NO-sensitive guanylyl cyclase discriminates pericyte-derived interstitial from intra-alveolar myofibroblasts in murine pulmonary fibrosis.

BACKGROUND: The origin of αSMA-positive myofibroblasts, key players within organ fibrosis, is still not fully elucidated. Pericytes have been discussed as myofibroblast progenitors in several organs including the lung. METHODS: Using tamoxifen-inducible PDGFRß-tdTomato mice (PDGFRß-CreERT2; R26tdTomato) lineage of lung pericytes was traced. To induce lung fibrosis, a single orotracheal dose of bleomycin was given. Lung tissue was investigated by immunofluorescence analyses, hydroxyproline collagen assay and RT-qPCR. RESULTS: Lineage tracing combined with immunofluorescence for nitric oxide-sensitive guanylyl cyclase (NO-GC) as marker for PDGFRß-positive pericytes allows differentiating two types of αSMA-expressing myofibroblasts in murine pulmonary fibrosis: (1) interstitial myofibroblasts that localize in the alveolar wall, derive from PDGFRß+ pericytes, express NO-GC and produce collagen 1. (2) intra-alveolar myofibroblasts which do not derive from pericytes (but express PDGFRß de novo after injury), are negative for NO-GC, have a large multipolar shape and appear to spread over several alveoli within the injured areas. Moreover, NO-GC expression is reduced during fibrosis, i.e., after pericyte-to-myofibroblast transition. CONCLUSION: In summary, αSMA/PDGFRß-positive myofibroblasts should not be addressed as a homogeneous target cell type within pulmonary fibrosis.

Gastrointestinal dysfunction in autism displayed by altered motility and achalasia in Foxp1+/- mice.

Gastrointestinal dysfunctions in individuals with autism spectrum disorder are poorly understood, although they are common among this group of patients. FOXP1 haploinsufficiency is characterized by autistic behavior, language impairment, and intellectual disability, but feeding difficulties and gastrointestinal problems have also been reported. Whether these are primary impairments, the result of altered eating behavior, or side effects of psychotropic medication remains unclear. To address this question, we investigated Foxp1+/- mice reflecting FOXP1 haploinsufficiency. These animals show decreased body weight and altered feeding behavior with reduced food and water intake. A pronounced muscular atrophy was detected in the esophagus and colon, caused by reduced muscle cell proliferation. Nitric oxide-induced relaxation of the lower esophageal sphincter was impaired and achalasia was confirmed in vivo by manometry. Foxp1 targets (Nexn, Rbms3, and Wls) identified in the brain were dysregulated in the adult Foxp1+/- esophagus. Total gastrointestinal transit was significantly prolonged due to impaired colonic contractility. Our results have uncovered a previously unknown dysfunction (achalasia and impaired gut motility) that explains the gastrointestinal disturbances in patients with FOXP1 syndrome, with potential wider relevance for autism.

Soluble guanylate cyclase as an alternative target for bronchodilator therapy in asthma.

Asthma is defined by airway inflammation and hyperresponsiveness, and contributes to morbidity and mortality worldwide. Although bronchodilation is a cornerstone of treatment, current bronchodilators become ineffective with worsening asthma severity. We investigated an alternative pathway that involves activating the airway smooth muscle enzyme, soluble guanylate cyclase (sGC). Activating sGC by its natural stimulant nitric oxide (NO), or by pharmacologic sGC agonists BAY 41-2272 and BAY 60-2770, triggered bronchodilation in normal human lung slices and in mouse airways. Both BAY 41-2272 and BAY 60-2770 reversed airway hyperresponsiveness in mice with allergic asthma and restored normal lung function. The sGC from mouse asthmatic lungs displayed three hallmarks of oxidative damage that render it NO-insensitive, and identical changes to sGC occurred in human lung slices or in human airway smooth muscle cells when given chronic NO exposure to mimic the high NO in asthmatic lung. Our findings show how allergic inflammation in asthma may impede NO-based bronchodilation, and reveal that pharmacologic sGC agonists can achieve bronchodilation despite this loss.

Phosphodiesterase 3A expression and activity in the murine vasculature is influenced by NO-sensitive guanylyl cyclase.

