Role of oestrogens in male erectile function 3

Therefore, the primary objective of this series of investigation was to characterise the direct in vitro tissue responses of oestradiol and phytoestrogen daidzein on the receptoral affin

Chapter 6 PHARMACOLOGICAL EFFECTS OF OESTRADIOL AND PHYTOESTROGEN (DAIDZEIN) ON RABBIT CORPUS

CAVERNOSAL SMOOTH MUSCLE IN VITRO

6 PHARMACOLOGICAL EFFECTS OF OESTRADIOL AND PHYTOESTROGEN (DAIDZEIN) ON RABBIT CORPUS

CAVERNOSAL SMOOTH MUSCLE IN VITRO 6.1 Objectives

Pharmacological studies on penile cavernosum have greatly improved our understanding

of the erectile process and the pathophysiology of erectile dysfunction (Adaikan et al., 1991a) During penile erection, the two haemodynamic processes viz., increased arterial inflow and restricted venous outflow are well-coordinated with the corpus cavernosal smooth muscle (CCSM) relaxation and inhibited contraction This system is generally accepted to be under neuroregulatory control and involves the adrenergic, cholinergic and non-adrenergic and non-cholinergic systems as described earlier However, biochemical substances including hormonal activity can modify these functions in their own specific ways Therefore, the primary objective of this series of investigation was to characterise the direct in vitro tissue responses of oestradiol and phytoestrogen daidzein on the receptoral affinities to corpus cavernosal contractile and relaxant agents and to compare these effects with the response of tissue strips from different treatment groups of rabbits

to these agents To more completely evaluate the cavernosal smooth muscle reactivity in these experimental states, isometric tension studies were performed on isolated strips of

CC This is in an attempt to determine if some of the effects observed in vivo could be explained more clearly from changes at the receptoral level in vitro As such, it is conceivable that a detailed pharmacological analysis of corporal tissue is required to reveal significant hormone-mediated alterations on these pathways

6.2 Materials and Methods

Genetically pure-bred strain of New Zealand White (NZW) healthy male rabbits were utilised in this investigation All the animals used in the investigation were supplied and

maintained by the Laboratory Animal Centre, Animal Holding Unit, National University

of Singapore, Singapore They were housed in a stable air-conditioned atmosphere with adequate daily care and ready access to standard rabbit diet (pellets) and water

6.2.1 Animal Groups

Two groups of rabbits (n=6, each) acted as untreated controls for acute incubation studies with oestradiol and phytoestrogen They were housed under stable conditions for 1 week prior to experimentation Rabbits belonging to the oral treatment groups (also n=6 each) received 0.1mg of oestradiol valerate (Progynova, Schering) or phytoestrogen, soybean isoflavone daidzein (Sigma) 0.01mg or 0.1mg as suspensions in water (fixed volume of 1ml) through oral gavage daily for 12 weeks Control rabbits of this study consumed the same volume of the vehicle by gavage daily The different treatment groups have been described in Table 2

6.2.2 In Vitro Experimental Design

6.2.2.1 Collection and Storage of Penile Tissue

The rabbits from different experimental groups were sacrificed with an inhalational overdose of carbon dioxide in a sealed gas chamber connected to carbon dioxide tank The penile tissue removed in toto from the surrounding tissues was transferred immediately into freshly prepared Tyrode’s solution The storage time was kept to the minimum and in vitro experiments begun within one hour after removal of the specimen The tissue from each animal yielded four strips of corpora cavernosa which were adequate for testing various parameters including responses to agonists and electrical stimulation In order to have adequate time for conducting in vitro experiments in the laboratory, the experimental protocol was adjusted in such a way that the treatment of

6.2.2.2 Preparation of Isolated Strips of the CCSM

Through fine dissection, the penile tissue was separated from the surrounding tissue

remnants The corpus spongiosum was removed and discarded and the tunica albuginea

slit open through a midline incision The corpora cavernosa were carefully dissected from

the closely adherent tunica and the surrounding fascia During the dissection, adequate

care was taken to bathe the tissue in fresh Tyrode in order to prevent apoptosis

6.2.2.3 The Organ Bath

Four 0.4-0.6cm long strips were cut from the corpora cavernosa and secured vertically in

25ml organ baths containing freshly prepared Tyrode’s solution The physiological

solution was adequately aerated with carbogen (95% oxygen and 5% carbon dioxide)

