EFFECTS OF ISOPROTERENOL ON SPONTANEOUS EXCITATIONS IN DETRUSOR SMOOTH MUSCLE CELLS OF THE GUINEA PIG

0022-5347/01/1661-0335/0 THE JOURNAL OF UROLOGY® Copyright © 2001 by AMERICAN UROLOGICAL ASSOCIATION, INC.®

Vol. 166, 335–340, July 2001 Printed in U.S.A.

EFFECTS OF ISOPROTERENOL ON SPONTANEOUS EXCITATIONS IN DETRUSOR SMOOTH MUSCLE CELLS OF THE GUINEA PIG YOKO NAKAHIRA, HIKARU HASHITANI, HIROYASU FUKUTA, SHOICHI SASAKI, KENJIRO KOHRI AND HIKARU SUZUKI From the Departments of Physiology and Urology, Nagoya City University Medical School, Nagoya, Japan

ABSTRACT

Purpose: Because ␤-adrenoceptor agonists would be a useful tool for the pharmacological treatment of unstable bladder, we investigated the cellular mechanisms underlying ␤-adrenoceptor mediated inhibition on spontaneous excitation in detrusor smooth muscle. Materials and Methods: Detrusor smooth muscle bundles were isolated from guinea pig bladders. Changes in membrane potential were recorded using an intracellular recording technique. In preparations loaded with the calcium indicator fura-PE3 changes in the concentration of intracellular calcium ions were measured simultaneously with membrane potential. Effects of isoproterenol on spontaneous changes in the membrane potential and intracellular Ca2⫹ were examined Results: Detrusor smooth muscle cells exhibited spontaneous action potentials that were associated with transient increases in intracellular Ca2⫹ (calcium transients). Isoproterenol, which hyperpolarized the membrane, prevented action potentials and calcium transients. This induced inhibition of calcium transients was not affected by cyclopiazonic acid. Isoproterenol induced hyperpolarization was inhibited by inhibitors of protein kinase A, N-[2-((p-bromocinnamyl)amino)ethyl]-5isoquinolinesulfonamide, hydrochloride and Rp-adenosine-3⬘,5⬘-cyclic phosphorothioate. Hyperpolarization was blocked by a solution containing 30 mM. potassium but not by a range of potassium channel blockers. Ouabain and a solution of 0.5 mM. potassium also inhibited hyperpolarization. Conclusions: Our results suggest that isoproterenol prevented spontaneous action potential discharges and associated calcium transients through the activation of protein kinase A. The isoproterenol induced inhibition of intracellular Ca2⫹ largely depends on the prevention of spontaneous action potentials since the contribution of the intracellular calcium store was small. Isoproterenol hyperpolarizes the membrane, probably by stimulating sodium pump activity. KEY WORDS: bladder; muscle, smooth; isoproterenol; action potentials; calcium

Since human detrusor smooth muscle contraction is almost solely mediated by acetylcholine release from the parasympathetic nerves,1 muscarinic receptor antagonists have been used in the pharmacological treatment of unstable bladder to suppress an overactive micturition reflex.2, 3 The antimuscarinic agents currently used lack selectivity for the bladder, and so their clinical usefulness is diminished due to side effects.2, 3 Pharmacological treatment of unstable bladder requires an agent that reduces the excitability of detrusor smooth muscle without preventing normal micturition. Potassium channel openers, which inhibit spontaneous contractions and underlying action potentials in bladder smooth muscle,4, 5 have not been useful clinically because of side effects on the cardiovascular system.2, 3 Alternatively calcium channel antagonists without antimuscarinic effects have been suggested as a treatment but again clinical trials demonstrated that such drugs are unsatisfactory.2, 6, 7 The activation of ␤-adrenoceptors has been shown to relax bladder smooth muscle in many mammals, including humans. This effect is considered to be mediated by a cyclic adenosine monophosphate dependent kinase termed protein kinase A.1 Despite the potent relaxing effect of ␤-agonists on bladder smooth muscle strips the clinical usefulness of ␤-agonists for the treatment of unstable bladder remains to be established.8 The lack of interest in ␤-agonists may be due to the atypical character of ␤-adrenoceptors in the human bladder, which have been identified as ␤3-adrenoceptors.9, 10 Recent advances into ␤3-subtype adrenoceptors in the bladAccepted for publication January 5, 2001.

