Loss of ryanodine receptor calcium-release channel expression associated with overactive urinary bladder smooth muscle contractions in a detrusor instability model

OBJECTIVE: To investigate the changes in spontaneous bladder smooth muscle contractions that occur during detrusor instability (DI), and to test the possibility that altered function or expression of ryanodine receptors (RyRs) could account for the increased bladder contractions. MATERIALS AND METHODS: After 8 weeks of partial bladder outlet obstruction, DI was confirmed in female experimental rats by filling cystometry. Muscle strips were dissected from freshly isolated bladders, and isometric tension recorded in strips from DI and normal bladders. The contractions were recorded during electrical stimulation or exposure to various agents. Western blot analysis was used to determine RyR expression in DI and normal bladder muscle. RESULTS: In DI bladder muscle, spontaneous contractile activity persisted in the presence of blockers for known neurotransmitter receptors in the bladder wall. The RyR blocker ryanodine significantly increased the spontaneous contractile frequency in normal bladder strips, but failed to affect spontaneous contractions in DI muscle. Caffeine inhibited spontaneous contractile activity in both the DI and normal strips. After administering the l-type Ca(2+) channel antagonist nimodipine, the myogenic contractile activity was abolished in normal strips; in contrast, in DI strips, the amplitude of contractions was reduced but the frequency of contractions was unchanged. Western blot analysis showed that RyR expression was lower in DI muscle than in normal bladder muscle. CONCLUSION: These results provide the first characterization of a loss of regulation of spontaneous contractile activity by RyRs in DI muscle associated with a significant decrease in RyR expression. RyRs in normal detrusor muscle act as negative-feedback regulators of spontaneous contractile activity, presumably by releasing Ca(2+) that activates Ca(2+)-dependent K(+) channels to decrease contractility. This mechanism might be weakened in DI muscle, resulting in spontaneous contractile overactivity.     

Protein kinase c regulates purinergic component of neurogenic contractions in mouse bladder

PURPOSE: We evaluate the role of protein kinase C in excitatory purinergic neurotransmission in the mouse bladder. MATERIALS AND METHODS: In isolated mouse detrusor strips contractile responses to electrical field stimulation were mostly mediated by neural released acetylcholine and adenosine triphosphate (ATP). The changes in neurotransmission were measured indirectly by recording the contraction of detrusor strips in response to repetitive electrical field stimulation by trains of electrical pulses at 8 Hz. 1 second in duration. RESULTS: A protein kinase C activator, 1 to 2.5 nM. (beta-phorbol-12,13-dibutyrate (beta-PDBu), but not the inactive form alpha-phorbol-12,13-dibutyrate, significantly enhanced neurogenic detrusor contractions. The purinergic component of the evoked detrusor contractions in the presence of atropine was specifically sensitive to this enhancing effect by beta-PDBu but the cholinergic component in the alpha,beta-methylene ATP treated detrusors remained unaffected. This enhancing effect of beta-PDBu was dependent on the extracellular calcium (Ca2+) concentration. A P and/or Q type Ca2+ channel blocker, 0.1 and 0.3 microM. omega-conotoxin-MVIIC, and protein kinase C inhibitors, 0.3 and 1 microM. staurosporine and 0.3 and 1 microM. bisindolylmaleimide I but not 0.1 and 0.3 microM. omega-conotoxin-GVIA, an N type Ca2+ channel blocker, abolished the effect of beta-PDBu. Moreover, beta-PDBu did not affect the muscle responses induced by the exogenous agonists carbachol or alpha,beta-methylene ATP and potassium chloride. CONCLUSIONS: These results suggest that the activation of Ca2+ channel, especially the P and/or Q type, may be involved in the enhancing effect of protein kinase C activator beta-PDBu on muscle contractions elicited by excitatory purinergic neurotransmission in the mouse detrusor strips.     

Effects of isoproterenol on spontaneous excitations in detrusor smooth muscle cells of the guinea pig.

PURPOSE: Because beta-adrenoceptor agonists would be a useful tool for the pharmacological treatment of unstable bladder, we investigated the cellular mechanisms underlying beta-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 Ca(2+) were examined RESULTS: Detrusor smooth muscle cells exhibited spontaneous action potentials that were associated with transient increases in intracellular Ca(2+) (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]-5-isoquinolinesulfonamide, 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 Ca(2+) 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.     

