Cholinergic input to, and cholinergic mechanisms within the lower lumbar (L6) and upper sacral (S1) spinal cord of rat may influence neuronal excitability and afferent transmission (Thor et al, 2000) and may provide the environment necessary for appropriate central nervous system control of bladder and bowel function. It is unclear, however, if cholinergic terminals and receptors are present in the L6 & S1 spinal segments of rat and when this may develop. Cholinergic mechanisms have been shown to alter sensory afferent transmission, enhance motoneuron excitability, induce plateau potentials via non-linear membrane properties in motoneurons and reveal oscillations in locomotor-related interneurons. The enhanced activity of sphincter motoneurons was attributed to non-linear properties during the continence phase of distention-evoked voiding in the decerebrate cat (Paroschy & Shefchyk, 2000). Candidate neurotransmitters inducing non-linear properties in cat sphincter motoneurons are 5-HT (Paroschy & Shefchyk, 2000) and acetylcholine via motoneuron axon collaterals (Sasaki, 1994) and other spinal sources. We have established using the antibody to the vesicular acetylcholine transporter (VAChT) that cholinergic terminals are present on ventrolateral Onuf (VLO), dorsomedial Onuf (DMO) motoneurons and parasympathetic preganglionic motoneurons (PGN) in the L6 and S1 rat spinal cord segments. Muscarinic receptor (M2), nicotinic-α4 and α7 receptor subunit immunoreactivity was also present on Onuf motoneurons and in regions dorsal to the PGN. One source of the cholinergic puncta on Onuf motoneurons may be from motoneuron axon collaterals which we observed on a postnatal day 15 VLO motoneuron. Cholinergic terminals were observed on vasoactive intestinal polypeptide-immunoreactive (VIP) afferents, interneurons in the intermediolateral (IML) region and perhaps on other afferents in the lateral and medial collateral pathway of L6 and S1 spinal segments. In the ventral horn, the cholinergic puncta and receptors appear to have a mature distribution around two weeks postnatal and the cholinergic terminals appeared to have a mature distribution in the IML region by three weeks postnatal. Using whole cell patch clamp recording techniques and thick slices of the L6 and S1 rat spinal cord, we observed excitatory responses of ventral horn neurons and motoneurons to carbachol (10-50 μM), a non-specific cholinergic agonist. Ventral horn neurons (postnatal day 8- 16) exhibited prolonged firing and prolonged depolarizations (plateau potentials) beyond the duration of the applied excitatory input from cholinergic (n=6/33) and other (n= 4/37) neurotransmitter systems. In a selection of the neurons with plateau potentials, the L-type calcium current played a role in the plateau production (n=5/5) and low frequency oscillations (n=2/2) as revealed by nifedipine. Postnatally, the voiding reflex changes from a perineal-evoked reflex, to the adult bladder-bladder reflex. Cholinergic input may be responsible in part for the bursting activity of the external urethral sphincter and the activation of the bladder, which is required for complete voiding reflexes in the adult rat. Plateau potentials and enhanced excitability due to cholinergic mechanisms could render inessential a constant excitatory drive that is required in the perineal-evoked voiding reflex in the neonatal rat and may underlie changes in the voiding reflexes that occur during postnatal development.

