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This thesis is missing pages 87, 172 and 255. No other copies of the thesis have these pages. -Digitization Centre / This thesis examined the effects of exhaustive exercise on acid-base and metabolite status in the intracellular and extracellular compartments of two very different fish species: the active, pelagic rainbow trout, and the sluggish, benthic starry flounder. In both species, exhaustive exercise resulted in an acidosis in the extracellular compartment of mixed respiratory and metabolic origin. Despite the reduction in pHe, red blood cell pHi was well regulated, though more precisely in trout than in flounder. Catecholamines were mobilized into the blood after exercise in trout but not in flounder. Circulating catecholamines may play an important role in regulating red blood cell (RBC) pHi in trout after exercise. In trout, lactate appeared in the blood in excess of H+ ; the reverse pattern was observed in flounder. H+ appearance was similar in both species. Differential release of lactate from the muscle mass was apparently responsible for this discrepancy. After exhaustive exercise, both trout and flounder experienced a severe intracellular acidosis in the white muscle, as measured by 14c-DMO (5,5-dimethyl -2,4-oxazolidinenione) distribution. H+ and lactate were not produced in equimolar quantities, with H+ produced in excess of lactate. Muscle lactate and H+ production was about 3-fold lower in flounder than in trout. The muscle intracellular acid-base disturbance was corrected more rapidly in flounder (4-8h) than in trout (8-12h). In flounder, this occurred prior to , but in trout after , correction of the extracellular acidosis. In flounder, a more rapid correction of muscle metabolite status was associated with the more rapid correction of the intracellular acidosis. After exercise there was a reduction in the whole body extracellular fluid volume and expansion of the intracellular fluid volume, largely reflecting changes within the muscle. This fluid shift resulted in a general hemoconcentration. Exercise led to a transient increase in net H+ excretion in both trout and flounder. Negligible amounts of lactate were transferred to the water. In flounder, about 20% of the total H+ load produced passed through the extracellular space and was transiently stored in the water, which appeared to hasten correction of the intracellular acid-base disturbance. In contrast, in trout, a much smaller portion of the acid load (about 6%), though about the same absolute amount as in flounder, was transferred to the water. This appeared to expedite correction of the extracellular acidosis.The results of this thesis argue against a prominent role for the Cori cycle in the final disposition of the lactate burden produced during exercise. Instead, it is suggested that the bulk of the lactate was metabolized in situ, either by oxidation or glyconeogenesis. In flounder, this was almost the sole fate of lactate, as very little appeared in the blood space. In trout, a significant portion of the lactate was exported to the blood, which was taken up and metabolized by aerobic tissues. / Thesis / Doctor of Philosophy (PhD)

Plyšinės jungtys (PJ) yra tarpląstelinę sąveiką užtikrinantys kanalai, svarbūs ląstelių dauginimuisi, diferenciacijai, migracijai bei koordinacijai. Skirtinguose žmogaus organizmo audiniuose atrastas 21 koneksino (Cx) tipas. Tam tikros ligos yra susijusios su Cx mutacijomis. PJ kanalų savybes įtakoja jungties įtampa, viduląstelinis pH (pHi), viduląstelinė dvivalenčių katijonų koncentracija ([Ca2+]i, [Mg2+]i), fosforinimas, įvairios cheminės medžiagos. Disertacinio darbo metu pirmą kartą parodėme, kad plačiai naudojamų skirtingų grupių PJ slopiklių, tokių kaip alkanolių, forano, meflokvino, veikimas priklauso nuo pHi ir Cx tipo. Iškėlėme ir eksperimentiškai patikrinome hipotezę, kad tam tikri PJ slopikliai veikia, sudarydami vandenilinius ryšius su Cx45 baltymo histidinais. Ištyrėme Cx36, Cx40, Cx43, Cx45 ir Cx47 PJ kanalų laidumo priklausomybę nuo pHi. Nustatėme [Mg2+]i įtaką