Potassium channel KV alpha1 subunit expression and function in human detrusor muscle

PURPOSE: We determined whether voltage gated K+ (KV) channels are expressed and functional in human detrusor smooth muscle MATERIALS AND METHODS: Information on KV channels was obtained using electrophysiological patch clamp, immunofluorescence, Western blot and isometric tension recording techniques RESULTS: Patch clamp recordings from detrusor cells revealed a Ca2+ independent K+ current that was activated by depolarization in a voltage range near the resting potential of detrusor smooth muscle. The current was inhibited by 3,4-diaminopyridine, a blocker of KV channels. Antibodies targeted to KValpha1 subunits revealed KV1.3 and KV1.6 expression in whole bladder tissue samples and specifically in detrusor smooth muscle cells. New specific blockers of KValpha1 channel currents (correolide and recombinant agitoxin-2) had a myogenic effect, characterized by increased amplitude of spontaneous contractions without an effect on the frequency of contractions or on resting baseline tension. CONCLUSIONS: KValpha1 subunits are expressed and functionally important in human detrusor muscle.     

Electromyographic detection of purinergic activity in Guinea pig detrusor smooth muscle

PURPOSE: We recorded nerve mediated extracellular electrical activity from guinea pig detrusor smooth muscle strips using suction electrodes and determined the electrophysiological origins of this signal and its relationship to contractile activity. MATERIALS AND METHODS: Mucosa-free detrusor strips were prepared from male guinea pigs sacrificed under Home Office license, physiologically superfused, attached to a pressure transducer and electrically stimulated (0.1 millisecond pulses). Electrical signals recorded using a bipolar reversible suction electrode were processed and recorded simultaneously with changes in strip tension. The effect of superfusion with alpha, beta-methylene adenosine triphosphate (ATP), atropine, extracellular [CaCl(2)] depletion and pharmacological Ca2+ channel blockade on the electrical and mechanical signals was determined. RESULTS: A biphasic electrical signal was consistently recorded from 37 detrusor strips. The signal was sensitive to graded reduction in [CaCl(2)] of the superfusate and abolished by tetrodotoxin in 7 preparations. The signal was also abolished in 12 preparations by alpha, beta-methylene ATP in association with an attenuated contraction but not significantly reduced in amplitude (p = 0.77) despite a significant reduction in tension with atropine (mean plus or minus SD 74% +/- 14% of control, p <0.001). The signal was attenuated to a mean maximum of 9% +/- 3% of control by pharmacological Ca2+ channel blockade and the remaining signal was abolished by alpha, beta-methylene ATP. CONCLUSIONS: The extracellular electrical signal recorded from guinea pig detrusor strips using suction electrodes originates from a purinergic mechanism. Although an atropine sensitive component may be present, the signal does not depend on cholinergic neuromuscular transmission and would not be expected to be generated by normal human detrusor. Provided that the electrophysiological basis of purinergic neurotransmission in guinea pig and human bladders is similar suction electrodes may be a valuable tool with which to evaluate in vitro and clinically by electromyography the pathological purinergic neuromuscular transmission that can be expressed in addition to normal cholinergic mechanisms in detrusor from dysfunctional human bladders.     

The role of Ni(2+)-sensitive T-type Ca(2+) channels in the regulation of spontaneous excitation in detrusor smooth muscles of the guinea-pig bladder