Cholinergic input to, and cholinergic mechanisms within the lower lumbar (L6) and upper sacral (S1) spinal cord of rat may influence neuronal excitability and afferent transmission (Thor et al, 2000) and may provide the environment necessary for appropriate central nervous system control of bladder and bowel function. It is unclear, however, if cholinergic terminals and receptors are present in the L6 & S1 spinal segments of rat and when this may develop. Cholinergic mechanisms have been shown to alter sensory afferent transmission, enhance motoneuron excitability, induce plateau potentials via non-linear membrane properties in motoneurons and reveal oscillations in locomotor-related interneurons. The enhanced activity of sphincter motoneurons was attributed to non-linear properties during the continence phase of distention-evoked voiding in the decerebrate cat (Paroschy & Shefchyk, 2000). Candidate neurotransmitters inducing non-linear properties in cat sphincter motoneurons are 5-HT (Paroschy & Shefchyk, 2000) and acetylcholine via motoneuron axon collaterals (Sasaki, 1994) and other spinal sources. We have established using the antibody to the vesicular acetylcholine transporter (VAChT) that cholinergic terminals are present on ventrolateral Onuf (VLO), dorsomedial Onuf (DMO) motoneurons and parasympathetic preganglionic motoneurons (PGN) in the L6 and S1 rat spinal cord segments. Muscarinic receptor (M2), nicotinic-α4 and α7 receptor subunit immunoreactivity was also present on Onuf motoneurons and in regions dorsal to the PGN. One source of the cholinergic puncta on Onuf motoneurons may be from motoneuron axon collaterals which we observed on a postnatal day 15 VLO motoneuron. Cholinergic terminals were observed on vasoactive intestinal polypeptide-immunoreactive (VIP) afferents, interneurons in the intermediolateral (IML) region and perhaps on other afferents in the lateral and medial collateral pathway of L6 and S1 spinal segments. In the ventral horn, the cholinergic puncta and receptors appear to have a mature distribution around two weeks postnatal and the cholinergic terminals appeared to have a mature distribution in the IML region by three weeks postnatal. Using whole cell patch clamp recording techniques and thick slices of the L6 and S1 rat spinal cord, we observed excitatory responses of ventral horn neurons and motoneurons to carbachol (10-50 μM), a non-specific cholinergic agonist. Ventral horn neurons (postnatal day 8- 16) exhibited prolonged firing and prolonged depolarizations (plateau potentials) beyond the duration of the applied excitatory input from cholinergic (n=6/33) and other (n= 4/37) neurotransmitter systems. In a selection of the neurons with plateau potentials, the L-type calcium current played a role in the plateau production (n=5/5) and low frequency oscillations (n=2/2) as revealed by nifedipine. Postnatally, the voiding reflex changes from a perineal-evoked reflex, to the adult bladder-bladder reflex. Cholinergic input may be responsible in part for the bursting activity of the external urethral sphincter and the activation of the bladder, which is required for complete voiding reflexes in the adult rat. Plateau potentials and enhanced excitability due to cholinergic mechanisms could render inessential a constant excitatory drive that is required in the perineal-evoked voiding reflex in the neonatal rat and may underlie changes in the voiding reflexes that occur during postnatal development.

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.