Cx43 ir Cx45 PJ kanalų laidumui bei Cx36 PJ kanalų laidumo priklausomybei nuo pHi, kas iki šiol nebuvo tirta. Cx45 ir Cx36 mutantinių baltymų tyrimai suteikė naujų žinių apie šių baltymų amino rūgštis, kurios gali būti reikšmingos PJ kanalų laidumo reguliavimui viduląsteliniu pH. / Gap junctions (GJ) are intercellular channels, which provide a direct pathway for electrical and metabolic cell-to-cell communication. GJ play an important role in cell proliferation, differentiation, migration, and coordination. The family of connexin (Cx) genes consists of 21 members in the human genome, and a variety of diseases is associated with Cx mutations. Electrical conductance (gj) of gap junctions is regulated by transjunctional voltage, intracellular pH (pHi), intracellular divalent cation concentration ([Ca2+]i and [Mg2+]i), phosphorylation and different chemical compounds. In this work we demonstrate for the first time that the potency of widely used GJ inhibitors such as alkanols, forane and mefloquine in GJ channel uncoupling is pHi- and Cx type-dependent. This type of modulation may occur through the formation of hydrogen bonds between uncouplers and histidines of Cx45 protein. We determined gj-pHi dependence of Cx36, Cx40, Cx43, Cx45 and Cx47 GJ channels, and evaluated the influence of [Mg2+]i on the conductance of Cx43 and Cx45 GJs and on the sensitivity of Cx36 GJs to pHi. The investigation of the Cx45 and Cx36 mutants provided with new knowledge on the structural elements, which may be responsible for the sensitivity of Cxs to pHi.

The master's thesis is focused on the study of response of the intracellular pH of the yeast cells on various external environments, primarily in a relation to the protonophore carbonyl cyanide m-chlorophenylhydrazone, CCCP. To measure the intracellular pH of the yeast cells we used a genetically coded fluorescent probe the ratiometric pHluorin. Using the method of synchronously scanned fluorescent spectra we were able to measure the intracellular pH of the cells with high precision. As a part of these experiments we also studied the influence of ionic strength of the cell suspensions buffers on the surface potential as well as the influence of the mineral salt KCl on the depolarization of the yeast membranes and cytosolic acidification induced by the protonophore CCCP. We examined the changes of cytosolic pH as such but we also used the measured pH as an indicator of the processes and the state of environment outside the cell. One of the most notable outcomes of this thesis is a new method of monitoring the value of the surface potential of the yeast cells by measuring the titration curves of cytosolic acidification induced by the protonophore CCCP.

Einleitung: Monocarboxylattransporter (MCT) 1 & 4 sind in zahlreichen Geweben als Kotransporter für Monocarboxylate und Protonen beschrieben. Auch im Pansenepithel werden MCT benötigt, um kurzkettige Fettsäuren (SCFA) aus dem Pansenlumen in die Pansenepithelzelle aufzunehmen (MCT4) und um SCFA und deren Metabolite aus der Pansenepithelzelle in das Blut auszuschleusen (MCT1). Die transepitheliale Permeation von SCFA über die Pansenwand ist von enormer Bedeutung, da sie die wichtigste Energiequelle der Wiederkäuer darstellen. Die beteiligten Transportprozesse müssen dementsprechend einer Anpassung an variierende Mengen von SCFA unterliegen. Bisherige Studien bei anderen Spezies deuten auf eine Regulation des MCT1 auf mRNA Ebene über den Peroxisom-Proliferator-aktivierten Rezeptor α (PPARα) hin. Ziele der Untersuchung: Das Ziel dieser Arbeit war herauszufinden, ob MCT1 in ovinen Pansenepithelzellen über PPARα reguliert wird und ob auch MCT4 dieser Regulation unterliegt. Eine gleichzeitige Regulation beider Transporter läge nahe, da sie gemeinsam an der transepithelialen Permeation beteiligt sind. Die Auswirkungen solch einer Regulation auf die Proteinexpression und die Transportleistung der MCT sollte charakterisiert werden. Ebenfalls war das Potenzial der bei erhöhter Kraftfutterfütterung vermehrt anfallenden SCFA Butyrat auf die MCT1 Expression zu untersuchen. Material & Methoden: Aus dem Vorhof von Schafen wurden Pansenepithelzellen gewonnen und entsprechend einer bereits etablierten Methode kultiviert. Nach einer Subkultivierung wurden die Zellen immunzytochemisch mit Antikörpern gegen MCT1, MCT4 und Na+/K+-ATPase untersucht, um deren Lokalisation in den kultivierten Pansenepithelzellen zu bestimmen. Weiterhin erfolgte eine Behandlung mit WY 14.643, einem spezifischen, synthetischen PPARα Agonisten, sowie mit GW 6471, einem Antagonisten des PPARα. Mittels qPCR wurden die relativen mRNA Mengen von MCT1, MCT4, ACO, CPT1A und CACT bestimmt und auf die Referenzgene GAPDH und Na+/K+-ATPase normalisiert. Die Proteinexpression von MCT1 und MCT4 wurde mittels Western Blot bestimmt. Zur funktionellen Quantifizierung wurde der intrazelluläre pH-Wert der Zellen mittels Spektrofluorometrie gemessen und der laktatabhängige Protonentransport als Vergleichswert zwischen den Behandlungen genutzt. Um den MCT-abhängigen Teil des Transportes zu bestimmen, wurde ein spezifischer MCT1 & 4 Inhibitor, die p-Hydroxymercuribenzensulfonsäure (pHMB) eingesetzt. Die Zellen wurden mit Butyrat über einen Zeitraum von 6 und 48 h induziert. Die Erfassung der MCT1 Expression erfolgte mittels semiquantitativer PCR. Ergebnisse: MCT1 & 4 sind sowohl in der Zellmembran als auch intrazellulär in den Pansenepithelzellen lokalisiert. Die mRNA Expressionsdaten konnten zeigen, dass MCT1 und die PPARα Zielgene durch WY 14.643 hochreguliert werden konnten, wohingegen die MCT4 Expression keine eindeutige Antwort auf die Stimulation zeigt. Die Behandlung mit den Antagonisten zeigt eine Abhängigkeit der MCT1 Expression von PPARα, die MCT4 Expression konnte dagegen nicht beeinflusst werden. Mittels pHMB gelang es, den laktatabhängigen Protonenexport fast vollständig zu blocken. Sowohl laktatabhängiger Protonenexport als auch die Proteinexpression zeigten keine Änderung durch WY 14.643 Stimulation. Die Butyratexposition veränderte die Morphologie der Pansenepithelzellen und schien nicht geeignet für Untersuchungen der mRNA Expression zu sein. Schlussfolgerungen: Es konnte in dieser Arbeit erstmals gezeigt werden, dass MCT1 in Pansenepithelzellen über PPARα reguliert wird, nicht aber MCT4. PPARα scheint demnach einer der entscheidenden Angriffspunkte für die Regulation des SCFA Transportes zu sein, dessen natürliche Liganden im Pansen aber noch nicht bekannt sind. Damit legt diese Arbeit den Grundstein für regulative Studien am intakten Pansenepithel. / Introduction: Monocarboxylate transporters (MCT) 1 & 4 are cotransporters of monocarboxylates and protons in a variety of mammalian cell types. In the ruminal epithelium MCT are necessary to transport short-chain fatty acids (SCFA) from the lumen into the ruminal epithelial cell (MCT4) and to discharge SCFA and their metabolites from the cell into the blood (MCT1). Transepithelial permeation of SCFA is of great importance, because they are the main source of energy for ruminants. The regulation of appropriate transport proteins should thus be subject to the adaptation to varying SCFA amounts. Previous studies in other species suggested that gene expression of MCT1 is regulated by peroxisome proliferator-activated receptor α (PPARα), a ligand-activated nuclear receptor. Aims: The aim of the study was to examine if MCT1 in ruminal epithelial cells is regulated by PPARα and furthermore if MCT4 can be regulated by PPARα, as well. A simultaneous regulation seems likely, because both are acting jointly in the transepithelial transporting of SCFA. The implications of such a regulation on protein expression and transport capacity of MCT should be characterized. The effect of butyrate, a SCFA which increases under concentrate feeding, on MCT1 expression was determined. Materials & Methods: Ruminal epithelial cells of sheep were cultivated according to methods previously established. After subcultivation, immunocytochemistry with antibodies against MCT1, MCT4 and Na+/K+-ATPase was performed to determine their localization in ruminal epithelial cells. For studying the influence of PPARα, WY 14.643, a synthetic