The buffer in the solution (sodium dihydrogen phosphate 2H2O) maintained the pH

constant at 7.4 whereas the thermostatic control in the circulator pump (Churchill)

minimised fluctuations and maintained the temperature constant at 37°C The Tyrode’s

solution provided the milieu required for optimal tissue responses and had the following

composition (Table 10) The organ bath set up used in the study is shown in Figure 37

magnesium sulphate 7H2O 38g/L 259.9

calcium chloride 2H2O 32g/L 264.9

sodium dihydrogen phosphate 2H2O 6.5g/L 0.04

Table 10: Chemical Composition of Tyrode’s Solution

Figure 37: The Organ Bath Set up for In Vitro Pharmacological Studies

6.2.2.4 G Tension and Equilibration

The smooth muscle strips were allowed to equilibrate for 60 minutes at the organ bath conditions including an initial tension of 1.5g During this equilibration period, the resting tone decreased progressively to stabilise between 1 and 1.2g In addition, the bath fluid was replenished periodically

6.2.2.5 Contact Time and Dose Cycle for Drugs

The contact time and dose cycle for drugs were kept constant for all experiments In case

of noradrenaline, the drug contact time consisted of 2-5 minutes and the cycle time, 45-60 minutes For acetylcholine and nitroglycerine, they were 1-2 minutes and 30 minutes respectively The duration of contact (incubation) for the oestradiol and daidzein was 60 minutes and that required for unmasking nitrergic relaxation, 30 minutes, for all the experiments The tissue strips were washed twice with Tyrode’s solution after each drug

6.2.2.6 Electrical Field Stimulation

Transmural stimulation of rabbit corpus cavernosal smooth muscle strips evoked nerve action potential that was observed as a relaxant response in a precontracted tissue Through a preliminary study on CC strips from control animals, the ideal frequency, duration and voltage that brought about the optimal response in the tissue were ascertained and then adhered to Isolated strips of corpora cavernosa were mounted between two platinum wire electrodes (28mm long and 8mm apart) in 25ml organ baths containing the Tyrode’s solution A Grass S88 stimulator was used to provide square wave pulses that stimulated the CC strips for 10 seconds at 2 minutes intervals

6.2.2.7 Drugs and Dilutions

Stock solutions of the drugs were stored at -20°C They were diluted to the required concentration with distilled water / physiological saline (0.9% w/v) just prior to use, on the day of experimentation All the concentrations of drugs were expressed as the final concentration of the agent in the organ bath fluid The chemicals and drugs used in the study and their sources are listed in Appendix 3

6.2.3 Isolated Tissue Experiments

6.2.3.1 Response to Noradrenaline

Adrenergic (contractile, excitatory and anti-erectile) response of CC strips was obtained through cumulative additions of increasing concentrations of noradrenaline (NA) Complete concentration-response graphs were obtained in all the strips with the drug concentration ranging from 0.24µM to 1052µM The effective concentration that produced 50% of the maximum contractile response (EC50) was calculated for each strip The contractile effects of NA at different concentrations were measured as the peak

tension of the tonic contraction developed when the strip was exposed to the neurotransmitter and the response expressed as a percentage of the maximum contraction

6.2.3.2 Responses to Acetylcholine and Nitroglycerine

The relaxant, inhibitory (pro-erectile) responses to acetylcholine (ACh) and the directly acting NO donor, nitroglycerine (NTG) were recorded after adequate precontraction of the CC strips with the EC50 of noradrenaline The effects of different concentrations in the range of 0.22µM to 659µM of ACh and 0.18µM to 78µM in case of NTG were studied and compared with those obtained in different experimental groups The relaxant response to both the agents was expressed as the percentage of the tone secondary to the

EC50 of noradrenaline

6.2.3.3 Nitrergic Neurotransmission

Nonadrenergic, noncholinergic (relaxant, inhibitory and pro-erectile) responses (NANC)

of the CC strips to electrical field stimulation (EFS) were recorded at frequencies 2, 5, 10,

20 and 40hz with 90v square waves for 1ms duration and 10 seconds exposure Prior to this, the CC strips were incubated with 3.5µM of atropine and 5.1µM of guanethidine for

