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Pharmacological Research 64 (2011) 235– 241

Contents lists available at ScienceDirect

Pharmacological Research

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xtracellular Ca2+ entry and mobilization of inositol trisphosphate-dependenta2+ stores modulate histamine and electrical field stimulation inducedontractions of the guinea-pig prostate

ichelle Lama, Karen Kerrb, Sabatino Venturaa, Betty Exintarisa,∗

Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, AustraliaSchool of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, New South Wales 2308, Australia

r t i c l e i n f o

rticle history:eceived 31 March 2011eceived in revised form 18 April 2011ccepted 18 April 2011

eywords:nositol triphosphatemooth musclelectrical field stimulationistamine

a b s t r a c t

This investigation aimed to examine the source of Ca2+ mobilization that leads to the contractile responseto either exogenously added histamine (1 �M–1 mM) or electrical field stimulation (10 Hz, 0.5 ms, 60 V).Removal of extracellular Ca2+ by removal of Ca2+ from the bathing medium reduced histamine (1 mM)induced responses by 34% and responses induced by electrical field stimulation by 94%. Similarly, block-ade of L-type Ca2+ channels by nifedipine (1 �M) reduced histamine (1 mM) induced responses by 43% andresponses induced by electrical field stimulation by 77%. Application of cyclopiazonic acid (CPA) (10 �M)to inhibit Ca2+ reuptake to the sarcoplasmic reticulum enhanced both histamine-induced and electricalfield stimulation induced responses to a small degree, while the addition of the inosotol triphosphate(IP3) receptor antagonist, 2-aminophenoxyethane borane (2-APB) (100 �M) inhibited histamine induced

rostate responses by 70% and electrical field stimulation induced responses by 57%. Ryanodine (1 �M) did notaffect contractile responses to either histamine or electrical field stimulation, either in the absence orpresence of 2-APB (100 �M). During both histamine and electrical field stimulation induced contractions,prostate smooth muscle generates IP3 receptor mediated Ca2+ release in conjunction with Ca2+ entry fromthe extracellular environment. Ryanodine receptors on the other hand, appear not to play a role in thisphysiological mechanism.

. Introduction

Phenylephrine-induced contractile responses of the guinea-ig [1], canine [2] and human [3] prostate have demonstratedhe presence of �1-adrenoceptors which are generally coupled tohe Gq/11 protein. �1-Adrenoceptor – Gq/11 activation produceshe second messengers inositol triphosphate (IP3) and diacyl-lycerol (DAG), which promote Ca2+ release from the internalarco-endoplasmic reticulum and Ca2+ entry from voltage-gateda2+ channels, respectively. Enhanced intracellular Ca2+ levels[Ca2+)]i) facilitate contractility of the smooth muscle.

Other agonists that induce contractility of the guinea-pig anduman prostate through membrane bound G protein coupledeceptors or ligand gated ion channels include: acetylcholine

4–6], adenosine 5′-triphosphate (ATP) [7], endothelin [8,9] anderotonin [4].

∗ Corresponding author. Tel.: +61 3 9903 9071; fax: +61 3 9903 9638.E-mail addresses: betty.exintaris@pharm.monash.edu.au,

etty.exintaris@monash.edu (B. Exintaris).

043-6618/$ – see front matter. Crown Copyright © 2011 Published by Elsevier Ltd. All rioi:10.1016/j.phrs.2011.04.009

Crown Copyright © 2011 Published by Elsevier Ltd. All rights reserved.

For example, acetylcholine acts on M1 muscarinic G proteincoupled receptors in the guinea-pig prostate gland to enhance elec-trical field stimulated contractions [31] whereas ATP acts on P2X1purinoceptors, which are ligand gated channels, that cause an influxof Ca2+ and resultant contraction in the guinea-pig prostate gland;IP3 dependent Ca2+ release may also be involved [7].

Another example of an agonist which acts at prostatic G pro-tein coupled receptors is the biogenic amine, histamine. Histamineproduces contractile responses in guinea-pig [4], rabbit [10] andhuman prostates [5]. In addition, histamine has been shown topotentiate electrical field stimulated contractions, as well as nora-drenergic, cholinergic and purinergic contractions in the guinea-pigprostate [11]. Studies of histamine release in the human prostatereveal that histamine concentrations are amplified in benign pro-static hyperplasia (BPH) due to an increase in mast cell numberwhich is proportional to age and the degree of hyperplasia [12].Histamine-containing mast cells typically cluster around vascu-lar structures in hyperplastic human prostates [5]. Conversely,

histamine has also been reported to be released from neuroen-docrine cells found in the glandular component of the prostateunder the control of �1-adrenoceptors [13]. Thus, it appears thatthe role of histamine in the prostate may be centred upon stimulat-

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ng production of glandular secretory fluids while also promotingontractility; however, little is known about the mechanismsnderlying the histamine-induced contractions of the prostateland.

