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Keywords:

  • agmatine;
  • ATP-sensitive K+ channels;
  • imidazoline I2-receptors;
  • prostate;
  • spontaneously hypertensive rat

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENT
  8. CONFLICT OF INTEREST
  9. REFERENCES

What's known on the subject? and What does the study add?

Neurotransmitters are known to control prostate contractility. Agmatine is one of them and induces relaxation through imidazoline receptors.

The paper shows that the action of agmatine is reduced in hypertensive rats, and that this change is related to the decrease of ATP-sensitive potassium channels in the prostate. The findings can increase our understanding of the possible underlying mechanism for the development of clinical benign prostatic hyperplasia.

OBJECTIVES

  • • 
    To compare agmatine-induced prostatic relaxation in hypertensive and control rats.
  • • 
    To investigate the responsible mechanism(s) and the role of the ATP-sensitive potassium channel.

METHODS

  • • 
    Prostate strips were isolated from male spontaneously hypertensive (SH) rats and normal Wistar-Kyoto (WKY) rats for measurement of isometric tension. The strips were precontracted with 1 µmol/L phenylephrine or 50 mmol/L KCl. Dose-dependent relaxation of the prostatic strips was studied by cumulative administration of agmatine, 1 to 100 µmol/L, into the organ bath.
  • • 
    Effects of specific antagonists on agmatine-induced relaxation were studied.
  • • 
    Western blotting analysis was used to measure the gene expression of the ATP-sensitive potassium channel in the rat prostate.

RESULTS

  • • 
    Prostatic relaxation induced by agmatine was markedly reduced in SH rats compared with WKY rats.
  • • 
    The relaxation caused by agmatine was abolished by BU224, a selective imidazoline I2-receptor antagonist, but was not modified by efaroxan at a dose sufficient to block imidazoline I1-receptors.
  • • 
    The relaxation induced by diazoxide at a concentration sufficient to activate ATP-sensitive potassium channels was markedly reduced in the SH rat prostate.
  • • 
    Expressions of ATP-sensitive potassium channel sulphonylurea receptor and inwardly rectifying potassium channel (Kir) 6.2 subunits were both decreased in the prostate of SH rats.

CONCLUSION

  • • 
    The decrease of agmatine-induced prostatic relaxation in SH rats is related to the change in ATP-sensitive potassium channels.

Abbreviations
I1, I2 and I3

imidazoline receptor types

KATP channel

ATP-sensitive potassium channel

SH

spontaneously hypertensive

WKY

Wistar-Kyoto

PE

phenylephrine

IBMX

3-isobutyl-1-methylxanthine

PKA

protein kinase A

SUR

sulphonylurea receptors

Kir 62

inwardly rectifying potassium channel 6.2 subunits

INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENT
  8. CONFLICT OF INTEREST
  9. REFERENCES

The BPH that occurs frequently in older men is associated with LUTS caused by the obstruction of the proximal urethra and of urine flow. The two mechanisms leading to BPH obstruction are increase in prostate volume and increase in prostate contractility. It has long been known that α-adrenergic receptors play in important role in controlling smooth muscle contraction in the human prostate [1]. Hence α-blockers have been a mainstay medical treatment in symptomatic BPH [2]. However the common adverse effects such as postural hypotension and ejaculatory disorder associated with α-blockers have limited their clinical usefulness [3]. Hence, investigation of alternative pathways controlling prostate contractility can improve our understanding of BPH pathophysiology as well as open up new therapeutic options for male LUTS.

Agmatine is the decarboxylation product of the amino acid arginine and is considered a putative neurotransmitter for binding with imidazoline receptors [4,5]. The expression of imidazoline receptors in rat prostates has been identified [6,7] and studies have revealed the relaxant effect of agmatine in smooth muscle [8,9]. Our preliminary study showed agmatine-induced prostate relaxation in the rat (submitted for publication [10]). Currently, imidazoline receptors are divided into three subtypes: I1-, I2- and I3-receptors [11]. According to previous reports, imidazoline I1-receptors were mainly distributed in the brain and modulated blood pressure [12], and imidazoline I3-receptors were found in the pancreas for regulation of insulin secretion [13,14]. Therefore, it is unlikely that the agmatine-induced prostatic relaxation is induced by activation of imidazoline I1-receptors or I3-receptors. On the other hand, it is known that ATP-sensitive potassium (KATP) channels are involved in the relaxation of urethral smooth muscle [15]. Opening of the KATP channel lowers the intracellular Ca+ concentration [16,17] and impairment of KATP channel activity can be associated with dysfunction of the lower urinary tract [18]. Hence, the KATP channel may play an important role in agmatine-induced prostatic relaxation.