Die Präeklampsie ist eine schwangerschaftsspezifische Bluthochdruck-Erkrankung, die im Allgemeinen nach der 20. Schwangerschaftswoche auftritt. Neben der Hypertonie sind die Proteinurie und die Ödembildung charakteristische Symptome der Präeklampsie. Obwohl heute die Pathophysiologie der Präeklampsie zum großen Teil verstanden ist, ist die Ätiologie dieser Erkrankung noch unklar. 1999 konnten wir in den Seren von Präeklampsie-Patientinnen agonistische Autoantikörper, die gegen den Angiotensin II AT1-Rezeptor gerichtet sind (AT1-AAK), nachweisen. Diese AT1-AAK gehören zur Antikörpersubklasse IgG3.<br /> Die AT1-AAK führen in Kulturen neonataler Rattenkardiomyozyten AT1-Rezeptor spezifisch zu einem positiv chronotropen Effekt. Mittels Immunpräzipitation wurde gezeigt, dass AT1-AAK spezifisch den AT1-Rezeptor präzipitieren. Kontrollproben, aus denen die AT1-AAK entfernt wurden, führen zu keiner Präzipitation des AT1-Rezeptors. Die Präzipitation des AT1-Rezeptors bleibt ebenfalls aus, wenn die AT1-AAK mit einem Peptid, welches der Aminosäuresequenz des zweiten extrazellulären Loops des humanen AT1-Rezeptors entspricht, behandelt wurden. Eine Langzeitbehandlung der Kulturen neonataler Rattenherzzellen mit AT1-AAK vermindert die funktionelle Ansprechbarkeit der Zellen auf einen erneuten AT1-Rezeptor-Stimulus.<br /> Eine veränderte AT1-Rezeptorexpression wurde nicht nachgewiesen. In guter Übereinstimmung mit den in vitro-Expressionsdaten wurde gezeigt, dass die plazentare AT1-Rezeptorexpression bei Präeklampsie-Patientinnen nicht verschieden von der plazentaren AT1-Rezeptorexpression gesunder Schwangerer mit nicht pathogen verändertem Blutdruck ist. Im Zellsystem der neonatalen Rattenherzzellen führen die AT1-AAK zur Aktivierung von Gi-Proteinen und zu verringerten intrazellulären cAMP-Spiegeln.<br /> Des Weiteren wurde gezeigt, dass die AT1-AAK in Kulturen neonataler Rattenherzzellen die Transkriptionsfaktoren AP-1 und NFkB aktivieren. Die Aktivierung des Transkriptionsfaktors NFkB wurde vornehmlich in den Nicht-Myozyten der Rattenherzzellkultur nachgewiesen. Generell wurde festgestellt, dass sich die AT1-AAK pharmakologisch wie der natürliche Agonist des AT1-Rezeptors, Angiotensin II, verhalten.<br /> Erste Daten dieser Arbeit deuten auf einen eventuellen Einfluss der AT1-AAK auf die Expression von Komponenten der extrazellulären Matrix bzw. assoziierter Faktoren (Kollagen III, MMP-2, TIMP-2, Colligin) hin. In allen in dieser Arbeit untersuchten Seren von klinisch diagnostizierten Präeklampsie-Patientinnen wurden agonistische AT1-AAK nachgewiesen. Wir vermuten daher, dass die AT1-AAK möglicherweise bedeutend in der Pathogenese der Präeklampsie sind. / Preeclampsia is a serious, pregnancy-specific disorder that usually occurs after week 20 of gestation and is characterized by hypertension, proteinuria, and oedema. While the pathophysiology is clear, little is known about etiology of preeclampsia.<br /> In 1999, we showed that sera from preeclamptic patients contain autoantibodies directed against angiotensin II AT1 receptor (AT1-AAB). These autoantibodies are immunoglobuliens of the IgG3 subclass.<br /> AT1-AAB accelerate the beating rate of neonatal rat cardiomyocytes. The agonistic effect can be blocked with the AT1 receptor blocker losartan.<br /> Co-immunoprecipitation studies have shown that AT1-AAB specifically precipitate the AT1 receptor while control samples lacking AT1-AAB do not. The AT1 receptor could not be precipitated following neutralization of the AT1-AAB by a peptide corresponding to the AT1 receptors second extracellular loop. In further studies on neonatal rat heart cells, we showed long-term stimulation of the AT1 receptor whereby AT1-AAB down-regulated the AT1 receptor-mediated response to a second agonistic receptor-stimulation.<br /> After long-term stimulation of neonatal rat heart cells, no changes in AT1 receptor expression could be identified. Corresponding to these in vitro-expression data, no difference was seen in placental AT1 receptor expression between patients with preeclampsia and healthy pregnant women. Next, we tested if the AT1-AAB lead to activation of AT1 receptor signaling in angiotensin II fashion. In neonatal rat heart cell cultures, AT1-AAB lead to activation of Gi-protein with reduced cAMP levels. AT1-AAB are able to activate the transcription factors AP-1 and NFkB in this cell system. In all observations, the agonistic AT1-AAB behave pharmacologically in a similar fashion to angiotensin II.<br /> Initial data suggest that AT1-AAB may have an effect on extracellular matrix components (ECM).<br /> We have found AT1-AAB in all women meeting the clinical criteria of preeclampsia and, therefore, suggest that AT1-AAB may be important to the pathogenesis of the disease.