Drugs such as tamsulosin and alfuzosin, which relax pro-tatic smooth muscle have proven to be the most beneficial inhe treatment of lower urinary tract symptoms caused by ure-hral obstruction due to BPH [35–37]. Identification of a novelarget within the Ca2+ signalling pathway, may be effectiven reducing smooth muscle tone, thereby providing an addi-ional mechanism for relieving lower urinary tract symptomsssociated with BPH. The aim of this study was to inves-igate the signalling pathways activated by histamine in theuinea-pig prostate, which regulate intracellular Ca2+ cycling.istamine induced contractile responses were compared to con-

ractile responses produced by electrical field stimulation in ordero determine whether the histamine and the neurotransmitterseleased by electrical field stimulation act via the same signallingathways.

. Methods

.1. Preparation of tissues

Sexually mature male Tri-colour guinea-pigs (500–700 g) werebtained from Monash University Animal Services. Guinea-pigsere killed by stunning and exsanguination following procedures

pproved by the Monash University Standing Committee of Animalthics in Animal Experimentation (Ethics number VCPA/2005/07).he ventral prostate was dissected out and each gland wasivided longitudinally in half to provide four preparations in total.he preparations were placed into 10 ml organ baths filled withrebs–Henseleit solution, which was composed of (mM): NaCl 118,Cl 4.7, MgSO4·7H2O 1.1, KH2PO4 1.18, NaHCO3 25.0, glucose 11.66nd CaCl2·2H2O 2.5. Each tissue was attached to a perspex tissueolder incorporating two parallel platinum electrodes at one endnd a force transducer at the other end. Tissues were placed under

g force and maintained at 37 ◦C and bubbled with 95% O2, 5%O2. Contractility was recorded isometrically with Grass FT03 forceransducers using a Powerlab system (AD Instruments, Castle Hill,SW, Australia) run on a personal computer.

During a 60 min equilibration period, tissues were electricallyeld stimulated using trains of 20 pulses at 10 Hz every 50 st 0.5 ms pulse duration and 60 V. After equilibration, electri-al field stimulation was turned off, tissues were washed andrebs solution containing KCl (0.1 M) was administered. As soons the response plateaued, tissues were washed three times over

15 min period and the addition of 0.1 M KCl was followed byashing.

.2. Drugs used

The drugs used were the IP3 receptor antagonist: 2-minophenoxyethane borane (2-APB) (Calbiochem Biosciences; Laolla, CA, USA), ryanodine (Tocris; Ellisville, MI, USA), the inhibitorf Ca2+ reuptake into the sarcoplasmic reticulum: cyclopiazoniccid (CPA), histamine diphosphate, the H1 histamine receptorntagonist: mepyramine maleate and the L-type Ca2+ channellocker: nifedipine (Sigma Aldrich; St. Louis, MO, USA). CPA andifedipine were dissolved in dimethyl sulfoxide (DMSO) to give

final bath concentration of DMSO that did not exceed 0.1%nd did not affect contractile responses. All other drugs wereissolved in distilled water. Ca2+-free Krebs was prepared by iso-onic replacement of CaCl2 with NaCl and the addition of EDTA1 mM).

search 64 (2011) 235– 241

2.3. Experimental procedure

Following the equilibration and KCl contractions, neurogeniccontractions were elicited by electrical field stimulation usingthe stimulation parameters previously described. Once contrac-tile responses stabilised, they were recorded for later analysis.Electrical field stimulation was then turned off and concentrationresponse curves to histamine were conducted on unstimulatedtissues. These were constructed by the addition of histamine inhalf log increments (0.1 �M–3 mM). Each concentration was addedto the organ bath as soon as the response to the previous con-centration had plateaued – roughly 20 s after addition of eachconcentration of histamine. The tissues were then thoroughlywashed with fresh Krebs–Henseleit intermittently over a periodof 30 min, during which time, the tension returned to the initialbaseline. A drug or treatment was then applied for a specific timeperiod (usually 30 min but 20 min for ryanodine) and electricalfield stimulation was re-commenced. Contractions in the pres-ence of the drug or treatment were recorded for comparison tothe initial pre-treatment electrical field stimulation induced con-tractions. Following this, a second concentration response curve tohistamine in the presence of the test drug or treatment was thenconstructed.