OBJECTIVE: To explore the role of Ni(2+)-sensitive T-type Ca(2+) channels in the generation of spontaneous excitation of detrusor smooth muscles. MATERIALS AND METHODS: In isolated detrusor smooth muscle bundles of the guinea-pig bladder, changes in the membrane potential and muscle tension were measured using intracellular microelectrodes and isometric tension recording. Changes in the intracellular Ca(2+) concentration were recorded from bundles loaded with the fluorescent dye fura-PE3. RESULTS: Detrusor smooth muscles had two types of spontaneous electrical activity, i.e. individual and bursting action potentials. Ni(2+) (30 microM), a blocker for T-type Ca(2+) channels, reduced the frequency of individual action potentials without changing their amplitude. Higher concentrations of Ni(2+) (100-300 microM) converted individual action potentials into the bursts, as did apamin (0.1 microM), a blocker of small-conductance Ca(2+)-activated K(+) channels (SK). They also increased the amplitudes of spontaneous Ca(2+) transients and corresponding contractions whilst reducing their frequencies. In preparations which generated bursting action potentials, nifedipine (1 microm) converted action potentials into spontaneous transient depolarizations (STDs), and subsequent applications of Ni(2+) (100 microm) abolished STDs. Gadolinium (100 microM) and SKF96365 (10 microM), blockers for nonselective cation channels, and niflumic acid (100 microm), a blocker for Ca(2+)-activated Cl- channels, had no effect on either the amplitude or frequency of spontaneous action potentials. CONCLUSIONS: The T-type Ca(2+) channel may have dual roles in generating spontaneous excitation in detrusor smooth muscles. First, activity of these channels may account for the preceding depolarizations that lead to action potentials. Second, Ca(2+) influx through T-type Ca(2+) channels may couple functionally to SK channels, contributing to the stability of the resting membrane potential in detrusor smooth muscle. Thus, pharmacological manipulation of T-type Ca(2+) channels in detrusor smooth muscles could be of potential value for treating the overactive bladder.     

脊髓损伤后逼尿肌细胞钙通道改变

目的 探讨脊髓损伤(SCI)后逼尿肌细胞钙通道改变及其与逼尿肌反射亢进(DH)的关系.方法 建立SD大鼠骶髓上损伤动物模型,进行逼尿肌条钙通道阻滞实验及逼尿肌细胞钙震荡激光共聚焦线性扫描.结果 相同前负荷下逼尿肌条的收缩频率SCI组(2.51±0.39)明显高于正常对照(C)组(1.95±0.58,P<0.05),滴加高浓度T型钙通道阻滞剂咪拉地尔后两者收缩频率降低至相同水平,滴加高浓度L型钙通道阻滞剂异搏定后两者收缩频率下降相同;逼尿肌细胞钙振荡频率SCI组(2.68±0.42)明显高于C组(1.87±0.48,P<0.05),滴加高浓度咪拉地尔后两者钙振荡频率降低至相同水平,滴加高浓度异搏定后两者振荡频率下降相同.结论 SCI后DH的发生与逼尿肌细胞T型钙通道性状改变密切相关.     

Relaxation mechanisms of neferine on the rabbit corpus cavernosum tissue in vitro

Aim： To investigate the relaxation mechanisms of neferine （Nef） on the rabbit corpus cavemosum tissue in vitro. Methods： Strips of rabbit corpus cavemosum were mounted in organ chambers. The effects of Nef were examined on isolated muscle strips precontracted with phenylephrine （PE） alone, in the presence of NW-nitro-L-arginine （LNNA, a nitric oxide synthase inhibitor）, 1-H-[ 1,2,4]oxadiazolo[4,3-tx]quinoxalin- 1-one （ODQ, a guanylyl cyclase inhibitor）, indomethacin （cyclooxygenase inhibitor）, tetraethylammonium （Ca^2＋ -activated K^＋ channel blocker）, 4-aminopiridine （4-AP ,voltage dependent K^＋ channel blocker） and glibenclamide （ATP sensitive K^＋channel blocker）. The effects of Nef on KCl-induced contraction of isolated muscle strips were also investigated. The procedure of calcium absencecalcium addition was designed to observe the effect of Nef on two components of the contractile responses to PE based on the source of Ca^2＋ （extracellular vs. intracellular）. Results： Corpus cavemosum strips relaxed in response to Nef （10-9-10-4 mol/L） in a concentration-dependent manner with an IC50 of 4.60 × 10^-6 mol/L. However, they were not affected by LNNA, ODQ, indomethacin or K^＋-channel blockers. Nef （10^-6 mol/L, 10^-5 mol/L） concentration dependently reduced the maximal contraction response of isolated strips induced by KC1 to 79.3% ± 5.5% and 61.5% ±3.2%, respectively （P 〈 0.01）. In the calcium absence-calcium addition procedure, Nef 10.5 mol/L inhibited both intracellular calcium-dependent and extracellular calcium-dependent contraction induced by PE （2 × 10^5 mol/L） （P 〈 0.05）. The inhibition ratios were 26.2% ± 5.4% and 48.3% ±7.6%, respectively. Conclusion： The results of the present study suggest that Nef possesses a relaxant effect on rabbit corpus cavemosum tissues, which is attributable to the inhibition of extracellular Ca^2＋ influx and the inhibition of release of intracellular stored Ca^2＋, but not mediated by the     

