Pavel I Nedvetsky

Publications of Pavel I Nedvetsky

• Reciprocal regulation of aquaporin-2 abundance and degradation by protein kinase A and p38-MAP kinase.

  Authors: Pavel I Nedvetsky, Vedrana Tabor, Grazia Tamma, Sven Beulshausen, Philipp Skroblin, Aline Kirschner, Kerim Mutig, Mareike Boltzen, Oscar Petrucci, Anna Vossenkämper, Burkhard Wiesner, Sebastian Bachmann, Walter Rosenthal, Enno Klussmann

  Journal of the American Society of Nephrology : JASN. 10/2010; 21(10):1645-56.

  Arginine-vasopressin (AVP) modulates the water channel aquaporin-2 (AQP2) in the renal collecting duct to maintain homeostasis of body water. AVP binds to vasopressin V2 receptors (V2R), increasingArginine-vasopressin (AVP) modulates the water channel aquaporin-2 (AQP2) in the renal collecting duct to maintain homeostasis of body water. AVP binds to vasopressin V2 receptors (V2R), increasing cAMP, which promotes the redistribution of AQP2 from intracellular vesicles into the plasma membrane. cAMP also increases AQP2 transcription, but whether altered degradation also modulates AQP2 protein levels is not well understood. Here, elevation of cAMP increased AQP2 protein levels within 30 minutes in primary inner medullary collecting duct (IMCD) cells, in human embryonic kidney (HEK) 293 cells ectopically expressing AQP2, and in mouse kidneys. Accelerated transcription or translation did not explain this increase in AQP2 abundance. In IMCD cells, cAMP inhibited p38-mitogen-activated protein kinase (p38-MAPK) via activation of protein kinase A (PKA). Inhibition of p38-MAPK associated with decreased phosphorylation (serine 261) and polyubiquitination of AQP2, preventing proteasomal degradation. Our results demonstrate that AVP enhances AQP2 protein abundance by altering its proteasomal degradation through a PKA- and p38-MAPK-dependent pathway.

• Regulation of aquaporin-2 trafficking.

  Authors: Pavel I Nedvetsky, Grazia Tamma, Sven Beulshausen, Giovanna Valenti, Walter Rosenthal, Enno Klussmann

  Handbook of experimental pharmacology. 02/2009;

  Principal cells lining renal collecting ducts control the fine-tuning of body water homeostasis by regulating water reabsorption through the water channels aquaporin-2 (AQP2), aquaporin-3 (AQP3), andPrincipal cells lining renal collecting ducts control the fine-tuning of body water homeostasis by regulating water reabsorption through the water channels aquaporin-2 (AQP2), aquaporin-3 (AQP3), and aquaporin-4 (AQP4). While the localization of AQP2 is subject to regulation by arginine-vasopressin (AVP), AQP3 and AQP4 are constitutively expressed in the basolateral plasma membrane. AVP adjusts the amount of AQP2 in the plasma membrane by triggering its redistribution from intracellular vesicles into the plasma membrane. This permits water entry into the cells and water exit through AQP3 and AQP4. The translocation of AQP2 is initiated by an increase in cAMP following V2R activation through AVP. The AVP-induced rise in cAMP activates protein kinase A (PKA), which in turn phosphorylates AQP2, and thereby triggers the redistribution of AQP2. Several proteins participating in the control of cAMP-dependent AQP2 trafficking have been identified; for example, A kinase anchoring proteins (AKAPs) tethering PKA to cellular compartments; phosphodiesterases (PDEs) regulating the local cAMP level; cytoskeletal components such as F-actin and microtubules; small GTPases of the Rho family controlling cytoskeletal dynamics; motor proteins transporting AQP2-bearing vesicles to and from the plasma membrane for exocytic insertion and endocytic retrieval; SNAREs inducing membrane fusions, hsc70, a chaperone, important for endocytic retrieval. In addition, cAMP-independent mechanisms of translocation mainly involving the F-actin cytoskeleton have been uncovered. Defects of AQP2 trafficking cause diseases such as nephrogenic diabetes insipidus (NDI), a disorder characterized by a massive loss of hypoosmotic urine.This review summarizes recent data elucidating molecular mechanisms underlying the trafficking of AQP2. In particular, we focus on proteins involved in the regulation of trafficking, and physiological and pathophysiological stimuli determining the cellular localization of AQP2. The identification of proteins and protein-protein interactions may lead to the development of drugs targeting AQP2 trafficking. Such drugs may be suitable for the treatment of diseases associated with dysregulation of body water homeostasis, including NDI or cardiovascular diseases (e.g., chronic heart failure) where the AVP level is elevated, inducing excessive water retention.