Phosphodiesterase 3 (PDE3) exists in two isoforms (PDE3A and PDE3B) and is known to act as cGMP-inhibited cAMP-degrading PDE. Therefore, PDE3 may likely be involved in the interaction between the two second messenger pathways. NO-sensitive guanylyl cyclase (NO-GC) is the most important cytosolic generator of cGMP. Here, we investigated the effect of NO-GC deletion on PDE3A-mediated signaling in animals lacking NO-GC either globally (GCKO) or specifically in smooth muscle cells (SMC-GCKO). PDE3A expression is detected in murine aortic smooth muscle, platelets, and heart tissue. Expression and activity of PDE3A in aortae from GCKO and SMC-GCKO mice was reduced by approx. 50% compared to that in control animals. PDE3A downregulation can be linked to the reduction in NO-GC and is not an effect of the increased blood pressure levels resulting from NO-GC deletion. Despite the different PDE3A expression levels, smooth muscle relaxation induced by forskolin to stimulate cAMP signaling was similar in all genotypes. Basal and forskolin-stimulated cAMP levels in aortic tissue were not different between KO and control strains. However, the potency of milrinone, a selective inhibitor of PDE3A, to induce relaxation was higher in aortae from GCKO and SMC-GCKO than that in aorta from control animals. These data were corroborated by the effect of milrinone in vivo, which led to an increase in systolic blood pressure in both KO strains but not in control mice. We conclude that NO-GC modulates PDE3A expression and activity in SMC in vivo conceivably to preserve functional cAMP signaling.

NO-GC in cells 'off the beaten track'.

Nitric oxide-sensitive guanylyl cyclase (NO-GC) has been shown to regulate a plethora of different functions in the body. These include, among many others, the fine-tuning of vascular tone, platelet reactivity and gastrointestinal motility. Evidence for the participation of NO-GC in these functions has been obtained from various species including humans, rodents, as well as insects. Clearly, individual cell types that express NO-GC contribute differentially to organ-specific NO/cGMP signaling in the body. Hence, identification of NO-GC-expressing cells and their individual involvement in NO/cGMP signaling constituted the focus of many studies over the last 40 years. Probably most information has been obtained from vascular smooth muscle cells and platelets, in which NO-GC is known to induce relaxation and inhibition of aggregation, respectively. Many other cell types that express the enzyme have been linked to certain functions, e.g. cardiomyocyte/inotropy or gastrointestinal smooth muscle cells/motility. However, in some cell types, e.g. myofibroblasts or pericytes, NO-GC expression is evident but individual functions of NO/cGMP signaling have yet to be assigned, whereas in other cell types, e.g. in erythrocytes, expression and role of NO-GC is still a matter of debate. This review discusses the current knowledge on 'less popular' cell types that express NO-GC (pericytes, myofibroblasts, cardiomyocytes, adipocytes, interstitial cells of Cajal, fibroblast-like cells and blood cells) and outlines possible further functions in cell types that have not gained strong attention so far.

Nitrergic signalling via interstitial cells of Cajal regulates motor activity in murine colon.

In the enteric nervous systems, NO is released from nitrergic neurons as a major inhibitory neurotransmitter. NO acts via NO-sensitive guanylyl cyclase (NO-GC), which is found in different gastrointestinal (GI) cell types including smooth muscle cells (SMCs) and interstitial cells of Cajal (ICC). The precise mechanism of nitrergic signalling through these two cell types to regulate colonic spontaneous contractions is not fully understood yet. In the present study we investigated the impact of endogenous and exogenous NO on colonic contractile motor activity using mice lacking nitric oxide-sensitive guanylyl cyclase (NO-GC) globally and specifically in SMCs and ICC. Longitudinal smooth muscle of proximal colon from wild-type (WT) and knockout (KO) mouse strains exhibited spontaneous contractile activity ex vivo. WT and smooth muscle-specific guanylyl cyclase knockout (SMC-GCKO) colon showed an arrhythmic contractile activity with varying amplitudes and frequencies. In contrast, colon from global and ICC-specific guanylyl cyclase knockout (ICC-GCKO) animals showed a regular contractile rhythm with constant duration and amplitude of the rhythmic contractions. Nerve blockade (tetrodotoxin) or specific blockade of NO signalling (L-NAME, ODQ) did not significantly affect contractions of GCKO and ICC-GCKO colon whereas the arrhythmic contractile patterns of WT and SMC-GCKO colon were transformed into uniform motor patterns. In contrast, the response to electric field-stimulated neuronal NO release was similar in SMC-GCKO and global GCKO. In conclusion, our results indicate that basal enteric NO release acts via myenteric ICC to influence the generation of spontaneous contractions whereas the effects of elevated endogenous NO are mediated by SMCs in the murine proximal colon.

