Urinary Bladder Smooth Muscle Research Articles

Novel BK channel regulatory mechanism by protein kinase C in guinea pig urinary bladder smooth muscle (865.1)

Large conductance Ca2+‐activated K+ (BK) channels are critical regulators of urinary bladder smooth muscle (UBSM) function. Here, we provide a novel mechanistic insight into BK channel regulation by protein kinase C (PKC) in UBSM. Voltage‐clamp experiments showed that pharmacological activation of PKC with phorbol 12‐myristate 13‐acetate (PMA), inhibited the spontaneous transient BK currents (TBKCs) in native freshly‐isolated guinea pig UBSM cells. Current‐clamp recordings revealed that PMA significantly depolarized UBSM membrane potential, and reduced the spontaneous transient hyperpolarizations in UBSM cells. The PMA inhibitory effects on UBSM membrane potential were abolished by the selective BK channel inhibitor paxilline and were not observed with its inactive analog, 4‐alpha‐PMA. PMA did not affect the amplitude of the whole cell steady‐state BK current or single BK channel open probability (recorded in cell‐attached mode) upon inhibition of all major Ca2+ sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PMA elevated the intracellular Ca2+ levels in UBSM cells, and increased spontaneous phasic and nerve‐evoked contractions of UBSM isolated strips. The results support the concept that PKC activation leads to a reduction in BK channel activity in UBSM via a Ca2+‐dependent mechanism, thus increasing UBSM contractility.Grant Funding Source: Supported by R01 DK084284 to G. V. Petkov

Prostaglandin E2 inhibits the large‐conductance voltage‐ and Ca2+‐activated K+ channels in guinea pig urinary bladder smooth muscle (865.7)

Prostaglandin E2 (PGE2) is an important signaling molecule that has been shown to be involved in the regulation of urinary bladder smooth muscle (UBSM) contractility in both rodents and humans. However, the underlying mechanism by which PGE2 evokes UBSM contractions is not well understood. Here, we investigated whether large conductance voltage‐ and Ca2+‐activated K+ (BK) channels mediate the stimulatory effects induced by PGE2 on guinea pig UBSM contractility. We used a combined experimental approach including patch‐clamp electrophysiology, live‐cell Ca2+ imaging, and isometric UBSM tension recordings to investigate BK channel regulation by PGE2 at both cellular and tissue levels. PGE2 increased the spontaneous phasic contractions of UBSM isolated strips in a concentration‐dependent manner (10 nM‐10 µM). BK channel inhibition with paxilline attenuated the PGE2‐induced UBSM phasic contractions, suggesting that BK channels are among the targets of PGE2. Our data also indicate that PGE2 (10 µM) caused an inhibition of the amplitude and frequency of spontaneous transient BK currents and increased the intracellular Ca2+ levels in freshly‐isolated UBSM cells. Thus, for the first time we have revealed a novel mechanism by which BK channels mediate the PGE2 stimulatory effects on guinea pig UBSM contractions.Grant Funding Source: Supported by NIH R01 DK084284 grant to Georgi V. Petkov.

Pharmacological dissection reveals functional roles for KCNQ channel subtypes in human urinary bladder smooth muscle contractility (1057.12)

This study is aimed at investigating the molecular expression of voltage‐gated potassium (KCNQ) channels in urinary bladder smooth muscle (UBSM) and to reveal their functional role in regulating the contractility of this tissue. Using native, freshly‐isolated human UBSM single cells and whole UBSM tissue, we examined the mRNA and protein expression for all KCNQ channel subtypes and elucidated their functional role in human UBSM contractility using RT‐PCR, western blot, and isometric UBSM tension recordings of isolated human UBSM strips. Human UBSM tissues were collected from open bladder surgeries and represent patients without preoperative symptoms of overactive bladder. Expression of KCNQ channel subtypes were identified at both the mRNA and protein levels in human UBSM. Functional studies revealed that spontaneous phasic contractility was significantly increased by the KCNQ channel inhibitor, XE991. On the contrary, the KCNQ channel activators retigabine, L‐364,373 (R‐L3), and ICA‐069673 attenuated UBSM spontaneous phasic contractions. This study demonstrates the novel findings that KCNQ channels are functionally expressed in human UBSM, whereby they serve an important functional role in regulating the spontaneous contractility of this tissue.Grant Funding Source: Supported by NIH R01 DK084284 to Georgi V. Petkov