• Heat shock protein 90 regulates stabilization rather than activation of soluble guanylate cyclase.

  Authors: Pavel I Nedvetsky, Sabine Meurer, Nils Opitz, Tatiana Y Nedvetskaya, Helmut Müller, Harald H H W Schmidt

  FEBS letters. 02/2008; 582(2):327-31.

  Endothelium-derived nitric oxide (NO) activates the heterodimeric heme protein soluble guanylate cyclase (sGC) to form cGMP. In different disease states, sGC levels and activity are diminishedEndothelium-derived nitric oxide (NO) activates the heterodimeric heme protein soluble guanylate cyclase (sGC) to form cGMP. In different disease states, sGC levels and activity are diminished possibly involving the sGC binding chaperone, heat shock protein 90 (hsp90). Here we show that prolonged hsp90 inhibition in different cell types reduces protein levels of both sGC subunits by about half, an effect that was prevented by the proteasome inhibitor MG132. Conversely, acute hsp90 inhibition affected neither basal nor NO-stimulated sGC activity. Thus, hsp90 is a molecular stabilizer for sGC tonically preventing proteasomal degradation rather than having a role in short-term activity regulation.

  Download

• Microtubules are needed for the perinuclear positioning of aquaporin-2 after its endocytic retrieval in renal principal cells.

  Authors: Anna Vossenkämper, Pavel I Nedvetsky, Burkhard Wiesner, Jens Furkert, Walter Rosenthal, Enno Klussmann

  American journal of physiology. Cell physiology. 10/2007; 293(3):C1129-38.

  Water reabsorption in the renal collecting duct is regulated by arginine vasopressin (AVP). AVP induces the insertion of the water channel aquaporin-2 (AQP2) into the plasma membrane of principalWater reabsorption in the renal collecting duct is regulated by arginine vasopressin (AVP). AVP induces the insertion of the water channel aquaporin-2 (AQP2) into the plasma membrane of principal cells, thereby increasing the osmotic water permeability. The redistribution of AQP2 to the plasma membrane is a cAMP-dependent process and thus a paradigm for cAMP-controlled exocytic processes. Using primary cultured rat inner medullary collecting duct cells, we show that the redistribution of AQP2 to the plasma membrane is accompanied by the reorganization of microtubules and the redistribution of the small GTPase Rab11. In resting cells, AQP2 is colocalized with Rab11 perinuclearly. AVP induced the redistribution of AQP2 to the plasma membrane and of Rab11 to the cell periphery. The redistribution of both proteins was increased when microtubules were depolymerized by nocodazole. In addition, the depolymerization of microtubules prevented the perinuclear positioning of AQP2 and Rab11 in resting cells, which was restored if nocodazole was washed out and microtubules repolymerized. After internalization of AQP2, induced by removal of AVP, forskolin triggered the AQP2 redistribution to the plasma membrane even if microtubules were depolymerized and without the previous positioning of AQP2 in the perinuclear recycling compartment. Collectively, the data indicate that microtubule-dependent transport of AQP2 is predominantly responsible for trafficking and localization of AQP2 inside the cell after its internalization but not for the exocytic transport of the water channel. We also demonstrate that cAMP-signaling regulates the localization of Rab11-positive recycling endosomes in renal principal cells.

• A Role of myosin Vb and Rab11-FIP2 in the aquaporin-2 shuttle.

  Authors: Pavel I Nedvetsky, Eduard Stefan, Sebastian Frische, Katja Santamaria, Burkhard Wiesner, Giovanna Valenti, John A Hammer, Søren Nielsen, James R Goldenring, Walter Rosenthal, Enno Klussmann

  Traffic (Copenhagen, Denmark). 03/2007; 8(2):110-23.

  Arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells. Its binding to Gs-coupled vasopressin V2 receptors increases cyclic AMP (cAMP) and subsequentlyArginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells. Its binding to Gs-coupled vasopressin V2 receptors increases cyclic AMP (cAMP) and subsequently elicits the redistribution of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the plasma membrane (AQP2 shuttle), thereby facilitating water reabsorption from primary urine. The AQP2 shuttle is a paradigm for cAMP-dependent exocytic processes. Using sections of rat kidney, the AQP2-expressing cell line CD8, and primary principal cells, we studied the role of the motor protein myosin Vb, its vesicular receptor Rab11, and the myosin Vb- and Rab11-binding protein Rab11-FIP2 in the AQP2 shuttle. Myosin Vb colocalized with AQP2 intracellularly in resting and at the plasma membrane in AVP-treated cells. Rab11 was found on AQP2-bearing vesicles. A dominant-negative myosin Vb tail construct and Rab11-FIP2 lacking the C2 domain (Rab11-FIP2-DeltaC2), which disrupt recycling, caused condensation of AQP2 in a Rab11-positive compartment and abolished the AQP2 shuttle. This effect was dependent on binding of myosin Vb tail and Rab11-FIP2-DeltaC2 to Rab11. In summary, we identified myosin Vb as a motor protein involved in AQP2 recycling and show that myosin Vb- and Rab11-FIP2-dependent recycling of AQP2 is an integral part of the AQP2 shuttle.

• Targeting the heme-oxidized nitric oxide receptor for selective vasodilatation of diseased blood vessels.

  Authors: Johannes-Peter Stasch, Peter M Schmidt, Pavel I Nedvetsky, Tatiana Y Nedvetskaya, Arun Kumar H S, Sabine Meurer, Martin Deile, Ashraf Taye, Andreas Knorr, Harald Lapp, Helmut Müller, Yagmur Turgay, Christiane Rothkegel, Adrian Tersteegen, Barbara Kemp-Harper, Werner Müller-Esterl, Harald H H W Schmidt

  The Journal of clinical investigation. 10/2006; 116(9):2552-61.

  ROS are a risk factor of several cardiovascular disorders and interfere with NO/soluble guanylyl cyclase/cyclic GMP (NO/sGC/cGMP) signaling through scavenging of NO and formation of the strongROS are a risk factor of several cardiovascular disorders and interfere with NO/soluble guanylyl cyclase/cyclic GMP (NO/sGC/cGMP) signaling through scavenging of NO and formation of the strong oxidant peroxynitrite. Increased oxidative stress affects the heme-containing NO receptor sGC by both decreasing its expression levels and impairing NO-induced activation, making vasodilator therapy with NO donors less effective. Here we show in vivo that oxidative stress and related vascular disease states, including human diabetes mellitus, led to an sGC that was indistinguishable from the in vitro oxidized/heme-free enzyme. This sGC variant represents what we believe to be a novel cGMP signaling entity that is unresponsive to NO and prone to degradation. Whereas high-affinity ligands for the unoccupied heme pocket of sGC such as zinc-protoporphyrin IX and the novel NO-independent sGC activator 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino) methyl [benzoic]acid (BAY 58-2667) stabilized the enzyme, only the latter activated the NO-insensitive sGC variant. Importantly, in isolated cells, in blood vessels, and in vivo, BAY 58-2667 was more effective and potentiated under pathophysiological and oxidative stress conditions. This therapeutic principle preferentially dilates diseased versus normal blood vessels and may have far-reaching implications for the currently investigated clinical use of BAY 58-2667 as a unique diagnostic tool and highly innovative vascular therapy.

  Download

• Effects of chronic endothelin ET(A) receptor blockade on blood pressure and vascular formation of cyclic guanosine-3',5'-monophosphate in spontaneously hypertensive rats.

  Authors: Michael Kirchengast, Klaus Witte, Kerstin Stolpe, Lothar Schilling, Pavel I Nedvetsky, Harald H H W Schmidt, Björn Lemmer

  Arzneimittel-Forschung. 02/2005; 55(9):498-504.

