cGMP-dependent kinase I (cGKI) is a major mediator of smooth muscle relaxation and exists in two isoforms, α and β. Both isoforms are supposed to mediate their effects via different intracellular signaling pathways. To verify this concept, the kinetics of relaxation mediated by either isoform was analyzed in gastric fundus smooth muscle from mice. Muscles from mice that express selectively the Iα or Iβ isoform of cGKI in smooth muscle (sm-cGKIα or sm-cGKIβ mice) were compared to muscles from conventional cGKI−/− mice. Fundus muscles were contracted by carbachol and then relaxed by 8-Br-cGMP or by electrical field stimulation (EFS). The time course of relaxation by 8-Br-cGMP was not different between muscles from sm-cGKIα and sm-cGKIβ mice. EFS induced a fast transient relaxation in muscles from sm-cGKIα and sm-cGKIβ mice that was blocked by the NO synthase inhibitor L-NAME. Recovery from this relaxation was about 4-times slower in muscles from sm-cGKIα mice than in muscles from sm-cGKIβ mice. The different kinetic of recovery from relaxation after EFS in sm-cGKIα and sm-cGKIβ mice suggests that different signaling pathways exist for each cGKI isoform in vivo in fundus muscles.

Nitric oxide (NO) and atrial natriuretic peptide (ANP) signalling via cGMP controls smooth muscle tone. One important signalling pathway of cGMP-dependent protein kinase type I (cGKI) is mediated by IRAG (IP(3) receptor associated cGKI substrate) which is highly expressed in smooth muscle tissues. To elucidate the role of IRAG for NO- and ANP-mediated smooth muscle tone regulation, cGKI localization, and for its possible function in blood pressure adjustment, we generated IRAG-knockout mice by targeted deletion of exon 3.IRAG deletion prevented stable interaction of IP(3) receptor type I (IP(3)RI) with cGKI beta determined by cGMP affinity chromatography. Confocal microscopy in vascular smooth muscle cells (VSMCs) showed that localization of cGKI beta and cGKI alpha did not change in absence of IRAG. NO-, ANP-, and cGMP-dependent relaxation of hormone-contracted aortic vessels and colon was significantly affected in IRAG-knockout mice. The suppression of cGMP-induced relaxation was not rescued by selective expression of cGKI beta in smooth muscle from cGKI beta-transgenic mice. NO-, ANP-, and cGMP-mediated inhibition of the hormone-induced increase in intracellular calcium concentration measured by Fura2 was suppressed in IRAG-deficient VSMC. Telemetric measurements revealed that IRAG-deficient animals exhibited normal basal tone, but were resistant to blood pressure reduction induced by lipopolysaccharide-treatment.These findings indicate that signalling of cGKI beta via IRAG is an essential functional part for regulation of smooth muscle tone and of intracellular calcium by NO (exogenously applicated or endogenously synthesized) and by ANP. IRAG signalling does not modulate basal tone but might be important for blood pressure regulation under pathophysiological conditions.

OBJECTIVES:To investigate the expression of isoforms of the cyclic AMP (cAMP)-dependent protein kinase (cAK) in the transition zone of the human prostate and the functional significance of the enzyme in the control of prostate smooth muscle. METHODS:Using Western blot analysis and immunohistochemistry, the expression and distribution in the prostate of cAKIalpha, cAKIbeta, cAKIIalpha, and cAKIIbeta in relation to alpha-actin and the phosphodiesterase PDE4 (types A and B) were investigated. The effects of the cAK inhibitor Rp-8-CPT-cAMPS on the reversion of the adrenergic tension of isolated prostate tissue induced by forskolin, rolipram, sodium nitroprusside (SNP), and tadalafil were examined by means of the organ bath technique. RESULTS:Immunosignals specific for cAKIalpha, cAKIIalpha, and cAKIIbeta were observed in the smooth musculature and glandular structures of the prostate. Double stainings revealed the colocalization of alpha-actin and PDE4 with the cAK isoforms. The expression of the cAK isoforms was confirmed by Western blot analysis. The relaxation of the tension induced by norepinephrine brought about by forskolin, rolipram, SNP, and tadalafil was significantly attenuated by Rp-8-CPT-cAMPS. CONCLUSIONS:The colocalization of smooth muscle alpha-actin and PDE4 with cAK, as well as the results from the organ bath experiments, provide further evidence for a pivotal role of the cAMP-dependent signaling in the regulation of prostate smooth muscle contractility. Compounds interacting with the cAMP/cAK pathway might represent a new therapeutic avenue to treat symptoms of benign prostatic hyperplasia and lower urinary tract symptomatology.

