Mauro Giorgi

Type 5 phosphodiesterase expression in the human vagina

OBJECTIVES It has been demonstrated that clitoral and vaginal tissues express nitric oxide synthase isoforms in a way that parallels that of the penile corpus cavernosum. Considering the role of the vagina in the female sexual response and the anatomic connection between the clitoris and the anterosuperior vaginal wall, our aim was to study the distribution of type 5 phosphodiesterase (PDE5) in the anterosuperior wall of the human vagina. METHODS Immunohistochemistry was performed on the vaginal tissue of 14 women obtained at autopsy and on exfoliated cells of the vaginal epithelium obtained from 5 healthy female donors. Specific antibodies against PDE5 were tested on both paraffin sections and cytologic smears. Immunoblotting experiments were performed in parallel with the same antibodies. RESULTS The histologic analysis of human cadaveric vaginal tissue revealed that PDE5 immunoreactivity was mostly localized in the smooth muscle of vessels, forming a pseudocavernous tissue in the vaginal wall and endothelium. The Skene periurethral glands and vaginal epithelium were also positive for the antibody. The latter finding was confirmed using exfoliated cells of the vaginal epithelium harvested in vivo. CONCLUSIONS The presence and tissue distribution of PDE5 in the human vagina suggest that the integrated system of nitric oxide synthase-PDE5 may play a physiologic role not only in the male sexual response but also in female sexual arousal.

Expression of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in mouse tissues and cell lines using an antibody against the enzyme amino-terminal domain

We have produced a polyclonal antibody that specifically recognizes cGMP-binding cGMP-specific phosphodiesterase (PDE5). The antibody was raised in rabbit using as immunogen a fusion protein, in which glutathione S-transferase was coupled to a 171 amino acid polypeptide of the N-terminal region of bovine PDE5. The antibody is able to immunoprecipitate PDE5 activity from mouse tissues and neuroblastoma extracts while it has no effect on all other PDE isoforms present in the extracts. PDE5 activity recovered in the immunoprecipitates retains its sensitivity to specific inhibitors such as zaprinast (IC(50)=0.6 microM) and sildenafil (IC(50)=3.5 nM). Bands of the expected molecular mass were revealed when solubilized immunoprecipitates were analysed in Western blots. The antibody selectively stained cerebellar Purkinje neurones, which are known to express high levels of PDE5 mRNA. Western blot analysis of mouse tissues revealed the highest expression signal in mouse lung, followed by heart and cerebellum, while a lower signal was evident in brain, kidney and a very low signal was present in the liver. In the hybrid neuroblastoma-glioma NG108-15 cells the antibody revealed a high PDE5 induction after dibutyryl-cAMP treatment.

Down-regulation of nitrergic transmission in the rat striatum after chronic nigrostriatal deafferentation.

Dopamine and NO are physiological stimulators of synthesis of cAMP and cGMP, respectively, and NO synthase‐containing interneurons in the striatum are physiologically activated by dopamine‐containing neurons in the substantia nigra. This study investigated whether lesioning dopamine neurons has multiple consequences in the striatum consistent with the reported sensitization of cAMP synthesis, including alteration of the NO–cGMP pathway and phosphodiesterase‐dependent metabolism of cyclic nucleotides. The substantia nigra of adult Sprague‐Dawley rats was unilaterally lesioned with 6‐hydroxydopamine. Two months later, we determined expression of NO synthase and evaluated cGMP and cAMP levels of intact and deafferented striatum. Moreover, we evaluated cAMP– and cGMP–phosphodiesterase activities in basal conditions and after Ca2+–calmodulin stimulation and determined the expression of the phosphodiesterase‐1B isoform and the levels of phosphodiesterase‐1B mRNA. Using immunocytochemistry we characterized the distribution of NO synthase and phosphodiesterase‐1B within striatal neurons. In the dopamine‐deafferented striatum, NO synthase levels were decreased by 42% while NO synthase‐immunopositive intrastriatal fibres but not NO synthase neuronal bodies were reduced in number. In the deafferented striatum basal cGMP levels were reduced, and cAMP levels were increased, but cGMP–phosphodiesterase and cAMP–phosphodiesterase activities were both increased in basal and Ca2+–calmodulin‐stimulated conditions. Accordingly, phosphodiesterase‐1B expression and phosphodiesterase‐1B mRNA were upregulated while a large population of medium‐sized striatal neurons showed increased phosphodiesterase‐1B immunoreactivity. Dopamine deafferentation led to a complex down‐regulation of the NO–cGMP pathway in the striatum and to an up‐regulation of phosphodiesterase‐1B‐dependent cyclic nucleotide metabolism, showing new aspects of neuronal plasticity in experimental hemiparkinsonism.

