β3-adrenoceptor agonists inhibit carbachol-evoked Ca oscillations in murine detrusor myocytes.

Objective: To test if carbachol (CCh)-evoked Ca oscillations in freshly isolated murine detrusor myocytes are affected by β3-adrenoceptor (β-AR) modulators.

Materials And Methods: Isometric tension recordings were made from strips of murine detrusor, and intracellular Ca measurements were made from isolated detrusor myocytes using confocal microscopy. Transcriptional expression of β-AR sub-types in detrusor strips and isolated detrusor myocytes was assessed using reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time quantitative PCR (qPCR).

The role of Ca-activated Cl current in tone generation in the rabbit corpus cavernosum.

Rabbit corpus cavernosum smooth muscle (RCCSM) cells express ion channels that produce Ca-activated Cl () current, but low sensitivity to conventional antagonists has made its role in tone generation difficult to evaluate. We have reexamined this question using two new generation blockers, T16A-A01 and CaCC-A01. Isolated RCCSM cells were studied using the perforated patch method.

Effects of new-generation TMEM16A inhibitors on calcium-activated chloride currents in rabbit urethral interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit Ca-activated Cl currents (I ) that are important for the development of urethral tone. Here, we examined if TMEM16A (ANO1) contributed to this activity by examining the effect of "new-generation" TMEM16A inhibitors, CACC-A01 and T16A-A01, on I recorded from freshly isolated rabbit urethral ICC (RUICC) and on contractions of intact strips of rabbit urethra smooth muscle. Real-time quantitative PCR experiments demonstrated that TMEM16A was highly expressed in rabbit urethra smooth muscle, in comparison to TMEM16B and TMEM16F.

Muscarinic Receptor Induced Contractions of the Detrusor are Mediated by Activation of TRPC4 Channels.

Purpose: Muscarinic receptor mediated contractions of the detrusor rely on Ca influx through voltage-gated Ca channels but to our knowledge the mechanism linking stimulation of M3Rs to the activation of voltage dependent Ca channels has not been established. TRPC4 channels are receptor operated cation channels that couple muscarinic receptor activation to depolarization of intestinal smooth muscle cells, voltage-activated Ca influx and contraction. We investigated whether TRPC4 channels are involved in cholinergic mediated contractions of the detrusor.

Differential efficacy of GoSlo-SR compounds on BKα and BKαγ channels.

Large conductance, voltage and Ca activated K channels (BK channels) are abundantly expressed throughout the body and are important regulators of smooth muscle tone and neuronal excitability. Their dysfunction is implicated in various diseases including overactive bladder, hypertension and erectile dysfunction. Therefore, BK channel openers bear significant therapeutic potential to treat the above diseases.

Regulation of nerve-evoked contractions of rabbit vas deferens by acetylcholine.

Stimulation of intramural nerves in the vas deferens of many species yields a classical biphasic contraction comprised of an initial fast component, mediated by P2X receptors and a second slower component, mediated by α1-adrenoceptors. It is also recognized that sympathetic nerve-mediated contractions of the vas deferens can be modulated by acetylcholine (Ach), however there is considerable disagreement in the literature regarding the precise contribution of cholinergic nerves to contraction of the vas deferens. In this study we examined the effect of cholinergic modulators on electric field stimulation (EFS)-evoked contractions of rabbit vas deferens and on cytosolic Ca(2+) levels in isolated vas deferens smooth muscle cells (VDSMC).

The role of Ca(2+) influx in spontaneous Ca(2+) wave propagation in interstitial cells of Cajal from the rabbit urethra.

Tonic contractions of rabbit urethra are associated with spontaneous electrical slow waves that are thought to originate in pacemaker cells termed interstitial cells of Cajal (ICC). ICC pacemaker activity results from their ability to generate propagating Ca(2+) waves, although the exact mechanisms of propagation are not understood. In this study, we have identified spontaneous localised Ca(2+) events for the first time in urethral ICC; these were due to Ca(2+) release from the endoplasmic reticulum (ER) via ryanodine receptors (RyRs) and, while they often remained localised, they sometimes initiated propagating Ca(2+) waves.

Molecular mechanisms underlying the effect of the novel BK channel opener GoSlo: involvement of the S4/S5 linker and the S6 segment.

GoSlo-SR-5-6 is a novel large-conductance Ca(2+)-activated K(+) (BK) channel agonist that shifts the activation V1/2 of these channels in excess of -100 mV when applied at a concentration of 10 μM. Although the structure-activity relationship of this family of molecules has been established, little is known about how they open BK channels. To help address this, we used a combination of electrophysiology, mutagenesis, and mathematical modeling to investigate the molecular mechanisms underlying the effect of GoSlo-SR-5-6.

The role of cAMP dependent protein kinase in modulating spontaneous intracellular Ca²⁺ waves in interstitial cells of Cajal from the rabbit urethra.