30 minutes in order to block the contractile cholinergic and adrenergic responses and to unmask the relaxant response

6.2.3.4 Incubation with Oestrogen and Phytoestrogen

In order to characterise the direct response of the CC strips to oestradiol and the naturally occurring phytoestrogen daidzein, the CC strips were incubated with 2.5µg of the respective agent (100ng/ml) for 60 minutes and the full concentration-response to each neurotransmitter and modulator repeated and recorded The time taken for tissue recovery after thorough clearance of the agent through repeated washing with Tyrode’s solution

response to ascending doses of 10ng/ml, 100ng/ml, 1µg/ml and 10µg/ml of oestradiol and phytoestrogen were studied after adequate precontraction of the CC strips with the EC50

of noradrenaline

6.2.4 Data Analysis

Data was analyzed by univariate analysis of variance and Tukey’s test for multiple comparisons All the results were expressed as mean ± SEM and levels of P<0.05 indicated statistical significance

6.3 Results

6.3.1 Standard Responses

Spontaneous contractions were readily seen in the tracings of the CC strips during equilibration with continuous maintenance of about 1g basal tone (Figure 38)

1 minute

Figure 38: Spontaneous Contractions in the CCSM

Inherent contractions recorded from the corpus cavernosal smooth

muscle strips in 4 channels (4 organ baths) during equilibration

The basal tone was maintained at 1g tension

Noradrenaline (0.2-1052 µM) produced concentration related contractions of the cavernosal smooth muscle This contractile response tested on all 4 strips from each animal was consistent At lower concentrations of this neurotransmitter, the response consisted of an increase in basal tone, superimposed on the regular rhythmic contractions

At the higher concentrations, the rhythmic activity diminished and the tonic contracture prevailed The peak of the tonic contracture occurred at about 1 minute after addition of the agonist The threshold concentration of noradrenaline that stimulated the corpora cavernosa was 0.2µM Noradrenaline did not show relaxant effect in any of the

experiments Typical cumulative concentration-response of CC strips to NA is shown in Figure 39

0.24µM 0.95µM 3µM 10µM 34µM 105µM 341µM 1052µM wash

Figure 39: Typical Tracing of Rabbit Cavernosal Response

to Noradrenaline

Sample tracing of the contractile (anti-erectile) response of

cavernosal strips from an untreated control rabbit to cumulative

concentrations of noradrenaline

The relaxant response to acetylcholine was tested within the concentration range of 0.2µM to 659µM on CC strips precontracted with the EC50 of noradrenaline calculated for the individual experiment The typical response to acetylcholine was a slow onset relaxation reaching a maximum within 1 minute and was generally comparable to that seen with nitroglycerine It was different from the inhibitory response to electrical field stimulation which was immediate both in onset and recovery The threshold concentration of ACh for eliciting this relaxant effect on these strips ranged from 0.2µM

to 0.9µM ACh did not produce any contractile effect on the rabbit cavernosal strips (Figure 40)

NA 9.46µM 1 minute 0.22 0.88 3 10 32 97 220 659

Figure 40: Typical Tracing of Rabbit Cavernosal Response

to Acetylcholine

Sample tracing of the relaxant (pro-erectile) response of

noradrenaline precontracted cavernosal strips from control rabbit

to increasing concentrations of acetylcholine (µM)

The relaxant response to directly acting NO donor, nitroglycerine was studied on CC strips adequately precontracted with the EC50 of NA The threshold concentration for the relaxant effect was 0.2µM The effect was slow in onset and recovery At higher concentrations there was a drop in basal tone by as much as 50% (Figure 41)

NA 9.46µM 1 minute 0.18 0.70 2.47 8 25 78.24 wash

Figure 41: Typical Tracing of Rabbit Cavernosal Response

to Nitroglycerine

Sample tracing of the relaxant (pro-erectile) response of

noradrenaline precontracted cavernosal strips from control rabbit

to increasing concentrations of nitroglycerine (µM)

Electrical stimulation of CC strips precontracted with the EC50 of NA at pulse width 1ms,

90 volts and a frequency range of 2 - 40 Hz for 10 seconds produced a frequency dependent relaxation response Prior to this stimulation, the CC strips were incubated for