der may lead to new developments in the treatment of unstable bladder. In addition, investigating the cellular mechanisms underlying ␤-adrenoceptor mediated relaxation may provide us with novel targets for pharmacological treatment of unstable bladder. It has been demonstrated in many smooth muscle tissues that protein kinase A hyperpolarizes the membrane of smooth muscle cells and, thus, inhibits calcium entry through L-type calcium channels, which leads to a reduction in intracellular Ca2⫹.11–15 However, to our knowledge the intracellular pathway involved in the ␤-adrenoceptor mediating relaxation of bladder smooth muscle has not been identified. To clarify the cellular mechanisms of ␤-adrenergic relaxation of bladder smooth muscle we examined the effects of isoproterenol, a ␤-adrenoceptor agonist, on spontaneous electrical and calcium responses. METHODS

The procedures described were approved by the animal experimentation ethics committee at Nagoya City University. Male guinea pigs weighing 200 to 300 gm. were sacrificed by a blow to the head after by cervical exsanguination. Single bundles of 1 to 2 mm. ⫻ 0.2 to 0.5 mm. detrusor smooth muscle were then prepared. Preparations were pinned out on a silicone elastomer at the bottom of a recording chamber with an approximate volume of 1 ml., which was mounted on the stage of an inverted microscope. The preparations were superfused with physiological saline warmed to 35C at a constant flow rate of 2 ml. per minute. Individual bladder smooth muscle cells

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were impaled with glass capillary microelectrodes filled with 0.5 M. KCl (tip resistance 150 to 250 M⍀). Membrane potential changes were recorded using a high input impedance Axoclamp-2B amplifier (Axon Instruments, Inc., Foster City, California) and displayed on a SS-9622 cathode ray oscilloscope (Iwatsu, Tokyo, Japan). After low pass filtering at a cutoff frequency of 1 kHz. membrane potential changes were digitized and stored on a personal computer for later analysis. For measurements of the concentration of intracellular calcium ion preparations were loaded with fluorescent furaPE3 dye by incubating with 1 mM. Ca2⫹ physiological saline containing 10 ␮M. fura-PE3 AM for 90 minutes at room temperature. After loading preparations were superfused with dye-free warmed physiological saline for 30 minutes. Preparations were illuminated with 2 periods of ultraviolet light at a wavelength of 340 and 380 nm. with an alternating frequency of higher than 40 Hz. The ratio of the emission fluorescence (F340/F380) was measured through a barrier filter of 510 nm. at a sampling rate of less than 100 ms. using the ARGUS/HiSCA micro photoluminescence measurement system (Hamamatsu Photonics, Hamamatsu, Japan). Physiological saline composition was 122 mM. NaCl, 4.7 mM. KCl, 1.2 mM. MgCl2, 2.5 mM. CaCl2, 15.5 mM. NaHCO3, 1.2 mM. KH2PO4 and 11.5 mM. glucose. The solution was aerated with 95% O2 and 5% CO2. The drugs used were cyclopiazonic acid, nifedipine, isoproterenol hydrochloride, propranolol hydrochloride, ouabain, apamin, 4-amynopyridine, barium chloride, glibenclamide (Sigma Chemical Co., St. Louis, Missouri), adenosine-3⬘,5⬘-cyclic phosphorothiolate-Rp (Rp-cAMPS), fura-PE3 AM, N-[2-((pbromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride (H-89) (Calbiochem, San Diego, California) and charybdotoxin (Peptide Institute, Osaka, Japan). Nifedipine was dissolved in 100% ethanol, and cyclopiazonic acid, glibenclamide, H-89 and fura-PE3-AM were dissolved in dimethyl sulfoxide. The remaining drugs were dissolved in distilled water. Measured values are expressed as the mean plus or minus standard deviation (SD).

