysed through Western blot and translocation of SGLTs was evaluated through Imunofluorescence. Results: In the control situation, the dpHi/dt was 0,169 ± 0,020 units pH/min (n=6). This parameter was modulated by glucose in a concentration and time dependent manner. Chronic treatment increased the dpHi/dt and this stimulatory effect was inhibited by Phlorizin (SGLTs inhibitor), H-89 (PKA inhibitor) and BAPTA (intracellular Ca2+ cheleator - Ca2+i). The chronic treatment induced internalization of SGLT1, increased the expression of SGLT2 and kept it in the cytosol. Conclusions: In chronic treatment, the internalization of SGLT1 involves a PKA-dependent and Ca2+i- independent mechanism. The maintenance of SGLT2 in the cytosol depends on PKA and Ca2+i. Thus, the cellular distribution of SGLT2 is associated with NHEs activity.

O objetivo do presente trabalho foi analisar o papel do ANP na ação não genômica da Aldosterona sobre o trocador Na+/H+ no segmento S3 do túbulo proximal de rato, isolado, in vitro. Os resultados indicam que o pHi basal do segmento S3 proximal de ratos é 7.20 + ou - 0.009 (n = 47/209). O valor médio da velocidade de extrusão celular de H+ na condição controle é de 0.195 + ou - 0.012 pHi/min (n = 16/96). Os dados confirmam que a aldosterona apresenta um efeito bifásico sobre o NHE1: em baixas doses (10-12 M) o estimula, enquanto que em altas doses (10-6M), o inibe. O ANP (10-6 M) não possui efeito sobre o NHE1; contudo, o ANP previne ambos os efeitos da aldosterona sobre esse trocador. O valor médio da concentração do cálcio no citosol ([Ca2+]i) na condição controle é 100 ± 1 (n = 5) hM Adicionalmente, nossos estudos mostram que o ANP diminui a [Ca2+]i e inibe o efeito estimulatório de ambas as doses de aldosterona sobre esse parâmetro. / The effects of aldosterone and ANP(2 min preincubation) on the intracellular pH recovery rate (pHirr) after the acid load induced by NH4Cl and on the [Ca2+]i were investigated in isolated rat S3 segment. The basal pHi was 7.20 + ou - 0.009(n=47/209) and the basal pHirr via the Na+/H+ exchanger was 0.195 + ou - 0.012 pHi/min(n=16/96). Aldosterone(10-12M) caused an increase in the pHirr, but aldosterone(10-6M) decreased it. ANP(10-6M) alone or plus aldosterone(10-12 or 10-6 M) had no effect on pHirr. The basal [Ca2+]i was 100 + ou - 1(n=5)hM. After 1 min of Aldosterone pi there was a transient and dose-dependent increase of the [Ca2+]i and after 6 min pi there was a new increase of [Ca2+]i. ANP alone decreased the [Ca2+]i and prevented the stimulatory effects of aldosterone(10-12 or 10-6M) on this parameter. The data indicate a nongenomic action of aldosterone and ANP on the Na+/H+ exchanger and on [Ca2+]i and are compatible with stimulation of the this exchanger by increases in [Ca2+]i in the lower range (at10-12M aldosterone) and inhibition by increases at high levels (at10-6M aldosterone).