2.4. Data analysis

2.4.1. Analysis of concentration response curvesThe peak force (g) induced by each concentration of histamine

was measured and expressed as a percentage of the mean responsesinduced by two applications of KCl.

2.4.2. Analysis of electrical field stimulation-induced contractionsThe mean peak force of the three twitches immediately prior to

the addition of drugs or treatments were measured and the mean ofthe last three twitches prior to the second CRC were also measured.These were then expressed as a percentage of the internal standard,KCl.

All results are expressed as mean ± s.e.m. (standard error ofmean). The potency of histamine under various conditions isexpressed as the pEC50 value and the efficacy is expressed as themagnitude of the response elicited by histamine at a concentrationof 1 mM, where 100% indicates the same response as the internalstandard, KCl.

These values were estimated from nonlinear regression plotsfitted to sigmoidal concentration–response curves (variable slope)based on a four parameter logistic equation. Differences betweencontrol concentration–response curves and those in the presence ofvarious drugs/treatments were calculated using repeated measurestwo-way analysis of variance (ANOVA) followed by Bonferroni posttest for comparisons between each histamine concentration. Allstatistical analyses and graphics were performed using GraphPadPrism version 5.0 (GraphPad Software, San Diego, CA, USA). Dif-ferences between the magnitudes of neurogenic responses werecompared using a Student’s two tailed t-test for paired data. Alldifferences were considered significant when P < 0.05.

3. Results

3.1. Effect of histamine

Histamine evoked tonic contractile responses to give a tension

at a concentration of 1 mM of 89 ±8% KCl in ventral portions ofthe guinea pig prostate. The responses were abolished in the pres-ence of the selective H1 receptor antagonist, mepyramine (0.1 �M),(P < 0.0001, n = 6) (Fig. 1).

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.2. Effects of Ca2+-free Krebs and nifedipine

In the absence of external Ca2+, histamine (1 mM) inducedesponses were reduced by 34% (from 105 ± 6 to 69 ± 22% KCl,

< 0.0001, n = 6) (Fig. 2A). Similarly, nifedipine (1 �M), an L-ype Ca2+ channel inhibitor, reduced histamine (1 mM) inducedesponses by 43%, to 60 ± 11% KCl (P < 0.0001, n = 6) (Fig. 2A).

The removal of external Ca2+ reduced electrical field stimula-ion induced responses from an amplitude of 53 ± 7 to 3 ± 1% KClP = 0.0005, n = 7), a reduction of 94% (Fig. 2Bi). In the presence ofifedipine (1 �M) electrical field stimulation induced contractionsecreased by 77% (90 ± 6 to 21 ± 7% KCl in control and nifedipine,espectively) (P = 0.0002, n = 9) (Fig. 2Bii).

.3. Effects of CPA, 2-APB and ryanodine

Contributions to [Ca2+]i by internal Ca2+ store release mech-nisms were further investigated with the addition of CPA10 �M), a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA)

ig. 2. Mean concentration response curves to the exogenous administration of histamiree Krebs (�) and in the presence of the L-type Ca2+ channel blocker nifedipine (1 �M)

ontraction from each concentration of histamine derived from 6 to 8 experiments. Verticaontractile responses to electrical field stimulation (20 pulses at 10 Hz, 0.5 ms duration af extracellular Ca2+ (panel Bi) and in the absence and presence of nifedipine (1 �M) (patimulation derived from 9 experiments. Vertical bars represent standard error of mean.

search 64 (2011) 235– 241 237

pump inhibitor. Responses to both exogenously administered his-tamine and electrical field stimulation were significantly enhanced(Fig. 3), The contractile response to histamine (0.1 mM) increasedfrom 113 ± 7 to 145 ± 16% KCl (P < 0.0096, n = 6) (Fig. 3A). Subse-quent removal of external Ca2+ reduced the contractile response tohistamine (1 mM) back down to 106 ± 35% KCl (P < 0.0001, n = 6)(Fig. 3A). Responses to electrical field stimulation in the pres-ence of CPA (10 �M), increased by 21% (103 ± 17 to 124 ± 16%KCl) (Fig. 3Bi). In Ca2+-free Krebs, responses in the presence ofCPA (10 �M) reduced from control levels of 88 ± 14 to 7 ± 1% KCl(P = 0.0005, n = 8), an inhibition of 92% (Fig. 3Bii).