RESULTS

Effects of isoproterenol on spontaneous action potentials recorded from smooth muscle cells of the guinea pig bladder. Bladder smooth muscle cells had a resting membrane potential of approximately ⫺40 mV. and exhibited spontaneous action potentials that were abolished by 10 ␮M. nifedipine, as reported previously in larger preparations of bladder smooth muscle.6, 8 A low concentration of 1 nM. isoproterenol reduced the frequency of spontaneous action potentials with or without slight hyperpolarization of the membrane (fig. 1, A). Higher concentrations of 10 nM. to 10 ␮M. isoproterenol hyperpolarized the membrane and abolished spontaneous action potentials (fig. 1, A). Hyperpolarization amplitude increased in a concentration dependent manner and reached a mean maximum plus or minus SD of 10.3 ⫾ 2.2 mV. in 25 experiments at 1 ␮M. isoproterenol (fig. 1, B). Involvement of cyclic adenosine monophosphate in the hyperpolarization induced by isoproterenol. Forskolin (10 ␮M.), an adenylate cyclase activator, hyperpolarized the membrane by a mean of 10.9 ⫾ 2.3 mV. in 7 experiments and prevented the generation of action potentials (fig. 1, A). In the presence of propranolol 1 ␮M. isoproterenol failed to cause detectable hyperpolarization in 4 experiments, while in 3 forskolin still caused an amplitude of hyperpolarization that was similar to that observed in the absence of propranolol. To examine the contribution of cyclic adenosine monophosphate to isoproterenol induced hyperpolarization we studied the effects of H-89 and Rp-cAMPS, which are inhibitors of protein kinase A. In preparations exposed to 1 ␮M. H-89 for 10 minutes the amplitude of isoproterenol induced hyperpolarization was reduced to a mean of 44.5% ⫾ 7.9% the control value in 4 experiments (fig. 2). In the presence of 10 ␮M. H-89 isoproterenol failed to cause detectable hyperpolarizations and did not block spontaneous action potentials in 3 experiments (fig. 2). In 3 preparations treated with 25 ␮M. RpcAMPS for 20 minutes isoproterenol caused hyperpolariza-

FIG. 1. Effects of isoproterenol and forskolin on changes in membrane potential recorded in guinea pig bladder smooth muscle cells. A, in same cell 1 nM. isoproterenol reduced frequency of spontaneous action potentials without detectable hyperpolarization and 0.1 ␮M. isoproterenol hyperpolarized membrane and prevented generation of spontaneous action potentials, while in other cell 10 ␮M. forskolin hyperpolarized membrane and abolished spontaneous action potentials. B, hyperpolarization amplitude versus agent concentrations. Black circles indicate isoproterenol induced responses in 8 to 25 preparations. Open circle indicates responses in 4 preparations in presence of 1 ␮M. propranolol, triangle forskolin induced responses in 7 preparations and vertical error bars SD.

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FIG. 2. Effects of H-89 on isoproterenol induced hyperpolarizations recorded in guinea pig bladder smooth muscle cells. In control solution 1 ␮M. isoproterenol hyperpolarized membrane of single cell and abolished spontaneous action potential discharges. In presence of 1 ␮M. H-89, 1 ␮M. isoproterenol still prevented generation of spontaneous action potentials in same cell but produced smaller hyperpolarization than in control condition. In presence of 10 ␮M. H-89, 1 ␮M. isoproterenol failed to hyperpolarize membrane of same cell and did not inhibit spontaneous action potentials.