Epidemiological evidence has shown a positive relationship between BPH and hypertension [19]. However, the underlying mechanism linking the two entities is not well established. In an attempt to investigate alterations of prostatic relaxation activity in hypertension, we used agmatine as the agonist to induce relaxation in isolated rat prostate. Then, we compared the differences of responses to agmatine in prostates isolated from normal and hypertensive rats. Also, signal expressions were investigated to understand the potential mechanism(s) of this change.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENT
  8. CONFLICT OF INTEREST
  9. REFERENCES

Twelve-week-old male spontaneously hypertensive (SH) rats and Wistar-Kyoto (WKY) rats were obtained from the animal centre of the National Cheng Kung University Medical College. Rats were maintained in a temperature-controlled room (25 ± 1 °C) under a 12 h light–dark cycle (lights on at 06:00). All rats were given water and fed standard chow (Purina Mills, LLC, St Louis, MO, USA) ad libitum. All animal-handling procedures were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, Bethesda, MD, USA, as well as the guidelines of the Animal Welfare Act. In total, 38 WKY rats as control groups and 182 SH rats as hypertensive groups were used in the present study.

Experiments were performed using strips from isolated prostates from SH and WKY rats. Each rat was killed by decapitation under anaesthesia with pentobarbital (50 mg/kg). Altogether 32 control tissue samples and 176 hypertensive tissue samples were used for organ bath studies. Prostate strips about 8 × 5 mm were mounted in an organ bath filled with 10 ml oxygenated Krebs' buffer (95% O2, 5% CO2) at 37 °C containing (in mmol/L): NaCl 135; KCl 5; CaCl2 2.5; MgSO4 1.3; KH2PO4 1.2; NaHCO3 20; d-glucose 10 (pH 7.4). Each preparation was connected to strain gauges (FT03; Grass Instrument, Quincy, MA, USA). Isometric tension was recorded using chart software (MLS 023, Powerlab; ADInstruments, Bella Vista, NSW, Australia). Strips were mounted and allowed to stabilize for 2 h. Each preparation was then gradually stretched to achieve an optimal resting tension of 0.5 g.

PROSTATIC RELAXATION CAUSED BY AGMATINE

After the resting tension had stabilized, a solution of phenylephrine (PE) (Sigma-Aldrich, St Louis, MO, USA) or KCl prepared in distilled water was added into the bathing buffer to induce a rapid increase in prostatic tone followed by a plateau phase tonic contraction. The final concentration in the organ bath for PE was 1 µmol/L and for KCl was 50 mmol/L. The PE or KCl pre-contracted prostate strips were then exposed to agmatine (1–100 µmol/L, dissolved in distilled water) to observe for the decrease in tonic contraction (relaxation). Relaxation was expressed as the percentage decrease of maximal tonic contraction. Concentration–relaxation curves were generated in cumulative fashion.

To study the imidazoline receptor subtypes, PE or KCl pre-contracted prostate strips were separately exposed to glibenclamide, a KATP channel inhibitor (Research Biochemical, Wayland, MA, USA); BU224, an imidazoline I2-receptor antagonist; or efaroxan, an imidazoline I1-receptor antagonist (Tocris Cookson, Bristol, UK) for 15 min before the addition of agmatine into the organ bath. To study the signal transduction pathway, 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cyclic AMP phosphodiesterase and H-89, an inhibitor of protein kinase A (PKA), were tested in the same manner. Control groups were treated with distilled water instead of these blockers. In addition, the relaxant effect on PE or KCl pre-contracted prostatic strips of diazoxide (Sigma-Aldrich), a potassium channel opener, was also studied in a dose-dependent manner.

