Effects of imatinib mesylate on the spontaneous activity generated by the guinea-pig prostate

Authors


Correspondence: Betty Exintaris, Department of Pharmaceutical Biology and Pharmacology, Monash Institute of Pharmaceutical Sciences, Parkville, Vic. 3052, Australia.

e-mail: betty.exintaris@monash.edu

Abstract

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

  • Several studies have examined the functional role of tyrosine kinase receptors in the generation of spontaneous activity in various segments of the gastrointestinal and urogenital tracts through the application of its inhibitor, imatinib mesylate (Glivec®), but results are fairly inconsistent.
  • This is the first study detailing the effects of imatinib mesylate on the spontaneous activity in the young and ageing prostate gland. As spontaneous electrical activity underlies the spontaneous rhythmic prostatic contractions that occur at rest, elucidating the mechanisms involved in the regulation of the spontaneous electrical activity and the resultant phasic contractions could conceivably lead to the identification of better targets and the development of more specific therapeutic agents to treat prostate conditions.

Objective

  • To investigate the effect of imatinib mesylate, a tyrosine kinase receptor inhibitor, in the generation of spontaneous electrical and contractile activity in the young and ageing guinea-pig prostate.

Materials and Methods

  • Standard tension and intracellular recording were used to measure spontaneous contractions and slow waves, respectively from the guinea-pig prostate at varying concentrations of imatinib mesylate (1–50 μm).

Results

  • Imatinib mesylate (1–10 μm), did not significantly affect slow waves recorded in the prostate of both age groups but at 50 μm, the amplitude of slow waves from the ageing guinea-pig prostate was significantly reduced (P < 0.05, n = 5).
  • In contrast, the amplitude of contractions across all concentrations in the young guinea-pig prostate was reduced to between 35% and 41% of control, while the frequency was reduced to 15.7% at 1 μm (n = 7), 49.8% at 5 μm (n = 10), 46.2% at 10 μm (n = 7) and 53.1% at 50 μm (n = 5).
  • Similarly, imatinib mesylate attenuated the amplitude and slowed the frequency of contractions in ageing guinea-pigs to 5.15% and 3.3% at 1 μm (n = 6); 21.1% and 20.8% at 5 μm (n = 8); 58.4% and 8.8% at 10 μm (n = 11); 72.7% and 60% at 50 μm (n = 5).

Conclusions

  • A significant reduction in contractions but persistence of slow waves suggests imatinib mesylate may affect the smooth muscle contractile mechanism.
  • Imatinib mesylate also significantly reduced contractions in the prostates of younger guinea pigs more than older ones, which is consistent with the notion that the younger guinea-pig prostate is more reliant on the tyrosine-dependent pacemaker ability of interstitial cells of Cajal-like prostatic interstitial cells.

Introduction

A highly developed system is required to generate and synchronise spontaneous contractions in hollow smooth muscle organs. In the gastrointestinal tract (GIT), interstitial cells of Cajal (ICC) are the established pacemaker cells that drive peristaltic contractions and several variations of these cells (ICC-like cells) have been reported in other smooth muscle organs around the body, e.g. gallbladder, urethra, bladder, renal pelvis and prostate [1-5].

Spontaneous activity in the prostate is thought to arise from ICC-like prostatic interstitial cells (PICs). In the human and guinea-pig prostate, PICs are generally localised in a fine layer sandwiched between the glandular epithelium and smooth muscle stroma but are also sparsely dispersed within the prostate stroma [5-7].

Spontaneous contractions begin with the generation of a pacemaker potential. It is thought that PICs conduct electrical pulses in the form of pacemaker potentials into neighbouring smooth muscle cells, which trigger the firing of slow waves and subsequently cause smooth muscle contraction. Therefore, it is reasonable to suggest that PICs contribute to the resting prostatic smooth muscle tone and ultimately to benign prostatic hyperplasia (BPH) by altering the generation of spontaneous activity with age. Additionally, mutations in c-Kit positive ICC-like cells contribute towards the generation of stromal tumours in the GIT [8] and possibly prostate [9, 10]. Thus their significance in potentially contributing to an enhanced prostate smooth muscle tone and the development of both irritative and obstructive lower urinary tract symptoms (LUTS) such as urgency, hesitancy and nocturia as seen in BPH, in addition to stromal tumour development may provide a novel target that could have dual action with fewer adverse effects than current treatments.