Role of phosphodiesterase‐1 in muscarinic receptor‐induced human urinary bladder smooth muscle excitability and contractility (865.4)

Role of phosphodiesterase‐1 in muscarinic receptor‐induced human urinary bladder smooth muscle excitability and contractility Wenkuan Xin1, Ning Li1, Eric S. Rovner2, Qiuping Cheng1, and Georgi V. Petkov1,2 1Drug Discovery & Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC 29208 and 2Medical University of South Carolina, Charleston, SC 29425 Activation of muscarinic M3 receptors (M3Rs) leads to an inhibition of the large conductance Ca2+‐activated K+ (BK) channels and an increase in human urinary bladder smooth muscle (UBSM) excitability. M3R‐mediated Ca2+ influx activates phosphodiesterase‐1 (PDE1) and reduces cellular cAMP levels. We investigated the mechanism by which the integrated PDE1 and MR signaling pathways stimulate human UBSM excitability and contractility. Pharmacological activation of M3Rs with carbachol (1 µM) decreased the frequency and amplitude of transient BK currents (TBKCs) in UBSM cells and subsequent inhibition of PDE1 with 8MM‐IBMX (10 µM) recovered TBKC activity. In the absence of M3R stimulation, inhibition of PDE1 increased TBKCs frequency, and subsequent inhibition of protein kinase A (PKA) with H‐89 (10 µM) reduced TBKCs frequency. 8MM‐IBMX (10 µM) hyperpolarized UBSM membrane potential, an effect which was blocked upon PKA inhibition. 8MM‐IBMX suppressed spontaneous phasic contractions and nerve‐evoked contractions of UBSM isolated strips. In conclusion, PDE1 enhances the M3R‐mediated inhibition of BK channels. Inhibition of PDE1 attenuates UBSM excitability by increasing BK channel activity via a PKA‐dependent mechanism. A combination of a PDE1 inhibitor and a MR antagonist can be an effective novel approach for the treatment of bladder overactivity.Key words: phosphodiesterase, muscarinic receptor, detrusorGrant Funding Source: NIH R01 DK084284 to Georgi V. Petkov

The large conductance Ca2+‐activated K+ channel: a key intermediate in cholinergic regulation of human urinary bladder smooth muscle excitability (865.5)

We investigated whether muscarinic receptor (MR) activation increases human urinary bladder smooth muscle (UBSM) excitability by directly or indirectly inhibiting the large conductance Ca2+‐activated K+ (BK) channels. We used the MR agonist, carbachol, to determine the changes in BK channel activity in freshly‐isolated human UBSM cells using the perforated whole cell and single BK channel patch‐clamp recordings. Carbachol inhibited the amplitude and frequency of BK channel‐mediated spontaneous transient outward currents and spontaneous transient hyperpolarizations, both triggered by ryanodine receptor‐dependent Ca2+ release. Carbachol also caused UBSM cell depolarization, which was not observed in the presence of iberiotoxin, a BK channel inhibitor. The potential direct effects of carbachol on BK channels were examined under conditions of removing all major cellular Ca2+ sources for BK channel activation with pharmacological inhibitors (thapsigargin, ryanodine, and nifedipine). In the presence of these inhibitors, carbachol did not affect the single BK channel open probability and mean BK channel conductance (cell‐attached configuration) or the whole cell BK currents. The data support the concept that MR activation triggers indirect BK channel inhibition mediated by intracellular Ca2+, thus increasing human UBSM cell excitability.Grant Funding Source: Supported by NIH R01 DK084284 to Georgi V. Petkov.