For Western blotting analysis, six control tissue samples and six hypertensive tissue samples were used. The prostate tissues were put in ice-cold homogenized buffer containing 10 mm Tris–HCl (pH 7.4), 20 mm EDTA, 10 mm EGTA, 20 mmβ-glycerolphosphate, 50 mm NaF, 50 mm sodium pyrophosphate, 1 mm phenylmethylsulfphonyl fluoride, and the protease inhibitors 25 µg/mL leupeptin and 25 µg/mL aprotinin. The mixture was centrifuged at 1000 g for 10 min at 4 °C. The supernatant containing the membrane fraction was centrifuged at 48 000 g for 30 min at 4 °C. The supernatant was removed, and the pellet was re-suspended in Triton-X-100 lysis buffer on ice for 30 min, homogenized and then centrifuged at 14 010 g for 20 min at 4 °C. Finally, the supernatant was transferred to a new Eppendorf tube and stored at −80 °C. The membrane extracts (20–80 µg) were separated by performing SDS–PAGE, and the proteins were transferred onto a BioTrace polyvinylidene fluoride membrane (Pall Corporation, Pensacola, FL, USA). Following blocking, the blots were developed using antibodies for imidazoline receptors (Abcam, Cambridge, UK), sulphonylurea receptor (SUR) (Millipore, Billierica, MA, USA) or inwardly rectifying potassium channel 6.2 subunits (Kir 6.2) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The blots were subsequently hybridized using horseradish peroxidase-conjugated goat anti-goat IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA), and developed using the Western Lightning Chemiluminescence Reagent PLUS (PerkinElmer Life Sciences Inc., Boston, MA). Densities of the obtained immunoblots at 170 kDa for SUR, 40 kDa for Kir 6.2 and 43 kDa for actin were quantified using Gel-Pro analyser software 4.0 (Media Cybernetics, Silver Spring, MD, USA).

All values are presented as the mean ±sem for a given number of animals or samples. Analysis of variance and Dunnett's post hoc test were used to evaluate the significance between groups. A P value <0.05 was considered a significant difference.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENT
  8. CONFLICT OF INTEREST
  9. REFERENCES

At the concentrations used, both PE and KCl induced a similar initial strong phasic contraction followed by a sustained tonic contraction in the prostatic strips. As shown in Fig. 1A, agmatine relaxed the PE-contracted prostate strips from WKY and SH rats in a concentration-dependent manner. The effect of agmatine was reversible after washout and was repeatable with a second application. Compared with that in WKY rats, the relaxation of PE-induced prostatic contraction by agmatine in SH rats was less. Also, the agmatine-induced relaxation in prostate strips contracted with KCl isolated from SH rats was less than that in WKY rats (Fig. 1B). In addition the effect of distilled water alone was investigated and the result showed no significant changes in prostatic relaxation (data not shown).

image

Figure 1. Concentration-dependent relaxation induced by agmatine in isolated prostate strips pre-contracted with 1 µmol/L phenylephrine (PE) (A) or 50 mmol/L KCl (B) in Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), respectively. Data represent mean ±sem of eight animals. *P < 0.05, and **P < 0.01 compared with the WKY group.

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The effect of imidazoline receptor blockade on agmatine-induced prostatic relaxation is shown in Fig. 2, Agmatine relaxed both PE and KCl pre-contracted prostate strips. At the maximum concentration tested (100 µmol/L), agmatine significantly attenuated the tonic contraction of prostate strips induced by PE to 66.03 ± 1.57% of the maximal contraction. Similarly, 100 µmol/L agmatine also lowered KCl-induced tonic constriction to 65.45 ± 0.92% of the maximum contraction. Then, BU224 (0.1–1 µmol/L) produced a significant and concentration-dependent attenuation of the relaxant effect of agmatine on tonic contraction of PE-contracted prostate strips. The prostatic relaxation due to agmatine in the KCl-pretreated prostate strips was also abolished in a similar manner in the presence of BU224 (Fig. 2B). However, efaroxan failed to abolish the relaxant effect of agmatine on tonic contraction in PE-contracted prostate strips even at the higher concentration (1 µmol/L). As shown in Fig. 2B, the prostatic relaxation by agmatine in KCl-contracted prostate strips was not reversed by efaroxan at a higher concentration. In the present study, we only showed the effects of blockers on agmatine at the maximum concentration (100 µmol/L). Indeed, a concentration inhibition study for the blockers would have provided additional information.