Several studies have examined the functional role of tyrosine kinase receptors in the generation of spontaneous activity through the application of its inhibitor, imatinib mesylate (Glivec®), but results are fairly inconsistent. A recent study using whole-cell patch-clamp techniques showed that imatinib mesylate abolished the pacemaker potentials recorded in the ICC of the small intestine by opening KATP channels [11]. In the guinea-pig bladder, the effects were concentration-dependent: low concentrations converted slow wave activity to individual action potentials while high concentrations abolished action potentials [12]. In contrast, another study has shown that imatinib mesylate reduces spontaneous contractile activity, but not the slow wave activity arising from the guinea-pig stomach [13], leading the authors to suggest that imatinib mesylate also has non-specific effects, in addition to blocking pacemaker activity.

As tyrosine kinase receptors may be responsible for the development, maintenance and function of PICs in the prostate, this study investigates the effect of imatinib mesylate on spontaneous electrical and contractile activities arising in the guinea-pig prostate gland.

Materials and Methods

This study required Tri-colour guinea-pigs obtained from Monash University Animal Services at two weight ranges: 350–450 g, which were considered sexually mature but juvenile (young: 43 guinea-pigs) and 750–850 g, which were considered to be ageing (ageing: 57 guinea-pigs). Guinea-pigs were humanely killed by stunning and exsanguination after procedures approved by the Monash University Standing Committee of Animal Ethics in Animal Experimentation, which complies with National Institutes of Health (NIH) ethical guidelines.

Intracellular Recording

An acinus was removed from the prostate and placed into a Petri dish filled with Krebs–Henseleit solution. An incision was made along the acinus and subsequently opened up to form a sheet. The tissue was firmly secured on the Sylgard elastomer lining of the organ bath using pins and was bathed in 1 mL Krebs–Henseleit that was constantly circulated and bubbled with 95% O2, 5% CO2 at 37 °C to maintain physiological conditions.

At the end of equilibration, standard intracellular recording techniques were used as previously described by Exintaris et al. [5]. Once a cell was impaled and slow wave activity was displayed, imatinib mesylate at predetermined concentrations was superfused through the preparation.

Tension Recording

After dissection of the prostate, four lobes were extracted and tied to separate pieces of thread. Tissue preparation dimensions varied by age: young guinea-pigs were 4 mm in length, 2 mm in width and 1.5 mm in height while ageing guinea-pigs were larger at 6 mm in length, 4 mm in width and 2.5 mm in height. Each end was tied to a metal hook fixed to the organ bath with one of the hooks attached to a force displacement transducer (Electronics Workshop, Monash University), allowing the tissue to be horizontally submersed in Krebs–Henseleit bathing solution. Contractile activity was isometrically recorded by the transducer, which was connected to a PowerLab 4/30 system (AD Instruments, Castle Hill, NSW, Australia) and a ML221 Bridge Amp (AD Instruments, Castle Hill, NSW, Australia). Tissues were placed under tension and equilibrated for 60 min and the circulating medium was bubbled with 95% O2: 5% CO2 at 37 °C.

After equilibration, imatinib mesylate was added to the circulating Krebs–Henseleit and the solution superfused through the tissue for 1 h.

Analysis

Intracellular recording

In a typical trace of slow wave activity, parameters, e.g. membrane potential (mV), the amplitude of the slow wave (mV), the number of spikes superimposed on the slow wave, the half-amplitude duration (s) and frequency (slow waves/min), were measured. The parameters of five consecutive electrical events were analysed in the absence and presence of imatinib (1–50 μm) and/or phenylephrine (1 μm) or a raised K+ saline (20 mm).

Tension recording

Various parameters were measured to characterise spontaneous contractions including: basal tone (N/g), amplitude (N/g), average rising slope (standard rise/run, N/s), duration (s), area under the curve (AUC) (N/s) and frequency (contractions/min). The basal tone and amplitude recorded was normalised over the mean tissue preparation weight (young: 24.3 mg; ageing: 30.2 mg). Contractions obtained in the presence of drugs were compared and expressed as a percentage of control contractions obtained before the addition of drugs.