Effect of Vitis vinifera Leaf Hydroalcoholic Extract on Rat Urinary Bladder Contractility

Introduction: Several reports have shown the various medicinal effects of grape (Vitis vinifera) seed and skin extracts, such as antioxidant, hypotensive and vasodilatory effects. It has been recently shown the relaxatory effect of grape leaf hydroalcoholic extract on rat ileum, uterus, aorta, trachea and vas deferens as well as on frog isolated heart. The aim of the present study was to investigate the effect of Vitis vinifera leaf hydroalcoholic extract (VLHE) on contractility of urinary bladder smooth muscle in male rats and to study the mechanism(s) of its action. Materials and Methods: The extract of dried grape leaf was prepared by macerated method using 70% ethanol for 72h in room temperature. The solvent was then evaporated. Urinary bladder was removed from male adult wistar rats after induction of deep anaesthetize by di-ethyle-ether. Tissues were then suspended in the organ bath containing physiologic solution (37oC, pH 7.4) bubbled with oxygen. Contractions were recorded isometrically under 1g resting tension. Results: VLHE at 1, 2, 4 and 8 mg/ml significantly reduced the urinary bladder contractions evoked by KCl (60 mM P<0.05) and (0.5, 1, 2 and 4 mg/ml) also reduced the contractions evoked by Acetylcholine (5 µM) dose dependently (P<0.05). In calcium free physiologic solution, KCl-induced (120 mM P<0.05) contractions were occurred only after adding calcium (0.312, 0.625, 1.25, 2.5 and 5 mM) to the organ bath solution. VLHE (2 mg/ml) also reduced different concentration of calcium-induced contraction in the presence of KCl (P<0.05). The VLHE- induced urinary bladder relaxation was unaffected by propranolol (1 µM for 20 mins) while the presence of tetraethylamonium (10 mM for 20 mins) could strongly reduce the relaxatory effect of VLHE. Conclusion: The data suggest that VLHE induces spasmolytic effect in rat urinary bladder, possibly via blocking voltage dependent calcium channels and activation of calcium-activated potassium channels.

BK Channel-Mediated Relaxation of Urinary Bladder Smooth Muscle: A Novel Paradigm for Phosphodiesterase Type 4 Regulation of Bladder Function

Elevation of intracellular cAMP and activation of protein kinase A (PKA) lead to activation of large conductance voltage- and Ca(2+)-activated K(+) (BK) channels, thus attenuation of detrusor smooth muscle (DSM) contractility. In this study, we investigated the mechanism by which pharmacological inhibition of cAMP-specific phosphodiesterase 4 (PDE4) with rolipram or Ro-20-1724 (C(15)H(22)N(2)O(3)) suppresses guinea pig DSM excitability and contractility. We used high-speed line-scanning confocal microscopy, ratiometric fluorescence Ca(2+) imaging, and perforated whole-cell patch-clamp techniques on freshly isolated DSM cells, along with isometric tension recordings of DSM isolated strips. Rolipram caused an increase in the frequency of Ca(2+) sparks and the spontaneous transient BK currents (TBKCs), hyperpolarized the cell membrane potential (MP), and decreased the intracellular Ca(2+) levels. Blocking BK channels with paxilline reversed the hyperpolarizing effect of rolipram and depolarized the MP back to the control levels. In the presence of H-89 [N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide dihydrochloride], a PKA inhibitor, rolipram did not cause MP hyperpolarization. Rolipram or Ro-20-1724 reduced DSM spontaneous and carbachol-induced phasic contraction amplitude, muscle force, duration, and frequency, and electrical field stimulation-induced contraction amplitude, muscle force, and tone. Paxilline recovered DSM contractility, which was suppressed by pretreatment with PDE4 inhibitors. Rolipram had reduced inhibitory effects on DSM contractility in DSM strips pretreated with paxilline. This study revealed a novel cellular mechanism whereby pharmacological inhibition of PDE4 leads to suppression of guinea pig DSM contractility by increasing the frequency of Ca(2+) sparks and the functionally coupled TBKCs, consequently hyperpolarizing DSM cell MP. Collectively, this decreases the global intracellular Ca(2+) levels and DSM contractility in a BK channel-dependent manner.