tion with a mean amplitude of 44.4% ⫾ 4.4% that of control values (data not shown). Effects of isoproterenol on spontaneous changes in intracellular Ca2⫹ and membrane potential in guinea pig bladder smooth muscle cells. In a separate series of experiments changes in intracellular Ca2⫹ were measured simultaneously with membrane potential. In detrusor smooth muscle cells each spontaneous action potential was accompanied by a calcium transient. Spontaneous action potentials and calcium transients were abolished by 10 ␮M. nifedipine in 5 experiments (fig. 3, A). Nifedipine also markedly reduced the resting level of intracellular Ca2⫹ (fig. 3, B). A low concentration of 1 nM. isoproterenol reduced the frequency of action potentials with slight hyperpolarization in 3 preparations (fig. 3, C). Isoproterenol (1 nM.) also reduced the frequency of calcium transients and the resting level of intracellular Ca2⫹ (fig. 3, D). Higher concentrations of 10 nM. to 1 ␮M. isoproterenol prevented the generation of action potentials and calcium transients (fig. 3, E). Higher concentrations of isoproterenol also hyperpolarized the membrane and reduced the resting level of intracellular Ca2⫹ (fig. 3, F). Effects of cyclopiazonic acid on isoproterenol induced hyperpolarization and intracellular Ca2⫹ oscillations. To investigate the contribution of intracellular calcium stores to isoproterenol induced inhibitions in intracellular Ca2⫹ we studied the effects of cyclopiazonic acid on the membrane potential and intracellular Ca2⫹. Cyclopiazonic acid (10 ␮M.) increased the frequency of spontaneously action potential discharges with slight depolarization in 4 experiments (mean 5.6 ⫾ 2.1 mV.). Cyclopiazonic acid also increased the frequency of spontaneous increases in intracellular Ca2⫹ and increased baseline intracellular Ca2⫹. In 4 preparations exposed to 10 ␮M. cyclopiazonic acid for 20 minutes 1 ␮M. isoproterenol hyperpolarized the membrane and abolished spontaneous action potentials. Isoproterenol also prevented calcium transients and reduced baseline intracellular Ca2⫹ (fig. 4). Effects of K⫹ channel blockers and increasing extracellular K⫹ on isoproterenol induced hyperpolarizations. To investigate the ionic mechanisms of isoproterenol induced hyperpolarization we examined the effects of a series of potassium channel blockers on isoproterenol induced hyperpolarization. The K⫹ channel blockers used were 0.1 ␮M. charybdotoxin in

5 preparations for large or intermediate conductance Ca2⫹ activated potassium channels, 0.1 ␮M. apamin in 4 for small conductance Ca2⫹ activated K⫹ channels, 10 ␮M. glibenclamide in 4 for adenosine triphosphate sensitive K⫹ channels, 1 mM. 4-aminopyridine in 4 for voltage dependent K⫹ channels and 0.1 mM. barium ions in 3 for inward rectifier K⫹ channels.16 Surprisingly none of the blockers blocked hyperpolarization. If the opening of K⫹ channels, which were not sensitive to the blockers examined, contributed to isoproterenol induced hyperpolarization, increasing the extracellular concentration of K⫹ would still be expected to reduce the hyperpolarization amplitude. Increasing extracellular K⫹ from 5 to 30 mM. depolarized the membrane by a mean of 19.2 ⫾ 3.1 mV. in 10 preparations and dramatically increased the frequency of action potentials (fig. 5, A). In high K containing solution 1 ␮M. isoproterenol did not cause detectable hyperpolarizations but gradually reduced the amplitude of action potentials and eventually abolished them in 8 experiments (fig. 5, A). Effects of ouabain and lowering extracellular K⫹ on isoproterenol induced hyperpolarization. To examine the contribution of the sodium pump to isoproterenol induced hyperpolarization we studied the effect of ouabain on hyperpolarization. Ouabain (10 ␮M.) increased the frequency of spontaneous action potentials, which was associated with a slight mean depolarization of 3.8 ⫾ 0.5 mV. in 7 experiments (fig. 5, B). In the presence of 10 ␮M. ouabain 1 ␮M. isoproterenol gradually reduced the amplitude of spontaneous action potentials and abolished them without detectable hyperpolarization in 7 preparations (fig. 5, B). After ouabain washout in 5 experiments the membrane slowly hyperpolarized to reach a mean peak hyperpolarization level of ⫺82.8 ⫾ 4.1 mV. The effect on isoproterenol induced hyperpolarization of lowering extracellular K⫹ from 5 to 0.5 mM. was also examined. Lowering extracellular K⫹ increased spontaneous action potentials with a slight mean depolarization of 4.1 ⫾ 1.1 mV. in 6 preparations (fig. 5, C). In the presence of low K containing solution 1 ␮M. isoproterenol again gradually inhibited spontaneous action potentials without detectable hyperpolarization in 6 experiments (fig. 5, C). After switching to normal physiological saline in 5 preparations the membrane

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FIG. 4. Effects of cyclopiazonic acid on isoproterenol induced hyperpolarization and intracellular Ca2⫹ inhibition. A, in preparation previously exposed to 10 ␮M. cyclopiazonic acid for 20 minutes 1 ␮M. isoproterenol hyperpolarized membrane and abolished action potential discharges. B, 1 ␮M. isoproterenol also prevented generation of calcium transients and reduced baseline intracellular Ca2⫹.