A Student's t-test for paired data was used unless stated otherwise and differences were considered statistically significant at P < 0.05. All values are expressed as mean (sem). Differences between the two age groups for both types of spontaneous activity were calculated using repeated two-way anova followed by Bonferroni post-test for comparisons between each imatinib mesylate concentration.

Drugs and Solutions Used

The drugs used in this study were: imatinib mesylate (LC Laboratories, MA, USA) and phenylephrine (Sigma-Aldrich, MO, USA). Both drugs were made to stock solutions of 0.01 m in distilled water. Krebs–Henseleit solution was composed of (mm): NaCl 120.0, KCl 5.0, MgSO4.7H20 1.2, KH2PO4 1.0, NaHCO3 25.0, Glucose 11.0 and CaCl2 2.5.

Results

Spontaneous Activity of the Guinea-Pig Prostate

Spontaneous slow-wave activity

Spontaneous slow-wave activity was recorded at a faster frequency of 5.4 (1.3) cycles/min in the ageing guinea-pig prostate (n = 23, Fig. 1Bii) compared with 3.5 (0.7) cycles/min in the young guinea-pig prostates (n = 21) (unpaired t-test, P < 0.05, Fig. 1Aii). However, there were no significant differences in the resting membrane potential (mean [sem] –53.7 [2.6] mV in the young guinea-pig prostate and –53.9 [3.3] mV in ageing guinea-pig prostates, unpaired t-test, P > 0.05) or the amplitude of slow wave activity (51.4 [2.6] mV in the young guinea-pig prostate and 50.2 [3.6] mV in ageing guinea-pig prostates, unpaired t-test, P > 0.05). In addition, the number of spikes superimposed on slow waves was greater in the young guinea-pigs compared with ageing guinea-pigs, at 3.3 (0.4) spikes vs 2.3 (0.5) spikes (unpaired t-test, P > 0.05).

Figure 1.

There was little difference between the slow-wave activity of both age groups. In the young guinea-pigs, the prostate displayed slow-wave activity (Aii, n = 21) with mean (sem) amplitudes of 51.4 (2.7) mV at a frequency of 3.6 (0.7) cycles/min and generated contractions (Ai, n = 26) with amplitudes of 0.16 (0.05) N/g at a frequency of 1.8 (0.5) contractions/min. The ageing guinea-pig prostate exhibited slow waves (Bii, n = 23) with a mean (sem) amplitude of 50.2 (3.6) mV at a higher frequency of 5.4 (1.3) cycles/min and contractions (Bi, n = 28) with an amplitude of 0.19 (0.06) N/g at a frequency of 2.4 (0.5) contractions/min.

Spontaneous contractile activity

There was little difference between control contractile activity in young (n = 26, Fig. 1Ai) and ageing (n = 28, Fig. 1Bi) guinea-pig prostates. While the ageing guinea-pig prostate appeared to generate contractions at a higher frequency (2.4 [0.5] contractions/min) than young guinea-pigs (1.8 [0.5] contractions/min) this was not considered significant. This was the same for the contractile amplitude between both ages, at 0.19 (0.06) N/g in ageing guinea-pigs and 0.16 (0.05) N/g in young guinea-pigs (unpaired t-test, P > 0.05).

The Effects of Imatinib Mesylate on Spontaneous Slow-Wave Activity in the Guinea-Pig Prostate

Imatinib mesylate appeared to have little effect on slow-wave activity of both young and ageing guinea-pig prostates. In the presence of imatinib mesylate at 1 μm, there were no significant changes in the slow-wave parameters of both ages (young: n = 4 and ageing: n = 6, Fig. 2). At 5 μm, imatinib mesylate again did not significantly affect slow-wave activity in both age groups (young: n = 4 and ageing: n = 5). This was also the case at 10 μm, where there was no significant effect on all parameters of slow-wave activity in both age groups (young: n = 3 and ageing: n = 6).

Figure 2.

Imatinib did not affect the total amplitude of slow-wave activity (i) but the frequency was significantly reduced by 50 μm imatinib in the young guinea-pig prostate (ii).