Este estudo investigou o efeito da glicose sobre a atividade e expressão da isoforma 1 do trocador Na+/H+ (NHE1) e da H+-ATPase do tipo vacuolar, em células MDCK (Mardin Darby Canine Kidney), linhagem derivada de rim de cão, que apresenta características similares às células principais e intercalares das porções distais do néfron. Por microscopia de fluorescência, se avaliou a velocidade de recuperação do pHi (dpHi/dt) e a capacidade tamponante (<font face=\"symbol\">bi). A partir desses parâmetros, se calculou o efluxo de H+ (JH+). Por Western blot, se avaliou a expressão de NHE1 e da subunidade E da H+-ATPase do tipo vacuolar. Resultados: Na condição controle o efluxo de H+ foi de 6.27 ± 0.51 mM/min (n = 9). O tratamento agudo com glicose (25 mM) aumentou o efluxo de H+ via NHE1, o qual foi modulado pela PI3 cinase. Na mesma condição, não se observou alterações na atividade da H+-ATPase. O tratamento crônico com glicose (25 mM) induziu significante aumento do efluxo de H+, via NHE1 e H+-ATPase. O efeito estimulador da glicose sobre a atividade de NHE1 e H+-ATPase foi dependente da atividade da p38 MAP cinase. Além disso, o tratamento crônico com glicose (25 mM) induziu fosforilação do sistema ezrin/radixin/moesin (ERM) e Akt. Conclusões: Nossos resultados indicam que no tratamento agudo com glicose (25 mM), o NHE1 foi modulado pela PI3 cinase. Contudo, no tratamento crônico com glicose (25 mM), a atividade do NHE1 foi modulada pelo sistema ERM/Akt e a atividade da H+-ATPase foi modulada pela p38 MAP cinase. / This study investigated the effect of glucose on the activity and expression of Na+/H+ exchanger isoform 1 (NHE1) and vacuolar H+-ATPase, in Mardin Darby Canine Kidney (MDCK) cells from dog kidney, with similar characteristics to principal and intercalated cells of the distal nephron. The pHi recovery rate (dpHi/dt) and the buffering capacity (<font face=\"symbol\">bi) was evaluated through fluorescence microscopy. From these parameters the H+ efflux (JH+) was calculated. By Western blot, the NHE1 and H+-ATPase (E subunit) expression was evaluated. Results: In the control situation the H+ efflux was 6.27 ± 0.51 mM/pH units (n = 9). Acute treatment with glucose (25 mM) increased the H+ efflux via NHE1, which was modulated by PI3 kinase. In the same condition, the H+-ATPase activity did not change. Chronic treatment with glucose (25 mM) induced significant increase in H+ efflux via NHE1 and H+-ATPase. The stimulatory effect of glucose on the NHE1 and H+-ATPase activity was dependent on p38 MAP kinase activity. Furthermore, chronic treatment with glucose (25 mM) induced Ezrin/radixin/moesin (ERM) and Akt phosphorylation. Conclusions: Our results indicate that during the acute treatment with glucose (25 mM), the NHE1 is modulated by PI3 kinase. However, during chronic treatment with glucose (25 mM), NHE1 activity was modulated by the ERM/Akt system and of H+-ATPase activity was modulated by p38 MAP Kinase.

Este estudo investigou o efeito da glicose sobre a atividade e expressão da isoforma 1 do trocador Na+/H+ (NHE1) e da H+-ATPase do tipo vacuolar, em células MDCK (Mardin Darby Canine Kidney), linhagem derivada de rim de cão, que apresenta características similares às células principais e intercalares das porções distais do néfron. Por microscopia de fluorescência, se avaliou a velocidade de recuperação do pHi (dpHi/dt) e a capacidade tamponante (<font face=\"symbol\">bi). A partir desses parâmetros, se calculou o efluxo de H+ (JH+). Por Western blot, se avaliou a expressão de NHE1 e da subunidade E da H+-ATPase do tipo vacuolar. Resultados: Na condição controle o efluxo de H+ foi de 6.27 ± 0.51 mM/min (n = 9). O tratamento agudo com glicose (25 mM) aumentou o efluxo de H+ via NHE1, o qual