Barbara Lies

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.

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.

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

•  Erectile dysfunction may result from reduced or non‐functional nitric oxide (NO)/cGMP‐mediated signalling. Mice lacking NO synthases are fertile whereas mice deficient in cGMP‐dependent protein kinase I suffer from erectile dysfunction. •  To clarify this discrepancy we performed studies on the corpus cavernosum of male mice lacking the NO receptor NO‐sensitive guanylyl cyclase (NO‐GC) either globally or specifically in smooth muscle cells. •  NO released from NO donors as well as from nitrergic neurons failed to relax precontracted corpus cavernosum from mice lacking NO‐GC either globally or specifically in smooth muscle; to our surprise, males from both knockout lines were fertile. •  Our data show that deletion of the NO receptor specifically in smooth muscle cells abolishes NO‐induced corpus cavernosum relaxation but does not lead to infertility.

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

•  Diseases of the lower urinary tract are associated with dysfunctions of cellular mechanisms that regulate smooth muscle tone. Nitric oxide (NO) mediates relaxation of most smooth muscle‐containing tissues via NO‐sensitive guanylyl cyclase (NO‐GC). Correlation of cellular localization with function of NO‐GC in the murine lower urinary tract has not been previously performed. •  Using cell‐specific knock‐out mice, we demonstrate that NO‐GC is expressed exclusively in smooth muscle cells of the urethral sphincter and mediates NO‐induced relaxation. •  In bladder detrusor, NO‐GC is not detected in smooth muscle cells but rather in platelet‐derived growth factor receptor α‐positive interstitial cells. NO‐GC in these cells does not contribute to NO‐induced relaxation; therefore, bladder detrusor smooth muscle appears to be unique as it is not relaxed by NO. •  The correlation of NO‐GC localization and function regarding smooth muscle relaxation allows the clinical use of compounds acting within NO/cGMP signalling to be assessed.

Radioimmunoassay for the Quantification of cGMP Levels in Cells and Tissues

Radioimmunoassay is an established method to determine the amount of a specific substance in a given cell or tissue sample. Commercially available RIA or Elisa are very cost intensive. Here, we describe the generation of radioactive cGMP tracer and the quantification of cGMP. Although working with radioactive material requires experience and care, this method is very sensitive and rather cheap, once it is established.

Nitrergic relaxation of gastrointestinal smooth muscle: role of interstitial cells of Cajal and smooth muscle cells

Background The signaling molecule nitric oxide (NO) is known to activate the enzyme NO-sensitive guanylyl cyclase (NOGC). By generation of the intracellular second messenger cGMP NO-GC regulates many physiological processes. In the gastrointestinal (GI) tract nitrergic neurons are part of the enteric nervous system which regulates GI motility. In addition, interstitial cells of Cajal (ICC) are thought to be involved in nitrergic relaxation. In this study, we intended to clarify the role of NO-GC regarding GI motility in mice by using cellspecific knockout strains.

Modulation of cGMP/cAMP crosstalk on the level of PDE3 in animals lacking NO-sensitive guanylyl cyclase

NO-sensitive guanylyl cyclase (NO-GC) is accepted to be the major receptor for the signaling molecule NO. Deletion of NO-GC in mice leads to disturbed NO/cGMP signaling. As a result, these mice show abolished NO-dependent relaxation of smooth muscle-containing tissues in the cardiovascular and gastrointestinal systems. Mice with general deletion (GCKO) suffer from increased blood pressure, reduced bleeding time, erectile dysfunction and die prematurely due to gastrointestinal dysmotility. Mice lacking NO-GC specifically in smooth muscle cells (SMC) show an elevated blood pressure similar to that seen in GCKO animals. Crosstalk between cGMP and cAMP signaling has been reported to occur in many different cells. Phosphodiesterase 3 (PDE3) is thought to be one of the prominent mediators of this crosstalk. Through its inhibitory action on PDE3, cGMP may regulate cAMP levels and cAMP-mediated signaling. Here, we reasoned that, in animals deficient in NO-GC, lack of NO-induced cGMP synthesis might affect cGMP/cAMP crosstalk on the level of PDE3. Smooth muscle relaxation induced by forskolin to stimulate adenylyl cyclase was similar in WT, GCKO and SMC-GCKO aortae. RIA-based detection of basal cAMP levels in aortic tissue from these mice did not reveal differences. Similarly, FRET measurements showed that PGE1-induced cAMP synthesis in isolated smooth muscle cells was unaffected by NO-GC deletion. However, aortae from GCKO and SMC-GCKO animals were more sensitive to milrinone, a specific inhibitor of PDE3, as the concentration curve for relaxation was shifted to the left. Analysis of PDE3 expression in aorta showed an approximately 50% reduction in PDE3 protein and specific enzyme activity. Similar reduction was detected in platelets but not in heart tissue of GCKO animals. Induction of SMC-specific NO-GC deletion by tamoxifen led to a parallel decline of NO-GC and PDE3 expression. Both proteins were strongly reduced already 5 days after the last tamoxifen injection. In contrast, systolic blood pressure in these animals started to rise only after 10 days and was maximal after 50 days. These data indicate that cGMP, not the increase in blood pressure, is responsible for the expression regulation of PDE3. We conclude that cGMP modulates PDE3 expression in SMC in order to keep up functional cAMP signaling.

Cell-specific modulation of gastrointestinal NO-induced relaxation by phosphodiesterases

Background In the gastrointestinal (GI) tract NO is produced by nNOS and released from nerve varicosities to relax GI smooth muscle. NO-sensitive guanylyl cyclase (NO-GC), the receptor for NO, is expressed in at least two cell types, smooth muscle cells (SMC) and interstitial cells of Cajal (ICC). It is still unclear which of these cell types acts as primary target for nerve-released NO. ICC express much higher amounts of NO-GC than SMC and may therefore scavenge NO. In these cells, the amount of NO-GC as well as the type(s) of phosphodiesterase(s) (PDE) present will influence strength and duration of the cGMP signal. Thereby, the cGMP/ cAMP crosstalk in these cells will be affected.