Role of oestrogens in male erectile function 4

Chapter EFFECT OF OESTRADIOL AND DAIDZEIN ON CYCLIC NUCLEOTIDES IN RAT CORPUS CAVERNOSAL SMOOTH MUSCLE CELL CULTURES 165 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 166 (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. 167 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 CO2 chamber. 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, 168 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 169 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 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 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 170 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 serumfree 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 171 out with the respective conjugates and antibodies. The levels of the cyclic nucleotides were then evaluated by enzyme immunoassay (R&D Systems USA, Appendix 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[...]... level of 97.2±7pg/ml was clearly in the female range (44 -153pg/ml) with E2/T increasing correspondingly to 3.3 Review of these patients’ profiles indicated known causes of hyperoestrogenism viz., diabetes mellitus in three patients and hypercholesterolaemia in one of them 188 Group A (15 cases) Group B (15 cases) Normal Range in Males Age (years) 41 .6±2.6 49 .7±2. 14 - Testosterone (ng/ml) 4. 2±0 .4 2.7±0.2... important clinical investigation based on the presenting complaints Detailed history on sexual function characterizing the onset and duration of ED, comparison of the status of present erectile function, average frequency of sexual activity in the past and present, incidences of nocturnal penile tumescence and morning erections, degree of sexual interest, partner’s level of interest and presence of co-morbid... the Andrology clinic of this centre indicates the role of oestrogen per se in the causation and / or maintenance of this dysfunction In this investigation, the hormone profile of patients provided the important supportive evidence towards a broader protocol that involved several objective and basic investigations in animal models for delineation of changes in the regulatory mechanisms of erectile process;... 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,... respect, other important findings of this thesis that followed include: Whole animal, in vivo and in vitro studies: Treatment caused significant prolongations of intromission and post-ejaculatory mounting latencies in male rats during sexual behavioural testing with similar dysfunctional trends for mount latency, mount and intromission frequencies, indicating a negative role of oestradiol on sexual performance... 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., 19 94) At the cellular level, the final levels of the... 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... androgen-oestrogen 195 balance in the male will emerge in the near future At this preliminary stage, the identity of the oestrogen receptors in the cavernosum was a major impetus for basic research using oestrogenic supplements in male animal models to evaluate their exact role on erectile function and for the comparative analysis of hormone profile of ED patients as components of this investigation In this respect,... 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... (Chapter 4) Significant decrease in intracavernous pressure response to nerve stimulation following intravenous as well as oral administration of oestradiol in rats indicating the detrimental effect of E2 on cavernous perfusion and tumescence (Chapter 5) Significant increase in adrenergic tone of the cavernosum during in vitro pharmacological studies in rabbits treated with oestradiol and phytoestrogen indicating . component of the blood vessels. The most convincing evidence for the role of prostaglandins in erectile function is the fact that intracavernosal injection of PGE 1 is an effective form of ED. 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. activation of guanylate cyclase and formation of cyclic GMP thereby inducing penile erection. In turn, impairment of the integrity of this system is widely implicated in the pathophysiology of