No description available.

Hypertrophie bezeichnet eine zelluläre Anpassungsleistung, die durch vermehrte Arbeitsbelastung ausgelöst wird und durch Zunahme von Zellgröße und Proteinsynthese sowie durch Veränderungen der Genexpression bei konstanter Zellzahl gekennzeichnet ist. Beim Ubiquitin-Proteasom-System handelt es sich um den wichtigsten intrazellulären Proteinabbaumechanismus eukaryontischer Zellen. Darüber hinaus spielt es eine wichtige Rolle im regulierten Abbau zellulärer Signalmediatoren und Transkriptionsfaktoren. In einem Hypertrophiemodell mit neonatalen Rattenkardiomyozyten wurde die Wirkung von Proteasominhibitoren auf die Ausbildung einer Hypertrophie untersucht. Behandlung mit Proteasominhibitoren (MG132, MG262) führte dabei zu einer dosisabhängigen Reduktion des Effekts der eingesetzten hypertrophieinduzierenden Agonisten (Isoproterenol, Angiotensin II, Phenylephrin). So konnte mit Hilfe morphometrischer Analysen Phalloidin-gefärbter Kardiomyozyten eine Verringerung des Zellwachstums gezeigt werden. Western Blots belegten eine verringerte Expression von Hypertrophiemarkerproteinen (beta-myosin heavy chain, alpha-sarcomeric actin, alpha-smooth muscle actin). Analog zu diesen Befunden konnte in einem Reportergenassay die Abnahme der Expression des brain natriuretic peptide (BNP) gezeigt werden. Eine reduzierte RNA- und Proteinsynthese konnte mit Hilfe der Inkorporation radioaktiver Substrate nachgewiesen werden. Als Nachweis für die effiziente Inhibition des Proteasoms durch MG132 dienten Western Blots akkumulierter, polyubiquitinierter Proteine, die reduzierte proteasomale Degradation fluorogener Substrate sowie die Akkumulation eines grün fluoreszierenden Proteins nach Transfektion mit einem Ubiquitin-GFP-Konstrukt. Als mögliche Mechanismen des antihypertrophen Effekts der Proteasominhibitoren konnten eine verringerte Aktivierbarkeit der MAP Kinasen ERK 1/2 (Western Blots) sowie eine reduzierte Aktivität des Transkriptionsfaktor NFkappaB (Reportergenassay) identifiziert werden. / Myocardial hypertrophy is an important adaptive response of the heart to increased workload. It is characterized by an increase in cell size and protein synthesis, and alterations in gene expression. The ubiquitin-proteasome-system is the major pathway for intracellular protein degradation in eucaryotic cells. It plays a major role in the regulated degradation of central signal mediators and transcription factors. In a model system of neonatal rat cardiomyocytes we investigated the effects of proteasome inhibitors on myocardial hypertrophy. Treatment with specific proteasome inhibitors reduced the hypertrophic effects of all used agonists (e.g. isoproterenol, phenylephrin) dose-dependently: 0.05-1 µM MG132 resulted in a marked reduction of cell size as determined by morphometric analysis of phalloidin-stained myocytes. Moreover, western blot analysis showed a concentration-dependently reduced expression of hypertrophic marker proteins (beta-myosin heavy chain, alpha-sarcomeric actin, alpha-smooth muscle actin). This correlated well with a suppressed expression of brain natriuretic peptide in reportergene assays. Reduced RNA and protein synthesis was determined by incorporation of radioactively labeled substrates. Efficient inhibition of the proteasome by MG132 was confirmed by increased accumulation of multi-ubiquitinated proteins in western blot analysis, by reduced degradation of fluorogenic substrates and by accumulation of a ubiquitin-conjugated variant of the green fluorescent protein. Suppression of cardiomyocyte hypertrophy by proteasome inhibition corresponded to reduced ERK 1/2 activation (determined by phospho-specific antibodies) and decreased NFkappaB activation (determined by luciferase assays).