and selective ligand of PPARα, and GW 6471, a synthetic antagonist of PPARα, were applied to the culture medium of the cells. After processing the specimens, the relative amount of mRNA of MCT1, MCT4 and the target genes ACO, CPT1A and CACT were analyzed by qPCR and normalized on the reference genes GAPDH and Na+/K+-ATPase. Protein abundance of MCT1 & 4 was measured by using the Western Blot method. Functional quantification was measured by the intracellular pH (pHi) of cells using spectrofluorometry as well as comparing the effect of WY 14.643 treatment on lactate-dependent proton export. To determine the MCT-dependent part of the pHi recovery, p-hydroxymercuribenzoic acid (pHMB), a specific inhibitor of MCT1 & 4, was applied. Cells were also treated with butyrate for 6 h and 48 h and the mRNA abundance of MCT1 was analyzed by semiquantitative PCR. Results: Both MCT1 and MCT4 were localized in the cell membrane as well as in the cytoplasm of ruminal epithelial cells. By qPCR it could be demonstrated that the mRNA abundance of MCT1 and PPARα target genes in the ruminal epithelial cells was increased by WY 14.643 in comparison to untreated cells, whereas the response of MCT4 did not yield distinct results. Treatment with the PPARα antagonist pointed out, that MCT1 is influenced by PPARα, but not MCT4. Lactate-dependent proton export was blocked almost completely by pHMB. Both lactate-dependent proton export and protein expression were not altered by WY 14.643 treatment. Butyrate exposure changed the morphology of ruminal epithelial cells and seemed unsuitable for the analysis of mRNA expression. Conclusion: For the first time, it could be demonstrated, that MCT1 in ruminal epithelial cells is regulated by PPARα, but not MCT4. PPARα seems to be a vital target in the rumen for SCFA transport regulation, whose natural triggers have yet to be identified. Furthermore, this study provides the basis for regulative studies on intact ruminal epithelium.

O objetivo do trabalho foi determinar o pHi, a capacidade tamponante do citosol na ausência de CO2/HCO3- (βi) de neurônios dos gânglios das raízes dorsais e investigar a expressão de trocadores Na+/H+ e sua função na regulação do pHi. O pHi foi estimado com o indicador fluorescente BCECF. A operação do trocador era quantificada na recuperação da acidose intracelular induzida experimentalmente. Na ausência do tampão CO2/HCO3- a taxa de alcalinização (k) deve-se, por hipótese, ao transporte de H+ pelo trocador. A hipótese foi confirmada pela ação de agentes farmacológicos, e.g., amiloride. Em soluções tamponadas por CO2/HCO3- as células tem pHi de 7,24 e, em soluções tamponadas com HEPES, 7,04. A βi foi de 8,17 mM/pH. As células se recuperam da acidose com k médio de 0,0138 s-1. O efeito inibitório do amiloride em concentração de 1 mM deve-se ao fato dos fenótipos celulares expressarem diferentes isoformas do trocador. Segundo RT-PCR, todas as 5 isoformas do trocador são expressas e a quantidade de RNAm, avaliada por qPCR, é maior para a NHE1, seguida de NHE5. / The objective here was to determine intracellular pH (pHi), cytosolic buffering power in CO2/HCO3- free medium (βi) of neurons from dorsal root ganglia and to investigate the functional expression of the Na+/H+ exchangers in the regulation of pHi. pHi was estimated with fluorescence indicator BCECF. Exchanger operation was quantified during recovery from intracellular acidification induced experimentally. In CO2/HCO3- free medium the alkalinization rate (k) is due, hypothetically, H+ extrusion by the exchanger. This assumption was confirmed by action of pharmacologic agents, e.g., amiloride. In medium buffered with CO2/HCO3- cells have pHi of 7.24 and, in medium buffered with HEPES, 7.04. βi calculated was 8.17 mM/pH. Cells recovery from acidosis with mean k of 0.0138 s-1. Inhibitory effect of amiloride in 1 mM concentration is due to cellular phenotypes expressing different Na+/H+ exchanger isoforms. According to RT-PCR, all the five exchanger isoforms are expressed and mRNA quantity, evaluated by qPCR, is greater to NHE1, followed by NHE5.