INTRODUCTION:The cyclic adenosine monophosphate-dependent protein kinase (cAK) is considered a key protein in the control of smooth muscle tone in the cardiovascular system. There is evidence that erectile dysfunction might be linked to systemic vascular disorders and arterial insufficiency, subsequently resulting in structural changes in the penile tissue. The expression and significance of cAK in human cavernous arteries (HCA) have not been evaluated.AIMS:To evaluate the expression of cAK isoforms in HCA and examine the role of cAK in the cyclic adenosine monophosphate (cAMP)- and cyclic guanosine monophosphate (cGMP)-mediated control of penile vascular smooth muscle.METHODS:The expression and distribution of phosphodiesterase type 4 (PDE4) and cAK isoforms in sections of HCA were investigated by means of immunohistochemistry and Western blot analysis. The effects of the cAK inhibitor Rp-8-CPT-cAMPS on the relaxation of isolated preparations of HCA (diameter > 100 µm) induced by rolipram, sildenafil, tadalafil, and vardenafil were studied using the organ bath technique.MAIN OUTCOME MEASURES:Investigate the expression of cAK in relation to α-actin and PDE4 in HCA and evaluate the effects of an inhibition of cAK on the relaxation induced by inhibitors of PDE4 and PDE5 of isolated penile arteries.RESULTS:Immunosignals specific for cAKIα, IIα, and IIβ were observed within the wall of HCA. Double stainings revealed colocalization of cAK with α-actin and PDE4. The expression of cAK isoforms was confirmed by Western blot analysis. The reversion of tension induced by inhibitors of PDE4 and PDE5 of isolated penile vascular tissue were attenuated significantly by Rp-8-CPT-cAMPS.CONCLUSIONS:Our results demonstrate the expression of cAK isoforms in the smooth musculature of HCA and its colocalization with PDE4. A significant role for cAK in the regulation mediated by cAMP and cGMP of vascular smooth muscle tone in HCA can also be assumed.

Klinische Studien zeigten positive Effekte der Phosphodiesterase- (PDE-)5-Inhibitoren Sildenafil, Tadalafil und Vardenafil auf die irritative Symptomatik des benigen Prostatasyndroms. Unklar ist bisher, ob diese Substanzen auf die glatte Muskulatur in der Transitionalzone der Prostata und/oder auf den Detrusormuskel wirken. Das Ziel der Studie war die Darstellung der Expression der PDE-Isoenzyme 1, 4 und 5 in der Detrusormuskulatur und die Untersuchung der Effekte selektiver PDE-Inhibitoren auf die muskarinerge Tension isolierter glatter Muskulatur des humanen Detrusors.

Objectives. The use of inhibitors of phosphodiesterase (PDE) isoenzymes 1 and 5 to treat overactive bladder has been suggested. To further evaluate the significance of PDE isoenzymes in detrusor smooth muscle relaxation, we investigated the effects of selective PDE inhibitors on the tension induced by carbachol of isolated human detrusor tissue. Using immunohistochemical methods, the expression of PDE1, PDE4, and PDE5 in human detrusor was also investigated. Material and Methods. The expression of PDE1, PDE4, and PDE5 was evaluated by means of conventional immunohistochemistry (IHC). Using the organ bath technique, the effects of the PDE inhibitors vinpocetine, rolipram, sildenafil, tadalafil, and vardenafil on the tension induced by the muscarinic agonist carbachol (1 mu M) were investigated. Results. The tension induced by carbachol was dose-dependently reversed by the PDE inhibitors; the maximum reversal of tension ranged from 7% (tadalafil) to 34% (vardenafil). IHC revealed that the expression of PDE isoenzymes was limited to the smooth musculature of the detrusor. While there was prominent expression of PDE4 and PDE5, immunoreactions indicating the presence of PDE1 were less abundant. Conclusion. Despite the fact that inhibitors of PDE1, PDE4, and PDE5 exerted only a weak relaxant response on detrusor strips precontracted by carbachol, our findings indicate that both the cAMP and cGMP pathways might be involved in the relaxation mechanism of human detrusor smooth muscle.