AcT-2: A Novel Myotropic and Antimicrobial Type 2 Tryptophyllin from the Skin Secretion of the Central American Red-Eyed Leaf Frog,Agalychnis callidryas

Tryptophyllins are a diverse family of amphibian peptides originally found in extracts of phyllomedusine frog skin by chemical means. Their biological activities remain obscure. Here we describe the isolation and preliminary pharmacological characterization of a novel type 2 tryptophyllin, named AcT-2, from the skin secretion of the red-eyed leaf frog, Agalychnis callidryas. The peptide was initially identified during smooth muscle pharmacological screening of skin secretion HPLC fractions and the unique primary structure—GMRPPWF-NH2—was established by both Edman degradation and electrospray MS/MS fragmentation sequencing. A. cDNA encoding the biosynthetic precursor of AcT-2 was successfully cloned from a skin secretion-derived cDNA library by means of RACE PCR and this contained an open-reading frame consisting of 62 amino acid residues with a single AcT-2 encoding sequence located towards the C-terminus. A synthetic replicate of AcT-2 was found to relax arterial smooth muscle (EC50 = 5.1 nM) and to contract rat urinary bladder smooth muscle (EC50 = 9.3 μM). The peptide could also inhibit the growth of the microorganisms, Staphylococcus aureus, (MIC = 256 mg/L) Escherichia coli (MIC = 512 mg/L), and Candida albicans (128 mg/L). AcT-2 is thus the first amphibian skin tryptophyllin found to possess both myotropic and antimicrobial activities.

Modulation of nerve-evoked contractions by β3-adrenoceptor agonism in human and rat isolated urinary bladder

Activation of β3-adrenoceptors has been shown to have a direct relaxant effect on urinary bladder smooth muscle from both rats and humans, however there are very few studies investigating the effects of β3-adrenoceptor agonists on nerve-evoked bladder contractions. Therefore in the current study, the role of β3-adrenoceptors in modulating efferent neurotransmission was evaluated. The effects of β3-adrenoceptor agonism on neurogenic contractions induced by electrical field stimulation (EFS) were compared with effects on contractions induced by exogenous acetylcholine (Ach) and αβ-methylene adenosine triphosphate (αβ-meATP) in order to determine the site of action. Isoproterenol inhibited EFS-induced neurogenic contractions of human bladder (pD2=6.79; Emax=65%). The effect of isoproterenol was selectively inhibited by the β3-adrenoceptor antagonist L-748,337 (pKB=7.34). Contractions induced by exogenous Ach (0.5–1μM) were inhibited 25% by isoproterenol (3μM) while contractions to 10Hz in the same strip were inhibited 67%. The selective β3-adrenoceptor agonist CL-316,243 inhibited EFS-induced neurogenic contractions of rat bladder (pD2=7.83; Emax=65%). The effects of CL-316,243 were inhibited in a concentration dependent manner by L-748,337 (pA2=6.42). Contractions induced by exogenous Ach and αβ-meATP were significantly inhibited by CL-316,243, 29% and 40%, respectively. These results demonstrate that the activation of β3-adrenoceptors inhibits neurogenic contractions of both rat and human urinary bladder. Contractions induced by exogenously applied parasympathetic neurotransmitters are also inhibited by β3-agonism however the effect is clearly less than on neurogenic contractions (particularly in human), suggesting that in addition to a direct effect on smooth muscle, activation of prejunctional β3-adrenoceptors may inhibit neurotransmitter release.

Large-conductance voltage- and Ca2+-activated K+ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle

Large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. PKC modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the PKC-BK channel interaction in DSM remains unknown. We provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca(2+) imaging, and functional studies of DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with PMA inhibited the spontaneous transient BK currents in native freshly isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode) upon inhibition of all major Ca(2+) sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca(2+) levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. Our results support the concept that PKC activation leads to a reduction of BK channel activity in DSM via a Ca(2+)-dependent mechanism, thus increasing DSM contractility.