Dominant role of interstitial cells of Cajal in nitrergic relaxation of murine lower oesophageal sphincter.

Oesophageal achalasia is a disease known to result from reduced relaxation of the lower oesophageal sphincter (LES). Nitric oxide (NO) is one of the main inhibitory transmitters. NO-sensitive guanylyl cyclase (NO-GC) acts as the key target of NO and, by the generation of cGMP, mediates nitrergic relaxation in the LES. To date, the exact mechanism of nitrergic LES relaxation is still insufficiently elucidated. To clarify the role of NO-GC in LES relaxation, we used cell-specific knockout (KO) mouse lines for NO-GC. These include mice lacking NO-GC in smooth muscle cells (SMC-GCKO), in interstitial cells of Cajal (ICC-GCKO) and in both SMC/ICC (SMC/ICC-GCKO). We applied oesophageal manometry to study the functionality of LES in vivo. Isometric force studies were performed to monitor LES responsiveness to exogenous NO and electric field stimulation of intrinsic nerves in vitro. Cell-specific expression/deletion of NO-GC was monitored by immunohistochemistry. Swallowing-induced LES relaxation is strongly reduced by deletion of NO-GC in ICC. Basal LES tone is affected by NO-GC deletion in either SMC or ICC. Lack of NO-GC in both cells leads to a complete interruption of NO-induced relaxation and, therefore, to an achalasia-like phenotype similar to that seen in global GCKO mice. Our data indicate that regulation of basal LES tone is based on a dual mechanism mediated by NO-GC in SMC and ICC whereas swallow-induced LES relaxation is mainly regulated by nitrergic mechanisms in ICC.

Cell-specific impact of nitric oxide-dependent guanylyl cyclase on arteriogenesis and angiogenesis in mice.

Nitric oxide (NO) acts as essential regulator of vasculogenesis and angiogenesis and is critical for arteriogenesis. Whether NO's effects in vivo are mediated through NO-sensitive guanylyl cyclase (NO-GC) and thus by cGMP-dependent mechanisms has been only poorly addressed. Mice lacking NO-GC globally or specifically in smooth muscle cells (SMC) or endothelial cells (EC) were subjected to two established models for arteriogenesis and angiogenesis, namely hindlimb ischemia and oxygen-induced retinopathy. Our data clearly show the involvement of NO-GC in the recovery of blood flow after hindlimb ischemia, and this effect could be attributed to NO-GC in SMC. In the retina, global deletion of NO-GC led to reduced oxygen-induced vessel loss and hypoxia-induced capillary regrowth, whereas pathological neovascularization was increased. These effects were also seen in mice with SMC-specific NO-GC deletion but not in animals lacking NO-GC in EC. Intriguingly, NO-GC was found to be strongly expressed in retinal pericytes. Our data prove the involvement of NO-GC in growth and plasticity of hindlimb and retinal vasculature after ischemic/hypoxic insult.

Interstitial cells of Cajal mediate nitrergic inhibitory neurotransmission in the murine gastrointestinal tract.

