Role of oestrogens in male erectile function 4

Chapter 9 EFFECT OF OESTRADIOL AND DAIDZEIN ON CYCLIC NUCLEOTIDES IN RAT CORPUS CAVERNOSAL SMOOTH MUSCLE CELL CULTURES... EFFECT OF OESTRADIOL AND DAIDZEIN ON CYCLIC NUCLEOTIDES IN RAT

Chapter 9 EFFECT OF OESTRADIOL AND DAIDZEIN ON CYCLIC

NUCLEOTIDES IN RAT CORPUS CAVERNOSAL

SMOOTH MUSCLE CELL CULTURES

9 EFFECT OF OESTRADIOL AND DAIDZEIN ON CYCLIC

NUCLEOTIDES IN RAT CORPUS CAVERNOSAL SMOOTH MUSCLE CELL CULTURES

9.1 Objectives

Cyclic nucleotides, cAMP and cGMP are well known second messengers for a number of intra and extracellular mechanisms involving neurotransmitters, hormones, chemical and biochemical agents (Soderling et al., 1998) At the cellular level, the responses of these second messenger systems are mediated through their respective intracellular targets such

as kinases, ion channels, transcriptional activators and limited by the activity of specific phosphodiesterase (PDE) isoforms (Beavo, 1995) including those within the cavernosum (Kuthe et al., 2001) The net tissue response is a function of the balance between the rates

of synthesis by respective cyclases and rate limiting degradation through PDEs to inert 5’ monophosphate nucleosides together with possible cross-talks in the two signal transduction pathways (Stief et al., 2000) In this regard, the aim of this investigation was to identify the role of oestrogens as a possible modulator of these two important intracellular cyclic nucleotides of the penile cavernosum and elucidate its relevance to other in vitro and in vivo findings from this study

9.2 Materials and Methods

9.2.1 Primary Cell Culture

9.2.1.1 Preparation of the Medium

Commercially available Dulbecco’s modified eagle medium (DMEM) was used for establishing the primary cavernosal smooth muscle cell culture The medium was

composed of 1g/L of D-glucose, L-glutamine, pyridoxine hydrochloride and 110mg/L of

sodium pyruvate (GIBCO BRL) It was subsequently reconstituted with 10% fetal bovine serum (Summit Biotechnology), antibiotics - penicillin (100units/ml) and streptomycin

(100µg/ml) (Sigma) and antifungal agent (amphotericin B, 250ng/ml Sigma) and stored between 2°C and 8°C The required volume was warmed to 25°C-30°C prior to use

9.2.1.2 Laminar Flow Hood and CO2 Incubator

Class II biology safety cabinet which provides a sterile working atmosphere was used in this investigation The built-in high efficiency antibacterial filter of the cabinet helped to remove gross contaminants The filtered air was then compressed and channeled through

a high efficiency particulate air (HEPA) filter in a laminar flow fashion This provided a continuous flow of purified air over the working area in parallel lines at a uniform velocity The HEPA filter also served to remove the bacteria from the circulating air Simultaneously, the aerosols generated during experimentation within the hood were also filtered out before reaching the surrounding environment The working area of the hood was sterilized with short wave ultraviolet light for ten minutes before procedures and the laminar hood was left turned on for at least twenty minutes before use The working

surface was constantly cleansed with 70% ethanol before, in between and after use

A carbon dioxide gas chamber was utilized to grow and maintain tissue explants plated in culture flasks The chamber was maintained at optimal conditions needed for the growth

of cells (5% - 10% CO2) The culture flasks were lightly closed to allow sufficient exchange of gases The temperature in the incubator remained constant at 37°C maintained through a water-jacketed heating system The chamber was additionally

humidified using a tray filled with ultra pure water The culture flasks were left

undisturbed for 48 – 72 hours and the reconstituted DMEM was replenished based on tissue utilization

9.2.1.3 Tissue Collection and Processing

Corpus cavernosal smooth muscle cells were cultured from the penile tissue of SD rats The animals were procured from Laboratory Animal Centre and were maintained at the Animal Holding Unit The rats were sexually mature adult males and weighed between 300g – 350g They were anaesthetized by an intraperitonial injection of pentobarbital sodium (45mg/kg body weight) After cleansing the perigenital area with iodine solution (Betadine®), a transverse skin incision of about 1cm was made over the perineum and the subcutaneous tissues were cleared by blunt dissection to expose the crus of the penis Penile tissue extending from the crus to mid shaft was then removed and quickly transferred to a sterile petridish containing Hank’s balanced salt solution free of calcium and magnesium (HBSS, GIBCO BRL)