Background: A role for the transient receptor potential (TRP) A1 ion channel in rat lower urinary tract (LUT) sensation and disease has been proposed, but in the human LUT no information on TRPA1 activity is available.Objectives: To investigate the distribution of TRPA1 in the human urethra and to study the effect of TRPA1 agonists on isolated urethral strip preparations.Design, settings, and participants: Urethral specimens were obtained preoperatively from 10 patients and were freshly prepared for Western blot, immunohistochemistry, and functional in vitro investigations.Measurements: The expression patterns of TRPA1 were studied with Western blot and immunohistochemistry. The effects of allyl isothiocyanate (A1), cinnamaldehyde (CA), and NaHS (donor of H2S) on tension of urethral strips were investigated in tissue baths.Results and limitations: TRPA1 immunoreactivity (-IR) was found in nerve fibres in the suburothelial space and was also located to nerve fibres of the muscle layer. Single TRPA1-IR nerves extended into the urothelium. A majority, but not all TRPA1-IR nerves also expressed immunoreactivity for CGRP or TRPV1. In the urothelium, TRPV1 was located to the outer layers whereas TRPA1 was observed in basal urothelial cells. Interspersed between strands of smooth muscle cells of the urethral wall, TRPA1- and vimentin-IR cells containing central nuclei and slender cytoplasmatic extensions were observed. In functional experiments, TRPA1-agonists had no contractile effect in urethral preparations. After precontraction with phenylephrine, AI, CA, and NaHS caused concentration-dependent relaxations of urethral strip preparations.Conclusions: The localization of TRPA1 to nerves that also express TRPV1 and CGRP, and in urothelial cells and interstitial cells, as well as the findings that TRPA1 agonists can modify tone of urethral preparations, propose a role for TRPA1 in afferent and efferent sensory signaling of the human outflow region. (C) 2008 European Association of Urology. Published by Elsevier B.V. All rights reserved

You have accessJournal of Urology1 Apr 2008EXPRESSION OF PHOSPHODIESTERASE (PDE) ISOENZYMES IN THE HUMAN SEMINAL VESICLE (SV) AND EFFECTS OF PDE INHIBITORS ON THE CONTRACTILE RESPONSES OF ISOLATED HUMAN SEMINAL VESICLE SMOOTH MUSCLE Stefan Uckert, Joachim E Sonnenberg, Katja Sigl, Margit E Mayer, Udo Jonas, Christian G Stief, and Markus A Kuczyk Stefan UckertStefan Uckert More articles by this author , Joachim E SonnenbergJoachim E Sonnenberg More articles by this author , Katja SiglKatja Sigl More articles by this author , Margit E MayerMargit E Mayer More articles by this author , Udo JonasUdo Jonas More articles by this author , Christian G StiefChristian G Stief More articles by this author , and Markus A KuczykMarkus A Kuczyk More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(08)60678-3AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "EXPRESSION OF PHOSPHODIESTERASE (PDE) ISOENZYMES IN THE HUMAN SEMINAL VESICLE (SV) AND EFFECTS OF PDE INHIBITORS ON THE CONTRACTILE RESPONSES OF ISOLATED HUMAN SEMINAL VESICLE SMOOTH MUSCLE." The Journal of Urology, 179(4S), p. 235 © 2008 by American Urological AssociationFiguresReferencesRelatedDetails Volume 179Issue 4SApril 2008Page: 235 Advertisement Copyright & Permissions© 2008 by American Urological AssociationMetricsAuthor Information Stefan Uckert More articles by this author Joachim E Sonnenberg More articles by this author Katja Sigl More articles by this author Margit E Mayer More articles by this author Udo Jonas More articles by this author Christian G Stief More articles by this author Markus A Kuczyk More articles by this author Expand All Advertisement PDF downloadLoading ...