Nitric oxide (NO) is a major inhibitory neurotransmitter in the gastrointestinal (GI) tract. Its main effector, NO-sensitive guanylyl cyclase (NO-GC), is expressed in several GI cell types, including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), and fibroblast-like cells. Up to date, the interplay between neurons and these cells to initiate a nitrergic inhibitory junction potential (IJP) is unclear. Here, we investigate the origin of the nitrergic IJP in murine fundus and colon. IJPs were determined in fundus and colon SMC of mice lacking NO-GC globally (GCKO) and specifically in SMC (SM-GCKO), ICC (ICC-GCKO), and both SMC/ICC (SM/ICC-GCKO). Nitrergic IJP was abolished in ICC-GCKO fundus and reduced in SM-GCKO fundus. In the colon, the amplitude of nitrergic IJP was reduced in ICC-GCKO, whereas nitrergic IJP in SM-GCKO was reduced in duration. These results were corroborated by loss of the nitrergic IJP in global GCKO. In conclusion, our results prove the obligatory role of NO-GC in ICC for the initiation of an IJP. NO-GC in SMC appears to enhance the nitrergic IJP, resulting in a stronger and prolonged hyperpolarization in fundus and colon SMC, respectively. Thus NO-GC in both cell types is mandatory to induce a full nitrergic IJP. Our data from the colon clearly reveal the nitrergic IJP to be biphasic, resulting from individual inputs of ICC and SMC.

Cell-specific deletion of nitric oxide-sensitive guanylyl cyclase reveals a dual pathway for nitrergic neuromuscular transmission in the murine fundus.

BACKGROUND & AIMS: It is not clear how nitric oxide (NO) released from enteric neurons relaxes gastrointestinal (GI) smooth muscle. In analogy to the vascular system, NO might directly induce relaxation of smooth muscle cells (SMCs) by acting on its receptor, NO-sensitive guanylyl cyclase (NO-GC). Alternatively, intermediate cells, such as the interstitial cells of Cajal (ICCs), might detect nitrergic signals to indirectly regulate smooth muscle tone, and thereby regulate the motor function of the GI tract. We investigated the role of ICCs and SMCs in nitrergic relaxation using mice with cell-specific disruption of the gene encoding the ß1 subunit of NO-GC (GUCY1B3). METHODS: We created mice that lack NO-GC specifically in SMCs (SM-guanylyl cyclase knockout [GCKO]), ICCs (ICC-GCKO), or both (SM/ICC-GCKO). We investigated the effects of exogenous and endogenous NO on murine fundus using isometric force studies. Total gut transit time was measured to monitor the functional consequences of NO-GC deletion on GI motility in vivo. RESULTS: NO-GC is expressed in ICC and SMC. Deletion of the NO receptor from SMCs incompletely reduced NO-induced fundus relaxation, which was hardly affected after ICC-specific deletion. Gut transit time did not change in SM-GCKO or ICC-GCKO mice compared with control mice. However, nitrergic relaxation was not observed in SM/ICC-GCKO mice, which had increased gut transit time compared with controls. CONCLUSIONS: In mice, NO-GC is the only NO receptor to relax the fundus; deletion of NO-GC from the combination of SMCs and ICCs blocks nitrergic signaling. Therefore, ICCs and SMCs jointly mediate the relaxant effect of enteric NO.

Correlation of cellular expression with function of NO-sensitive guanylyl cyclase in the murine lower urinary tract.

The action of nitric oxide (NO) to stimulate NO-sensitive guanylyl cyclase (NO-GC), followed by production of cGMP, and eventually to cause smooth muscle relaxation is well known. In the lower urinary tract (LUT), in contrast to the cardiovascular system and the gastrointestinal tract, specific localization in combination with function of NO-GC has not been investigated to date. Consequently, little is known about the mechanisms regulating relaxation of the lower urinary tract in general and the role of NO-GC-expressing cells in particular. To study the distribution and function of NO-GC in the murine lower urinary tract, we used internal urethral sphincter and bladder detrusor from global (GCKO) and smooth muscle cell-specific (SM-GCKO) NO-GC knock-out mice for immunohistochemical analyses and organ bath experiments. In urethral sphincter, NO-GC-positive immunofluorescence was confined to smooth muscle cells (SMCs). Deletion of NO-GC in SMCs abolished NO-induced relaxation. In bladder detrusor, exposure to NO did not cause relaxation although immunohistochemistry uncovered the existence of NO-GC in the tissue. In contrast to the urethral sphincter, expression of NO-GC in bladder detrusor was limited to platelet-derived growth factor receptor α (PDGFRα)-positive interstitial cells. In conclusion, NO-GC found in SMCs of the urethral sphincter mediates NO-induced relaxation; bladder detrusor is unique as NO-GC is not expressed in SMCs and, thus, NO does not induce relaxation. Nevertheless, NO-GC expression was found in PDGFRα-positive interstitial cells of the murine bladder with an as yet unknown function. Further investigation is needed to clarify the role of NO-GC in the detrusor.