Through fine dissection, the penile tissue was separated from the surrounding connective tissues and the corpus spongiosum The tissue was washed well with HBSS to remove blood, tissue debris and other contaminants and transferred to another sterile petridish containing the reconstituted DMEM Under the strict aseptic conditions of the laminar hood, the tunica albuginea was slit open to expose the corpus cavernosum smooth muscle, which was then excised The tissue was immediately minced to minute pieces of less than 1mm diameter (Chamley-Campbell J et al., 1979) and washed 2-3 times with the reconstituted medium It was then plated into a 75cm2 tissue culture flask (Nunc, USA) containing 15ml of the reconstituted DMEM, partially closed and kept in the CO2chamber

The culture flasks were examined under an inverted phase contrast microscope regularly

to monitor cell growth and attachment Before taking out of the chamber, the flasks were tightly closed to avoid contamination during transport to the microscope As a follow up,

the outer surface of the flasks and their caps were duly cleansed with ethanol before placing them back in the chamber and partially opening the caps The light source and condenser of the inverted microscope are above the stage and downwardly directed The objective and the turret are pointed from below the stage to facilitate direct and clear visualization of cell attachment to the substratum through the bottom of the flask Smooth muscle cell characterization and confirmation were done through immunohistochemistry

in an earlier investigation (Gauthaman, 2002)

9.2.1.4 Trypsinization

Continuous proliferation of cells from these explant tissues will result in confluence associated with saturation of all available space with attached cells Any overgrowth at this stage will lead to cell death due to lack of space for migration This is the appropriate time for further propagation of these cells through detachment from the flask and subsequent transfer This process is referred to as trypsinization since it uses trypsin – ethylene diamine tetra acetic acid (EDTA) for enzymatic dissociation of these cells from adherent cell lines without loss of cellular integrity Trypsin-EDTA solution (Sigma) used in this investigation contained 5.0g/L of Trypsin (1: 250), 2g/L of EDTA.4Na in 0.9% NaCl Prior to this procedure, the culture medium from the flask was discarded and the cells were washed with HBSS by gently rocking the flasks After discarding the wash solution, the trypsin-EDTA solution (6-9ml/75cm2 flask) was added and the flasks were gently rocked and placed in 37°C incubators for 5-15 minutes to ensure complete cell dissociation; the elongated cells would appear as rounded illuminant bodies under the inverted microscope Following complete detachment of all the cells visualised as floating clumps, an equal volume of the reconstituted medium was added to stop

continuing trypsin activity and the flasks were kept upright for the cells to settle at the bottom The cells were then collected by pipetting from the surface of the monolayer

9.2.1.5 Subculture and Cell Counts

The detached cells were then transferred to 15ml tubes and centrifuged at 500g in 15°C for 5 minutes using a bench top centrifuge (MSE Mistral 2000, Sanyo) The supernatant medium was discarded and the pellet was reconstituted in 1ml of fresh DMEM From this volume, fixed numbers of cells were taken out using micropipettes and plated into fresh 75cm2 flasks for further propagation

Cell viability in subcultures was routinely monitored using the Trypan blue exclusion principle In this staining method, the living cells would exclude the dye due to the presence of a negatively charged intact membrane and appear clear and transilluminant

In contrast, the dead cells with damaged membranes would transmit the dye and appear blue To count the number of viable cells, a cell suspension (20µl) following trypsinization was treated with an equal volume of Trypan blue (2 fold dilution) and mixed well It was left undisturbed for 5 minutes Haemocytometer (Neubauer’s chamber) and the cover slip were gently cleansed with 70% ethanol before use and a drop

of the Trypan blue-cell mixture was carefully added to the edge of the cover slip placed

on the Neubauer’s chamber The fluid was seen to immediately fill up the counting chamber through capillary suction Under the microscope, the cells present in the middle 1mm square and the four corner squares were counted leaving out the dead cells The procedure was repeated if more than 10% of cells appeared in clumps By convention, only the cells on the top and left side grids were included in a square and those on the bottom and right side grids were excluded