Role of the T-type Ca2+ current on the contractile performance of guinea pig detrusor smooth muscle

AIMS: The importance of the T-type Ca(2+) current in determining detrusor contractile function was investigated by using guinea pig muscle in vitro. METHODS: NiCl(2) (200 microM) was used to block selectively the T-type Ca(2+) current, and 20 microM verapamil was used to block the L-type Ca(2+) current in this tissue. The selectivity of these agents at such concentrations has been previously demonstrated. RESULTS: In normal extracellular solution (4 mM KCl) 200 microM NiCl(2) and 20 microM verapamil reduced electrically stimulated contractions by 17 +/- 6% and 65 +/- 10%, respectively. At high concentrations of the two agents, the contraction was completely abolished by NiCl(2) but by only 74 +/- 18% in the case of verapamil; this finding suggests that NiCl(2) has additional negative inotropic actions at higher concentrations. Carbachol and KCl contractures were attenuated to a similar extent to that of electrically stimulated contractions by NiCl(2) and verapamil, which suggests that they act on the muscle rather than the motor nerve. The dependence of the membrane potential on the relative ability of NiCl(2) and verapamil to attenuate the contraction was tested by varying the extracellular [KCl], [KCl](o). Varying [KCl](o) between 2 and 10 mM depolarised detrusor myocytes from (-65.1 +/- 4.7 mV to -42.7 +/- 4.0 mV (a slope of 32 mV per 10-fold change of [KCl](o)). In low [KCl](o),blockade by NiCl(2) was more effective and that of verapamil less effective; at high [KCl](o), the reverse potency was recorded. CONCLUSIONS: The data are consistent with the hypothesis that Ca(2+) influx through both T-type and L-type Ca(2+) channels determines the contractile status of detrusor smooth muscle and that T-type channel activity is more important at membrane potentials near the resting level. A significant role for T-type channel activity in the resting state was evident in that spontaneous contractions were attenuated to a greater extent than evoked contractions.     

Comparative effects of five different calcium channel blockers on the atropine-resistant contraction in electrically stimulated rabbit urinary bladder

The effects of the calcium channel blockers nifedipine, verapamil, diltiazem, flunarizine, and lidoflazin on electrically induced contractions of isolated rabbit detrusor were compared in the absence and presence of muscarinic receptor blockade with scopolamine. All drugs had a depressant effect on the contractions both before and after scopolamine addition, nifedipine being the most potent of the calcium channel blockers. However, only nifedipine showed a significant preference for the nonmuscarinic part of the contraction, which was almost abolished at a concentration of 10^?6 M. It is concluded that combined muscarinic receptor and calcium channel blockade is an effective means of inhibiting bladder contraction. Differences between calcium channel blockers may be of importance in this respect.     

Esophageal blood flow in the rabbit: response to calcium channel blockers

Calcium channel blockers have recently been added to the therapeutic regimen for patients who have chest pain of esophageal origin. Although relief of symptoms has been reported, this has not always been associated with changes in esophageal contraction pressures or luminal pH. Myoischemia has been proposed as one possible mechanism for esophageal chest pain. We have investigated the effect of the calcium channel blockers verapamil, nifedipine, and diltiazem on esophageal blood flow in the rabbit model. Esophageal blood flow was measured three times in each rabbit with use of the radiolabeled microsphere technique after a 30-minute continuous infusion of (1) saline solution (baseline), (2) a low dose, and (3) a high dose of each agent. Esophageal mucosal blood flow significantly decreased with nifedipine but was unchanged with verapamil and diltiazem. Esophageal muscle blood flow significantly increased--approximately 100% after administration of each of the calcium channel blockers. Thus esophageal muscle blood flow is enhanced after administration of calcium channel blockers, and this may be one therapeutic mechanism of the calcium channel blockers in the relief of esophageal chest pain in some esophageal diseases.     