Preserved fertility despite erectile dysfunction in mice lacking the nitric oxide receptor.

Nitric oxide (NO) and cGMP have been shown to be important mediators of penile erection. Erectile dysfunction may result from reduced or non-functional signal transduction within this cascade. There is, however, some inconsistency in the available data as mice lacking NO synthases (endothelial and neuronal nitric oxide synthase, or both) appear to be fertile whereas mice deficient in cGMP-dependent protein kinase I (PKGI) suffer from erectile dysfunction. To clarify this discrepancy we performed studies on mice lacking the NO receptor NO-sensitive guanylyl cyclase (NO-GC). In addition, we generated cell-specific NO-GC knockout (KO) lines to investigate the function of NO in individual cell types. NO-GC was specifically deleted in smooth muscle or endothelial cells (SM-guanylyl cyclase knockout (SM-GCKO) and EC-GCKO, respectively) and these KO lines were compared with total knockouts (GCKO) and wild-type animals. We investigated expression of NO-GC, NO-induced relaxation of corpus cavernosum smooth muscle and their ability to generate offspring. NO-GC-positive immunostaining was detected in smooth muscle and endothelial cells of murine corpus cavernosum but not in interstitial cells of Cajal. NO released from NO donors as well as from nitrergic neurons failed to relax precontracted corpus cavernosum from GCKO mice in organ bath experiments. Similar results were obtained in corpus cavernosum from SM-GCKO mice whereas deletion of NO-GC in endothelial cells did not affect relaxation. The lack of NO-induced relaxation in GCKO animals was not compensated for by guanosine 3,5-cyclic monophosphate (cGMP) signalling. To our surprise, GCKO males were fertile although their ability to produce offspring was decreased. Our data show that deletion of NO-GC specifically in smooth muscle cells abolishes NO-induced corpus cavernosum relaxation but does not lead to infertility.

Biomimetic Connection of Transcutaneous Implants with Skin.

Bacterial infection is a crucial complication in implant restoration, in particular in permanent skin-penetrating implants. Therein, the resulting gap between transcutaneous implant and skin represents a permanent infection risk, limiting the field of application and the duration of application. To overcome this limitation, a tight physiological connection is required to achieve a biological and mechanical welding for a long-term stable closure including self-healing probabilities. This study describes a new approach, wherein the implant is connected covalently to a highly porous electrospun fleece featuring physiological dermal integration potential. The integrative potential of the scaffold is shown in vitro and confirmed in vivo, further demonstrating tissue integration by neovascularization, extracellular matrix formation, and prevention of encapsulation. To achieve a covalent connection between fleece and implant surface, self-initiated photografting and photopolymerization of hydroxyethylmethacrylate is combined with a new crosslinker (methacrylic acid coordinated titanium-oxo clusters) on proton-abstractable implant surfaces. For implant modification, the attached fleece is directed perpendicular from the implant surface into the surrounding dermal tissue. First in vitro skin implantations demonstrate the implants' dermal integration capability as well as wound closure potential on top of the fleece by epithelialization, establishing a bacteria-proof and self-healing connection of skin and transcutaneous implant.

Cardiovascular Functions of Ena/VASP Proteins: Past, Present and Beyond.

Actin binding proteins are of crucial importance for the spatiotemporal regulation of actin cytoskeletal dynamics, thereby mediating a tremendous range of cellular processes. Since their initial discovery more than 30 years ago, the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family has evolved as one of the most fascinating and versatile family of actin regulating proteins. The proteins directly enhance actin filament assembly, but they also organize higher order actin networks and link kinase signaling pathways to actin filament assembly. Thereby, Ena/VASP proteins regulate dynamic cellular processes ranging from membrane protrusions and trafficking, and cell-cell and cell-matrix adhesions, to the generation of mechanical tension and contractile force. Important insights have been gained into the physiological functions of Ena/VASP proteins in platelets, leukocytes, endothelial cells, smooth muscle cells and cardiomyocytes. In this review, we summarize the unique and redundant functions of Ena/VASP proteins in cardiovascular cells and discuss the underlying molecular mechanisms.