image

Figure 2. The inhibitory effect of efaroxan or BU224 on the relaxation of agmatine (100 µmol/L) in prostates isolated from spontaneously hypertensive rats contracted with 1 µmol/L phenylephrine (PE) (A) or 50 mmol/L KCl (B). Data represent mean ±sem of eight animals. **P < 0.01 and ***P < 0.001 compared with vehicle-treated control.

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In prostatic strips of SH rats pre-contracted by PE or KCl, as shown in Fig. 3, agmatine-induced relaxation was abolished by treatment with glibenclamide (1 µmol/L). Moreover, prostatic relaxation by agmatine was increased by IBMX at a concentration (10 µmol/L) sufficient to inhibit phosphodiesterase [20] and was decreased by H-89 at a concentration (1 µmol/L) sufficient to abolish PKA action [21].

image

Figure 3. The effects of inhibitors for signals on the relaxation induced by agmatine (100 µmol/L) in prostates isolated from spontaneously hypertensive rats contracted with 1 µmol/L phenylephrine (PE) (A) or 50 mmol/L KCl (B). Data represent mean ±sem of eight animals. **P < 0.01 and ***P < 0.001 compared with vehicle-treated control, respectively.

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The role of potassium channels in agmatine-induced prostatic relaxation is shown in Fig. 4A; diazoxide produced concentration-dependent (1–100 µmol/L) relaxation of the tonic contraction of PE-contracted prostate strips. Compared with that in WKY rats, the relaxation of the PE-induced prostatic contraction by diazoxide was lower in SH rats. Also, the diazoxide-induced relaxation in prostate strips contracted with KCl isolated from SH rats was lower than that in WKY rats (Fig. 4B). Histological examination of the prostate found no evidence of tissue fibrosis or smooth muscle cell damage in the SH rats.

image

Figure 4. Concentration-dependent relaxation induced by diazoxide (1–100 µmol/L) in isolated prostates pre-contracted with 1 µmol/L phenylephrine (PE) (A) or 50 mmol/L KCl (B) in Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), respectively. Data represent mean ±sem of eight animals. **P < 0.01 and ***P < 0.001 compared with the WKY group.

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The expressions of SUR and Kir 6.2 in prostates from SH rats were significantly decreased compared with those in prostates from WKY rats (Fig. 5). Quantification of the protein levels is also shown in Fig. 5. The quantification of the protein levels included at least six samples from the SH rats and six samples from the WKY group.

image

Figure 5. The difference in the protein levels of sulphonylurea receptors (SUR) (A) and inwardly rectifying K+ channel subunit 6.2 (Kir 6.2) (B) obtained from prostates between Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Data represent mean ±sem of six animals. *P < 0.05 compared with the WKY group.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENT
  8. CONFLICT OF INTEREST
  9. REFERENCES

In the present study, we found that prostatic relaxation caused by agmatine is markedly reduced in SH rats compared with WKY rats. The concentration-dependent relaxation induced by agmatine was observed in prostate strips contracted with PE or KCl. Hence, it is of special interest to understand the potential mechanism(s) of this difference.