The induction of cyclic nucleotide phosphodiesterase 4 gene (PDE4D) impairs memory in a water maze task.

In this study, the effects on memory of intraperitoneal post-training administration of cyclic nucleotide phosphodiesterase (PDE) inhibitors, DC-TA 46 and rolipram, were tested using a visible/hidden-platform water maze task. The effects of these compounds on cyclic nucleotide levels in the hippocampal formation (HF) and striatum (CP) were also assessed, by enzymatic immunoassay (EIA). The results obtained from rats trained in the visible-platform task were not significantly different from controls. On the contrary, the animals trained in the hidden-platform water maze task showed a memory impairment, when injected with DC-TA 46 at maximal dose of 20mg/kg and with rolipram at 3 and 30 mg/kg doses. The effects of these drugs on cyclic nucleotide levels in HF and CP were observed at 30 min and at 24h after drug administration. Thirty minutes after drug injection, we observed an increase of cAMP level, both in HF and in CP. Twenty-four hours after the retention test, we observed that in CP the cAMP intracellular level remained high, while in the HF at effective doses both inhibitors induced cAMP PDE activity, determining a decrease of cyclic nucleotide. Semi-quantitative RT-PCR analysis, together with Western blot immunodetection, showed a mRNA and protein induction of PDE4D PDE isoforms, that may account for the increase of PDE activity observed. Our data suggest that, despite cyclic nucleotide increase at 30 min, the fundamental event causing memory impairment, came from the subsequent long time decrease of cAMP levels, due to the post-translational PDE4D induction.

PDE10A and PDE10A-dependent cAMP catabolism are dysregulated oppositely in striatum and nucleus accumbens after lesion of midbrain dopamine neurons in rat: a key step in parkinsonism physiopathology.

Loss of dopamine neurons in experimental parkinsonism results in altered cyclic nucleotide cAMP and cGMP levels throughout the basal ganglia. Our objective was to examine whether expression of phosphodiesterase 10A (PDE10A), an isozyme presenting a unique distribution in basal ganglia, is altered after unilateral injection of 6-hydroxydopamine in the medial forebrain bundle, eliminating all midbrain dopaminergic neurons, such that cyclic nucleotide catabolism and steady state could be affected. Our study demonstrates that PDE10A mRNA levels were decreased in striatal neurons 10 weeks after 6-hydroxydopamine midbrain lesion. Such changes occurred in the striatum ipsilateral to lesion and were paralleled by decreased PDE10A protein levels and activity in striatal neurons and in striato-pallidal and striato-nigral projections. However, PDE10A protein and activity were increased while PDE10A mRNA was unchanged in the nucleus accumbens ipsilateral to the 6-hydroxydopamine midbrain lesion. Accordingly, cAMP levels were down-regulated in the nucleus accumbens, and up-regulated in the striatum ipsilateral to the lesion, but they were not significantly changed in substantia nigra and globus pallidus. Unlike cAMP, cGMP levels were decreased in all dopamine-deafferented regions. The opposite variations of cAMP steady state in striatum and nucleus accumbens are concordant and likely dependent, at least in part, on the down-regulation of PDE10A expression and activity in the former and its up-regulation in the latter. On the other hand, the down-regulation of cGMP steady state in the striato-nigral and striato-pallidal complex is not consistent with and is likely independent from the concomitant down-regulation of PDE10A. Therefore, dopamine loss inversely regulates PDE10A gene expression in the striatum and PDE10A post-transcription in the nucleus accumbens, therein differentially modulating PDE10A-dependent cAMP catabolism.