Identification de la première mutation non-sens de la cornéodesmosine avec expression d’une protéine tronquée et causant un peeling skin syndrome, type B

The large-conductance, calcium-activated potassium channels (BK, also termed BKCa, Slo, or MaxiK) distributed in both excitable and non-excitable cells are involved in many cellular functions such as action potential repolarization; neuronal excitability; neurotransmitter release; hormone secretion; tuning of cochlear hair cells; innate immunity; and modulation of the tone of vascular, airway, uterine, gastrointestinal, and urinary bladder smooth muscle tissues. Because of their high conductance, activation of BK channels has a strong effect on membrane potential. BK channels differ from all other potassium (K+) channels due to their high sensitivity to both intracellular calcium (Ca2+) concentrations and voltage. These features make BK channels ideal negative feedback regulators in many cell types by decreasing voltage-dependent Ca2+ entry through membrane potential hyperpolarization. The current review aims to give a comprehensive understanding of the structure and molecular biology of BK channels and their relevance to various pathophysiological conditions. The review will also focus on the therapeutic potential and pharmacology of the various BK channel activators and blockers.

Control of urinary bladder smooth muscle excitability by the TRPM4 channel modulator 9-phenanthrol

The Ca2+-activated monovalent cation selective transient receptor potential melastatin 4 (TRPM4) channel has been recently identified in detrusor smooth muscle (DSM) of the urinary bladder. Two recent publications by our research group provide evidence in support of the novel hypothesis that TRPM4 channels enhance DSM excitability and contractility. This is a critical question as prior studies have primarily targeted hyperpolarizing currents facilitated by K+ channels, but the depolarizing component in DSM cells is not well understood. For the first time, we utilized the selective TRPM4 channel inhibitor, 9-phenanthrol, to investigate TRPM4 channel functional effects in DSM at both cellular and tissue levels in rodents. Our new data presented here showed that in rat DSM cells, 9-phenanthrol attenuates spontaneous inward currents in the presence of the muscarinic receptor agonist, carbachol, thus reducing DSM cell excitability. In support of our original hypothesis, we found that TRPM4 channel mRNA levels are much higher in DSM vs. vascular smooth muscle and that inhibition of TRPM4 channels can potentially attenuate DSM excitability. Thus, we postulate the novel concept that selective pharmacological inhibition of TRPM4 channels can limit both excitability and contractility of DSM.

Ethanol-mediated relaxation of guinea pig urinary bladder smooth muscle: involvement of BK and L-type Ca2+ channels

Mechanisms underlying ethanol (EtOH)-induced detrusor smooth muscle (DSM) relaxation and increased urinary bladder capacity remain unknown. We investigated whether the large conductance Ca(2+)-activated K(+) (BK) channels or L-type voltage-dependent Ca(2+) channels (VDCCs), major regulators of DSM excitability and contractility, are targets for EtOH by patch-clamp electrophysiology (conventional and perforated whole cell and excised patch single channel) and isometric tension recordings using guinea pig DSM cells and isolated tissue strips, respectively. EtOH at 0.3% vol/vol (~50 mM) enhanced whole cell BK currents at +30 mV and above, determined by the selective BK channel blocker paxilline. In excised patches recorded at +40 mV and ~300 nM intracellular Ca(2+) concentration ([Ca(2+)]), EtOH (0.1-0.3%) affected single BK channels (mean conductance ~210 pS and blocked by paxilline) by increasing the open channel probability, number of open channel events, and open dwell-time constants. The amplitude of single BK channel currents and unitary conductance were not altered by EtOH. Conversely, at ~10 μM but not ~2 μM intracellular [Ca(2+)], EtOH (0.3%) decreased the single BK channel activity. EtOH (0.3%) affected transient BK currents (TBKCs) by either increasing frequency or decreasing amplitude, depending on the basal level of TBKC frequency. In isolated DSM strips, EtOH (0.1-1%) reduced the amplitude and muscle force of spontaneous phasic contractions. The EtOH-induced DSM relaxation, except at 1%, was attenuated by paxilline. EtOH (1%) inhibited L-type VDCC currents in DSM cells. In summary, we reveal the involvement of BK channels and L-type VDCCs in mediating EtOH-induced urinary bladder relaxation accommodating alcohol-induced diuresis.