Intracellular calcium in hypertrophic smooth muscle from rat urinary bladder

OBJECTIVE: To explore whether infravesical outlet obstruction is associated with alterations in calcium activation of detrusor smooth muscle. MATERIAL AND METHODS: Outlet obstruction was created by partial ligature of the urethra in female rats. Western blotting was performed using an antibody against the cytoplasmatic region of the alpha1c subunit of the L-type Ca2+ channel. Intracellular calcium was measured using Fura-2 in detrusors that had been obstructed for 10 days and activated by high K+ concentrations at different extracellular Ca2+ concentrations. The rate of force development after rapid opening of L-type Ca2+ channels was measured in contractions initiated by flash photolysis of nifedipine in Ca2(+)-containing depolarizing solution. RESULTS: Bladder weight increased from 62 +/- 3 to 254 +/- 43 mg after 10 days of obstruction. Expression of the alpha1c subunit increased after 3 days and continued to increase until it was about fourfold greater after 10 days; however, it had not increased further at 6 weeks. This change was reversible after removal of obstruction. Activation with K+ produced a stable force at different extracellular Ca2+ concentrations, with no difference in response between controls and rats that had been obstructed for 10 days. Intracellular Ca2+ concentrations were lower in the obstructed group, showing that the calcium sensitivity of the contraction force had increased. The delay between the opening of L-type channels and the onset of contraction was longer in obstructed detrusors. CONCLUSIONS: Growth of detrusor muscle following obstruction is accompanied by attenuated calcium transients following activation, despite upregulation of L-type Ca2+ channels. The Ca2+ sensitivity of contraction was increased in obstructed detrusors. We suggest that the decreased surface: volume ratio in hypertrophic smooth muscle cells is partly involved in the lowered Ca2+ transients. The increases in L-type calcium channels and in calcium sensitivity may be compensatory mechanisms.     

Thiopental induces contraction of rat aortic smooth muscle through Ca(2+) release from the sarcoplasmic reticulum

Little is known about the mechanism of thiopental-induced contraction in vascular smooth muscle. This study aimed to clarify this question by conducting isometric tension experiments and (45)Ca(2+) flux measurements in endothelium-denuded rat aortic rings. Thiopental induced a concentration-dependent contraction under basal tension. This contraction was enhanced when rings were precontracted with phenylephrine in the presence of verapamil. In Ca(2+)-free solution, thiopental-induced contraction was reduced but not abolished with high concentrations. Ca(2+) store depletion with a maximum dose of caffeine in Ca(2+)-free solution further reduced the contraction by subsequent thiopental. Ca(2+) store depletion with thapsigargin completely abolished contraction by thiopental. (45)Ca(2+) influx experiment in the presence of verapamil showed that thiopental could not induce any Ca(2+) influx with or without phenylephrine prestimulation. The (45)Ca(2+) efflux experiment showed more evidence of thiopental-induced Ca(2+) release, which was abolished by thapsigargin. In conclusion, thiopental induces contraction in rat aortic smooth muscle by releasing Ca(2+) from the sarcoplasmic reticulum without Ca(2+) influx. IMPLICATIONS: This is the first study providing evidence that thiopental-induced vascular contraction is caused by Ca(2+) release from the sarcoplasmic reticulum of the smooth muscle.     

Spontaneous slow wave and contractile activity of the guinea pig prostate

PURPOSE: We characterized the electrical events underlying spontaneous contractions of the stroma of the guinea pig prostate. MATERIALS AND METHODS: Membrane potential of the stroma was recorded using standard electrophysiological recording techniques. The structure of the prostate was viewed using confocal or electron microscopy. RESULTS: In stromal cells spontaneous depolarizing membrane transients (12 mV. in amplitude) occurred at 5 minutes-1 and triggered 1 or more spikes. The membrane potential, and frequency and duration of the potential transients were not affected by the calcium channel blocker nifedipine (1 microM. for greater than 5 minutes), or blockers of neuronal propagation (tetrodotoxin), and the effects of cholinergic (atropine), adrenergic (guanethidine or prazosin) and sensory blockers (capsaicin) of neurotransmission. However, the amplitude of the superimposed spikes was significantly reduced by nifedipine. A network of c-Kit immunoreactive cells was evident in the interstitial layer between the stroma and glandular lined lumen. These prostatic interstitial cells contained many morphological features distinguishing interstitial cells of Cajal, the pacemaker cells of the gastrointestinal tract. Prostatic interstitial cells formed close appositions with each other, with neighboring smooth muscle cells and with varicose axon bundles. CONCLUSIONS: Movement of the contents of the peripheral prostatic acini into the minor and major prostatic ducts is likely to occur via spontaneous contractions triggered by myogenic slow wave activity in the stromal wall. By analogy with the intestine and urethra prostatic interstitial cells may well act as the pacemaker for prostatic slow waves as well as form an intercellular communication network interacting with the intrinsic nerves and stromal cells.     