Immunohistochemical localisation of PDE5 in Leydig and myoid cells of prepuberal and adult rat testis

Expression of phosphodiesterase 5 (PDE5) in the rat testis at several pre and postnatal developmental stages was investigated by immunohistochemistry. The enzyme was localised in vascular smooth muscle cells, as well as in Leydig and peritubular cells. The latter were identified as myoid, based on their immunoreactivity to desmin and α-smooth muscle actin. The presence of PDE5 in myoid cells was confirmed by Western blot analysis and immunohistochemistry performed on highly purified cell fractions, obtained from 16-day-old rats. The expression of PDE5 in these somatic cells of rat testis is discussed in view of the roles played by cGMP signal transduction pathways in the mammalian male reproductive function.

Aflatoxin B1 is an inhibitor of cyclic nucleotide phosphodiesterase activity

Aflatoxin B1 (AFB1) action on cyclic nucleotide phosphodiesterase (PDE) activity has been tested on tissue extracts of various organs. In the presence of 100 microM AFB1 a significant inhibition of cAMP and cGMP hydrolytic activity is observed in all tested tissue extracts. However, cGMP hydrolytic activity appears more sensitive to AFB1 inhibition than cAMP hydrolytic activity and a considerably higher inhibition is observed in lung and spleen, than in liver, brain, kidney, and heart. When cGMP is used as substrate, the inhibitory response reaches 72% in lung and spleen extracts. We have also tested AFB1 effects on lung and liver PDE activity peaks separated by DEAE-cellulose chromatography. These data confirm the poor sensitivity to the toxin of all PDE activities present in liver, while the lung peak (where PDE V in present) shows a higher sensitivity to AFB1. In order to establish whether PDE V is in fact more sensitive to AFB1, we have used mouse neuroblastoma cells, in which cGMP hydrolytic activity has been shown to be due to PDE V only. In this case, the calculated IC50 is 24 microM and Dixon plot analysis shows a competitive inhibitory effect with a Ki of 16.7 microM. We have also used aflatoxin B2 and M2, and they proved to be much less effective than AFB1: AFB2 inhibits PDE V with an IC50 of 117 microM, while AFM2 does not show any effect. These results provide the first evidence of a competitive inhibition of AFB1 on an enzymatic activity and suggest that an alteration of cellular cyclic nucleotide levels may play a role in the mechanism of aflatoxin action.

Regulation of phosphodiesterase 5 expression and activity in human pregnant and non-pregnant myometrial cells by human chorionic gonadotropin

Objectives: This study has a twofold aim: 1) to investigate whether protein expression and enzyme activity of phosphodiesterase 4 (PDE5) can be detected in human myometrium and undergo changes in relation to the presence of pregnancy and/or labor; 2) to evaluate whether PDE 5 expression and activity in myometrial cells can be influenced by human chorionic gonadotropin (HCG). Methods: Primary cultures of myometrial cells, obtained from non-pregnant women and from pregnant women at tem, either before or during labor, were carried out in the presence of HCG or dibutyryl-cyclic AMP (ad-cAMP), the non-hydrolizable analogue of cAMP. PDE5 expression in cultures of myometrial cells was detected by immunocytochemistry and western blot. PDE5 activity was detected in cell extracts by enzyme assay. Results: PDE5 is expressed and is functionally active in smooth myscle cells. Treatment of cell cultures with HCG and db-cAMP results in a reduction of PDE5 expression and activity. The effects of HCG and db-cAMP are exerted irrespective of the functional status of the myometrium (n0n-pregnant, pregnant not in labor, pregnant in labor). Conclusions: PDE5 protein is expressed in human non-pregnant and pregnant myometrium. HCG reduces PDE5 expression and enzyme activity in smooth muscle cells, possibly through a pathway involving cAMP

Differential expression and localization of calmodulin-dependent phosphodiesterase genes during ontogenesis of chick dorsal root ganglion.