However, at 50 μm, the frequency of slow waves from the young guinea-pig prostate was reduced from 2.9 (0.2) to 1.9 (0.4) slow waves/min (P < 0.05, n = 4, Fig. 2ii). In the ageing guinea-pig prostate, the resting membrane potential was depolarised from –56 (3.3) to –50.6 (2) mV (P < 0.05, n = 5). There was no significant difference in the responses to imatinib mesylate between the two age groups but in two preparations taken from the ageing guinea-pig prostate, slow-wave activity was abolished in the presence of 50 μm imatinib mesylate; this was not included in the data presented.

Effects of Imatinib Mesylate on Spontaneous Contractile Activity in the Guinea-Pig Prostate

In contrast to the relative insensitivity of slow-wave activity to imatinib mesylate (except at the highest concentration used), spontaneous contractile activity was reduced to varying degrees in the presence of all concentrations of imatinib mesylate with greater reductions in amplitude, frequency and duration occurring at the lower concentrations for both age groups. At 1 μm, imatinib mesylate reduced the amplitude of contractions in the young guinea-pig prostate (0.2 [0.05] N/g in control to 0.07 [0.06] N/g in imatinib mesylate, P < 0.05, n = 7, Fig. 3i) and reduced the frequency to 24.7 (1.8)% of control (P < 0.05, Fig. 3ii). There was a greater degree of inhibition in the ageing guinea-pig prostate (n = 5), where the amplitude was suppressed from 0.13 (0.05) to 0.01 (0.01) N/g (P < 0.05, Fig. 3i), the duration was reduced from 7.6 (1.6) s to 1.4 (1.2) s (P < 0.01, Fig. 3iii) and frequency slowed down to 2.7 (2.6)% of control (P < 0.01, Fig. 3ii). The difference between the inhibited frequencies of the two age groups was very significant (two-way anova, P < 0.001).

Figure 3.

Imatinib significantly inhibited the contractile amplitude at all concentrations in young but not ageing prostates (i). Across all imatinib concentrations, contractions were significantly slowed down (ii) and the duration was only inhibited at low concentrations (iii).

The amplitude and frequency of contractions at 5 μm were significantly reduced from 0.2 (0.03) to 0.07 (0.04) N/g (P < 0.05, n = 9, Fig. 3i) and 1.6 (0.4) to 0.6 (0.3) contractions/min (P < 0.05, Fig. 3ii). Similarly, the responses in the ageing guinea-pig prostate were also reduced to 35.3 (7.4)% (from 0.13 [0.02] to 0.03 [0.01] N/g, P < 0.01, n = 8, Fig. 3i) and slowed down from 2.1 (0.4) contractions/min in control to 0.4 (0.2) contractions/min in imatinib mesylate (P < 0.05, Fig. 3ii).

At 10 μm imatinib mesylate, the contractile amplitude of the young guinea-pig prostate was significantly reduced to 26.4 (12.9)% (from 0.12 [0.016] to 0.03 [0.015] N/g, P < 0.05, n = 7). In the ageing guinea-pig prostate, only the duration and frequency of contractions were significantly reduced from 8 (1.3) to 2 (1) s and from 3 (0.8) to 0.3 (0.1) contractions/min, respectively (both P < 0.01, n = 9, Fig. 3iii and ii, respectively).

The difference in contractile amplitude between the two age groups was significant at the highest concentration of 50 μm imatinib mesylate (two-way anova, P < 0.05). Contractions generated from the young guinea-pig prostate (n = 5, Fig. 3i) were reduced to 27.1 (16.6)% of control amplitude (0.2 [0.09] N/g in control, 0.08 [0.060] N/g in imatinib mesylate, P < 0.01). Imatinib mesylate did not reduce contractile activity to the same extent in the ageing guinea-pig prostate (n = 8); this difference was considered significant (two-way anova, P < 0.05).

Imatinib Mesylate on Agonist or High K+-Induced Activity

Contractions in response to phenylephrine (1 μm) and high K+ (20 mm) was also examined in the absence and presence of imatinib mesylate (1 μm) to determine whether the prostate smooth muscle cells were still viable after imatinib mesylate incubation. In the absence of imatinib, the contractile frequencies and AUC were significantly augmented in the young (phenylephrine: n = 8; K+: n = 6) and ageing guinea-pig prostate (phenylephrine: n = 8; K+: n = 9). Although spontaneous contractions were significantly suppressed after 60 min exposure to 1 μm imatinib, this was not seen in responses to phenylephrine and high K+ in both young (phenylephrine: n = 7; K+: n = 5) and ageing guinea-pigs (phenylephrine: n = 8; K+: n = 9), where contractile responses were similar to contractions without imatinib (Fig. 4).