  Endothelin (ET) mediates vasoconstriction in intact arterial blood vessels with functional endothelium via stimulation of ET(A) receptors, while ET(B) receptor stimulation leads to vasodilation viaEndothelin (ET) mediates vasoconstriction in intact arterial blood vessels with functional endothelium via stimulation of ET(A) receptors, while ET(B) receptor stimulation leads to vasodilation via nitric oxide (NO) release and formation of cyclic guanosine-3',5'-monophosphate (cGMP). In spontaneously hypertensive rats (SHR) the cGMP-forming NO-receptor guanylyl cyclase (sGC) is downregulated. It is unclear whether ET contributes to the hypertensive phenotype of SHR, and whether this involves the disturbed cGMP signaling. The selective ETA receptor antagonist darusentan (CAS 171714-84-4), given orally via drinking water (10 mg kg(-1) d(-1)) for 12 weeks, significantly lowered systolic blood pressure of SHR as determined by radiotelemetry. Neither impaired endothelium-dependent relaxation to acetylcholine was restored nor sGC expression and activity affected when compared to control SHR. While these findings show a role for ETA receptors in blood pressure regulation in genetically elevated blood pressure, downregulation of sGC expression and cGMP-mediated vasorelaxant response in SHR were shown to be independent of ETA receptors. The findings suggest distinct mechanisms of gene expression affecting ET and cGMP mediated vasomotor functions.

• Distribution of soluble guanylyl cyclase in the rat brain.

  Authors: Jin-Dong Ding, Alain Burette, Pavel I Nedvetsky, Harald H H W Schmidt, Richard J Weinberg

  The Journal of comparative neurology. 06/2004; 472(4):437-48.

  The diffusible messenger nitric oxide (NO) acts in the brain largely through activation of soluble guanylyl cyclase (sGC), a heterodimer comprising alpha and beta subunits. We usedThe diffusible messenger nitric oxide (NO) acts in the brain largely through activation of soluble guanylyl cyclase (sGC), a heterodimer comprising alpha and beta subunits. We used immunohistochemistry to study the distribution of both sGC subunits in the brain of adult rats. alpha and beta subunits gave similar widespread staining throughout the CNS, which was strongest in neostriatum, olfactory tubercle, and supraoptic nucleus. Double-labeling experiments showed striking cellular colocalization in most brain regions, suggesting that the two subunits may be organized into enzymatically active alpha/beta heteromers. Mismatches were observed in cerebellar cortex: Purkinje cells and Bergmann glia were positive for both subunits, whereas granule cells and interneurons in the molecular layer were strongly immunopositive for beta but only weakly stained for the alpha subunit. By using multiple labeling, we compared the localization of sGC with neuronal nitric oxide synthase (NOS-I, the NO-producing enzyme in neurons). In forebrain, the distribution of sGC and NOS-I was complementary, with only occasional colocalization. In contrast, cellular colocalization was common in midbrain and cerebellum. These data support a widespread role for the NO/sGC/cGMP pathway in the CNS and suggest that, in addition to its role as paracrine messenger, NO may also be an intracellular autocrine agent.

• There's NO binding like NOS binding: protein-protein interactions in NO/cGMP signaling.

  Authors: Pavel I Nedvetsky, William C Sessa, Harald H H W Schmidt

  Proceedings of the National Academy of Sciences of the United States of America. 01/2003; 99(26):16510-2.

• Regional distribution of protein and activity of the nitric oxide receptor, soluble guanylyl cyclase, in rat brain suggests multiple mechanisms of regulation.

  Authors: Pavel I Nedvetsky, Christoph Kleinschnitz, Harald H H W Schmidt

  Brain research. 10/2002; 950(1-2):148-54.