FIG. 3. Effects of isoproterenol on spontaneous changes in membrane potential and intracellular Ca2⫹ in same preparation of guinea pig bladder smooth muscle cells. Under control condition detrusor smooth muscle cells exhibited spontaneous action potentials and associated calcium transients. A, 10 ␮M. nifedipine depolarized membrane and abolished spontaneous action potentials. B, 10 ␮M. nifedipine also prevented calcium transients and reduced resting level of intracellular Ca2⫹. C, 1 nM. isoproterenol reduced frequency of spontaneous action potentials with slight hyperpolarization. D, 1 nM. isoproterenol also reduced frequency of calcium transients and baseline intracellular Ca2⫹. E, 0.1 ␮ M. isoproterenol hyperpolarized membrane and abolished spontaneous action potentials. F, 0.1 ␮M. isoproterenol also prevented calcium transients and reduced baseline intracellular Ca2⫹.

rapidly hyperpolarized and reached a mean peak negative potential of ⫺89.3 ⫾ 2.8 mV. DISCUSSION

This study demonstrated that isoproterenol prevented the generation of spontaneous action potentials and increases in intracellular Ca2⫹ in guinea pig bladder detrusor smooth muscle. Isoproterenol also hyperpolarized the membrane and reduced the resting level of intracellular Ca2⫹. Isoproterenol induced hyperpolarization was inhibited by ouabain and by lowering or increasing extracellular K⫹ but not by a range of potassium channel blockers. The stimulation of ␤-adrenoceptors has been shown to inhibit contractions of bladder smooth muscle in many mammals, including human.1 Generally the activation of ␤-adrenoceptors is considered to stimulate adenylate cyclase to increase cyclic adenosine monophosphate, which activate protein kinase A to express its biological effects.1, 2 Increases in cyclic adenosine monophosphate induced by forskolin or

phosphodiesterase inhibitors suppress contractility in the guinea pig bladder.17 In our study forskolin, an adenylate cyclase activator, hyperpolarized the membrane and abolished spontaneous action potentials. Furthermore, isoproterenol induced hyperpolarization was inhibited by the protein kinase A inhibitors H-89 and Rp-cAMPS, suggesting that the inhibitory actions of isoproterenol are mediated by protein kinase A. Since the effects of isoproterenol on intracellular Ca2⫹ paralleled its effects on changes in the membrane potential, it may be suggested that the effects of isoproterenol on electrical activity mainly account for the inhibition of intracellular Ca2⫹. Moreover, the inhibitory effects of isoproterenol on intracellular Ca2⫹ remained unchanged in the presence of cyclopiazonic acid, suggesting that Ca2⫹ uptake into intracellular calcium stores may not have a major role in the isoproterenol induced inhibition of intracellular Ca2⫹. This finding is consistent with those of a previous report indicating that the pumping of Ca2⫹ into intracellular stores has a major role in cyclic guanosine monophosphate but not in cyclic adenosine monophosphate induced relaxation in canine tracheal smooth muscle.18 In many smooth muscles protein kinase A induced hyperpolarization results from the activation of several types of potassium channels.12–15 However, none of the potassium channel blockers examined, including apamin, charybdotoxin, 4-aminopyridine, glibenclamide or barium ions, blocked isoproterenol induced hyperpolarization in bladder smooth muscle. Therefore, the activation of potassium channels may not underlie isoproterenol induced hyperpolarization in guinea pig detrusor smooth muscles. Ouabain inhibited isoproterenal induced hyperpolarization, suggesting that sodium pump activity contributes to hyperpolarization. This result confirms those in previous reports in which the activation of ␤-adrorenoceptors or the cyclic adenosine monophosphate-protein kinase A pathway stimulated sodium pump activity.19, 20 The great hyperpolarization observed after ouabain washing or low potassium containing solution may represent over activation of the sodium pump, probably due to an accumulation of Na⫹ during pump blocking. If isoproterenol hyperpolarizes the membrane through the opening of potassium channels, which were not sensitive to the blockers examined, hyperpolarization amplitude would be expected to be increased by lowering extracellular K⫹. In contrast, if the sodium pump contributes to hyperpolarization, lowering extracellular K⫹ would reduce the hyperpolarization amplitude by inhibiting sodium pump activity. Since isoproterenol did not cause detectable hyperpolarization in low extracellular K⫹, hyperpolarization is more likely to be