Molecular Expression and Pharmacological Evidence for a Functional Role of Kv7 Channel Subtypes in Guinea Pig Urinary Bladder Smooth Muscle

Voltage-gated Kv7 (KCNQ) channels are emerging as essential regulators of smooth muscle excitability and contractility. However, their physiological role in detrusor smooth muscle (DSM) remains to be elucidated. Here, we explored the molecular expression and function of Kv7 channel subtypes in guinea pig DSM by RT-PCR, qRT-PCR, immunohistochemistry, electrophysiology, and isometric tension recordings. In whole DSM tissue, mRNAs for all Kv7 channel subtypes were detected in a rank order: Kv7.1~Kv7.2Kv7.3~Kv7.5Kv7.4. In contrast, freshly-isolated DSM cells showed mRNA expression of: Kv7.1~Kv7.2Kv7.5Kv7.3~Kv7.4. Immunohistochemical confocal microscopy analyses of DSM, conducted by using co-labeling of Kv7 channel subtype-specific antibodies and α-smooth muscle actin, detected protein expression for all Kv7 channel subtypes, except for the Kv7.4, in DSM cells. L-364373 (R-L3), a Kv7.1 channel activator, and retigabine, a Kv7.2-7.5 channel activator, inhibited spontaneous phasic contractions and the 10-Hz electrical field stimulation (EFS)-induced contractions of DSM isolated strips. Linopiridine and XE991, two pan-Kv7 (effective at Kv7.1-Kv7.5 subtypes) channel inhibitors, had opposite effects increasing DSM spontaneous phasic and 10 Hz EFS-induced contractions. EFS-induced DSM contractions generated by a wide range of stimulation frequencies were decreased by L-364373 (10 µM) or retigabine (10 µM), and increased by XE991 (10 µM). Retigabine (10 µM) induced hyperpolarization and inhibited spontaneous action potentials in freshly-isolated DSM cells. In summary, Kv7 channel subtypes are expressed at mRNA and protein levels in guinea pig DSM cells. Their pharmacological modulation can control DSM contractility and excitability; therefore, Kv7 channel subtypes provide potential novel therapeutic targets for urinary bladder dysfunction.

Altered expression of caveolin 2 and 3 in smooth muscle of rat urinary bladder by 17β-estradiol

BackgroundThe purpose of this study was to investigate the effect of estrogen alteration on the expression of caveolin 2 and 3 in rat smooth muscle of urinary bladder.MethodsFemale Sprague–Dawley rats were divided into three groups: control, bilateral ovariectomy (Ovx), and bilateral ovariectomy followed by subcutaneous injections of 17β-estradiol (Ovx?+?Est). After 4 weeks, urodynamic measurements were taken to ascertain the contraction interval and contraction pressure. The expression and cellular localization of caveolin 2 and 3 were determined by Western blot and immunohistochemistry in rat urinary bladder smooth muscle.ResultsIn cystometrograms, the contraction interval (min) was significantly lower in the Ovx group (3.1?±?1.5) than in the control group (5.6?±?1.2), but was increased after estrogen treatment (9.3?±?1.0). Conversely, the average contraction pressure (mmHg) was higher in the Ovx group (26.2?±?2.3) than in the control group (21.9?±?3.1), and was decreased after estrogen treatment (23.8?±?3.5). Caveolin 2 and 3 expression was localized in the cell membrane of the smooth muscle. The protein expression of both caveolin 2 and 3 was significantly lower after ovariectomy and was restored to the control levels after 17β-estradiol treatment.ConclusionsHormonal alteration causes a significant change in the expression of caveolin 2 and 3 in smooth muscle of rat urinary bladder. These findings suggest that these molecules might have functional roles in the detrusor overactivity that occurs in association with hormonal alteration.