30 minutes in adequate concentrations of atropine (3.5µM) and guanethidine (5.1µM) to selectively bring about the NANC response that is mediated through nitric oxide In the presence of both guanethidine (adrenergic neuronal blocker) and atropine (a muscarinic receptor blocker), EFS resulted in excitation of only the NANC neurons and the response

is rapid and marked corporal smooth muscle relaxation (Figure 42); this was abolished by

the sodium channel blocker tetrodotoxin (Tejada et al., 1988) indicating the involvement

of neuronal response

NA 9.46µM 1 minute 2Hz 5Hz 10Hz 20Hz 40Hz wash

Figure 42: Typical Tracing of Rabbit Cavernosal Response

to Electrical Field Stimulation

Pro-erectile nitrergic (NANC) relaxation response of noradrenaline

precontracted cavernosal strips from control rabbit during

electrical field stimulation with increasing frequencies

6.3.2 Acute Incubation Study

6.3.2.1 Incubation with Oestrogen and Phytoestrogen

6.3.2.1.1 Response to Noradrenaline

The contractile response to NA was potentiated in all the strips obtained from 12 rabbits incubated respectively with 2.5µg of oestradiol valerate / daidzein suspension compared with the control This accentuation in response was evident from 0.2µM of NA to 1052µM The contractile response to NA in the oestradiol and daidzein treated groups

were 5.5±0.2% and 25.7±0.2% more than the maximal control response taken as 100% and the differences were statistically significant (Figure 43)

0 30 60 90 120 150

Figure 43: Contractile Response to Noradrenaline

during Acute Incubation

Cumulative concentration-response curves to noradrenaline (NA) following

incubation of CC strips from normal rabbits (n=6) with 100ng/ml of

oestradiol valerate or phytoestrogen daidzein for 60min Both oestradiol and

daidzein produced a significant increase in the anti-erectile / contractile

response to NA (*p<0.05)

6.3.2.1.2 Response to Acetylcholine

The relaxant response to ACh (n=12) was slightly more than the control response following incubation with the two types of oestrogens at concentrations ranging from 0.2µM to 6.6µM At the particular concentration of 65.9µM of ACh, when the control relaxant effect was 100%, the increase in relaxation were 12.2±2.5% and 64.4±9.4% for oestradiol and phytoestrogen incubation; the latter difference was statistically significant (Figure 44)

0 30 60 90 120 150 180

Figure 44: Relaxant Response to Acetylcholine during Acute Incubation

Relaxation response of noradrenaline precontracted cavernosal strips from

normal rabbits (n=6) to acetylcholine (ACh) following incubation with

100ng/ml of oestradiol valerate or daidzein Phytoestrogen isoflavone

daidzein produced a significant increase in ACh-mediated relaxation

compared to control response (*p<0.05)

6.3.2.1.3 Response to Nitroglycerine

The relaxant effects (n=12) to this directly acting nitric oxide donor were 15.2±10% and 9.2±8.2% more than the control response at 78.24µM (100%) for the oestradiol and daidzein incubations respectively However, these differences were not statistically significant (Figure 45)

6.3.2.1.4 Nitrergic Response to Electrical Field Stimulation

NANC mediated relaxant response was statistically unaffected during incubation with oestradiol or phytoestrogen at all the frequencies tested For the control CC strips, the mean maximal (100%) relaxation was obtained at 20 Hz At this frequency, the measured relaxant response was for oestradiol incubation was 107.3±3.0% and that for daidzein was 97.7±3.6% (Figure 46)

0 30 60 90 120

Figure 45: Relaxant Response to Nitroglycerine

during Acute Incubation

Relaxation response of noradrenaline precontracted cavernosal strips from

normal rabbits to increasing concentrations of nitroglycerine This followed incubation of cavernous strips with 100ng/ml oestradiol valerate or daidzein

0 20 40 60 80 100 120

Figure 46: Nitrergic Relaxant Response during Acute Incubation

Nitrergic relaxation response of noradrenaline precontracted cavernosal

strips from normal rabbits to increasing frequencies of electrical field

stimulation This followed incubation of cavernous strips with 100ng/ml

oestradiol valerate or daidzein

6.3.2.2 Response to Ascending Concentrations of Oestrogen and Phytoestrogen

Direct effects of oestradiol and daidzein were tested at ascending concentrations of the respective agent viz., 10ng/ml, 100ng/ml, 1µg/ml and 10µg/ml on NA EC50 precontracted

