Sönke Behrends

Expression of nitric oxide-sensitive guanylyl cyclase subunits in human corpus cavernosum

The muscles of the corpus cavernosum of the penis relax in response to stimulation of non-adrenergic, non-cholinergic nerves or nitric oxide (NO)-donating drugs to elicit erection. It is generally assumed that NO mediates this effect via activation of soluble guanylyl cyclase and a subsequent increase in cyclic guanosine 3, 5-monophosphate concentration. However, there are no data on the expression of this enzyme in human corpus cavernosum. The purpose of the present study was the molecular characterization of NO-sensitive guanylyl cyclase in human corpus cavernosum. RNA was extracted from tissue samples obtained from seven patients undergoing penile prosthetic surgery or correction of penile deviation. Reverse transcriptase-polymerase chain reaction (RT-PCR) with specific primers for the subunits of NO-sensitive guanylyl cyclase was performed, and PCR products were subcloned and sequenced. Specific amplification products encoding the alpha(1), beta(1), alpha(2), and beta(2) subunits were detected. In addition, we isolated a transcript encoding a novel variant beta(2) subunit. To test whether this novel transcript arises by alternative splicing or whether it is encoded by a separate gene, a 4000-bp clone of the corresponding genomic DNA sequence was isolated. Sequence analysis suggests that the novel beta(2) variant arises by alternative splicing from the same gene as the beta(2) subunit on chromosome 13. In conclusion, our findings suggest the presence of different subunit mRNAs of NO-sensitive guanylyl cyclase in human corpus cavernosum.

Biliverdin IX is an endogenous inhibitor of soluble guanylyl cyclase

Heme oxygenase (HO) converts heme to carbon monoxide (CO) and biliverdin IX. CO is a weak activator of soluble guanylyl cyclase (SGC), the enzyme that catalyzes the conversion of GTP to the second messenger cGMP. HO overexpression has recently been shown to inhibit production of cGMP by SGC in vivo. The aim of the present study was to investigate a possible influence of biliverdin IX on SGC activity. Using recombinant alpha(1)/beta(1) isoform of SGC, we show an inhibitory effect of biliverdin IX in the micromolar range both on basal and NO stimulated guanylyl cyclase activity. Bilirubin IX which differs from biliverdin IX in two hydrogen atoms had no effect. Biliverdin IX reduced maximal guanylyl cyclase activity (V(max) values) while it had no effect on the K(M) values indicating unchanged affinity towards the substrate GTP. Concentration response experiments using the NO donor, 2,2-diethyl-1-nitroso-oxyhydrazine (DEA/NO), showed that enzyme activities at maximal DEA/NO concentration were reduced by biliverdin IX. The affinity of the NO-donor, DEA/NO, towards SGC was significantly reduced in the presence of biliverdin IX. Biliverdin IX lowered enzyme activity at maximal activator concentrations of YC-1 and protoporphyrin IX (PPIX) while it had no significant effect on the EC(50) values of these two NO independent activators. The inhibitory effect of biliverdin IX on PPIX activated enzyme activity is not shared by ODQ, which indicates that the inhibitory mechanism of biliverdin IX is different from ODQ.

Cloning and functional expression of the rat α2 subunit of soluble guanylyl cyclase

Nitric oxide-sensitive guanylyl cyclase is a heterodimeric enzyme consisting of one alpha and one beta subunit. Here, we clone the first alpha(2) subunit ortholog and functionally express the cDNA in Sf-9 cells. Our data indicate a high degree of conservation of the primary sequence and functional activity of the rat alpha(2) subunit.

Cerebral expression of the α2-subunit of soluble guanylyl cyclase is linked to cerebral maturation and sensory pathway refinement during postnatal development

Abstract Soluble guanylyl cylase (sGC) has been identified for being a receptor for the gaseous transmitters nitric oxide and cabon monoxide. Currently four subunits α 1 , α 2 , β 1 , and β 2 have been characterized. Heterodimers of α and β-subunits as well as homodimers of the β 2 -subunit are known to constitute functional sGC which use GTP to form cGMP a potent signal molecule in a multitude of second messenger cascades. Since NO-cGMP signaling plays a pivotal role in neuronal development we analyzed the maturational expression pattern of the newly characterized α 2 -subunit of sGC within the brain of Wistar rats by means of RNase protection assay and immunohistochemistry. α 2 -subunit mRNA as well as immunoreactive α 2 -protein increased during postnatal cerebral development. Topographical analysis revealed a selective high expression of the α 2 -subunit in the choroid plexus and within developing sensory systems involving the olfactory and somatosensory system of the forebrain as well as parts of the auditory and visual system within the hindbrain. In cultured cortical neurons the α 2 -subunit was localized to the cell membrane, especially along neuronal processes. During the first 11 days of postnatal development several cerebral regions showed a distinct expression of the α 2 -subunit which was not paralleled by the α 1 /β 1 -subunits especially within the developing thalamo-cortical circuitries of the somatosensory system. However, at later developmental stages all three subunits became more homogenously distributed among most cerebral regions, indicating that functional α 1 /β 1 and α 2/ β 1 heterodimers of sGC could be formed. Our findings indicate that the α 2 -subunit is an essential developmentally regulated constituent of cerebral sensory systems during maturation. In addition the α 2 -subunit may serve other functions than forming a functional heterodimer of sGC during the early phases of sensory pathway refinement.

Glyceryl trinitrate treatment up-regulates soluble guanylyl cyclase in rat dura mater

Nitric oxide (NO) is a key molecule in vascular headaches and the dura mater has been implicated as a tissue where vascular headache develops. Here we demonstrate expression, enzyme activity and cellular distribution of the intracellular receptor for NO, soluble guanylyl cyclase (sGC), in rat dura mater. Subcutaneous treatment of rats with the NO-donor glyceryl trinitrate (GTN) induced an increase of sGC expression and activity in dural blood vessels after 20–30 min. It has previously been shown that GTN induces headache in normal subjects after 20–30 min. Our findings suggest that an up-regulation of the NO target enzyme contributes to the pathogenesis of GTN-induced headache explaining the subacute rather than acute onset of symptoms.

The β2 subunit of nitric oxide-sensitive guanylyl cyclase is developmentally regulated in rat kidney

Abstract. We have recently shown that nitric oxide activates the β2 subunit of soluble guanylyl cyclase. In the present study, we show developmental regulation of this subunit. Analysis of mRNA expression by RT-PCR and RNase protection analysis in kidneys revealed no expression of the β2 subunit in neonatal and strong expression in adult rats. A reciprocal regulation with much lower expression levels was observed in rat lung. Further examination of kidneys from 3, 6, 16, 22, 25, 31 and 36-day-old rats showed that significant expression appears between postnatal day 16 and 22. Isolation of the rat β2 promoter by genome walking and cloning into a reporter gene vector showed promoter activity for the sense but not the antisense construct providing an in vitro assay for further analysis of the developmental β2 subunit regulation.