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.

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.

Einleitung: Monocarboxylattransporter (MCT) 1 & 4 sind in zahlreichen Geweben als Kotransporter für Monocarboxylate und Protonen beschrieben. Auch im Pansenepithel werden MCT benötigt, um kurzkettige Fettsäuren (SCFA) aus dem Pansenlumen in die Pansenepithelzelle aufzunehmen (MCT4) und um SCFA und deren Metabolite aus der Pansenepithelzelle in das Blut auszuschleusen (MCT1). Die transepitheliale Permeation von SCFA über die Pansenwand ist von enormer Bedeutung, da sie die wichtigste Energiequelle der Wiederkäuer darstellen. Die beteiligten Transportprozesse müssen dementsprechend einer Anpassung an variierende Mengen von SCFA unterliegen. Bisherige Studien bei anderen Spezies deuten auf eine Regulation des MCT1 auf mRNA Ebene über den Peroxisom-Proliferator-aktivierten Rezeptor α (PPARα) hin. Ziele der Untersuchung: Das Ziel dieser Arbeit war herauszufinden, ob MCT1 in ovinen Pansenepithelzellen über PPARα reguliert wird und ob auch MCT4 dieser Regulation unterliegt. Eine gleichzeitige Regulation beider Transporter läge nahe, da sie gemeinsam an der transepithelialen Permeation beteiligt sind. Die Auswirkungen solch einer Regulation auf die Proteinexpression und die Transportleistung der MCT sollte charakterisiert werden. Ebenfalls war das Potenzial der bei erhöhter Kraftfutterfütterung vermehrt anfallenden SCFA Butyrat auf die MCT1 Expression zu untersuchen. Material & Methoden: Aus dem Vorhof von Schafen wurden Pansenepithelzellen gewonnen und entsprechend einer bereits etablierten Methode kultiviert. Nach einer Subkultivierung wurden die Zellen immunzytochemisch mit Antikörpern gegen MCT1, MCT4 und Na+/K+-ATPase untersucht, um deren Lokalisation in den kultivierten Pansenepithelzellen zu bestimmen. Weiterhin erfolgte eine Behandlung mit WY 14.643, einem spezifischen, synthetischen PPARα Agonisten, sowie mit GW 6471, einem Antagonisten des PPARα. Mittels qPCR wurden die relativen mRNA Mengen von MCT1, MCT4, ACO, CPT1A und CACT bestimmt und auf die Referenzgene GAPDH und Na+/K+-ATPase normalisiert. Die Proteinexpression von MCT1 und MCT4 wurde mittels Western Blot bestimmt. Zur funktionellen Quantifizierung wurde der intrazelluläre pH-Wert der Zellen mittels Spektrofluorometrie gemessen und der laktatabhängige Protonentransport als Vergleichswert zwischen den Behandlungen genutzt. Um den MCT-abhängigen Teil des Transportes zu bestimmen, wurde ein spezifischer MCT1 & 4 Inhibitor, die p-Hydroxymercuribenzensulfonsäure (pHMB) eingesetzt. Die Zellen wurden mit Butyrat über einen Zeitraum von 6 und 48 h induziert. Die Erfassung der MCT1 Expression erfolgte mittels semiquantitativer PCR. Ergebnisse: MCT1 & 4 sind sowohl in der Zellmembran als auch intrazellulär in den Pansenepithelzellen lokalisiert. Die mRNA Expressionsdaten konnten zeigen, dass MCT1 und die PPARα Zielgene durch WY 14.643 hochreguliert werden konnten, wohingegen die MCT4 Expression keine eindeutige Antwort auf die Stimulation zeigt. Die Behandlung mit den Antagonisten zeigt eine Abhängigkeit der MCT1 Expression von PPARα, die MCT4 Expression konnte dagegen nicht beeinflusst werden. Mittels pHMB gelang es, den laktatabhängigen Protonenexport fast vollständig zu blocken. Sowohl laktatabhängiger Protonenexport als auch die Proteinexpression zeigten keine Änderung durch WY 14.643 Stimulation. Die Butyratexposition veränderte die Morphologie der Pansenepithelzellen und schien nicht geeignet für Untersuchungen der mRNA Expression zu sein. Schlussfolgerungen: Es konnte in dieser Arbeit erstmals gezeigt werden, dass MCT1 in Pansenepithelzellen über PPARα reguliert wird, nicht aber MCT4. PPARα scheint demnach einer der entscheidenden Angriffspunkte für die Regulation