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Regulatory importance of cyclic nucleotides in smooth muscle growth of the urogenital tract
Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Smooth muscle hyperplasia/hypertrophy is, if not responsible for, so at least involved in those diseases, which impair life quality for most people in today's society.

This thesis, presents a pharmacological investigation, related to the regulatory role of cyclic nucleotides, of smooth muscle hyperplasia/hypeitrophy in human uterine leiomyoma and benign prostate hyperplasia (BPH).

Four main aspects, with cAMP as a connecting thought, have been analyzed, namely expression and characterization of the adrenergic receptors (AR), determination of adenylyl cyclase (AC)- and phosphodiesterase (PDE)-activity, and finally the connection between mentioned issues and proliferation of cultured smooth muscle cells (smc).

In the frrst paper, characterization of the a 2-adrenergic receptor (az-AR) subtypes in human myometrium at term pregnancy was examined by combining radioligand binding-studies with reverse transcriptase-polymerase chain reaction (RT-PCR). Results demonstrated a significant eo-expression of α2A and α2B, and a weak indication of the α2C-AR, which however was identified at the mRNA level by the RT-PCR analysis.

In the next investigatio~ smooth muscle tissue of human uterine leiomyoma (benign smooth muscle tumor) was compared with surrounding myometrial tissue (control). The expression of AR, AC- and PDE-activity was analyzed, as well as the effect of cAMP with respect to growth regulation of cultured leiomyoma smc. Primarily, a significantly reduced ß2-AR expression and AC-activity was detected in leiomyoma compared to control tissue, whereas the PDE-activity was approximately 100% higher. In addition, the α2-AR population in leiomyoma was slightly increased. When cultured leiomyoma smc was treated with cAMP increasing agents as forskolin, an AC stimulating agent, or papaverin, a general PDE inhibitor, a considerable inhibition of DNA replication and protein synthesis was obtained.

In the thh·d paper, a proliferation study was made on cultured benign prostate hyperplasia smc, were the mitogen effect of lysophosphatidic acid (LPA) and cAMP/cGMP increasing agents was investigated. LPA generated a dose-dependent mitogen response, which was efficiently inhibited, both by forskolin, and by papaverin. In addition, sildenafil (Viagra®), which serve as a potent and selective PDE5 inhibitor, also decreased the LPA mediated growth promotion in a dose dependent manner.

The last study, demonstrate primarily the expression pattem of LPA receptors (Edg) in BPH smc. Further, the intracellular cAMP changes in LPA stimulated BPH smc and the proliferative effect of the LPA analogue sphingosine 1-phosphate (SIP) was considered. First, all Edg was identified with exception of Edg6. Moreover, the cAMP level was unchanged by LPA per se, whereas co-incubation with forskolin generated a rapid and transient response. Further, SIP generated a divergent response including a LPA equivalent mitogen effect at low concentrations whereas inhibition of DNA replication was obtained at higher concentrations.

In summary, this project demonstrates that cyclic nucleotides inhibit smooth muscle hyperplasia/hypertrophy in the luogenital tract. These results also suggest that manipulation of cyclic nucleotide level using tissue specific PDE inhibitors might constitute a new therapeutic approach for hyperplasia/hypertrophy related diseases in the urogenital tract.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2001. , 45 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 694
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-27452Local ID: 12105ISBN: 91-7373-136-6 (print)OAI: oai:DiVA.org:liu-27452DiVA: diva2:248004
Public defence
2001-11-02, Berzeliussalen, Universitetssjukhuset, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-22Bibliographically approved
List of papers
1. Characterization of α2-Adrenoceptor Subtypes in Pregnant Human Myometrium
Open this publication in new window or tab >>Characterization of α2-Adrenoceptor Subtypes in Pregnant Human Myometrium
1998 (English)In: Gynecologic and Obstetric Investigation, ISSN 0378-7346, E-ISSN 1423-002X, Vol. 45, no 3, 145-150 p.Article in journal (Refereed) Published
Abstract [en]