The number of cells and % viability were calculated as follows

Total number of viable cells = A x B x C x 104

Total number of dead cells = A x B x D x 104

Total cell count = Viable cell count + Dead cell count

(A: volume of cells (suspension), B: dilution factor, C: number of unstained cells and D: number of stained cells; 104 was the conversion factor for 0.1mm3 to ml)

% Viability: Viable cell count

x 100 Total cell count

9.2.2 cAMP and cGMP Assays

Intracellular levels of the two cyclic nucleotides, cAMP and cGMP were estimated in the cultured rat corpus cavernosal smooth muscle cells Using the calculation, a known number of CCSM cells (2.5 x 105 cells per well) were seeded in 24 well plates containing 0.5ml of DMEM The cultures were maintained overnight in the CO2 incubator for further proliferation and confluence Following confirmation under the inverted microscope, the cells were washed with HBSS and incubated in fresh, foetal calf serum-free DMEM for 30 minutes The cells were then incubated with a non-specific PDE inhibitor isobutyl methyl xanthine (IBMX) at a concentration of 250µM/L for 15 minutes before adding specific drugs This agent prevented the breakdown of intracellular cyclic nucleotides by the endogenous PDE activity Some of the wells were incubated with either nitroglycerine or prostaglandin E1 (3.5ng – 0.35µg) as positive controls to stimulate the synthesis of cGMP and cAMP respectively Others were incubated either with oestradiol valerate or daidzein suspension in sterile water (1µg - 100µg) to assess their individual roles on the synthesis of these intracellular nucleotides The CCSM cells were incubated with respective pharmacological agents for 15 minutes The cells were then transferred to antibody coated ELISA plates and standard assay protocol was carried

out with the respective conjugates and antibodies The levels of the cyclic nucleotides were then evaluated by enzyme immunoassay (R&D Systems USA, Appendix 6 and 7)

9.2.3 Data Analysis

The activity variables for different drug incubations were analyzed by paired samples T test for appropriate comparison All the results were expressed as mean ± SEM and the level of significance was taken at p<0.05

9.3 Results

9.3.1 Status of Primary Culture

The primary explant tissue from the rat corpus cavernosal smooth muscle grew to complete confluence without any incidence of contamination when cultured in reconstituted DMEM under the standard CO2 chamber conditions The culture flasks were viewed under the inverted microscope daily for the status of attachment to the substratum and consequent proliferation The smooth muscle cells were seen to radiate in different directions from the attached tissue and the medium on discolouration was appropriately replenished to aid unhindered growth

9.3.1.1 Cell Migration from Explants

Cell migration from explants was noticed around 7 – 10 days; this was followed by attachment to the substratum, further proliferation and gross increase in number of cells under all fields The cells were doubling approximately in 48 – 72 hours They were all typical smooth muscle cells characterized by spindle shapes and central nuclei with firm attachment to the substratum Extensive proliferation of these cells led to the formation of

a monolayer of gradual confluence (Figures 60-62)

Figure 60: Primary Explant Corpus Cavernosum Smooth

Muscle Cell Culture on Day 7

Typical spindle-shaped smooth muscle cells (black arrows)

growing and proliferating from the primary explant (white arrow)

at 1 week (x100)

Figure 61: Primary Explant Corpus Cavernosum Smooth

Muscle Cell Culture on Day 10

Indicates extensive proliferation and attachment of the smooth

muscle cells (black arrows) from the primary explant (white arrow)

to the substratum at 10 days (x100)

Figure 62: Primary Explant Corpus Cavernosum Smooth

Muscle Cell Culture on Day 14

Proliferation and confluence of smooth muscle cells (black arrows)

in the primary cell culture at the time of trypsinization in 2 weeks

(x100)

9.3.1.2 Subculture Viability

The cells on trypsinization were completely detached and appeared as rounded translucent bodies in multiple clumps in all fields The fixed number of viable cells counted by Trypan blue staining and plated into fresh 75cm2 flasks continued to proliferate and attached to the substratum firmly Upon reaching confluence and further subculturing, there were adequate cells for the assay procedure of estimation of cyclic nucleotides All the cells at subculture retained their original growth characteristics and only up to two subcultures were used for the study