前列冲剂对雄性大鼠离体膀胱逼尿肌作用的研究

目的观察前列冲剂对雄性大鼠离体膀胱逼尿肌运动的影响，并初步探讨其作用机制。方法运用BL-420型生物信号系统记录离体大鼠膀胱逼尿肌肌条的活动，观察不同剂量前列冲剂对大鼠膀胱逼尿肌自发活动的影响，并分析其作用机制。结果前列冲剂对大鼠膀胱逼尿肌有明显的兴奋作用，可增加其收缩时间、最大收缩张力及膀胱活动力，并呈量效关系。该作用可被异搏定（L型电压依从性Ca2＋通道阻断剂）和阿托品部分阻断（P〈0.01）。结论前列冲剂在体外可增强膀胱平滑肌的收缩，其作用是通过多条途径发挥的，尤其是通过L型钙通道和胆碱能M型受体而起作用的。     

The mechanism of contraction by 2-chloroadenosine in cat detrusor muscle cells

PURPOSE: Four adenosine receptors (ARs), designated A1AR (A1 adenosine receptor), A2aAR (A2a adenosine receptor), A2bAR (A2b adenosine receptor), and A3AR (A3 adenosine receptor), have been cloned from various species, but the contraction mechanism via A1ARs in cat detrusor muscle cell is not well known. MATERIALS AND METHODS: We examined the cellular mechanism using an A1AR agonist 2-chloroadenosine (2-CA) in cat detrusor cell isolated by enzymatic digestion. To examine which phospholipase mediates the contraction, we used phospholipase inhibitors. RESULTS: The adenosine analog potency order is R-N6-phenylisopropyladenosine (R-PIA) > 5'-N-ethylcarbosamine adenosine (NECA) > 2-chloroadenosine (2-CA) > S-N6-phenylisopropyladenosine (S-PIA). The ratio of equi-effective concentrations of R-PIA/S-PIA was 58.2. 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 300 nM) shifted to the right the concentration-response curves of 2-CA. These results indicate A1ARs mediate 2-CA induced contraction in cat detrusor muscle. G proteins (Gi1, Gi2, Gi3, Go, Gs, and Gq) in cat detrusor muscle were detected by immunoblot analysis. Pertussis toxin (PTX) inhibited 2-CA induced contraction. In permeabilized cells, antibodies against Galphai3 antagonized 2-CA induced contraction, suggesting that the contraction is mediated by Gi3 protein. A phosphatidylinositol-specific phospholipase C (PLC) inhibitor, neomycin, reduced 2-CA induced contraction, but a phospholipase D (PLD) inhibitor, p-chloromercuribenzoic acid, and a phospholipase A2 (PLA2) inhibitor, dimethyl-eicosa-dienoic acid (DEDA), had no effect. We found the presence of the main PLC isozymes, PLC-beta1, PLC-beta3, and PLC-gamma1. 2-CA induced contraction in permeabilized cells was inhibited by PLC-beta3 but not by PLC-beta1 or PLC-gamma1 antibody. These results imply that A1ARs are coupled to PLC-beta3 via PTX-sensitive Gi3 protein. Sr2+ medium and thapsigargin, which replaces intracellular Ca2+ and deplete intracellular calcium stores respectively, inhibited 2-CA induced contraction. CONCLUSIONS: These results suggest that A1ARs mediating 2-CA induced contraction exist in cat detrusor muscle and the contraction depends on a PTX-sensitive Gi3 protein, PLC-beta3 and the release of intracellular Ca2+.     