Renal and vascular studies of aqueous extract of Urtica dioica in rats and rabbits

Urtica dioica has a variety of uses in traditional medicine for genitourinary ailments kidney disorders, allergies, diabetes, anemia, gastrointestinal tract ailments, musculoskeletal aches and alopecia. However, only a few of these uses have scientific bases that support their clinical uses. This study was done to evaluate some of the in vivo and in vitro pharmacological actions of this plant. Eighteen local domestic rabbits (Oryctolagus cuniculus) were used for in vitro studies (effect of the plant extract on isolated pulmonary arteries and isolated urinary bladder smooth muscle) and in vivo studies (effect of the extract on renal function). Six male albino rats were used for studying the effects of the plant extract on blood pressure and heart rate. Urtica dioica extract produced a significant increase in urine volume and urinary Na + excretion without significant changes in K

Overactive bladder mediated by accelerated Ca2+ influx mode of Na+/Ca2+ exchanger in smooth muscle

The Na(+)/Ca(2+) exchanger (NCX) is thought to be a key molecule in the regulation of cytosolic Ca(2+) dynamics. The relative importance of the two Ca(2+) transport modes of NCX activity leading to Ca(2+) efflux (forward) and influx (reverse) in smooth muscle, however, remains unclear. Unexpectedly, spontaneous contractions of urinary bladder smooth muscle (UBSM) were enhanced in transgenic mice overexpressing NCX1.3 (NCX1.3(tg/tg)). The enhanced activity was attenuated by KB-R7943 or SN-6. Whole cell outward NCX current sensitive to KB-R7943 or Ni(2+) was readily detected in UBSM cells from NCX1.3(tg/tg) but not wild-type mice. Spontaneous Ca(2+) transients in myocytes of NCX1.3(tg/tg) were larger and frequently resulted in propagating events and global elevations in cytosolic Ca(2+) concentration. Significantly, NCX1.3(tg/tg) mice exhibited a pattern of more frequent urination of smaller volumes and this phenotype was reversed by oral administration of KB-R7943. On the other hand, KB-R7943 did not improve it in KB-R7943-insensitive (G833C-)NCX1.3(tg/tg) mice. We conclude that NCX1.3 overexpression is associated with abnormal urination owing to enhanced Ca(2+) influx via reverse mode NCX leading to prolonged, propagating spontaneous Ca(2+) release events and a potentiation of spontaneous UBSM contraction. These findings suggest the possibility that NCX is a candidate molecular target for overactive bladder therapy.

Activation of muscarinic M3 receptors inhibits large-conductance voltage- and Ca2+-activated K+ channels in rat urinary bladder smooth muscle cells

Large conductance voltage- and Ca(2+)-activated K(+) (BK) channels are key regulators of detrusor smooth muscle (DSM) contraction and relaxation during urine voiding and storage. Here, we explored whether BK channels are regulated by muscarinic receptors (M-Rs) in native freshly isolated rat DSM cells under physiological conditions using the perforated whole cell patch-clamp technique and pharmacological inhibitors. M-R activation with carbachol (1 μM) initially evoked large transient outward BK currents, followed by inhibition of the spontaneous transient outward BK currents (STBKCs) in DSM cells. Carbachol (1 μM) also inhibited the amplitude and frequency of spontaneous transient hyperpolarizations (STHs) and depolarized the DSM cell membrane potential. Selective inhibition of the muscarinic M3 receptors (M3-Rs) with 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; 0.1 μM), but not muscarinic M2 receptors with methoctramine (1 μM), blocked the carbachol inhibitory effects on STBKCs. Furthermore, blocking the inositol 1,4,5-triphosphate (IP3) receptors with xestospongin-C (1 μM) inhibited the carbachol-induced large transient outward BK currents without affecting carbachol inhibitory effects on STBKCs. Upon pharmacological inhibition of all known cellular sources of Ca(2+) for BK channel activation, carbachol (1 μM) did not affect the voltage-step-induced steady-state BK currents, suggesting that the muscarinic effects in DSM cells are mediated by mobilization of intracellular Ca(2+). In conclusion, our findings provide strong evidence that activation of M3-Rs leads to inhibition of the STBKCs, STHs, and depolarization of DSM cells. Collectively, the data suggest the existence of functional interactions between BK channels and M3-Rs at a cellular level in DSM.