Figure 4.

The application of phenylephrine (PE) and high K+ enhanced the amplitude, AUC and frequency of contractions both in the presence and absence of imatinib (1 μm). The lack of significant difference between the contractile responses with and without imatinib suggests smooth muscle cell integrity remains intact after incubation with imatinib.

Slow-wave frequency was enhanced with the application of phenylephrine and high K+ in both young (phenylephrine: n = 15; K+: n = 7) and ageing guinea-pig prostates (phenylephrine: n = 11; K+: n = 6) but similar to contractions in the presence of imatinib mesylate, these responses were not significantly different to responses in the absence of imatinib in both age groups.

Discussion

ICC-Like Cells in the Guinea-Pig Prostate

Ultrastructurally, c-Kit immunopositive PICs from the guinea-pig prostate have been described as containing numerous mitochondria, highly developed rough endoplasmic reticulum and both caveolae and an incomplete basal lamina [5, 12]. These observations taken together with the lack of contractile function [13] suggests that the PICs are distinct entities from other morphologically similar cells, e.g. fibroblasts and myofibroblasts.

c-Kit is a proto-oncogene that encodes for receptor tyrosine kinases that regulate the phenotype typical of ICC-like cells, e.g. cell proliferation, apoptosis and Ca2+ mobilisation. The presence of c-Kit on PICs in the guinea-pig prostate alludes to a functional aspect that may regulate electrical propagation similar to the ICCs of the GIT. Both c-Kit and its truncated form, Tr-Kit, lead to downstream activation of PLCγ1 (phospholipase Cγ1) and cell proliferation, an event that can be suppressed by the addition of imatinib mesylate [15]. Imatinib mesylate competitively inhibits the ATP binding site located on the receptor tyrosine kinase, preventing the phosphorylation of PLCγ1 and the cleavage of PIP2 (phosphatidylinositol 4,5-bisphosphate) into IP3 (inositol trisphosphate) and DAG (diacylglycerol), which can significantly reduce Ca2+ mobilisation and contractility.

Recently, various research groups have investigated the effect of imatinib mesylate on the spontaneous electrical and mechanical activity of smooth muscle organs including the guinea-pig stomach [13], urinary bladder [12] and gallbladder [1], to ascertain the role of ICC-like cells in the generation of their spontaneous activity. The responses produced were varied: contractile activity was suppressed but electrical activity persisted in the guinea-pig stomach and bladder. In the gallbladder, imatinib abolished all spontaneous activity. Therefore, in the present study we have investigated the role of tyrosine kinase receptors in the generation of spontaneous activity in the guinea-pig prostate. In addition, we used two age groups to ascertain whether there are any age-related differences to the effects of imatinib mesylate, especially as pacemaker potentials have not been recorded in the ageing prostate gland to date [14].

Imatinib Mesylate on Young vs Ageing Guinea-Pig Prostates

In the presence of imatinib mesylate, both age groups displayed little change in spontaneous slow-wave activity with the exception of the highest concentration (Fig. 2ii). A major difference between the two age groups occurred in the amplitude of contractile activity at all concentrations of imatinib mesylate. In the ageing guinea-pig prostate, a concentration-dependent inhibition was seen where low concentrations of imatinib mesylate produced the most inhibition whereas high concentrations produced less inhibition of contractile activity.

In contrast, contractile amplitude was generally reduced to 26–40% across all concentrations in the younger guinea-pig prostate. This difference between age groups may parallel the reduction in pacemaker potential activity and increase in spike potential activity with age, which was previously observed by Dey et al. [14]. Like the guinea-pig bladder [16], the guinea-pig prostate generates nifedipine-sensitive spike potentials, as well as slow-wave activity. Spike potentials arise from the opening of L-type Ca2+ channels by independent smooth muscle cells rather than electrical propagation from PICs leading to contractions without activating tyrosine kinases and bypassing the release of Ca2+ from intracellular stores. Hyperactive smooth muscle cells recorded in ageing guinea-pig prostates that have a higher probability of firing independently of propagating pulses from pacemaker cells could correlate to the slightly higher amplitude and frequency in spontaneous contractions of the ageing guinea-pig prostate. Furthermore, contractions caused by spike potentials may be more resistant to the effects of imatinib mesylate; this was seen in a preparation displaying spike potentials in the ageing guinea-pig prostate, where imatinib mesylate had no effect on amplitude or frequency (data not shown).