9.3.2 Effect on cAMP Levels

The CCSM cells on incubation with varying concentrations of prostaglandin E1, oestradiol and phytoestrogen daidzein produced an increase in the levels of intracellular cAMP PGE1, a known stimulator of cAMP production in the corpus cavernosum was used as a positive control in the concentration range of 0.0035µg/ml to 0.35µg/ml The increases in cAMP release observed for the PGE1 concentrations of 0.0035µg/ml, 0.035µg/ml and 0.35µg/ml were 32%, 51.5% and 38.3% respectively This was with reference to the inherent cAMP production in untreated control cells and the changes at 0.035µg/ml and 0.35µg/ml of PGE1 were statistically significant (Figure 63)

0 4 8 12 16 20

Cavernosum Smooth Muscle Cells

Indicates significant increase in intracellular mean cyclicadenosine monophosphate (cAMP) release at 0.035µg/ml and 0.35µg/ml of Prostaglandin E1 incubation (*P<0.05) of rat corpus cavernosal smooth muscle cell cultures

Both oestradiol and daidzein were usedin the concentration range of 1µg/ml to 100µg/ml

in the respective wells for stimulation of cAMP formation Oestrogenic activity seemed

to increase the level of cAMP production at almost all the dosages tested However, unlike the stimulatory effect of PGE1, these changes were less marked and statistically insignificant The mean maximal increases for oestradiol incubations in comparison with the control value were 2.3%, 32.8%, 9.7% and 3.6% respectively for 1, 3, 10 and 100µg/ml of oestradiol valerate used For the same concentrations of phytoestrogen, there was slight decrease in cAMP production at 1µg/ml (-4.2%) however at the other three higher concentrations, the increases were respectively 25.4% (3µg/ml), 41% (30µg/ml) and 34% (100µg/ml) None of these changes were statistically significant thereby indicating that further dosage titrations are required (Figure 64)

0 4 8 12 16

Figure 64: Cyclic Adenosine Monophosphate Level Secondary

to Oestradiol and Daidzein in Cultured Rat Corpus

Cavernosum Smooth Muscle Cells

Increase in intracellular cyclic adenosine monophosphate (cAMP)

release was seen following incubation of cultured smooth muscle

cells with oestradiol or daidzein The difference compared to the

cAMP production in normal culture (control) was not statistically

significant

9.3.3 Effect on cGMP Levels

The CCSM cells on incubation with varying concentrations of nitroglycerine, oestradiol and daidzein produced an increase in the levels of intracellular cGMP for NTG and phytoestrogen and a decrease in cGMP release for all doses except 3µg/ml of oestradiol However, none of these changes attained statistical significance thereby indicating that further titrations in concentrations of the individual agents are required

NTG, a known stimulator of cGMP production in the corpus cavernosum was used as a positive control in the concentration range of 0.0035µg/ml to 0.35µg/ml Compared to the inherent cGMP production in untreated control cells, there was a definite increase in the level of cGMP at 0.035µg/ml and 0.35µg/ml The mean maximal increases in cGMP

release observed for the NTGconcentrations of 0.0035µg/ml, 0.035µg/ml and 0.35µg/ml were 1.25%, 25.68% and 27.39% respectively (Figure 65)

0 2 4 6 8 10 12 14

Cavernosum Smooth Muscle Cells

Indicates increase in intracellular mean Cyclic GuanosineMonophosphate (cGMP) release at 0.035µg/ml and 0.35µg/ml

of nitroglycerine incubation of rat corpus cavernosal smooth muscle cell cultures

Both oestradiol and daidzein were used in the same concentration range of 1µg/ml to 100µg/ml as in cAMP studies, for testing their effects on cGMP formation Oestrogenic activity seemed to decrease the level of cGMP production at all but one of the doses tested The mean difference for oestradiol incubations in comparison with the control value were -3.3%, 22.4%, -9.6% and -47.8% respectively for 1, 3, 10 and 100µg/ml of oestradiol valerate used For the same concentrations of phytoestrogen used, there was slight decrease in cGMP production at 1µg/ml (-9.2%) however at the next three higher concentrations, there was a dose-dependent increase in cGMP as seen by the mean

maximal responses of 11.9% (3µg/ml), 14.8% (30µg/ml) and 18.6% (100µg/ml)

respectively However, none of these changes were statistically significant (Figure 66)