CC strips from these rabbits At the escalating doses tested, the two oestrogens did not produce any significant contraction or relaxation of the CC strips (Figures 47 & 48)

NA 9.46µM 1 minute E 2 :250ng 2.5µg 25µg 250µg

Figure 47: Concentration – Response to Oestradiol

Sample tracing from the study on acute incubation effect of

increasing concentration of oestradiol valerate on the noradrenaline

precontracted tone of cavernosal strips from normal rabbits in 4

organ bath chambers

NA 9.46µM 1 minute PE:250ng 2.5µg 25µg 250µg

Figure 48: Concentration – Response to Daidzein

Sample tracing from the study on acute incubation effect of

increasing concentration of phytoestrogen daidzein on the

noradrenaline precontracted tone of cavernosal strips from normal

rabbits

6.3.3 Long-Term Treatment Groups (0.1mg of Oestradiol / Daidzein)

Statistical comparisons were made of the responses from CC strips of 0.1mg oestradiol and 0.1mg daidzein treated rabbits with those from time matched and vehicle treated control rabbits at 12 weeks Essentially, the contractile response to NA was significantly potentiated in the cavernosum of oestradiol and daidzein treatment groups Acetylcholine elicited good endothelium-mediated relaxation in normal animals, which was

significantly attenuated in the treated groups Similarly, NO-mediated NANC neurotransmission was also decreased in the treatment groups while endothelium-independent relaxant response to directly acting NO donor NTG reduced significantly in

the oestradiol group and affected minimally in phytoestrogen group

6.3.3.1 Response to Noradrenaline

There was a trend towards increasing sensitivity and contractility (leftward shift) with the higher concentrations of NA used This increase in potency was accompanied by a 5.9±0.2% and 29.7±0.2% increase in maximal g tension generated in the groups of oestradiol and daidzein treated rabbits compared with the control groups (Figure 49)

6.3.3.2 Responses to Acetylcholine and Nitroglycerine

The EC50 of NA for precontraction of these strips for the relaxant effect ranged from 4.7µM to 14.2µM The relaxant response was significantly inhibited in the corporal tissues from the two oestrogen treated groups when compared to the control group The percentage of the relaxant response at 65.9µM of ACh was respectively 65.5±2.0% and 68.9±3.2% of the control response taken as 100% The direct smooth muscle relaxant nitroglycerine caused only 81.5±4.4% of the relaxant response in the oestradiol group at 78.2µM and this attenuation was statistically significant compared to 91.7±3.8% response

in the daidzein treated rabbits (Figures 50 & 51)

6.3.3.3 Nitrergic Response to Electrical Field Stimulation

The cavernosal neural relaxation response (nitrergic) to EFS was attenuated in both the groups of oestrogen and phytoestrogen treated rabbits The impairment at 2hz was 26.0% for oestradiol (0.1mg) and 13.6% for daidzein (0.1mg) and those at 20hz (the frequency that produced 100% relaxation in control strips) were 18.9% and 14% respectively; the

0 30 60 90 120 150

Figure 49: Contractile Response to Noradrenaline in

Long-Term Treatment Groups

Cumulative concentration-response of cavernosal strips from 0.1mg

oestradiol valerate or phytoestrogen daidzein treated rabbits compared to the

control at 12 weeks (n=6, each) Both oestradiol and daidzein pretreatment

produced significant increases in the anti-erectile / contractile response to

noradrenaline (*p=0.005 and p<0.001)

0 20 40 60 80 100 120

Figure 50: Relaxant Response to Acetylcholine in

Long-Term Treatment Groups

Relaxation response of noradrenaline precontracted cavernosal strips from

0.1mg oestradiol valerate or phytoestrogen daidzein treated rabbits

compared to the control at 12 weeks (n=6, each) Both oestradiol and

daidzein pretreatment produced a significant decrease (anti-erectile effect) in

relaxation to increasing concentrations of acetylcholine (*p<0.001)

0 20 40 60 80 100 120

Figure 51: Relaxant Response to Nitroglycerine in

Long-Term Treatment Groups

Relaxation response of noradrenaline precontracted cavernosal strips from

0.1mg oestradiol valerate or phytoestrogen daidzein treated rabbits

compared to the control at 12 weeks (n=6, each) Oestradiol pretreatment

produced a significant decrease (anti-erectile effect) in relaxation to

increasing concentrations of nitroglycerine (*p<0.005)