Interstitial cells of Cajal (ICC) serve as electrical pacemakers in the rabbit urethra. Pacemaking activity in ICC results from spontaneous intracellular Ca(2+) waves that rely on Ca(2+) release from endoplasmic reticulum (ER) stores. The purpose of this study was to investigate if the action of protein kinase A (PKA) affected the generation of Ca(2+) waves in ICC.

The effect of high [K(+)]o on spontaneous Ca(2+) waves in freshly isolated interstitial cells of Cajal from the rabbit urethra.

Interstitial cells of Cajal (ICC) act as putative pacemaker cells in the rabbit urethra. Pacemaker activity in ICC results from spontaneous global Ca(2+) waves that can be increased in frequency by raising external [K(+)]. The purpose of this study was to elucidate the mechanism of this response.

Pharmacological characterization of TMEM16A currents.

Recent studies have shown that transmembrane protein 16 A (TMEM16A) is a subunit of calcium-activated chloride channels (CACCs). Pharmacological agents have been used to probe the functional role of CACCs, however their effect on TMEM16A currents has not been systematically investigated. In the present study, we characterized the voltage and concentration-dependent effects of 2 traditional CACC inhibitors (niflumic acid and anthracene-9-carboxcylic acid) and 2 novel CACC / TMEM16A inhibitors (CACC(inh)A01 and T16A(inh)A01) on TMEM16A currents.

Development of GoSlo-SR-5-69, a potent activator of large conductance Ca2+-activated K+ (BK) channels.

We have designed, synthesised and characterised the effects of a number of novel anthraquinone derivatives and assessed their effects on large conductance, Ca(2+) activated K(+) (BK) channels recorded from rabbit bladder smooth muscle cells using the excised, inside/out configuration of the patch clamp technique. These compounds are members of the GoSlo-SR family of compounds, which potently open BK channels and shift the voltage required for half maximal activation (V1/2) negatively. The efficacy of the anilinoanthraquinone derivatives was enhanced when the size of ring D was increased, since the cyclopentane and cyclohexane derivatives shifted the V1/2, by -24 ± 6 mV and -54 ± 8 mV, respectively, whereas the cycloheptane and cyclooctane derivatives shifted the V1/2 by -61 ± 6 mV and -106 ± 6 mV.

ATP evokes inward currents in corpus cavernosum myocytes.

Introduction: Although adenosine triphosphate (ATP) has often been reported to relax the corpus cavernosum, this may be mediated by indirect effects, such as release of nitric oxide from the endothelium. Recent data suggest that P2X(1) receptors may be up-regulated in diabetes, and these exert an anti-erectile effect by causing the corpus cavernosum smooth muscle cells (CCSMCs) to contract. However, to date, there is no functional evidence that ATP can directly stimulate CCSMC.

Mechanisms underlying activation of transient BK current in rabbit urethral smooth muscle cells and its modulation by IP3-generating agonists.

We used the perforated patch-clamp technique at 37°C to investigate the mechanisms underlying the activation of a transient large-conductance K(+) (tBK) current in rabbit urethral smooth muscle cells. The tBK current required an elevation of intracellular Ca(2+), resulting from ryanodine receptor (RyR) activation via Ca(2+)-induced Ca(2+) release, triggered by Ca(2+) influx through L-type Ca(2+) (CaV) channels. Carbachol inhibited tBK current by reducing Ca(2+) influx and Ca(2+) release and altered the shape of spike complexes recorded under current-clamp conditions.

Structure-activity relationships of a novel group of large-conductance Ca(2+)-activated K(+) (BK) channel modulators: the GoSlo-SR family.

Opening up ion channels: We synthesised a series of anthraquinone analogues, called the GoSlo-SR family. Their effects on bladder smooth muscle BK channels were examined and, as shown, shifted voltage dependent activation >-100 mV (at 10 μM). They were more efficacious than NS11021 and could provide a new scaffold for the design of efficacious BK openers.

Effects of phenylephrine on spontaneous activity and L-type Ca2+ current in isolated corpus cavernosum myocytes.

Introduction: Norepinephrine is important in maintaining detumescent tone in the corpus cavernosum, although the mechanism is incompletely understood. As α-adrenoceptor-induced tone is antagonized by L-type Ca(2+) channel blockers, it is usually assumed that direct modulation of this current is involved. However, the effects of α-adrenoceptor agonists have never been directly examined on L-type current in corpus cavernosum myocytes (CCSMC), leaving open other possibilities.

Platelet-derived growth factor receptor-α cells in mouse urinary bladder: a new class of interstitial cells.