0 2 4 6 8 10 12

Figure 66: Cyclic Guanosine Monophosphate Level Secondary

to Oestradiol and Daidzein in cultured Rat Corpus

Cavernosum Smooth Muscle Cells

Increase in intracellular Cyclic Guanosine Monophosphate

(cGMP) release was seen following incubation of cultured smooth

muscle cells with higher doses of daidzein Oestradiol induced a

biphasic response consisting of increase at low doses (1µg and

3µg) and decrease at higher doses (30µg and 100µg) The

difference compared to the cGMP production in normal culture

(control) was not statistically significant

9.4 Discussion and Conclusion

It is presently evident that nitric oxide (NO) plays an important role in the mediation of penile erection Within the corpus cavernosum, it is released from the non-adrenergic non-cholinergic (NANC) neurons and also from the endothelium in response to activation

of the respective NO synthase (Adaikan et al., 1991a; Burnett et al., 1992) Nitric oxide causes relaxation of the cavernosal smooth muscle through activation of guanylate cyclase and formation of cyclic GMP thereby inducing penile erection In turn,

erectile dysfunction Erection is also mediated through an alternative system, the cAMP pathway by endogenous vasodilator prostaglandins (PGs) formed by the endothelium as well as the smooth muscle component of the blood vessels The most convincing evidence for the role of prostaglandins in erectile function is the fact that intracavernosal injection of PGE1 is an effective form of ED management (Adaikan et al., 1986b) and PGI2, formed in the human CC in response to activation by parasympathomimetics in turn stimulates the release of cyclic AMP (Jeremy et al., 1986; Miller et al., 1994) At the cellular level, the final levels of the two important nucleotide mediators of penile erection viz., cGMP and cAMP are under the control of phosphodiesterases which in turn metabolise them to their respective inactive forms (Jeremy et al., 1997)

Through studies attempting to understand the relative contribution to penile erection by the two pathways, it was found that although PGE1 induced penile tumescence through cAMP system in humans, it failed to produce an adequate erectile response in rats (Martinez-Pineiro et al., 1993) thereby indicating the possibility of species variations in cAMP system mediated penile erection In the same rat model, papaverine (a non-specific PDE inhibitor) induced penile erection in the presence of methylene blue, a known inhibitor of guanylate cyclase activity (needed for cGMP release) This finding confirmed that if cGMP pathway in the CC was inhibited, cAMP system may take over the functional role of inducing penile tumescence; this is probably similar to the inhibitory effect of cGMP on cAMP-mediated LH activity in the ovary (LaPolt et al., 2003)

The existence of synergism and functional crosstalk between NO-cGMP and cAMP was not well recognized in the human penile smooth muscle until recently An earlier in vitro study in which a combination of VIP and the NO donor linsidomine chlorhydrate (SIN-1) (respective stimulants of cAMP and cGMPpathways) was used in the human CC and the

cavernous arteryfailed to show such interactions (Hempelmann et al.,1995) However in

a clinical study conducted on 50 ED patients, a combination of PGE1with SIN-1 was more effective in treating EDthan either PGE1 or SIN-1 given alone (Tordjman, 1993), thereby indicating an additive therapeutic effect Further synergy of PGE1 with S-

nitrosoglutathione was demonstrated by Angulo et al (2000) to be present specifically at the cavernosal trabecular smooth muscle but not in penile arteries thereby indicating a tissue difference in response despite the fact that both components (CC and vasculature) are anatomically and functionally the key effectors of penile erection However, with the recent identity of the existence of cross talk between cAMP and cGMP in the cavernosal smooth muscle cells (Abdel-Latif, 2001; Kim et al., 2000; Stief et al., 2000), it is likely that both the systems may be complementing each other at the cellular level for the final common mediation of the erectile process Taken together, attempts to delineate the effect of oestrogens on these physiological processes will have definite bearing on its possible role in the pathophysiology of erectile dysfunction In this investigation on nucleotide levels in cultured cavernosal smooth muscle cells, both oestradiol and daidzein produced a concentration-related increase in the intracellular release of cAMP, while the effect of oestradiol on cGMP was variable and biphasic consisting of an increase at lower concentration followed by a decrease in cGMP at higher concentrations Phytoestrogen produced a mild increase in cGMP at all doses tested indicating that further titrations are required