EFFECTS OF ISOPROTERENOL ON SPONTANEOUS EXCITATION IN DETRUSOR SMOOTH MUSCLE ⫹

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2⫹

stimulate Na /Ca exchange to enhance sodium pump activity. Isoproterenol prevented the generation of spontaneous action potentials without detectable hyperpolarization in solutions containing ouabain, or low or high extracellular K⫹. These results suggested that isoproterenol is capable of directly blocking voltage dependent L-type calcium channels, unlike in other tissues, in which ␤-adrenoceptors have been reported to activate rather than inhibit L-type calcium channels.22–24 In the control solution low concentrations of isoproterenol reduced the frequency but not the amplitude of spontaneous action potentials. Therefore, only a higher concentration of isoproterenol may directly inhibit L-type calcium channels and this effect became obvious when hyperpolarization was prevented. CONCLUSIONS

The stimulation of ␤-adrenoceptors activates protein kinase A to hyperpolarize the membrane, probably through the activation of sodium pump activity. The inhibition of electrical activity mainly accounts for the ␤-adrenoceptor mediated reduction in intracellular Ca2⫹ for relaxing bladder smooth muscle. Our study indicates that not only ␤-adrenoceptor agonists, but also drugs that modify protein kinase A or the sodium pump would be useful targets as pharmacological treatment for detrusor instability. Dr. Rick Lang provided manuscript assistance. REFERENCES

FIG. 5. Continuous recordings from 3 preparations show effects of altering extracellular K⫹ and ouabain on isoproterenol induced hyperpolarization. A, increasing extracellular K⫹ ([K⫹]o) from 5 to 30 mM. depolarized membrane and increased frequency of spontaneous action potentials. In presence of high potassium solution 1 ␮M. isoproterenol did not cause detectable hyperpolarization but gradually reduced amplitude of action potentials and eventually abolished them. B, 10 ␮M. ouabain increased frequency of spontaneous action potentials with slight depolarization of membrane. In presence of 10 ␮M. ouabain 1 ␮M. isoproterenol did not cause detectable hyperpolarization but gradually suppressed spontaneous action potentials. C, lowering extracellular K⫹ from 5 to 0.5 mM. increased frequency of action potentials with slight depolarization. In presence of low potassium solution 1 ␮M. isoproterenol abolished spontaneous action potentials without detectable hyperpolarization.

induced by activation of the sodium pump rather than by potassium channels. However, the reduction in the amplitude of isoproterenol induced hyperpolarization in high extracellular K⫹ containing solution suggests involvement of the potassium channels rather than the sodium pump in isoproterenol induced hyperpolarization. A possible explanation of this discrepancy is that the sodium pump had already been activated in the high potassium containing solution preparation and, thus, isoproterenol failed to cause further hyperpolarization. In cardiac cells the sodium pump is simulated by an increase in extracellular K⫹.21 Alternatively increasing extracellular K⫹ may indirectly stimulate sodium pump activity through the activation of the Na⫹/Ca2⫹ exchanger. It has been reported that the sodium pump is largely co-distribute with the Na⫹/Ca2⫹ exchanger and intracellular calcium stores in smooth muscle.22 Increasing extracellular K⫹ depolarizes the membrane and dramatically increases the frequency of action potential discharges. This increased influx of Ca2⫹ through L-type channels would greatly stimulate calcium release from intracellular stores by calcium-induced calcium release and may

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