0 20 40 60 80 100 120

Figure 52: Nitrergic Relaxant Response in Long-Term Treatment Groups

Nitrergic (NANC-mediated) relaxation response of noradrenaline

precontracted cavernosal strips from 0.1mg oestradiol valerate or daidzein

treated rabbits compared to the control at 12 weeks (n=6, each) Both

oestradiol and daidzein pretreatment produced a significant decrease

(anti-erectile effect) in relaxation to increasing frequencies of electrical field

stimulation (*p<0.001)

6.3.4 Responses in 0.01mg Daidzein Treated Rabbits

Somewhat similar trend as in the higher concentration of daidzein treated rabbits was seen in this group especially with regard to increase in contractile response and sensitivity to all the concentrations of NA used The other responses viz., relaxant responses to neurotransmitter and modulators such as NO, ACh and NTG were statistically unaffected in this group when compared with the control response and the responses in the higher daidzein treated group (Table 11)

%Resp to NA %Resp to ACh %Resp to NTG %Resp to NANC

Table 11: Comparative Responses in 0.01mg and 0.1mg Daidzein Treated Rabbits

In vitro tissue responses to noradrenaline (NA), acetylcholine (ACh), nitroglycerine (NTG) and electrical field stimulation (EFS) of cavernosal strips from lower and higher daidzein treated rabbits compared to control at 12 weeks (n=6, each) For 0.01mg intake, the increase in contractile response to noradrenaline was significant (*p<0.05) whereas for 0.1mg of daidzein, in addition to the significantly increased contraction with noradrenaline, decreases in relaxation responses to acetylcholine and electrical stimulation were also statistically significant (*p<0.05)

6.4 Discussion and Conclusion

The corpus cavernosum contains specialized vascular smooth muscle that is under tonic contraction mediated by excitatory noradrenergic innervation that serves to maintain penile flaccidity (Adaikan and Karim, 1981) Penile erection results from relaxation of the CCSM, which in turn results from decreased excitatory (contractile) mediation, activation of inhibitory (relaxant) pathways and efficient corporal veno-occlusive trapping of blood in the cavernosum (Lue, 1998) Similar to processes in other smooth muscles, CCSM activation by excitatory neurotransmitters, hormones or depolarization will result in cytosolic Ca2+ influx and smooth muscle contraction (Chuang et al., 1998) Prerequisite to normal erectile function therefore, is the effective inhibition of tone in the CCSM which is maintained by the release of NA from sympathetic nerve terminals and activation of post-synaptic α1-adrenergic receptors (Adaikan et al., 1991a) It is well understood over the last decade that a NO-cGMP inhibitory pathway plays a dominant role in mediating penile erection; this sequentially involves sexual arousal, NO release

from NANC neurons and endothelium within the CC, NO activation of soluble guanylate cyclase, increase in cGMP and cGMP-mediated relaxation of the penile erectile tissue (Nakane, 2003)

The peripheral effects of sex hormones on these functions of the penile smooth muscle have not been fully understood Erectile tissue from patients undergoing gender reassignment surgery after oestrogen treatment have been used in a number of previous studies evaluating receptoral affinities of the CC with the understanding that hormonal treatment does not qualitatively change the inherent functional responses of these tissues(Adaikan, 1979; Hedlund and Andersson, 1985) The limitations in procuring human penile erectile tissue for such studies have led to the widespread use of cavernosum from animal models for evaluating the various postulates and hypotheses made on human erectile pathophysiology; such in vitro studies have indicated that basically, erectile dysfunction in many patients may be stemming from an increased contractility or impaired relaxation of the penile smooth muscle or both (Taub et al., 1993) Furthermore, important age related penile tissue changes include decrease in smooth muscle content, increased sensitivity to alpha adrenoceptors and decreased NO production (Wespes, 2002) and there is a high incidence of ED in this age group