Nitric oxide-sensitive guanylyl cyclase is dispensable for nitrergic signaling and gut motility in mouse intestinal smooth muscle.

BACKGROUND & AIMS: The nitric oxide-guanosine 3',5'-cyclic monophosphate (cGMP) signaling pathway has an important role in the control of smooth muscle tone. NO is produced by NO synthases and acts as a major inhibitory neurotransmitter in the gastrointestinal (GI) tract. The main target, NO-sensitive guanylyl cyclase (NO-GC), is stimulated by NO to produce the intracellular messenger cGMP. We investigated the role of NO-GC in nitrergic relaxation and GI motility. METHODS: We tested relaxation of GI smooth muscle in mice that do not express NO-GC or mice with disruption of NO-GC specifically in smooth muscle cells. Different segments of the GI tract (fundus, lower esophageal sphincter, pyloric sphincter, and duodenum) were used in isometric force studies. NO donors and electrical field stimulation were used to assess nitrergic signaling. Whole-gut transit time was measured as an indicator of GI motility. RESULTS: Mice that lack NO-GC do not have NO-induced relaxation of GI smooth muscle. Gut transit time was increased, resulting in GI dysfunction. Surprisingly, in mice that lack NO-GC specifically in smooth muscle, NO-induced relaxation was reduced only slightly, and whole-gut transit time was unchanged compared with wild-type mice. CONCLUSIONS: Lack of NO-GC in smooth muscle cells does not impair NO-induced relaxation of GI tissues or GI motility. The NO receptor guanylyl cyclase in GI smooth muscle is therefore dispensable for nitrergic signaling in mice.

Smooth muscle-specific deletion of nitric oxide-sensitive guanylyl cyclase is sufficient to induce hypertension in mice.

BACKGROUND: Arterial hypertension is one of the major diseases in industrial countries and a major cause of mortality. One of the main vascular factors responsible for the relaxation of blood vessels and regulation of blood pressure is nitric oxide (NO). NO acts predominantly via NO-sensitive guanylyl cyclase (NO-GC), which is made up of 2 different subunits (alpha and beta). Deletion of the beta(1) subunit leads to a global NO-GC knockout, and these mice are hypertensive. However, global deletion of NO-GC in mice does not allow identification of the cell/tissue type responsible for the elevated blood pressure. METHODS AND RESULTS: To determine the relative contribution of smooth muscle cells to the hypertension seen in NO-GC knockout mice, we generated smooth muscle-specific knockout mice for the beta(1) subunit of NO-GC using a tamoxifen-inducible system. Male mice were investigated because the Cre transgene used is located on the Y chromosome. Tamoxifen injection led to a rapid reduction of NO-GC expression in smooth muscle but did not affect that in other tissues. Parallel to a reduction in NO-induced cGMP accumulation, NO-induced relaxation of aortic smooth muscle was gradually lost after induction by tamoxifen. Concomitantly, these animals developed hypertension within 3 to 4 weeks. CONCLUSIONS: We generated a model in which the development of hypertension can be visualized over time by deletion of a single gene in smooth muscle cells. In sum, our data provide evidence that deletion of NO-GC solely in smooth muscle is sufficient to cause hypertension.

Cardioprotection by ischemic postconditioning and cyclic guanosine monophosphate-elevating agents involves cardiomyocyte nitric oxide-sensitive guanylyl cyclase.