  Nitric oxide (NO) is an unconventional neuromodulator that signals by intercellular diffusion. Its effects are often mediated by activation of its cytosolic receptor, the hemoprotein soluble guanylylNitric oxide (NO) is an unconventional neuromodulator that signals by intercellular diffusion. Its effects are often mediated by activation of its cytosolic receptor, the hemoprotein soluble guanylyl cyclase (sGC). Regional distribution of heterodimeric (alpha/beta) sGC at both the activity and protein level and its regulation are still unclear. Here, sGC was analyzed in rat brain by Western blot and NO donor-stimulated cyclic GMP accumulation. sGCalpha(1) and sGCbeta(1) immunoreactive protein signals strongly correlated with each other. However, V(max) values depended on the type of NO donor used. Sodium nitroprusside, the most widely used compound and formally an NO(+) donor, was up to 20-fold less effective in stimulating sGC activity than the NO donor diethylamine NONOate. In contrast to the rather even distribution of sGC proteins and SNP-stimulated cGMP accumulation in various regions of rat brain, diethylamine NONOate-stimulated sGC activity varied up to 8-fold between the different brain regions tested. In conclusion, we show that expression of both sGCalpha(1) and sGCbeta(1) subunits is tightly coregulated in rat brain, while yet unknown additional mechanisms affect the V(max) of sGC.

• Regional distribution of protein and activity of the nitric oxide receptor, soluble guanylyl cyclase, in rat brain suggests multiple mechanisms of regulation

  Authors: Pavel I Nedvetsky, Christoph Kleinschnitz, Harald H.H.W Schmidt

  Brain Research.

  Nitric oxide (NO) is an unconventional neuromodulator that signals by intercellular diffusion. Its effects are often mediated by activation of its cytosolic receptor, the hemoprotein soluble guanylylNitric oxide (NO) is an unconventional neuromodulator that signals by intercellular diffusion. Its effects are often mediated by activation of its cytosolic receptor, the hemoprotein soluble guanylyl cyclase (sGC). Regional distribution of heterodimeric (α/β) sGC at both the activity and protein level and its regulation are still unclear. Here, sGC was analyzed in rat brain by Western blot and NO donor-stimulated cyclic GMP accumulation. sGCα1 and sGCβ1 immunoreactive protein signals strongly correlated with each other. However, Vmax values depended on the type of NO donor used. Sodium nitroprusside, the most widely used compound and formally an NO+ donor, was up to 20-fold less effective in stimulating sGC activity than the NO donor diethylamine NONOate. In contrast to the rather even distribution of sGC proteins and SNP-stimulated cGMP accumulation in various regions of rat brain, diethylamine NONOate-stimulated sGC activity varied up to 8-fold between the different brain regions tested. In conclusion, we show that expression of both sGCα1 and sGCβ1 subunits is tightly coregulated in rat brain, while yet unknown additional mechanisms affect the Vmax of sGC.