The application of 2-APB (100 �M), an IP3 receptor antagonist,inhibited histamine (3 mM) induced responses by 70% (Fig. 4A and 5). Contractile responses dropping from 85 ± 4% KCl to 25 ± 4% KCl(P < 0.0001, n = 6) (Fig. 4A). When external Ca2+ was removed fromthe bathing medium in addition to the presence of 2-APB, responsesto histamine (3 mM) were reduced similarly by 71% with to 25 ± 8%KCl (P < 0.0001, n = 6) (Fig. 4A). Electrical field stimulation inducedresponses in the presence of 2-APB (100 �M) were also reducedfrom 62 ± 6 to 27 ± 2% KCl (P = 0.0004, n = 6) (Figs. 4Bi and 5). 2-APB(100 �M) without external Ca2+ inhibited electrical field stimula-tion induced responses from 55 ± 7 to 2 ± 1% KCl (P = 0.0038, n = 6)(Figs. 4Bii and 5).

Ryanodine (1 �M) had no significant effect on responses toeither responses to exogenously added histamine or electrical fieldstimulation as shown in Fig. 4 (P = 0.5008, n = 6). Similarly, theremoval of external Ca2+ in the presence of ryanodine (1 �M) pro-duced an inhibition of 44% to 48 ± 6% KCl (P < 0.0001, n = 6) (Fig. 4A),which was similar to the degree of inhibition caused by Ca2+ freeKrebs in the absence of ryanodine (Fig. 2A). No change in electricalfield stimulation induced twitch height was seen in the presenceof ryanodine (1 �M) (n = 9) (Fig. 4Biii) and the removal of exter-nal Ca2+ in the presence of ryanodine (1 �M) again reduced theheight of contraction by 88%, from a control of 61 ± 9 to 7 ± 2% KCl(P = 0.0003, n = 9) (Fig. 4B). This degree of inhibition of electricalfield stimulation induced contraction was similar to the inhibitionseen by Ca2+ Krebs alone (Fig. 2Bi).

3.4. Effects of 2-APB with ryanodine

2-APB (100 �M) was administered in combination with ryan-odine (1 �M) to determine whether residual contractions seen at

ne expressed as a percentage of KCl contraction (A) in control tissues (�), in Ca2+

in standard Krebs–Henseleit solution (�). Symbols represent mean peak height ofl bars represent standard error of mean. **P < 0.01, ***P < 0.001. Panel B shows meant 60 V) expressed as a percentage of KCl contraction in the absence and presencenel Bii). Columns represent mean peak height of contraction from electrical field

**P < 0.01, ***P < 0.001.

238 M. Lam et al. / Pharmacological Research 64 (2011) 235– 241

Fig. 3. Mean concentration response curves to the exogenous administration of histamine expressed as a percentage of KCl contraction (A) in control tissues (�), in thepresence of the inhibitor of Ca2+ reuptake into the sarcoplasmic reticulum: CPA (10 �M) (�) and in the presence of CPA (10 �M) in Ca2+ free Krebs (�). Symbols representmean peak height of contraction from each concentration of histamine derived from 6 experiments. Vertical bars represent standard error of mean. **P < 0.01, ***P < 0.001.P s at 1a of CPc cal ba

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anel B shows mean contractile responses to electrical field stimulation (20 pulsebsence and presence of CPA (10 �M) (panel Bi) and in the absence and presenceontraction from electrical field stimulation derived from 8 to 9 experiments. Verti

igh histamine concentrations (1–3 mM) were the result of a com-ined effect of IP3 and ryanodine receptors. However as previouslybserved (Fig. 4), responses were inhibited by 70% with a reduc-ion from 63 ± 4 to 19 ± 2% KCl (P < 0.0001, n = 6) (Fig. 6A). Thisas not different to the inhibition seen by 2-APB alone (Fig. 4A).owever, in Ca2+-free Krebs, responses were further reduced to