In the female physiology, oestrogen increased cAMP dependent protein kinase activity in the brain (Shingo and Kito, 2002) and the cAMP signal transduction pathway interacted with ER such that both oestradiol and protein kinase A co-promoted the functions of oestrogen response elements in target tissues such as breast and uterus (Lazennec et al.,

2001) This transcriptional activation of oestrogen results in subsequent phosphorylation

of cAMP response element-binding protein (CREB), this step can be blocked by faslodex, the antioestrogen ICI 182,780 (Wade and Dorsa, 2003) Furthermore, the importance of this correlation is shown by the finding in rat myometrium that oestrogen pretreatment caused a 2-5 fold increase in the intracellular levels of cAMP (Abdalla et al., 2000) and in turn an increase in cAMP (induced by forskolin and IBMX) activated ERα and to some extent ERβ even in the absence of the exogenous hormone (Coleman et al., 2003) Similarly, oestrogenic substances from plants including genistein and daidzein were shown in cardiac myocytes to potentiate the function of cAMP dependent chloride channels (Chiang et al., 1996) However, while the other female hormone progesterone inhibited the activity of cAMP-phosphodiesterase (thereby increasing cAMP levels further), oestradiol did not have any effect on this cAMP metabolizing pathway (Kofinas

et al., 1990) Taken together, these factors support the observed trend in cAMP activity in the penile cavernosal smooth muscle cells in this investigation secondary to both oestradiol and daidzein treatments although much less than the 2-5 fold increase reported

in the uterus (see Abdalla et al., 2000)

Nitroglycerine-induced increase in cGMP release was not statistically significant at the concentrations used in this study However, it has been shown that with further dose escalations / titrations (3µg/ml – 100µg/ml), the cGMP release secondary to NTG was statistically significant in the rat cavernosal smooth muscle cell cultures (Gauthaman, 2002) On the cGMP pathway, available data indicates that oestrogen-induced increase in permeability of cultured human cervical epithelial cells was mediated through the activation of NO-cGMP system (Gorodeski, 2000), oestradiol increased the levels of both

NO and cGMP in cardiac myocytes (White et al., 2002) and decreased GTPase activity in

the vasculature (Buhimschi et al., 2001) However, while oestradiol reduced the soluble guanylase cyclase activity as well as protein expression in rats (Krumenacker et al., 2001), phytoestrogen isoflavonoid such as quercetin (compound similar to daidzein in structure-activity) stimulated this enzyme in porcine renal cell lines (Chen et al., 2003) Furthermore and importantly, the effect of oestrogen on nNOS activity was shown to be dose-dependent as well as biphasic in neutrophils of male volunteers, with increase in nNOS activity at lower concentrations of oestradiol and inhibition of this activity at higher concentrations (Hayashi et al., 1994; Garcia-Duran et al., 1999) These observations indicate the possibility of a gender variation in tissue response to E2 (Miller, 1999); this may include the male erectile tissue (cavernosum) since these observations are similar to the trend in cGMP activity seen with oestradiol incubation in the cavernosal smooth muscle cell cultures in this investigation Although oestrogen receptors also mediate stimulation of vascular eNOS activity (Cho et al., 2003; Thompson and Khalil, 2003), the relative contribution to the erectile process by EDNO under conditions of impaired nNOS activity (especially at higher levels of oestradiol) needs further evaluation

Taken together, this oestradiol-induced decrease in cGMP release indicates a functional difference with testosterone since androgens are shown to stimulate NO-cGMP pathways through nNOS and eNOS activities in the penile tissue and vasculature (Reilly et al., 1997b; Tep-areenan et al., 2003); however, androgens also promote cAMP activity (Walker, 2003) Another pathway of interest is the stimulation of androgen release by oestrogenic activity; in cultured adrenal cortical cells both oestradiol and daidzein suppressed cortisol synthesis and increased cAMP-mediated dehydroepiandrosterone-sulphate production (Mesiano et al., 1999)