The aim of the present investigation was to determine which subtypes of the α2-adrenoceptors are being expressed in the human pregnant myometrium at term pregnancy. In radioligand binding studies, the specific binding of [3H]rauwolscine to human myometrial membranes was specific and of high affinity with Kd of 2.8 ± 0.6 nM and Bmax of 95 ± 5 fmol/mg protein. Results from competition for the binding of [3H]rauwolscine using subtype-selective ligands, oxymetazoline (α2A-subptype), chlorpromazine (α2B-subtype) and prazosin (α2B-α2C-subtype), suggested that the α2A- and α2B-subtypes are being co-expressed. In order to examine if also the α2C-subtype is being expressed we used an optimal concentration of oxymetazoline or chlorpromazine which would block the high-affinity site, equivalent to the α2A- and α2B-subtype respectively. Competition curves of both oxymetazoline and chlorpromazine still showed a significantly better fit using a two-site model, suggesting that the α2C-subtype also is being expressed. The expression of α2C-subtype mRNA was confirmed using reverse transcription-polymerase chain reaction on mRNA isolated from myometrial biopsies.

In conclusion, our results suggest that all three subtypes of α2-adrenoceptors are being coexpressed in the human myometrium at term pregnancy and that α2-expression is dominated by the α2A-subtype.

Keyword
α2-Adrenergic receptors, Subtype, Myometrium, Human, Pregnancy, [3H]rauwolscine, Reverse transcription-polymerase chain reaction
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-80081 (URN)10.1159/000009944 (DOI)
Available from: 2012-08-20 Created: 2012-08-20 Last updated: 2017-12-07Bibliographically approved
2. Changes in β2-adrenoceptor expression and in adenylyl cyclase and phosphodiesterase activity in human uterine leiomyomas
Open this publication in new window or tab >>Changes in β2-adrenoceptor expression and in adenylyl cyclase and phosphodiesterase activity in human uterine leiomyomas
2000 (English)In: Molecular human reproduction, ISSN 1360-9947, E-ISSN 1460-2407, Vol. 6, no 9, 835-842 p.Article in journal (Refereed) Published
Abstract [en]

Uterine leiomyoma is a very common benign tumour with unclear pathophysiology in adult women. In the present study we have investigated the expression level of α2- and β2-adrenoceptors, and the adenylyl cyclase and phosphodiesterase activity in leiomyoma tissue compared with adjacent myometrium. Our results show that the α22-adrenoceptor ratio is increased in leiomyoma, due to a significant decrease in β2-adrenoceptor expression. These changes were not due to an increased innervation, as the tumour tissue was completely devoid of nerve fibres. Moreover, the adenylyl cyclase activity of leiomyoma membranes was found to be ~50% lower, whereas the phosphodiesterase activity was significantly increased (by ~100%). We found that stimulating an increase in intracellular cyclic AMP, by adenylyl cyclase activity through β2-adrenoceptors (isoprenaline), by direct enzyme activation (forskolin), or by inhibition of phosphodiesterase activity (papaverine), potently blocked both protein and DNA synthesis in cultured leiomyoma smooth muscle cells. Our results imply the adrenoceptors might be involved in, or a consequence of, leiomyoma growth. The results also suggest a new interesting approach for leiomyoma pharmacotherapy.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25070 (URN)10.1093/molehr/6.9.835 (DOI)9500 (Local ID)9500 (Archive number)9500 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
3. Lysophosphatidic acid stimulates proliferation of cultured smooth muscle cells from human BPH tissue: Sildenafil and papaverin generate inhibition
Open this publication in new window or tab >>Lysophosphatidic acid stimulates proliferation of cultured smooth muscle cells from human BPH tissue: Sildenafil and papaverin generate inhibition
2002 (English)In: The Prostate, ISSN 0270-4137, E-ISSN 1097-0045, Vol. 51, no 1, 50-58 p.Article in journal (Refereed) Published
Abstract [en]

Background The endogenous substance lysophosphatidic acid (LPA) has been found to generate proliferation of cultured smooth muscle cells (SMC). Therefore, the effect of LPA on human benign prostate hyperplasia (BPH) could be of interest.

