Dieter Groneberg

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 (&agr; and &bgr;). Deletion of the &bgr;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 &bgr;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.

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.

Soluble Guanylate Cyclase Is Required for Systemic Vasodilation But Not Positive Inotropy Induced by Nitroxyl in the Mouse

Nitroxyl (HNO), the reduced and protonated form of nitric oxide (NO·), confers unique physiological effects including vasorelaxation and enhanced cardiac contractility. These features have spawned current pharmaceutical development of HNO donors as heart failure therapeutics. HNO interacts with selective redox sensitive cysteines to effect signaling but is also proposed to activate soluble guanylate cyclase (sGC) in vitro to induce vasodilation and potentially enhance contractility. Here, we tested whether sGC stimulation is required for these HNO effects in vivo and if HNO also modifies a redox-sensitive cysteine (C42) in protein kinase G-1&agr; to control vasorelaxation. Intact mice and isolated arteries lacking the sGC-&bgr; subunit (sGCKO, results in full sGC deficiency) or expressing solely a redox-dead C42S mutant protein kinase G-1&agr; were exposed to the pure HNO donor, CXL-1020. CXL-1020 induced dose-dependent systemic vasodilation while increasing contractility in controls; however, vasodilator effects were absent in sGCKO mice whereas contractility response remained. The CXL-1020 dose reversing 50% of preconstricted force in aortic rings was ≈400-fold greater in sGCKO than controls. Cyclic-GMP and cAMP levels were unaltered in myocardium exposed to CXL-1020, despite its inotropic-vasodilator activity. In protein kinase G-1&agr;C42S mice, CXL-1020 induced identical vasorelaxation in vivo and in isolated aortic and mesenteric vessels as in littermate controls. In both groups, dilation was near fully blocked by pharmacologically inhibiting sGC. Thus, sGC and cGMP-dependent signaling are necessary and sufficient for HNO-induced vasodilation in vivo but are not required for positive inotropic action. Redox modulation of protein kinase G-1&agr; is not a mechanism for HNO-mediated vasodilation.

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

Nitric oxide (NO) is an important inhibitory neurotransmitter in the gastrointestinal tract. Oesophageal achalasia may result from impairment of nitrergic relaxation. Smooth muscle cells (SMCs) have been accepted to be the major targets for neuronal NO to mediate relaxation. However, besides SMCs, the receptor for NO, NO‐sensitive guanylyl cyclase (NO‐GC), has been shown in interstitial cells of Cajal (ICC). Using cell‐specific knockout mice, this study shows that NO‐GC in SMC and ICC modulates lower oesophagus sphincter tone in vitro and in vivo. More importantly, NO‐GC in ICC possesses a dominant role in mediating swallowing‐induced relaxation. Lack of functional nitrergic signalling, thus, results in deficits in relaxation of the lower oesophagus sphincter as seen in achalasic patients.

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

Dysregulation of nitric oxide (NO) signalling is associated with GI motility dysfunctions like chronic constipation, achalasia or Hirschsprungs disease. The inhibitory effect of NO is mainly exerted 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). Here, we focus on the investigation of NO‐GC function in murine colon. Using cell‐specific knock‐out mice, we demonstrate that NO‐GC is expressed in myenteric ICC of murine colon and participates in regulation of colonic spontaneous contractions in longitudinal smooth muscle. We report a novel finding 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. These results help in understanding possible pathological mechanisms involved in slowed colonic action and colonic inertia.

Lack of effect of ODQ does not exclude cGMP signalling via NO-sensitive guanylyl cyclase.

Nitric oxide (NO) is known to activate NO‐sensitive guanylyl cyclase (NO‐GC) and to elicit cGMP production. However, NO has also been proposed to induce cGMP‐independent effects. It is accepted practice to use specific NO‐GC inhibitors, such as ODQ or NS2028, to assess cGMP‐dependent NO effects. Consequently, NO‐induced reactions seen in the presence of these inhibitors commonly serve as an affirmation of cGMP independence.

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

Significance Asthmatics depend on β-agonist bronchodilator drugs, but a majority develop resistance to these drugs in their lifetime, and new ways to bronchodilate are needed. We show that brochodilation can be triggered in normal human and asthmatic mouse airways through an alternative signaling pathway, using new pharmacologic agents that directly activate the soluble guanylate cyclase (sGC) enzyme. Because an sGC-based drug was recently approved to treat pulmonary arterial hypertension, our findings imply that such drugs could become new bronchodilators in asthma. Our work also provides insight on how the sGC signaling enzyme becomes desensitized toward NO in inflammatory asthma, and thus helps to explain why NO is an ineffective bronchodilator in this disease. 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.

Integrative Control of Gastrointestinal Motility by Nitric Oxide

In the gastrointestinal (GI) tract, nitric oxide (NO) has been shown over the last 25 years to exert a prominent function as inhibitory neurotransmitter. Apart from the regulation of secretion and resorption, NO from nitrergic neurons has been demonstrated to be crucial for GI smooth muscle relaxation and motility. In fact, several human diseases such as achalasia, gastroparesis, slow transit constipation or Hirschsprungs disease may involve dysfunctional nitrergic signaling. Most of NOs effects as neurotransmitter are mediated by NO-sensitive guanylyl cyclase (NO-GC) and further transduced by cGMP-dependent mechanisms. In contrast to the vascular system where NO from the endothelium induces relaxation by acting on NO-GC solely in smooth muscle cells, GI tissues contain several different NO-GCexpressing cell types that include smooth muscle cells, interstitial cells of Cajal and fibroblast-like cells. Based on this diverse localization of the NO receptor, the exact pathway(s) leading to NO-induced relaxation are still unknown. Global and cell-specific knockout mouse strains have been generated that lack enzymes participating in nitrergic signaling. These animals have been helpful in examining the role of NO in smooth muscle of the GI tract. Here, we discuss the current knowledge on NO-mediated mechanisms in the relaxation of GI smooth muscle in stomach, small and large intestine including sphincters. Special focus is placed on the integration of nitrergic signals by specialized cell types within the gut smooth muscle layers.

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.

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.