± 2% KCl, P < 0.0001, n = 6) (Fig. 4A). Contractile responses to elec-rical field stimulation were also reduced in the presence of aombination of 2-APB (100 �M) and ryanodine (1 �M) from 57 ± 6o 22 ± 3% KCl (P = 0.0038, n = 6) (Fig. 6Bi). This inhibition was

gain to a similar degree as observed with 2-APB (100 �M) aloneFig. 4Bi). As observed in previous tests of the effect of Ca2+ freerebs on contractile responses elicited by electrical field stimula-

ion (Figs. 2Bi, 3Bii and 4Bii, Biv), Ca2+ free Krebs along with 2-APB

ig. 4. Mean concentration response curves to the exogenous administration of histamresence of the IP3 receptor antagonist: 2-APB (100 �M) (�), in the presence of 2-APB (1resence of ryanodine (1 �M) in Ca2+ free Krebs (©). Symbols represent mean peak heighertical bars represent standard error of mean. **P < 0.01, ***P < 0.001. Panel B shows meuration at 60 V) expressed as a percentage of KCl contraction in the absence and prese

n Ca2+ free Krebs (panel Bii), in the absence and presence of ryanodine (1 �M) (panel Biolumns represent mean peak height of contraction from electrical field stimulation deri**P < 0.001.

0 Hz, 0.5 ms duration at 60 V) expressed as a percentage of KCl contraction in theA (10 �M) in Ca2+ free Krebs (panel Bii). Columns represent mean peak height ofrs represent standard error of mean. **P < 0.01, ***P < 0.001.

(100 �M) and ryanodine (1 �M) inhibited the majority of the con-traction (91%), reducing the twitch height from 54 ± 7 to 5 ± 1% KCl(P < 0.0001, n = 6) (Fig. 6Bii).

4. Discussion

4.1. Histamine in the guinea-pig prostate

It has been suggested that in BPH an enhanced prostatic smooth

muscle tone largely stems from increased �1-adrenoceptor activitywhich utilises the Gq/11 signalling transduction pathway [1]. His-tamine has also recently been shown to potentiate noradrenergiccontractions [11], emphasising a modulatory role for histamine in

ine expressed as a percentage of KCl contraction (A) in control tissues (�), in the00 �M) in Ca2+ free Krebs (�), in the presence of ryanodine (1 �M) (�), and in thet of contraction from each concentration of histamine derived from 6 experiments.an contractile responses to electrical field stimulation (20 pulses at 10 Hz, 0.5 ms

nce of 2-APB (100 �M) (panel Bi), in the absence and presence of 2-APB (100 �M)ii), in the absence and presence of ryanodine (1 �M) in Ca2+ free Krebs (panel Biv).ved from 6 experiments. Vertical bars represent standard error of mean. **P < 0.01,

M. Lam et al. / Pharmacological Re

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nd Bii) and 2-APB (100 �M) combined with Ca2+ free Krebs (Aiii and Biii) on bothistamine (A) and electrical field stimulation (20 pulses at 10 Hz, 0.5 ms duration at0 V) induced contractions (B).

he guinea-pig prostate. It has been previously established thatistamine initiates Ca2+ mobilization through activation of H1eceptors, which similarly employs Gq/11 signalling to produce con-ractility [14]. The activation of Gq/11-coupled H1 receptors initiateshe hydrolysis of phosphatidyl-inositol biphosphate (PIP2) to pro-uce the second messengers IP3 and DAG. These second messengerslevate [Ca2+]i through Ca2+ entry from outside the cell as well asnternal Ca2+ store release [15].

.2. External Ca2+ entry in histamine-induced contractions

External Ca2+ entry to the cytosol is largely regulated by voltage-perated channels, particularly L-type Ca2+ channels in smoothuscle cells. Ca2+ entry through L-type Ca2+ channels contribute

3% of the histamine-induced contractions and the removal of alla2+ from the external medium reduced histamine responses by a

omewhat similar amount of 34%. The lower degree of inhibition bya2+-free Krebs compared to nifedipine (1 �M) indicates that whilehe majority of Ca2+ from extracellular fluid enters the cell via L-ype voltage-gated Ca2+ channels, a small amount of Ca2+ entry may

ig. 6. Mean concentration response curves to the exogenous administration of histamresence of both 2-APB (100 �M) and ryanodine (1 �M) (�) and in the presence of both 2-Aeak height of contraction from each concentration of histamine derived from 6 experim

shows mean contractile responses to electrical field stimulation (20 pulses at 10 Hz, 0.nd presence of 2-APB (100 �M) and ryanodine (1 �M) (panel Bi) and in the absence andolumns represent mean peak height of contraction from electrical field stimulation deri**P < 0.001.

search 64 (2011) 235– 241 239

occur through other channels such as a T-type voltage-gated chan-nels [16]. More importantly, the large residual contraction in thepresence of nifedipine and or Ca2+ free Krebs indicates that intracel-lular Ca2+ stores are contributing to the majority of the contractionelicited by histamine.