Methods The proliferative effect of LPA on cultured human prostatic SMC from specimens obtained at trans-urethral resection of the prostate (TURP) because of BPH, was analyzed by [3H]-thymidine and [35S]-methionine incorporation. In addition, LPA stimulated BPH SMC were treated with papaverin, forskolin, sildenafil or zaprinast, well known to increase the intracellular level of cAMP or cGMP.

Results LPA produced a dose-dependent increase in BPH SMC, both regarding DNA- and protein-synthesis with EC50 values of 3 and 10 μM, respectively. Furthermore, both papaverin, a general phosphodiesterase inhibitor regarding cAMP hydrolyzes, and forskolin, an adenylyl cyclase stimulating agent, inhibited the LPA-stimulated DNA replication in a dose dependent manner with IC50  = 2.5, and 0.35 μM, respectively. cGMP increasing agents, such as the NO-donors SIN-1 and SNAP, produced a weak anti-proliferative response. However, both phosphodiesterase 5 inhibitors sildenafil (Viagra®) and zaprinast efficiently blocked DNA replication. In addition, when the protein synthesis was examined, we found that the LPA response was significantly inhibited by forskolin and papaverin.

Conclusions The major conclusion of this investigation is that the endogenous serum component LPA, is able to promote human BPH SMC growth. In addition, our study indicates that cyclic nucleotides can inhibit this effect. Future clinical studies will be needed to determine if different specific phosphodiesterase inhibitors per se or in combination could represent a new therapeutic possibility for the treatment of BPH.

Keyword
BPH, SMC, hyperplastic, PDE, AC, cAMP, cGMP, [H-3]-thymidine, [S-35]-methionine, proliferation
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-47887 (URN)10.1002/pros.10077 (DOI)
Note
On the day of the defence day the status of this article was submitted.Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13Bibliographically approved
4. Characterization of EDG receptor expression and proliferative response in cultured human BPH smooth muscle cells
Open this publication in new window or tab >>Characterization of EDG receptor expression and proliferative response in cultured human BPH smooth muscle cells
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The endogenous phospholipids, lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are both known to generate a Vvide variety of effects in various cell systems by the endothelial differentiation gene (Edg) receptor family, including 7 different G-protein coupled Edg receptors.

In this study, expression of LPA- and SlP Edg receptors was examined, and so was the effect with respect to proliferation on cultured BPH smooth muscle cells smc. Mmeover, theresponse on cAMP levels was examined. Finally, a potential link between activation of the MAP kinase cascade and the LPA stimulated proliferation was investigated.

First, the RT-PCR analysis of the Edg receptors in BPH smc, demonstrated a heterogeneous expression including all receptors except the Edg6 subtype. Further, in contrast to LPA, the mitogen effect of SIP, demonstrated a concentration-dependent biphasic response, including stimulation below 1μM, whereas inhibition was obtained at higher concentrations. Forskolin induced a rapid and transient cAMP response in LPA stimulated cells, with a peak-value after 3 minutes. After 15 minutes the cAMP level had retmned to base-line level. However a gradual increase to 15% of maximum value was obtained after additional 30 minutes, and thereafter a gradual reduction was observed. The mentioned antiproliferative response generated by SIP could not be conelated to an intracellular cAMP increase. Finally, when the LPA treated smc was co-incubated with the MAPK kinase inhibitor PD98059 (10 μM) the mitogen response was eliminated.

The cAIVIP increase, which was induced by forskolin, corresponds with mentioned antiproliferative effect whereas a similar con-elation was not obtained regarding SIP. The intracellular signal mechanisms triggered by LPA and S1P in BPH smc remain to be further investigated.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-80084 (URN)
Available from: 2012-08-20 Created: 2012-08-20 Last updated: 2012-08-20Bibliographically approved

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