foi modulado pela PI3 cinase. Na mesma condição, não se observou alterações na atividade da H+-ATPase. O tratamento crônico com glicose (25 mM) induziu significante aumento do efluxo de H+, via NHE1 e H+-ATPase. O efeito estimulador da glicose sobre a atividade de NHE1 e H+-ATPase foi dependente da atividade da p38 MAP cinase. Além disso, o tratamento crônico com glicose (25 mM) induziu fosforilação do sistema ezrin/radixin/moesin (ERM) e Akt. Conclusões: Nossos resultados indicam que no tratamento agudo com glicose (25 mM), o NHE1 foi modulado pela PI3 cinase. Contudo, no tratamento crônico com glicose (25 mM), a atividade do NHE1 foi modulada pelo sistema ERM/Akt e a atividade da H+-ATPase foi modulada pela p38 MAP cinase. / This study investigated the effect of glucose on the activity and expression of Na+/H+ exchanger isoform 1 (NHE1) and vacuolar H+-ATPase, in Mardin Darby Canine Kidney (MDCK) cells from dog kidney, with similar characteristics to principal and intercalated cells of the distal nephron. The pHi recovery rate (dpHi/dt) and the buffering capacity (<font face=\"symbol\">bi) was evaluated through fluorescence microscopy. From these parameters the H+ efflux (JH+) was calculated. By Western blot, the NHE1 and H+-ATPase (E subunit) expression was evaluated. Results: In the control situation the H+ efflux was 6.27 ± 0.51 mM/pH units (n = 9). Acute treatment with glucose (25 mM) increased the H+ efflux via NHE1, which was modulated by PI3 kinase. In the same condition, the H+-ATPase activity did not change. Chronic treatment with glucose (25 mM) induced significant increase in H+ efflux via NHE1 and H+-ATPase. The stimulatory effect of glucose on the NHE1 and H+-ATPase activity was dependent on p38 MAP kinase activity. Furthermore, chronic treatment with glucose (25 mM) induced Ezrin/radixin/moesin (ERM) and Akt phosphorylation. Conclusions: Our results indicate that during the acute treatment with glucose (25 mM), the NHE1 is modulated by PI3 kinase. However, during chronic treatment with glucose (25 mM), NHE1 activity was modulated by the ERM/Akt system and of H+-ATPase activity was modulated by p38 MAP Kinase.

No description available.

Einleitung: Ruminal werden Futterpflanzen zu kurzkettigen Fettsäuren (SCFAs) abgebaut. Diese bilden die Hauptenergiequelle für den Wiederkäuerorganismus. Da diese Fettsäuren jedoch auch maßgeblich die pH-Homöostase der Vormagenschleimhaut beeinflussen, muss das Pansenepithel in der Lage sein, Änderungen im Substrat- und Protonenangebot festzustellen und anschließend regulative Prozesse anzupassen, um Stoffwechselentgleisungen und so auch einer Pansenazidose vorzubeugen. In anderen Spezies erwiesen sich sogenannte „Freie Fettsäurerezeptoren“ (FFARs) als potenzielle Sensoren veränderter SCFA-Mengen im Darmlumen, die u. a. durch Modulation der intrazellulären Spiegel an zyklischem Adenosinmonophosphat (cAMP) ihre Wirkung vermitteln. Ziele der Untersuchungen: Es sollte in der vorliegenden Arbeit untersucht werden, ob FFARs im Pansenepithel des Schafes vorkommen und durch SCFAs aktiviert sowie intrazelluläre Signalwege über cAMP moduliert werden können. Im Anschluss sollte erarbeitet werden, inwiefern der nachgewiesene Einfluss von Butyrat auf die epithelialen cAMP-Spiegel Auswirkungen auf die epitheliale pH-Modulation infolge einer veränderten Aktivität von Monocarboxylattransportern (MCTs) und Na+/H+-Austauschern (NHEs) hat. Tiere, Material und Methoden: Sämtliche Untersuchungen wurden an Geweben des Vormagens von Schafen (Ovis gmelini aries) durchgeführt. Mittels Reverse-Transkriptase-Polymerase-Kettenreaktion (RT-PCR) und immunhistochemischer Färbungen wurde das Vorliegen verschiedener FFARs in nativem Pansengewebe untersucht. Zur funktionellen