Agonist-induced contraction and accumulation of inositol phosphates in the guinea-pig detrusor: evidence that muscarinic and purinergic receptors raise intracellular calcium by different mechanisms

In vitro contractile responses to electrical stimulation, ATP, histamine, and carbachol were measured in strips of guinea pig detrusor. The contractile responses were redetermined at intervals after replacement of the Krebs bicarbonate buffer with a nominally calcium-free medium. Agonist induced accumulation of [3H]-inositol phosphates was measured in a suspension of detrusor slices. Electrical stimulation (five second train; frequency 20 Hz; pulse width 100 microseconds) produced a contraction that was abolished by tetrodotoxin (10(-6) M) and reduced by approximately 50% in the presence of atropine (10(-8) M). This atropine resistant component was abolished by desensitization of the purine receptors with alpha, beta-methylene ATP, confirming that the response was mediated by nerves that released ATP and acetylcholine. Carbachol, ATP, and histamine produced concentration dependent contractions in guinea-pig detrusor strips. The response to ATP was much more dependent on extracellular calcium than the response to carbachol. Muscarinic, but not purine-receptor stimulation induced the accumulation of [3H]-inositol phosphates. These data suggest that ATP stimulates a purine receptor which opens a membrane ion channel and allows an influx of calcium while muscarinic receptor stimulation can mobilize intracellular calcium via hydrolysis of inositol phospholipid and production of the second messenger inositol triphosphate.     

Pathophysiology of idiopathic detrusor instability and detrusor hyper-reflexia. An in vitro study of human detrusor muscle

Muscle strips from urodynamically normal bladders and from bladders exhibiting idiopathic detrusor instability or detrusor hyper-reflexia were compared under isometric conditions in an organ bath. Spontaneous contractions developed more often in unstable and hyper-reflexic muscle and were of greater amplitude, frequency and basal tension. Electrical field stimulation caused a frequency-dependent contraction which was largely abolished by both tetrodotoxin (TTX) and atropine in all three muscle types. Comparison of their frequency response curves demonstrated a significantly greater sensitivity than that of unstable and hyper-reflexic muscle to low stimulation frequencies. Acetylcholine caused a dose-related contractile response in all muscle types. There were no significant differences between the dose response curves of unstable and hyper-reflexic muscle, and those of normal muscle. The results suggest that the pathophysiology of the involuntary detrusor contraction is common to both idiopathic detrusor instability and detrusor hyper-reflexia and that this is related to a disorder of an intrinsic neuromodulatory mechanism within the detrusor muscle.     

Effect of nifedipine on the contractile responses of the isolated rat bladder

The effect of the calcium channel blocker nifedipine on the motor transmission in isolated preparations of rat detrusor smooth muscle has been studied. Nifedipine blocked the major part (75 to 80%) of the contractile response to electrical field stimulation, while atropine only blocked 20 to 25%. In preparations pretreated with atropine, the response to electrical field stimulation was completely abolished by nifedipine. The converse was also true; in preparations pretreated with nifedipine the response was fully blocked by atropine. The nifedipine-resistant response was greatly potentiated by the anticholinesterase eserine. The blocking action of nifedipine on motor transmission was partially antagonised by raising Ca2(+)-concentration. Acetylcholine concentration-response curve was shifted to the right by nifedipine. It is concluded that the non-cholinergic motor neurotransmitter evokes contraction of the rat detrusor smooth muscle by activating external Ca2(+)-transport channels whereas the cholinergic contraction is mediated partly or wholly by alternative mechanisms.     

Calcium channel blockade and contractile responses in the isolated human vas deferens

The effects of removal of extracellular calcium and of the calcium channel blockers nifedipine, verapamil and diltiazem were studied on contractions induced by electrical field stimulation and high K+-solution in isolated preparations of the human vas deferens. Electrically induced contractions were blocked by tetrodotoxin and alpha-adrenoceptor blockade. They were abolished in calcium-deficient medium, and suppressed by the calcium channel blockers in the order of potency nifedipine greater than verapamil greater than diltiazem. The maximum blocking effect of nifedipine was approximately 40%. All the blockers practically abolished K+-induced contractions. It is concluded that even if the contractile response of the human vas deferens to electrical stimulation is dependent on extracellular calcium, calcium channel blockers seem to have only a limited effect on this contraction and their capability of impairing the function of the vas deferens in patients is questioned.