There is no doubt that agmatine is an agonist of imidazoline receptors. In the present study, the action of agmatine was effectively abolished by BU224 at a concentration sufficient to block imidazoline I2-receptors, suggesting an activation of imidazoline I2-receptors by agmatine in the prostatic relaxation in SH rats. However, the action of agmatine was not reversed by efaroxan even at a concentration sufficient to block imidazoline I1-receptors. Mediation of imidazoline I1-receptors seems unlikely in the prostatic relaxation of agmatine caused in SH rats. Imidazoline receptors have been divided into three subtypes: I1-, I2- and I3-receptors [11]. The central action through activation of imidazoline I1-receptors has been reported to exert an antihypertensive effect [22]. Moreover, imidazoline I3-receptors are mostly presented in pancreatic cells associated with the production of insulin [14]. Therefore, mediation of imidazoline I1- or I3-receptors in prostatic relaxation seems unlikely. Actually, imidazoline I2-receptors are expressed in rat prostates [7]. Activation of imidazoline I2-receptors is suggested to participate in the action of agmatine on the relaxation of isolated prostate strips. Moreover, possible changes in the receptor site should also be considered as a reason for the difference in response in SH rats. However, there is currently no suitable tool or agent to provide more evidence for this; it will be investigated in the future.

In addition, prostatic relaxation by agmatine in SH rats was attenuated by blockade of KATP channels, indicating the involvement of KATP channels in prostatic relaxation by agmatine. Potassium channels play an important role in the regulation of contractility in guinea-pig prostate smooth muscle cells [23]. The activation of KATP channels causes hyper-polarization of the cell membrane and consequently relaxes smooth muscle. It has been established that an activation of adenylyl cyclase can increase the intracellular cAMP to activate cAMP-dependent PKA for the opening of KATP channels [21]. As shown in Fig. 3, we characterized that agmatine-induced prostatic relaxation was blocked by glibenclamide. The prostatic relaxation by agmatine was abolished by H-89 at a concentration sufficient to block PKA [21] and was enhanced by IBMX at a concentration sufficient to inhibit cAMP-phosphodiesterase [20]. These data suggest that the possible mechanism for agmatine-induced rat prostatic relaxation is mediated through the cAMP–PKA pathway to open KATP channels.

Then, we focused on the role of KATP channels in the change of prostatic relaxation by agmatine in SH rats. We used diazoxide, the well-known agent, as a potassium channel opener [24] to investigate the changes in SH rats. Similar to a previous report [25], diazoxide induced a concentration-dependent relaxation in prostate tissue contracted with PE. Prostatic relaxation caused by diazoxide was also decreased in samples from SH rats compared with tissue from WKY rats (Fig. 4). The role of potassium channels in the change of prostatic relaxation by agmatine in SH rats can be considered.

Previous evidence showed reduced potassium channel expression in human prostate cancer [26]. Moreover, the KATP channels are composed of four Kir subunits and four SURs [27]. In the present study, we found that expressions of Kir and SUR in prostate tissues are both lowered in SH rats (Fig. 5). A decrease of KATP channels in the SH rat prostate was shown; this is consistent with the reduction of prostatic relaxation caused by diazoxide in SH rats. Also, the relaxation of agmatine in strips of prostate from SH rats was abolished by the pretreatment with glibenclamide at a concentration sufficient to block KATP channels. Therefore, the decrease in KATP channels is an important mechanism causing reduction of prostatic relaxation induced by agmatine in SH rats.

Clinical and epidemiological studies have shown hypertension to be a risk factor for the development of clinical BPH [19,28]. Autonomic nervous system hyperactivity has been shown to be significantly associated with male LUTS, so it may be a link between hypertension and BPH [29]. Furthermore, increased tissue concentration of norepinephrine was previously shown in the SH rat prostate, suggesting heightened sympathetic activity that could lead to increased prostate contractility [30]. On the other hand, the current study has uncovered an impaired prostate relaxation mechanism that could also lead to prostate obstruction. However, the translational value needs to be confirmed by future studies, because the present investigation was performed using animal models.

In conclusion, the findings suggest that a decrease of KATP channel expression leads to reduced rat prostatic relaxation induced by agmatine under hypertensive conditions. Therefore, prostatic KATP channels can become a new target in the development of agents for treating BPH, especially in hypertensive patients.

ACKNOWLEDGEMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENT
  8. CONFLICT OF INTEREST
  9. REFERENCES

We thank Mr K.F. Liu for technical assistance. The present study is supported in part by a grant from Chi-Mei Medical Centre (CLFHR9829).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENT
  8. CONFLICT OF INTEREST
  9. REFERENCES