Charakterisierung wurden Epithelstücke aus dem ventralen Pansensack in Ussing-Kammern inkubiert und anschließend die cAMP-Spiegel im Epithel mittels einer quantitativen, kompetitiven Analyse bestimmt. Dabei wurde der Einfluss von Forskolin (ein Stimulator der cAMP-synthetisierenden Adenylylzyklasen), von Butyrat sowie von Niacin (ein FFAR-Agonist) betrachtet. Mithilfe von radioaktiv markiertem Azetat wurde der Effekt variierender cAMP-Spiegel auf die Transportaktivität von MCTs unter Zuhilfenahme von zwei verschiedenen MCT-Hemmstoffen (Cyanohydroxyzimtsäure und p-Hydroxymercuribenzoesäure) in Ussing-Kammern evaluiert. Die Aktivität der NHEs wurde an kultivierten Pansenepithelzellen durch Messung des intrazellulären pH-Wertes mittels Spektrofluorometrie unter Einfluss des NHE-Inhibitors 5-N-Ethyl-N-Isopropyl Amilorid ermittelt. Auch hierbei wurden in den Zellen unterschiedliche cAMP-Spiegel durch Forskolin-Applikation induziert. Die Daten der verschiedenen Untersuchungen wurden an 5-8 Tieren je Versuchsansatz erhoben. Die Normalverteilung wurde mittels Kolmogorov–Smirnov-Test ermittelt. Ein Friedman-Test mit anschließendem Dunn-Test wurde für die Analyse der cAMP-Experimente genutzt. MCT und NHE Experimente wurden mithilfe einer einfachen, geblockten Varianzanalyse und anschließendem Tukey-Test ausgewertet. Ergebnisse: Die FFARs GPR109A und FFAR2 konnten an allen untersuchten Lokalisationen (Netzmagen, Pansenvorhof, dorsaler und ventraler Pansensack, Psalter) über RT-PCR bzw. im ventralen Pansensack auch über die immunhistochemischen Färbungen detektiert werden, wohingegen FFAR3 lediglich als mRNA im Vorhof nachweisbar war. Dies lässt die beiden Rezeptoren GPR109A und FFAR2 als mögliche Strukturen zur Detektion von SCFAs im Pansenepithel erscheinen. Die Analyse der intrazellulären cAMP-Spiegel in Epithelien aus dem ventralen Pansensack konnte einen hemmenden Einfluss von Butyrat auf diesen Botenstoff darlegen, was auf eine Beteiligung der genannten FFARs hindeutet. Die Applikation des GPR109A-Agonisten Niacin hatte jedoch keinen Effekt auf die cAMP-Spiegel, sodass eine Wirkungsvermittlung von Butyrat über diesen Rezeptor unwahrscheinlich scheint. Mit Blick auf die funktionellen Auswirkungen dieser cAMP-Modulation hatten variierende cAMP-Level im Kontrast zu Erkenntnissen aus Nicht-Wiederkäuerspezies keinen Einfluss auf die Transportaktivität des ruminalen MCT1 unter den gewählten in vitro-Versuchsbedingungen. Andererseits konnte die Regulation des intrazellulären pH-Wertes von kultivierten Pansenepithelzellen tendenziell durch erhöhte cAMP-Spiegel gehemmt werden, was auf einer Hemmung von NHEs durch den second messenger beruhen könnte. Schlussfolgerungen: Die Expression von GPR109A und FFAR2 lassen diese zwei FFARs als potenzielle Sensoren der intraruminalen bzw. intraepithelialen Nährstoffkonditionen erscheinen. Dabei deuten die vorliegenden Untersuchungen auf eine Aktivierung des FFAR2 durch Butyrat und dessen Metaboliten in den basalen Schichten des Pansenepithels hin. Infolge der Rezeptoraktivierung kommt es vermutlich zu einer Verminderung der intraepithelialen cAMP-Spiegel, welche wiederum einen (schwachen) Einfluss auf die Regulation des intrazellulären pH-Wertes mithilfe von NHEs zu haben scheinen. Entgegen unserer Ausgangshypothese scheinen aber die FFARs des ovinen Pansenepithels die pH-Homöostase des Epithels nur geringfügig zu beeinflussen. Ihre genaue physiologische Bedeutung – insbesondere des GPR109A – bleibt somit noch spekulativ.:1 Einleitung 1 2 Literaturübersicht 3 2.1 Bedeutung kurzkettiger Fettsäuren für den Wiederkäuer 3 2.2 Transport kurzkettiger Fettsäuren über das Pansenepithel 3 2.2.1 Apikale Aufnahme in das Pansenepithel 4 2.2.2 Basolaterale Ausschleusung in den Blutstrom 