Specific classes of interstitial cells exist in visceral organs and have been implicated in several physiological functions including pacemaking and mediators in neurotransmission. In the bladder, Kit(+) interstitial cells have been reported to exist and have been suggested to be neuromodulators. More recently a second interstitial cell, which is identified using antibodies against platelet-derived growth factor receptor-α (PDGFR-α) has been described in the gastrointestinal (GI) tract and has been implicated in enteric motor neurotransmission.

P2X receptor currents in smooth muscle cells contribute to nerve mediated contractions of rabbit urethral smooth muscle.

Purpose: Adenosine triphosphate is capable of relaxing and contracting urethral smooth muscle. The mechanisms responsible for the relaxing effects of adenosine triphosphate have been well studied but those involved in the contractile response are still unclear. We investigated the contributions of interstitial cells of Cajal and smooth muscle cells to nerve mediated, adenosine triphosphate dependent contractions of urethral smooth muscle.

Rho-associated kinase plays a role in rabbit urethral smooth muscle contraction, but not via enhanced myosin light chain phosphorylation.

The involvement of Rho-associated kinase (ROK) in activation of rabbit urethral smooth muscle contraction was investigated by examining the effects of two structurally distinct inhibitors of ROK, Y27632 and H1152, on the contractile response to electric field stimulation, membrane depolarization with KCl, and α1-adrenoceptor stimulation with phenylephrine. Both compounds inhibited contractions elicited by all three stimuli. The protein kinase C inhibitor GF109203X, on the other hand, had no effect.

Novel excitatory effects of adenosine triphosphate on contractile and pacemaker activity in rabbit urethral smooth muscle.

Purpose: Adenosine triphosphate is thought to be an important neurotransmitter in urethral smooth muscle but its physiological role is still unclear. We characterized the effects of adenosine triphosphate on contractile and pacemaker activity in rabbit urethral smooth muscle.

Materials And Methods: Tension recordings were made from strips of rabbit proximal urethral smooth muscle.

Voltage-dependent Ca2+ currents contribute to spontaneous Ca2+ waves in rabbit corpus cavernosum myocytes.

Introduction: Corpus cavernosum myocytes generate spontaneous tone that contributes to penile detumescence. It is essential to elucidate how tone is generated to fully understand the processes involved in erection. Tissue experiments have shown that blockers of voltage-dependent Ca2+ channels (VDCCs) reduce tone.

Spontaneous Ca2+ waves in rabbit corpus cavernosum: modulation by nitric oxide and cGMP.

Introduction: Detumescent tone and subsequent relaxation by nitric oxide (NO) are essential processes that determine the erectile state of the penis. Despite this, the mechanisms involved are incompletely understood. It is often assumed that the tone is associated with a sustained high cytosolic Ca(2+) level in the corpus cavernosum smooth muscle cells, however, an alternative possibility is that oscillatory Ca(2+) signals regulate tone, and erection occurs as a result of inhibition of Ca(2+) oscillations by NO.

Role of mitochondria in modulation of spontaneous Ca2+ waves in freshly dispersed interstitial cells of Cajal from the rabbit urethra.

Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit pacemaker activity that results from spontaneous Ca(2+) waves. The purpose of this study was to investigate if this activity was influenced by Ca(2+) uptake into mitochondria. Spontaneous Ca(2+) waves were recorded using a Nipkow spinning disk confocal microscope and spontaneous transient inward currents (STICs) were recorded using the whole-cell patch clamp technique.

Structure function relationships in the lymphatic system and implications for cancer biology.

The lymphatic system, composed of lymphatic vessels, lymph, lymph nodes, and lymphocytes, is a distinctive vasculature (discontinuous basement membrane, open endothelial junctions, anchoring filaments, valves, and intrinsic contractility), different yet similar to the blood vasculature; an integral component of the plasma-tissue fluid-lymph circulation (the "blood-lymph loop"); and the center of the immunoregulatory network. Lymphatics are involved in diverse developmental, growth, repair, and pathologic processes both analogous to and distinct from those affecting the blood vasculature. Interference with the blood-lymph loop produces swelling [an imbalance between lymph formation (regulated by Starling's law of transcapillary fluid exchange) and lymph absorption], scarring, nutritional and immunodysregulatory disorders, as well as disturbances in lymph(hem)angiogenesis (lymphedema-angiodysplasia syndromes).

Origin of spontaneous rhythmicity in smooth muscle.

Rhythmic electrical activity is a feature of most smooth muscles but the mechanical consequences can vary from regular rapid phasic contractions to sustained contracture. For many years it was thought that spontaneous electrical activity originated in smooth muscle cells but recently it has become apparent that there are specialized pacemaker cells in many organs that are morphologically and functionally distinct from smooth muscle and that the former cells are the source of spontaneous electrical activity. Such a pacemaker function is well documented for the ICC of the gastrointestinal tract but evidence is accumulating that ICC-like cells play a similar role in other types of smooth muscle.