des SCFA Transportes zu sein, dessen natürliche Liganden im Pansen aber noch nicht bekannt sind. Damit legt diese Arbeit den Grundstein für regulative Studien am intakten Pansenepithel. / Introduction: Monocarboxylate transporters (MCT) 1 & 4 are cotransporters of monocarboxylates and protons in a variety of mammalian cell types. In the ruminal epithelium MCT are necessary to transport short-chain fatty acids (SCFA) from the lumen into the ruminal epithelial cell (MCT4) and to discharge SCFA and their metabolites from the cell into the blood (MCT1). Transepithelial permeation of SCFA is of great importance, because they are the main source of energy for ruminants. The regulation of appropriate transport proteins should thus be subject to the adaptation to varying SCFA amounts. Previous studies in other species suggested that gene expression of MCT1 is regulated by peroxisome proliferator-activated receptor α (PPARα), a ligand-activated nuclear receptor. Aims: The aim of the study was to examine if MCT1 in ruminal epithelial cells is regulated by PPARα and furthermore if MCT4 can be regulated by PPARα, as well. A simultaneous regulation seems likely, because both are acting jointly in the transepithelial transporting of SCFA. The implications of such a regulation on protein expression and transport capacity of MCT should be characterized. The effect of butyrate, a SCFA which increases under concentrate feeding, on MCT1 expression was determined. Materials & Methods: Ruminal epithelial cells of sheep were cultivated according to methods previously established. After subcultivation, immunocytochemistry with antibodies against MCT1, MCT4 and Na+/K+-ATPase was performed to determine their localization in ruminal epithelial cells. For studying the influence of PPARα, WY 14.643, a synthetic and selective ligand of PPARα, and GW 6471, a synthetic antagonist of PPARα, were applied to the culture medium of the cells. After processing the specimens, the relative amount of mRNA of MCT1, MCT4 and the target genes ACO, CPT1A and CACT were analyzed by qPCR and normalized on the reference genes GAPDH and Na+/K+-ATPase. Protein abundance of MCT1 & 4 was measured by using the Western Blot method. Functional quantification was measured by the intracellular pH (pHi) of cells using spectrofluorometry as well as comparing the effect of WY 14.643 treatment on lactate-dependent proton export. To determine the MCT-dependent part of the pHi recovery, p-hydroxymercuribenzoic acid (pHMB), a specific inhibitor of MCT1 & 4, was applied. Cells were also treated with butyrate for 6 h and 48 h and the mRNA abundance of MCT1 was analyzed by semiquantitative PCR. Results: Both MCT1 and MCT4 were localized in the cell membrane as well as in the cytoplasm of ruminal epithelial cells. By qPCR it could be demonstrated that the mRNA abundance of MCT1 and PPARα target genes in the ruminal epithelial cells was increased by WY 14.643 in comparison to untreated cells, whereas the response of MCT4 did not yield distinct results. Treatment with the PPARα antagonist pointed out, that MCT1 is influenced by PPARα, but not MCT4. Lactate-dependent proton export was blocked almost completely by pHMB. Both lactate-dependent proton export and protein expression were not altered by WY 14.643 treatment. Butyrate exposure changed the morphology of ruminal epithelial cells and seemed unsuitable for the analysis of mRNA expression. Conclusion: For the first time, it could be demonstrated, that MCT1 in ruminal epithelial cells is regulated by PPARα, but not MCT4. PPARα seems to be a vital target in the rumen for SCFA transport regulation, whose natural triggers have yet to be identified. Furthermore, this study provides the basis for regulative studies on intact ruminal epithelium.