6 2.3 Metabolisierung kurzkettiger Fettsäuren im Pansenepithel 8 2.4 pH-Homöostase 9 2.4.1 pH-Regulation des Pansenlumens 9 2.4.2 pH-Regulation des Pansenepithels 10 2.5 Anpassungsmechanismen des Pansenepithels 12 2.6 Rolle des Butyrats 15 2.7 Fettsäurerezeptoren 16 2.7.1 G-Protein-gekoppelte Rezeptoren 17 2.7.2 GPRs für SCFAs 17 2.7.2.1 FFAR2 17 2.7.2.2 FFAR3 18 2.7.2.3 GPR109A 19 2.7.3 FFARs im Wiederkäuerorganismus 20 2.8 Monocarboxylattransporter 22 2.8.1 Die Familie der MCTs 22 2.8.2 Regulation der MCTs 23 2.8.3 MCTs im Pansenepithel 24 2.9 Natrium-Protonen-Austauscher 26 2.9.1 Die Familie der NHEs 26 2.9.2 Regulation der NHEs 27 2.9.3 NHEs im Pansenepithel 28 2.10 Fragestellungen der vorliegenden Arbeit 30 3 Publikationen 32 3.1 Publikation 1 32 3.2 Publikation 2 41 3.2.1 Supporting Information 56 4 Diskussion 57 4.1 Nachweis von FFARs im Pansenepithel 57 4.1.1 Regulation intrazellulärer Signalwege durch FFARs 59 4.1.2 GPR109A als potenzieller Butyrat-Rezeptor im Pansenepithel 62 4.1.3 FFAR2 als potenzieller Rezeptor für Butyrat 63 4.2 Seitenabhängigkeit der Butyrat-Effekte 64 4.3 pH-Abhängigkeit der cAMP-Spiegel 66 4.4 Einfluss von cAMP auf die Aktivität der MCTs 68 4.5 Einfluss von cAMP auf die NHE-Aktivität 70 4.6 Schlussfolgerungen 73 5 Zusammenfassung 75 6 Summary 77 7 Literaturverzeichnis 79 8 Anhang 101 8.1 Im Rahmen dieser Dissertation gehaltene Präsentationen 101 Danksagung 103 / Introduction: Forage plants are ruminally degraded to short chain fatty acids (SCFAs). These serve as the main energy source for ruminants. As SCFAs also influence the pH-homeostasis of the ruminal mucosa, the epithelium must be able to detect changes of both substrate and proton accumulation and adapt transport processes accordingly, in order to prevent metabolic dysfunction and thus the risk of ruminal acidosis. Studies in non-ruminant species detected so-called ‘free fatty acid receptors’ (FFARs) as potential SCFA-sensors in the gut lumen. It has been shown that these receptors transduce their information by modulation of intracellular levels of cyclic adenosine monophosphate (cAMP). Aim: This study intended to investigate if FFARs are located in the ovine ruminal epithelium. It should further be evaluated if FFARs can be stimulated by SCFAs leading to a modulation of intracellular pathways via cAMP. Finally, the study aimed to elucidate the influence of low epithelial cAMP-levels after butyrate application on the regulation of pH-homeostasis in the ruminal epithelium by modulating the activity of transport proteins such as monocarboxylate transporters (MCTs) and Na+/H+ exchangers (NHEs). Animals, material, and methods: All experiments were conducted with ovine (Ovis aries) ruminal tissues. The expression of different FFARs was investigated in native tissues using a reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining. For functional analysis, epithelial cAMP levels were determined by a quantitative and competitive assay after incubation of epithelia of the ruminal ventral sac in Ussing chambers. The influence of forskolin (a stimulator of the adenylyl cyclases), butyrate, as well as niacin (an FFAR agonist) was evaluated. Further, the effect of varying cAMP levels on transport activity of MCTs was characterised on Ussing chamber-mounted epithelia with radioactively labelled acetate and two MCT inhibitors (cyano-hydroxycinnamic acid and p-hydroxymercuribenzoic acid). Finally, the activity of NHEs was assessed in cultured ruminal epithelial cells. The intracellular pH was evaluated by spectrofluorometry while the cells were incubated with forskolin (to modify intracellular cAMP levels) or the NHE inhibitor 5-(N-ethyl-N-isopropyl)-amiloride. The data for the different set-ups were acquired from 