In support of this notion, previous studies by Torihashi et al. [17] reported a reduction in c-Kit expression with age in the murine GIT. In the guinea-pig prostate, cells still expressed c-Kit with age [6]; however, it is possible that a change in c-Kit function may occur in the guinea-pig prostate, particularly if PIC cell size and number of processes on the cell body can change with age. In the likelihood that c-Kit function is altered with age, it would correspond to the present findings that imatinib mesylate exerts a greater inhibitory effect on younger guinea-pig prostates, as they appear to be more reliant on PIC pacemaker activity. By contrast, the ageing guinea-pig prostate is more likely to generate imatinib mesylate-insensitive contractions, resulting from the opening of Ca2+ channels during spike discharge. Kubota et al. [18] speculated that low concentrations of imatinib mesylate (to 10 μm) specifically inhibited tyrosine kinase receptors, while high concentrations (50 μm) directly affected Ca2+ channels. This is fairly consistent with reports that suggest the tyrosine kinase receptor is closely associated with Ca2+ channels [19], especially as the channels could potentially play a greater role in generating spontaneous activity in the ageing guinea-pig prostate.

Imatinib Mesylate on Spontaneous Slow Waves and Contractions

Overall, contractile activity was reduced to a greater degree at all concentrations of imatinib mesylate in comparison to slow-wave activity in both age groups. This was also seen in studies of the guinea-pig stomach and detrusor [12, 13], where imatinib mesylate significantly reduced spontaneous contractions but not spontaneous electrical activity. These studies suggested that imatinib mesylate uncoupled synchronised smooth muscle bundles and affected Ca2+ mobilisation in smooth muscle cells rather than directly affecting the pacemaker activity of ICC-like cells [13, 18].

In the present study, the persistence of slow-wave activity in the guinea-pig prostate indicates that cell-to-cell coupling as well as Ca2+ mobilisation is still intact for slow-wave generation and propagation to occur and that pacemaker activity generated by PICs is relatively unaffected by the blockade of c-Kit. Rather, by inhibiting tyrosine kinase receptors, it is the contractile apparatus of smooth muscle cells that may be inadvertently affected by imatinib mesylate. Phosphatidylinositol-3-OH kinase (PI3-K) activation is one of the downstream effects of tyrosine kinase receptor activity that can influence the reorganisation of actin filaments [20]. It is possible that the impairment of tyrosine kinase receptors (and the resulting activation of PI3-K) by imatinib mesylate may prevent actin filament function in the prostate stroma thus impeding smooth muscle contractility. However, as contractile responses to phenylephrine and high K+ were unaffected by imatinib mesylate at the lowest concentration (Fig. 4), perhaps a separate signalling pathway is used for agonist- or high K+-induced contractions, as the activation of PI3-K may regulate contractions independently from Ca2+ and myosin light chain phosphorylation [21, 22].

To summarise, imatinib mesylate reduced the spontaneous contractile activity in the younger guinea-pigs to a greater extent than the ageing guinea-pig prostate, which is consistent with the notion that the ageing guinea-pig prostate is less reliant on the pacemaker ability of PICs and more reliant on spike activity generated by asynchronous smooth muscle bundles [14]. Ca2+ mobilisation from the PICs is not affected by c-Kit inhibition but tyrosine kinase receptor blockade does affect the spontaneous contractility of smooth muscle cells. Spontaneous activity in PICs appears to be regulated by another Ca2+ mobilisation pathway that is not associated with tyrosine kinase signalling.

Acknowledgements

This manuscript was supported by the NHMRC and a Monash Postgraduate Publication Award.

Conflict of Interest

None declared.

Abbreviations
AUC

area under the curve

GIT

gastrointestinal tract

ICC

interstitial cells of Cajal

PICs

ICC-like prostatic interstitial cells

PI3-K

phosphatidylinositol-3-OH kinase

Ancillary