• Regulation of the nitric oxide receptor, soluble guanylyl cyclase

  Authors: Pavel I. Nedvetsky

  Soluble guanylyl cyclase (sGC) is the best established receptor for nitric oxide (NO) and regulates a great number of important physiological functions. Surprisingly, despite the wellappreciatedSoluble guanylyl cyclase (sGC) is the best established receptor for nitric oxide (NO) and regulates a great number of important physiological functions. Surprisingly, despite the wellappreciated roles of this enzyme in regulation of vascular tone, smooth muscle cell proliferation, platelet aggregation, renal sodium secretion, synaptic plasticity, and other functions, extremely little is known about the regulation of sGC activity and protein levels. To date, the only well-proven physiologically relevant sGC regulator is NO. In the present study, some additional possibilities for sGC regulation were shown. Firstly, we evaluated the ability of different NO donors to stimulate sGC. Significant differences in the sGC stimulation by SNP and DEA/NO were found. DEA/NO stimulated sGC much stronger than did SNP. Interestingly, no correlation between the sGC protein and maximal activity distribution was found in rat brain regions tested, suggesting the existence of some additional regulatory mechanisms for sGC. The failure of SNP to stimulate sGC maximally might be one of the reasons why the lack of correlation between the distribution of sGC activity and proteins in brain was not detected earlier. Prolonged exposure of endothelial cells to NO donors produced desensitization of the cGMP response. This desensitization cannot be explained by increased PDE activity, since PDE inhibitors were not able to prevent the NO donor-induced decrease of the maximal cGMP response in endothelial cells. The failure of SH-reducing agents to improve the cGMP response after its desensitization by NO suggests that a SH-independent mechanism mediates NO effects. Demonstration that the potency of the recently described activator of oxidized (heme-free) sGC, BAY58-2667, to stimulate sGC increases after prolonged exposure of the cells to an NO donor, DETA/NO, suggests that oxidation of heme may be a reason for NOinduced desensitization of sGC and decrease in sGC protein level. Indeed, the well-known heme-oxidizing agent ODQ produces a dramatic decrease in sGC protein levels in endothelial cells and BAY58-2667 prevents this effect. Although the mechanism of sGC activation and stabilization by BAY58-2667 is unknown, this substance is an interesting candidate to modulate sGC under conditions where sGC heme iron is oxidized. Very little is known about regulation of sGC by intracellular localization or translocation between different intracellular compartments. In the present study, an increase in sGC sensitivity to NO under membrane association was demonstrated. Treatment of isolated lung with VEGF markedly increased sGC in membrane fractions of endothelial cells. Failure of VEGF to stimulate sGC membrane association in cultured endothelial cells allows us to propose a complex mechanism of regulation of sGC membrane association and/or a transient character of sGC membrane attachment. A very likely mechanism for the attachment of sGC to membranes is via sGCinteracting proteins. These proteins may participate also in other aspects of sGC regulation. The role of the recently described sGC interaction partner, Hsp90, was investigated. Shortterm treatment of endothelial cells with an Hsp90 inhibitor does not affect NO donor or calcium ionophore-stimulated cGMP accumulation in the cells. However, inhibition of Hsp90 results in a rapid and dramatic decrease in sGC protein levels in endothelial cells. These effects were unrelated to changes in sGC transcription, since inhibition of transcription had much slower effect on sGC protein levels. In contrast, inhibitors of proteasomes abolished the reduction in sGC protein levels produced by an Hsp90 inhibitor, suggesting involvement of proteolytic degradation of sGC proteins during inhibition of Hsp90. All these data together suggest that Hsp90 is required to maintain mature sGC proteins. In conclusion, in the present study it was demonstrated that multiple mechanisms are involved in the regulation of sGC activity and its sensitivity to NO. Oxidation of sGC heme by NO seems to be one of the mechanisms for negative regulation of sGC in the presence of high or prolonged stimulation with NO. Another possible means of regulating sGC sensitivity to NO is via the intracellular translocation of the enzyme. It has been also demonstrated here that attachment of sGC to the membrane fraction results in an apparent increase in the enzyme sensitivity to NO. Additionally, Hsp90 was required to maintain sGC protein in endothelial and other cell types. However, we could not find any acute affect of Hsp90 on sGC