You have accessJournal of Urology1 Apr 2008FUNCTIONAL SIGNIFICANCE OF cAMP-DEPENDENT PROTEIN KINASE A IN THE REGULATION OF SMOOTH MUSCLE TONE IN THE HUMAN PROSTATE Katja Sigl, Stefan Uckert, Udo Jonas, Christian G Stief, Petter Hedlund, and Eginhard S Waldkirch Katja SiglKatja Sigl More articles by this author , Stefan UckertStefan Uckert More articles by this author , Udo JonasUdo Jonas More articles by this author , Christian G StiefChristian G Stief More articles by this author , Petter HedlundPetter Hedlund More articles by this author , and Eginhard S WaldkirchEginhard S Waldkirch More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(08)61325-7AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "FUNCTIONAL SIGNIFICANCE OF cAMP-DEPENDENT PROTEIN KINASE A IN THE REGULATION OF SMOOTH MUSCLE TONE IN THE HUMAN PROSTATE." The Journal of Urology, 179(4S), p. 452 © 2008 by American Urological AssociationFiguresReferencesRelatedDetails Volume 179Issue 4SApril 2008Page: 452 Advertisement Copyright & Permissions© 2008 by American Urological AssociationMetricsAuthor Information Katja Sigl More articles by this author Stefan Uckert More articles by this author Udo Jonas More articles by this author Christian G Stief More articles by this author Petter Hedlund More articles by this author Eginhard S Waldkirch More articles by this author Expand All Advertisement PDF downloadLoading ...

Signaling by NO/cGMP relaxes various smooth muscles, modulating thereby vascular tone and gastrointestinal motility. An important signaling pathway of cGMPdependent protein kinase type I (cGKI)-dependent relaxation is mediated by IRAG (IP3 Receptor Associated cGKI substrate) which is highly expressed in smooth muscle tissues. In order to elucidate the physiological role of IRAG we generated IRAG knockout mice by targeted deletion of exon 3.

Signalling by cGMP-dependent protein kinase type I (cGKI) relaxes various smooth muscles modulating thereby vascular tone and gastrointestinal motility. cGKI-dependent relaxation is possibly mediated by phosphorylation of the inositol 1,4,5-trisphosphate receptor I (IP(3)RI)-associated protein (IRAG), which decreases hormone-induced IP(3)-dependent Ca(2+) release. We show now that the targeted deletion of exon 12 of IRAG coding for the N-terminus of the coiled-coil domain disrupted in vivo the IRAG-IP(3)RI interaction and resulted in hypomorphic IRAG(Delta12/Delta12) mice. These mice had a dilated gastrointestinal tract and a disturbed gastrointestinal motility. Carbachol- and phenylephrine-contracted smooth muscle strips from colon and aorta, respectively, of IRAG(Delta12/Delta12) mice were not relaxed by cGMP, while cAMP-mediated relaxation was unperturbed. Norepinephrine-induced increases in [Ca(2+)](i) were not decreased by cGMP in aortic smooth muscle cells from IRAG(Delta12/Delta12) mice. In contrast, cGMP-induced relaxation of potassium-induced smooth muscle contraction was not abolished in IRAG(Delta12/Delta12) mice. We conclude that cGMP-dependent relaxation of hormone receptor-triggered smooth muscle contraction essentially depends on the interaction of cGKI-IRAG with IP(3)RI.

ABSTRACT Given that a large proportion of the bacteria colonizing the roots of plants is capable of producing N -acyl- l -homoserine lactone (AHL) molecules, it appears likely that these bacterial pheromones may serve as signals for communication between cells of different species. In this study, we have developed and characterized novel Gfp-based monitor strains that allow in situ visualization of AHL-mediated communication between individual cells in the plant rhizosphere. For this purpose, three Gfp-based AHL sensor plasmids that respond to different spectra of AHL molecules were transferred into AHL-negative derivatives of Pseudomonas putida IsoF and Serratia liquefaciens MG1, two strains that are capable of colonizing tomato roots. These AHL monitor strains were used to visualize communication between defined bacterial populations in the rhizosphere of axenically grown tomato plants. Furthermore, we integrated into the chromosome of AHL-negative P . putida strain F117 an AHL sensor cassette that responds to the presence of long-chain AHLs with the expression of Gfp. This monitor strain was used to demonstrate that the indigenous bacterial community colonizing the roots of tomato plants growing in nonsterile soil produces AHL molecules. The results strongly support the view that AHL signal molecules serve as a universal language for communication between the different bacterial populations of the rhizosphere consortium.