4.3. Intracellular Ca2+ release in histamine-induced contractions

Ca2+ store release from the sarcoplasmic reticulum occurs viasecond messenger signalling and intracellular Ca2+ concentration([Ca2+]i)-dependent Ca2+ entry. Maintaining [Ca2+]i involves thereuptake of Ca2+ into (and possibly its release from) the sar-coplasmic reticulum using the SERCA pump [17]. We observed anaugmented histamine response in the presence of CPA, which hasalso been observed in the guinea-pig bladder and vas deferens [18]after incubation with CPA (10 �M). It has been suggested that CPAmay cause potentiation by indirectly reducing Ca2+-dependent K+

channel activity, preventing smooth muscle relaxation [19]. How-ever, blockade of Ca2+ reuptake into the sarcoplasmic reticulummay simply be causing prolonging the half life of Ca2+ in the cyto-plasm and thereby enhancing contractile activity. If Figs. 2 and 3 arecompared, it appears as though in the absence of external Ca2+, Ca2+

re-uptake inhibition by CPA is still capable of potentiating the his-tamine induced contraction which was observed in Ca2+ free Krebs.The removal of external Ca2+ and addition of the chelating agent,EDTA (1 mM), should have emptied intracellular Ca2+ stores in thepresence of CPA, yet residual contractions suggest other intracellu-lar stores such as the mitochondria may play a role in contributingto histamine-induced contractions.

Recent studies have investigated the roles of both ryanodineand IP3 receptor-induced Ca2+ release in the guinea-pig taeniacaeci, small intestine, bladder and vas deferens, producing compa-rable conclusions. In the guinea-pig taenia caeci, intracellular Ca2+

release is largely ryanodine receptor-dependent when stimulatedby histamine (100 �M) [20]. However, it has also been reported thatin the small intestine, histamine relies on IP3-mediated Ca2+ releaseto augment muscarinic contractions [21]. Similar findings have

ine expressed as a percentage of KCl contraction (A) in control tissues (�), in thePB (100 �M) and ryanodine (1 �M) in Ca2+ free Krebs (�). Symbols represent meanents. Vertical bars represent standard error of mean. **P < 0.01, ***P < 0.001. Panel

5 ms duration at 60 V) expressed as a percentage of KCl contraction in the absence presence of 2-APB (100 �M) and ryanodine (1 �M) in Ca2+ free Krebs (panel Bii).

ved from 6 experiments. Vertical bars represent standard error of mean. **P < 0.01,

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een reported in the guinea-pig bladder [22] and in the myocytes ofuinea-pig vas deferens [23]. Our studies with 2-APB in the guinea-ig prostate demonstrate that IP3-dependent Ca2+ release was theredominant intracellular Ca2+ source in histamine-induced con-ractions.

The addition of 2-APB has been shown to inhibit not onlyP3-dependent Ca2+ efflux but also capacitative Ca2+ entry [15].P3 receptors associated with store-operated channels regulateapacitative Ca2+ entry upon sarcoplasmic reticulum depletion.hese plasma membrane channels await binding from nearbyP3 receptors, which open store-operated channels, consequentlyeplenishing sarcoplasmic reticulum stores [24]. It has previouslyeen documented that 2-APB inhibits histamine responses and thathis is a result of direct IP3 receptor and store-operated channelnhibition [25]. The addition of 2-APB with Ca2+-free Krebs (Fig. 4)nhibited contractions but this was not different to the inhibi-ion produced by 2-APB alone, suggesting that external Ca2+ entrylays a secondary role to IP3-mediated intracellular Ca2+ release inistamine-induced contractions.

[Ca2+]i elevation from IP3-dependent Ca2+ release and exter-al Ca2+ entry may also regulate calcium-induced-calcium-releaseCICR), a mechanism that boosts [Ca2+]i by opening Ca2+-sensitiveyanodine receptors and more IP3 receptors. Upon agonist stimu-ation, Ca2+ waves produced by CICR can sustain high [Ca2+]i andropagate contractions via gap junctions to adjoining cells [15,24].