activity, as reported recently. All these findings demonstrate that the regulation of sGC activity and protein level is a much more complex process than had been assumed earlier. Lösliche Guanylylcyclase (sGC) ist der Hauptrezeptor für Stickstoffmonooxid (NO), der sich an der Regulation zahlreicher physiologischer Funktionen beteiligt. Trotz ihrer sehr gut untersuchten Rolle in der Regulation der Blutgefässenrelaxation, synaptische Plastizität, Aggregation der Trombozyten, renale Sekretion und anderen wichtigen Funktionen, ist die Regulation der sGC selber noch nicht ausreichend verstanden. Der einzige, zur Zeit bekannte, physiologische Regulator der sGC ist NO. In der vorgelegten Arbeit wurde die Existenz anderer Möglichkeiten der sGC Regulation gezeigt. Zuerst, wurde die Fähigkeit verschiedener NO Donoren sGC zu stimulieren untersucht. DEA/NO stimulierte sGC viel stärker als SNP. Interessanterweise, wurde keine Korrelation zwischen der Verteilung des sGC Proteins und der Enzymaktivität unter Vmax- Bedingungen in verschiedenen Rattenhirnregionen gefunden. Das deutet auf zusätzliche Regulationsmechanismen hin. Die fehlende Fähigkeit von SNP sGC maximal zu stimulieren könnte ein Grund dafür sein, warum dieses Phänomen nicht schon früher gezeigt wurde. Langfristige Behandlung von Endothelzellen mit NO Donoren produzierte eine Desensitisierung der nachfolgenden cGMP Antwort. Diese Desensitisierung kann nicht durch erhöhte Phosphodiesterase-Aktivität erklärt werden, da Phosphodiesterasenhemmer die durch NO Donor verursachte Abnahme der cGMP Antwort nicht rückgängig macht. SHreduzierende Substanzen waren nicht in der Lage die cGMP Antwort zu verbessern, was zur Annahme führt, dass SH-Gruppenoxidation keine wichtige Rolle bei der Wirkung von NO auf sGC spielt. Es müssen daher andere Regulationsmechanismen vorhanden sein. Oxidation des Häms scheint ein möglicher Mechanismus der NO-induzierten sGC Desensitisierung. Einkürzlich beschriebener Aktivator der oxidierten (bzw. Häm-freien) sGC, BAY58-2667, stimulierte sGC nach Vorbehandlung mit NO Donoreb stärker als ohne Vorbehandlung. Es wird vermutet, dass oxidierte sGC verstärkt abgebaut wird was die durch NO oder Häm oxidierende Substanzen induzierte sGC Proteinabnahme erklären würde. Tatsächlich, nahm sGC Proteinlevel nach der Behandlung mit der Häm oxidierenden Substanz, ODQ, ab. BAY58-2667 verhinderte diesen Effekt. Ferner erhöht die Membranassoziation von sGC derer Empfindlichkeit gegenüber NO. Die Membranassoziation der sGC in Endothelzellen ist reguliert. Behandlung isolierter Lunge mit VEGF erhöht den Anteil an membrangebundener sGC in Endothelzellen dramatisch. In kultivierten Endothelzellen könnte VEGF die Membranassoziation jedoch nicht stimulieren, was einen komplexen Mechanismus der Membranassoziation der sGC in vivo vermuten lässt. Wenig ist bekannt über die Interaktionen von sGC mit anderen Protein und der möglichen Rolle dieser Interaktionen bei der Regulation des Enzyms. Proteininteraktionen scheinen aber ein möglicher Mechanismus für die Membranassoziation der sGC zu sein. Aus diesem Grund wurde die Rolle eines vor kurzem beschriebenen sGC-bindenden Proteins, Hsp90, auf die sGC Regulation untersucht. Kurzfristige Behandlung der Endothelzellen mit Hsp90 Inhibitoren hat keine Auswirkung auf NO Donor- und Calciumionophore-stimulierte cGMP-Produktion. Langfristige Hemmung von Hsp90 führte dagegen zur schnellen und deutlichen Abnahme des sGC Proteins. Dieser Effekt ist nicht durch eine Veränderung der Translation zu erklären, weil Tranlationshemmer einen viel langsameren sGC Abfall verursachten. Im Gegenteil, konnte ein Proteasomeninhibitor, MG132, die Effekte von Hsp90 Hemmern rückgängig machen. Das lässt eine proteolytische Abbau der sGC für die Effekte von Hsp90 Hemmer verantwortlich machen. Diese Daten deuten darauf hin, dass Hsp90 für Aufrechterhaltung des Enzyms notwendig ist. Zusammenfassend, wurde in der vorliegenden Arbeit gezeigt, dass sGC Aktivität und ihre Empfindlichkeit gegenüber ihren Aktivator NO durch multiple Faktoren beeinflusst werden kann. Oxidation des Häms durch NO könnte ein Mechanismus der negativen Regulation der sGC bei dauernd erhöhter Konzentration von NO sein. Ein zusätzlicher Mechanismus der Regulation der Empfindlichkeit der sGC gegenüber NO scheint die intrazellulare Translokation zu sein. Wir konnten hier zeigen, das die Membranassoziation der sGC ihre Empfindlichkeit gegenüber NO erhöht. Auch dieProteinlevel der sGC scheinen unter Kontrolle verschiedener Faktoren zu sein. Einer davon ist Hsp90, der für die Aufrechterhaltung des sGC Proteins sowohl in Endothelzellen als auch in anderen Zelltypen notwendig ist. Alle diese Daten zeigen, dass Regulation der sGC ein viel komplexerer Vorgang ist als bis her angenommen wurde und eröffnen interessante neue Forschungsrichtungen innerhalb dieses wichtigen Signalweges.