Functional BK channels facilitate the β3-adrenoceptor agonist-mediated relaxation of nerve-evoked contractions in rat urinary bladder smooth muscle isolated strips

The large-conductance voltage- and Ca2+-activated K+ (BK) channel is a major regulator of detrusor smooth muscle (DSM) contractility thus facilitating urinary bladder function. Recent findings suggest that activation of β3-adrenoceptors causes DSM relaxation. However, it is unknown whether the β3-adrenoceptor-mediated DSM relaxation is BK channel-dependent during nerve-evoked contractions. To test this hypothesis, we induced nerve-evoked contractions in rat DSM isolated strips by using a tissue bath system equipped with platinum electrodes for electrical field stimulation (EFS). (±)-(R*,R*)-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy] acetic acid sodium hydrate (BRL37344), a β3-adrenoceptor agonist, significantly decreased the amplitude and muscle force of the 20Hz EFS-induced DSM contractions in a concentration-dependent manner. The BRL37344 inhibitory effect was significantly antagonized by 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride (SR59230A), a β3-adrenoceptor antagonist. We further isolated the cholinergic from the purinergic component of the 0.5–50Hz EFS-induced DSM contractions by using selective inhibitors, atropine as well as suramin and α,β-methylene-ATP. We found that BRL37344 inhibited both the purinergic and cholinergic components of the nerve-evoked contractions in rat DSM isolated strips. The pharmacological blockade of the BK channels with iberiotoxin, a selective BK channel inhibitor, increased the amplitude and muscle force of the 20Hz EFS-induced contractions in rat DSM isolated strips. In the presence of iberiotoxin, there was a significant reduction in the BRL37344-induced inhibition of the 20Hz EFS-induced contractions in rat DSM isolated strips. These latter findings suggest that BK channels play a critical role in the β3-adrenoceptor-mediated inhibition of rat DSM nerve-evoked contractions.

Diurnal Variation in Mouse Urinary Bladder Smooth Muscle (UBSM) Contractility

Physiological output varies by time of day, coordinated by the central circadian clock in the hypothalamus. Recently, a circadian rhythm in micturition was demonstrated in rodents, correlated with a day‐night change in the storage and voiding of the bladder. The goal of this study was to determine whether isolated UBSM exhibited an intrinsic difference in contractility between day and night. Mouse UBSM strips were harvested at 4 time points over the circadian cycle, zeitgeber time (ZT) 2, 8, 14, and 20, and phasic and nerve‐evoked (EFS) contractions were investigated using isometric tension recordings. We hypothesized that UBSM harvested from mice during the active period when micturition peaks (ZT8–14) would demonstrate stronger contractile activity than strips harvested during the rest period (ZT20–2), when the bladder relaxes to store urine. Compared to ZT20, at ZT8 UBSM strips exhibited larger KCl‐induced (10 ± 0.4 versus 7 ± 0.4 mN), phasic (0.08 ± 0.02 versus 0.03 ± 0.01 mN), and EFS‐induced contractions (28 ± 3 versus 22 ± 2 mN, n = 6 mice per condition). Increased contractile amplitudes at ZT8 were correlated with lower expression of the BK K+ channel, a regulator of UBSM excitability. These results identify a diurnal difference in UBSM contractility in the absence of central control, and suggest that daily regulation of bladder excitability occurs locally within smooth muscle tissue. Funded by NIDDK R21089337.