Ryanodine, an alkaloid derived from the Ryania speciosa Vahllant [26], targets the ryanodine receptor located on the sar-oplasmic reticulum to produce concentration-dependent Ca2+

elease. It was first reported in skeletal muscle that ryanodineeceptors partially open at low nanomolar ryanodine concentra-ions while greater than 10 �M reduced Ca2+ permeability [27].ater studies observed that ryanodine (10 �M) depleted Ca2+

tores in rabbit aortic smooth muscle rather than inhibiting Ca2+

elease [28]. As ryanodine binds to a partially opened channel, aonformational change occurs, ‘locking’ channels in an open con-guration – promoting Ca2+ leakage [29]. Under normal conditionsith Krebs–Henseleit, ryanodine (1 �M) did not affect histamine

esponses and indeed the inhibition seen with ryanodine in combi-ation with Ca2+ free Krebs was similar to the inhibition seen witha2+ free Krebs alone.

The addition of 2-APB together with ryanodine caused a reduc-ion of 70% – no different to the addition of 2-APB alone. Theemoval of external Ca2+ further reduced the residual response.hus, Ca2+ release via IP3 receptors constitutes the major portionequired for contractility with lesser but substantial roles playedy store-operated and L-type voltage-gated Ca2+ channels.

.4. Electrical field stimulation induced contractions of theuinea-pig prostate

It is believed that the H1 receptor activates the Gq/11 signallingathway but neurogenic stimulation of adrenergic and muscariniceceptors also produce contractions by this pathway [30–32]. Alose association between histamine and neurotransmitter releases therefore likely to exist so as to coordinate prostate contractility.euronal stimulation of the human glandular prostate mediatesistamine release from neuroendocrine and mast cells [13], whilendogenous histamine potentiates post-synaptic activity at recep-or sites such as �1-adrenoceptors and M1-muscarinic receptors inoth the vas deferens [33] and prostate [11] of guinea-pigs. The sec-ndary component of this study was to examine Ca2+ mobilizationesulting from nerve stimulation and whether this was comparable

o the results obtained in histamine induced contractile studies.

Although external Ca2+ entry plays an important role in con-ributing to histamine-induced contractions, it appears to play anven greater role in nerve mediated responses. It was observed that

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the degree of inhibition by nifedipine was less than that producedby Ca2+ free Krebs, which was not the case in histamine inducedcontractions. While histamine acts via the same pathways (Gq/11)as neurotransmitters, noradrenaline and acetylcholine, ATP-gatedP2X channels are an additional pathway for Ca2+ influx in nervemediated responses [7,34]; hence the greater inhibition in Ca2+-free Krebs.

Similar to observations in histamine-induced contractions, thedegree of twitch amplitude elevation (by CPA at 121%) and inhi-bition (by 2-APB at 57%) or non significant change (by ryanodine)imply that intracellular Ca2+ release follows a common signallingpathway in the guinea-pig prostate. Furthermore, the inhibitoryaction of 2-APB suggests that both histamine-induced and electri-cal field stimulation induced contractions occur primarily thoughIP3 receptor-dependent Ca2+ release. The synergistic relationshipbetween histamine and neurogenic responses, in addition to theestablished observation that histamine elicits contractions in theprostate smooth muscle in this study and previous studies [4] hasprovided substantial support that histamine may play a modulatoryrole in prostatic contractility and consequently contribute towardsthe smooth muscle tone of the prostate.

In conclusion, histamine and electrical field stimulation inducedcontractions use the same Gq/11 signalling pathway to mobilizeCa2+ from extracellular and intracellular stores. In addition, nerveevoked responses also generate an influx of Ca2+ through P2Xchannels to produce contractile responses. Histamine exerts con-siderable contractility on the guinea-pig prostate which appearsto be primarily dependent on IP3-mediated Ca2+ release while Ca2+

release through ryanodine receptors appear not to play a role in sus-taining [Ca2+]i within the prostate smooth muscle. IP3-dependentCa2+ release in the prostate smooth muscle may provide a noveltarget within the Ca2+ signalling pathway, which may be effectivein reducing smooth muscle tone, thereby relieving lower urinarytract symptoms as a result of BPH.

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