5-8 animals each. Kolmogorov–Smirnov test was used for testing normality. For cAMP level analyses, the Friedman test followed by Dunn's test was performed. MCT and NHE measurements were analysed using one-way randomized block analysis of variance followed by Tukey's test. Results: GPR109A and FFAR2 were detected in all ovine ruminal epithelia examined (reticulum, atrium ruminis, ruminal ventral and dorsal sac, omasum) by RT-PCR and in ruminal ventral sac also by immunohistochemical staining. FFAR3, however, was detected solely on mRNA level in tissues of the ovine atrium ruminis. Thus, the two immunohistochemically detected receptors may serve as potential sensors for SCFAs in the ruminal epithelium. The analysis of intraepithelial cAMP levels revealed an inhibiting influence of butyrate application on cAMP pointing to an activation of FFARs by this SCFA. Nonetheless, the incubation with the GPR109A agonist niacin did not show any effect on cAMP levels. This finding contradicts the theory of an activation of GPR109A by butyrate. Looking at functional consequences of varying cAMP levels, in contrast to studies on non-ruminant species ruminal MCT1 activity was not influenced by different cAMP levels, at least under the conditions chosen in this in vitro study. However, regulation of intracellular pH in cultured ruminal epithelial cells tended to decrease with augmented cAMP levels. This might be mediated by an inhibition of NHEs. Conclusions: The expression of GPR109A and FFAR2 points at a participation of these receptors in sensing intraruminal and intraepithelial energy status. The present data hint at an activation of FFAR2 by butyrate or its metabolites in the basal layers of the epithelium. Activation of the receptor leads to decreased cAMP levels. This in turn seems to slightly modify the regulation of intracellular pH via NHEs. Contradicting our initial hypothesis, ovine ruminal FFARs seem to play only a minor role in modulation of epithelial pH homeostasis. The main physiological role of ruminal FFARs – especially of GPR109A – remains to be clarified.:1 Einleitung 1 2 Literaturübersicht 3 2.1 Bedeutung kurzkettiger Fettsäuren für den Wiederkäuer 3 2.2 Transport kurzkettiger Fettsäuren über das Pansenepithel 3 2.2.1 Apikale Aufnahme in das Pansenepithel 4 2.2.2 Basolaterale Ausschleusung in den Blutstrom 6 2.3 Metabolisierung kurzkettiger Fettsäuren im Pansenepithel 8 2.4 pH-Homöostase 9 2.4.1 pH-Regulation des Pansenlumens 9 2.4.2 pH-Regulation des Pansenepithels 10 2.5 Anpassungsmechanismen des Pansenepithels 12 2.6 Rolle des Butyrats 15 2.7 Fettsäurerezeptoren 16 2.7.1 G-Protein-gekoppelte Rezeptoren 17 2.7.2 GPRs für SCFAs 17 2.7.2.1 FFAR2 17 2.7.2.2 FFAR3 18 2.7.2.3 GPR109A 19 2.7.3 FFARs im Wiederkäuerorganismus 20 2.8 Monocarboxylattransporter 22 2.8.1 Die Familie der MCTs 22 2.8.2 Regulation der MCTs 23 2.8.3 MCTs im Pansenepithel 24 2.9 Natrium-Protonen-Austauscher 26 2.9.1 Die Familie der NHEs 26 2.9.2 Regulation der NHEs 27 2.9.3 NHEs im Pansenepithel 28 2.10 Fragestellungen der vorliegenden Arbeit 30 3 Publikationen 32 3.1 Publikation 1 32 3.2 Publikation 2 41 3.2.1 Supporting Information 56 4 Diskussion 57 4.1 Nachweis von FFARs im Pansenepithel 57 4.1.1 Regulation intrazellulärer Signalwege durch FFARs 59 4.1.2 GPR109A als potenzieller Butyrat-Rezeptor im Pansenepithel 62 4.1.3 FFAR2 als potenzieller Rezeptor für Butyrat 63 4.2 Seitenabhängigkeit der Butyrat-Effekte 64 4.3 pH-Abhängigkeit der cAMP-Spiegel 66 4.4 Einfluss von cAMP auf die Aktivität der MCTs 68 4.5 Einfluss von cAMP auf die NHE-Aktivität 70 4.6 Schlussfolgerungen 73 5 Zusammenfassung 75 6 Summary 77 7 Literaturverzeichnis 79 8 Anhang 101 8.1 Im Rahmen dieser Dissertation gehaltene Präsentationen 101 Danksagung 103