An important hormonal imbalance observed in aging man is testosterone decline together with oestradiol elevation Similarly, hormonal changes in castration may be considered to include a change in the critical balance between androgens and oestrogen Studies on castrated dogs had indicated a functional impairment of erectile tissues consisting of a higher tone of the detumescent / contractile factors, which would result in an incomplete relaxation of the cavernosal smooth muscle (Muller et al., 1988) However, Lin et al

penile erectile ability mediated through peripheral neural or cavernosal mechanisms, while in the rabbit model, castration impaired both neurogenic and endothelium-dependent relaxations and subsequently increased sensitivity to NANC-mediated relaxation of the CC (up-regulation) due to a decrease in noradrenergic activity (Andersson et al., 1992; Holmquist et al., 1994) In rats, testosterone replacement was shown to improve ICP-mediated erectile response through facilitatory effects on the pro-erectile post ganglionic parasympathetic neurons rather than the end organ (Giuliano et al., 1993) Together, these changes may be considered to account for the erectile impairment of androgen deficiency states including andropause

In the present investigation, long-term treatment of male rabbits with oestradiol or phytoestrogen produced a significant increase in contractile response to NA and impaired ACh-endothelial and NO-NANC relaxations in the treatment groups With the evidence for decreased NANC-nerve fibre density and neurotransmitter staining, generalized changes in autonomic reactivity and higher basal catecholamine concentration in the CC

of the elderly (Wespes, 2002), age-associated changes of erectile function may consist of increased dominance of α1-adrenergic activity (increased corporal smooth muscle tone) with declines in β-adrenergic, cholinergic receptors and NANC fibres interfering with the corporal smooth muscle relaxation necessary for initiating and sustaining penile erection The oestrogen-mediated anti-erectile effects of this investigation conform to this pathophysiological situation with the possibility that the age-related tissue changes are indeed due to the higher oestradiol levels of the elderly? However, none of these earlier studies involving castration or low testosterone looked at E2 levels or the independent changes brought about by E2 increase Phytoestrogens vary from oestradiol in their receptoral affinities (Kostelac et al., 2003), for eg daidzein binds more readily than

oestradiol or even genistein to ERβ Additionally, they have multiple nongenomic effects including inhibition of tyrosine kinase and DNA topoisomerase activities, induction of apoptosis, suppression of angiogenesis and antioxidant changes (Ososki and Kennelly, 2003) They are also shown to inhibit human neuronal acetylcholine receptors (Nakazava and Ohno, 2003) This plethora of other cellular / biochemical effects of phytoestrogens might have caused the difference in results obtained in this group compared to the oestradiol treated animals

Although the present investigation was not designed to address any possible differences

in the neuromediator functions due to the hormonal imbalance with testosterone, similar data from acute incubation studies involving oestradiol / daidzein especially the increased adrenergic contraction with NA at all concentrations tested suggests a direct oestrogenic influence of these pathways These findings are in accordance also with increased vascular smooth muscle sensitivity to catecholamine and α-adrenergic affinity in oestrogen treated male rats (Colucci et al., 1982) and the impaired coronary flow response to ACh in human males in the presence of 17-β oestradiol (Collins et al., 1995) There was a significant potentiation of ACh mediated relaxation during preincubation with daidzein in this investigation Oestradiol may also mediate some vasodilatation through eNOS (Thompson and Khalil, 2003) and nNOS (Fadel et al., 2003) activities while inhibiting iNOS in bones (Cuzzocrea et al., 2003) Furthermore, while testosterone upregulated both eNOS and nNOS isoenzymes in the CC (Marin et al., 1999), oestrogen down-regulated nNOS activity in the female genital tract (Min et al., 2001) In the present investigation, oestrogenic effect (oestradiol and daidzein) impaired the NO-mediated pro-erectile tissue responsiveness to electrical field stimulation in the chronic study Taking

isoform in the mediation of penile erectile function (Cashen et al., 2002), the results in this investigation of oestradiol and daidzein induced impairment of NO-NANC response and the reduced sensitivity of erectile tissue to nitroglycerine (NO donor) for the final common pathway, changes in the NO-cGMP axis may be of particular clinical significance in the precipitation of erectile impairment by increased oestrogenic activity Additionally, the accentuated contractile response to noradrenaline may be a further contributory factor Based on similar results obtained from acute tissue incubation studies and oral treatment groups in the present investigation, it seems probable that the erectile impairment by oestrogens can arise from deterioration of neurotransmitter responses via NO-cGMP axis in addition to possible direct (genomic) effects mediated through

oestrogen receptors identified within the cavernosum (Chapter 2)