The level and characteristics of 3′−5′‐cyclic nucleotide phosphodiesterase (PDE) activity in chick dorsal root ganglion (DRG) extracts of 5‐day posthatching chicken (P5) and E10 and E18 embryos were studied. At all stages, PDE activity is stimulated by calcium and calmodulin. A 5‐fold increase in basal cAMP and cGMP PDE activity is evident from E10 to E18, while from E18 to P5 basal PDE activity remains constant. Ion exchange chromatography elution profile indicates that PDE1 isoforms represent the bulk of the PDE activity present. Inhibition studies were performed in order to distinguish the activity due to PDE1A, B and C. Western blot analysis using anti‐mammalian PDE1A, B and C specific antibodies was also performed. Densitometric analysis of the stained bands reveals that PDE1B and PDE1C display a prominent increase between day 10 and day 18 of development (eight‐ and 3.6 fold, respectively) while a more limited increase (1.6‐ and 1.5‐fold) is observed between E18 and P5; on the other hand PDE1A shows continuously increasing levels throughout development. Immunohistochemical analysis was performed with isoform specific antibodies used for western blot analysis. PDE1A immunoreactivity is found in the cytoplasm and fibers of several neurons differing in size and distributed throughout the ganglion. PDE1B staining is evident on all neurons, however, fibers appear very faintly labelled. All neurons appear stained by PDE1C antibody, although the intensity of immunostaining is always heterogeneous in different neuronal populations: no staining was evident on fibers or in non‐neural cells. The distinct spatial and temporal expression patterns of PDE1 isoforms may indicate their different physiological roles in developing and mature chick DRG.

Induction of cyclic AMP and cyclic GMP 3′:5′-cyclic nucleotide phosphodiesterase activities in neuroblastoma lines under differentiating conditions

It is now widely accepted that cyclic nucleotide phosphodiesterases (PDEs) play fundamental roles in signal transduction pathways; they show a remarkable molecular complexity, different tissue distribution and complex regulatory mechanisms. Here we report PDE isoforms expression in two dibutyryl cyclic AMP differentiated murine cell lines: the hybrid neuroblastoma‐glioma 108CC15 and the parental neuroblastoma N18TG2. They differ for the ability to establish functional synapses, a feature present only in the former. Ionic exchange chromatography elution profiles of N18TG2 and 108CC15 undifferentiated cell extracts show two main peaks of activity. The first one hydrolyzes cyclic GMP and is specifically inhibited by Zaprinast, thus representing a member of the PDE5 family. The second peak hydrolyzes cyclic AMP and is significantly inhibited by rolipram, as all the PDE4 family members. The induction of differentiation by dibutyryl cyclic AMP in both clonal lines results in an increase of PDE activities only after 3 hr of treatment, suggesting that protein neosynthesis is involved. Interestingly in both clones, besides the increase in cyclic AMP hydrolyzing specific activity (3.1 folds in 108CC15 and 2.5 folds in N18TG2), we also observed an increase in cyclic GMP hydrolyzing activity (1.7 folds in 108CC15 and 4.3 folds in N18TG2). While the induction of PDE4, previously reported also in other cellular systems, could be considered as a feedback response to the higher cyclic AMP levels, this is not true for the isoform that hydrolyzes cyclic GMP. These data suggest that the induction of PDE isoforms in neuroblastoma cells could be related to the activation of neuronal differentiative pathway.