Aims: It has been suggested that the nitric oxide-sensitive guanylyl cyclase (NO-GC)/cyclic guanosine monophosphate (cGMP)-dependent signalling pathway affords protection against cardiac damage during acute myocardial infarction (AMI). It is, however, not clear whether the NO-GC/cGMP system confers its favourable effects through a mechanism located in cardiomyocytes (CMs). The aim of this study was to evaluate the infarct-limiting effects of the endogenous NO-GC in CMs in vivo. Methods and results: Ischemia/reperfusion (I/R) injury was evaluated in mice with a CM-specific deletion of NO-GC (CM NO-GC KO) and in control siblings (CM NO-GC CTR) subjected to an in vivo model of AMI. Lack of CM NO-GC resulted in a mild increase in blood pressure but did not affect basal infarct sizes after I/R. Ischemic postconditioning (iPost), administration of the phosphodiesterase-5 inhibitors sildenafil and tadalafil as well as the NO-GC activator cinaciguat significantly reduced the amount of infarction in control mice but not in CM NO-GC KO littermates. Interestingly, NS11021, an opener of the large-conductance and Ca2+-activated potassium channel (BK), an important downstream effector of cGMP/cGKI in the cardiovascular system, protects I/R-exposed hearts of CM NO-GC proficient and deficient mice. Conclusions: These findings demonstrate an important role of CM NO-GC for the cardioprotective signalling following AMI in vivo. CM NO-GC function is essential for the beneficial effects on infarct size elicited by iPost and pharmacological elevation of cGMP; however, lack of CM NO-GC does not seem to disrupt the cardioprotection mediated by the BK opener NS11021.

The IgCAM CLMP regulates expression of Connexin43 and Connexin45 in intestinal and ureteral smooth muscle contraction in mice.

CAR-like membrane protein (CLMP), an immunoglobulin cell adhesion molecule (IgCAM), has been implicated in congenital short-bowel syndrome in humans, a condition with high mortality for which there is currently no cure. We therefore studied the function of CLMP in a Clmp-deficient mouse model. Although we found that the levels of mRNAs encoding Connexin43 or Connexin45 were not or were only marginally affected, respectively, by Clmp deficiency, the absence of CLMP caused a severe reduction of both proteins in smooth muscle cells of the intestine and of Connexin43 in the ureter. Analysis of calcium signaling revealed a disordered cell-cell communication between smooth muscle cells, which in turn induced an impaired and uncoordinated motility of the intestine and the ureter. Consequently, insufficient transport of chyme and urine caused a fatal delay to thrive, a high rate of mortality, and provoked a severe hydronephrosis in CLMP knockouts. Neurotransmission and the capability of smooth muscle cells to contract in ring preparations of the intestine were not altered. Physical obstructions were not detectable and an overall normal histology in the intestine as well as in the ureter was observed, except for a slight hypertrophy of smooth muscle layers. Deletion of Clmp did not lead to a reduced length of the intestine as shown for the human CLMP gene but resulted in gut malrotations. In sum, the absence of CLMP caused functional obstructions in the intestinal tract and ureter by impaired peristaltic contractions most likely due to a lack of gap-junctional communication between smooth muscle cells.

Optical control of a receptor-linked guanylyl cyclase using a photoswitchable peptidic hormone.

The optical control over biological function with small photoswitchable molecules has gathered significant attention in the last decade. Herein, we describe the design and synthesis of a small library of photoswitchable peptidomimetics based upon human atrial natriuretic peptide (ANP), in which the photochromic amino acid [3-(3-aminomethyl)phenylazo]phenylacetic acid (AMPP) is incorporated into the peptide backbone. The endogeneous hormone ANP signals via the natriuretic peptide receptor A (NPR-A) through raising intracellular cGMP concentrations, and is involved in blood pressure regulation and sodium homeostasis, as well as lipid metabolism and pancreatic function. The cis- and trans-isomers of one of our peptidomimetics, termed TOP271, exhibit a four-fold difference in NPR-A mediated cGMP synthesis in vitro. Despite this seemingly small difference, TOP271 enables large, optically-induced conformational changes ex vivo and transforms the NPR-A into an endogenous photoswitch. Thus, application of TOP271 allows the reversible generation of cGMP using light and remote control can be afforded over vasoactivity in explanted murine aortic rings, as well as pancreatic beta cell function in islets of Langerhans. This study demonstrates the broad applicability of TOP271 to enzyme-dependent signalling processes, extends the toolbox of photoswitchable molecules to all classes of transmembrane receptors and utilizes photopharmacology to deduce receptor activation on a molecular level.