Neurogenic Contractions Research Articles

Influence of Age on Anticontractile Effect of Perivascular Adipose Tissue in Normotensive and Hypertensive Rats.

Perivascular adipose tissue (PVAT) and its vasomodulatory effects play an important role in the physiology and pathophysiology of blood vessels. Alterations in PVAT associated with reduction in its anticontractile influence are proven to contribute to vascular dysfunction in hypertension. The aim of this study was to examine whether the changes in PVAT properties could participate in progression of vascular abnormalities in developing spontaneously hypertensive rats (SHR). Normotensive Wistar-Kyoto (WKY) rats and SHR, both in 5th and in 12th week of age, were used. Systolic blood pressure was similar between WKY rats and SHR in 5th week of age; however, in 12th week, it was significantly increased in SHR comparing to WKY rats. The amount of retroperitoneal fat was higher in WKY rats in both age groups, whereas body weight was higher in WKY rats only in 12th week, when compared to age-matched SHR. From isolated superior mesenteric arteries, two ring preparations were prepared for isometric tension recording, one with PVAT intact and other with PVAT removed. In WKY rats as well as in SHR, arterial contractile responses to noradrenaline, applied cumulatively on rings, were significantly inhibited in the presence of intact PVAT. In both age groups, anticontractile effect of PVAT was higher in WKY rats than in SHR. Neurogenic contractions, induced by electrical stimulation of perivascular sympathoadrenergic nerves, were significantly attenuated in the presence of PVAT in WKY mesenteric arteries from both age groups; however, in arteries from SHR, intact PVAT had no influence on this type of contractile responses. The results suggest that in SHR impairment of anticontractile effect of PVAT precedes hypertension and might contribute to its development.

Generation and Regulation of Spontaneous Contractions in theProstate.

Spontaneous myogenic contractions have been shown to be significantly upregulated in prostate tissue collected from men with Benign Prostatic Hyperplasia (BPH), an extremely common disorder of the ageing male. Although originally thought likely to be involved in 'housekeeping' functions, mixing prostatic secretions to prevent stagnation, these spontaneous myogenic contractions provide a novel opportunity to understand and treat BPH. This treatment potential differs from previous models, which focused exclusively on attenuating nerve-mediated neurogenic contractions. Previous studies in the rodent prostate have provided an insight into the mechanisms underlying the regulation of myogenic contractions. 'Prostatic Interstitial Cells' (PICs) within the prostate appear to generate pacemaker potentials, which arise from the summation of number of spontaneous transient depolarisations triggered by the spontaneous release of Ca2+ from internal stores and the opening of Ca2+-activated Cl- channels. Pacemaker potentials then conduct into neighbouring smooth muscle cells to generate spontaneous slow waves. These slow waves trigger the firing of 'spike-like' action potentials, Ca2+ entry and contraction, which are not attenuated by blockers of neurotransmission. However, these spontaneous prostatic contractions can be modulated by the autonomic nervous system. Here, we discuss the mechanisms underlying rodent and human prostate myogenic contractions and the actions of existing and novel pharmacotherapies for the treatment of BPH. Understanding the generation of human prostatic smooth muscle tone will confirm the mechanism of action of existing drugs, inform the identification and effectiveness of new pharmacotherapies, as well as predict patient outcomes.

Hypoactivity of rat detrusor muscle in a model of cystitis: exacerbation by non-selective COX inhibitors and amelioration by a selective DP1 receptor antagonist.

Various studies have confirmed that prostaglandins (PG) alter the bladder motor activity and micturition reflex in both human and animals. However, no sufficient data is reported about the effect of cyclooxygenase (COX) inhibitors neither in normal bladder physiology nor in pathological conditions. This study aims to compare the potential effects of some COX inhibitors with varying COX-1/COX-2 selectivities (indomethacin, ketoprofen, and diclofenac) with that of the selective COX-2 inhibitor (DFU) on bladder function. The role played by some PGs and their receptors in controlling detrusor muscle function in normal condition and in cystitis is also studied. Organ bath experiments were performed using isolated rat detrusor muscle. Direct and neurogenic contractions were induced using ACh and electric stimulation (EFS), respectively. A model of hemorrhagic cystitis was induced by single injection of cyclophosphamide (300mg/kg) in rats, and confirmed by histophathological examination. Results are expressed as mean ± SEM of 5-9 rats. Alprostadil and iloprost (1 nM- 10 µM) concentration-dependently potentiated ACh (100μM)- and EFS (4Hz)-induced contraction, with maximum potentiation of 40.01 ± 5.29 and 27.59 ± 6.64%, respectively, in case of ACh contractions. In contrast, ONO-AE1-259 (selective EP2 agonist, 1nM-10μM) inhibited muscle contraction. SC51322 (EP1-antagonist, 10μM) and RO1138452 (IP antagonist, 10μM) inhibited both direct and neurogenic responses. Hemorrhagic cystitis reduced both ACh and EFS responses as well as the potentiatory effect of iloprost and the inhibitory effect of RO1138452 on ACh contractions. ONO-AE3-237 (DP1 antagonist, 1μM) significantly potentiated contractions in cystitis but showed no effect in normal bladder. A significant inhibition of contractile response was observed in presence of indomethacin, ketoprofen, and diclofenac at all tested concentrations (20, 50, and 100μM). Highest effect was induced by diclofenac. The effect of these COX inhibitors on EFS contractions was intensified in case of cystitis, indomethacin being the most potent. Atropine (1nM) significantly reduced indomethacin effect on ACh contraction only in normal rats. On the other hand, DFU (10-6M) significantly potentiated the contractile effect of ACh in case of cystitis although it showed no effect in normal rats. EP1 receptors seem to play an important role in rat bladder contractility. DP1 receptors as COX-2, on the other hand, gain an important role only in case of cystitis. The use of non-selective COX inhibitors in cystitis may be associated with bladder hypoactivity; selective COX-2 inhibitors may be a safer option.

Healthcare Provider’s Guide to Kratom: Succinct Introduction to the Basics and the Questions

(1) Background: The leaves of Mitragyna speciosa (kratom), a tropical tree indigenous to Southeast Asia, have been used traditionally to increase stamina, as a digestive aid, and for pain relief. Defined as a novel psychoactive substance, it is becoming increasingly popular in the U.S., Europe, and Japan, aided by availability through the Internet. (2) Methods: PubMed and MedLine searches were conducted of published articles available in English. (3) Results: Mitragynine and 7-hydroxymitragynine are the primary psychoactive compounds of the over 25 alkaloids present. Subjective effects are dose dependent, i.e., stimulation at low doses, analgesia and sedative effects at moderate doses, and opioid-like effects at high doses. Fatalities are rare and may be linked to polypharmacy. Kratom can be abused; abrupt discontinuation may produce withdrawal symptoms, although kratom dependency does not necessarily imply overt dysfunction or decreased quality of life. Indeed, Kratom has been used in Asia by opioid addicts as a way of easing withdrawal during times when their drug of choice was scarce or unavailable or during intentional withdrawal; the possible use of kratom in the treatment of opioid addiction is worthy of further study, as is its potential as an analgesic agent. Mitragynine may also possess anti-inflammatory and possible anticancer properties. Kratom extracts and mitragynine have shown cytotoxicity to certain human cancer cell lines and may modulate muscle neurogenic contraction and gastric secretion. (4) Conclusions: Kratom use as a recreational drug is on the rise, but it likely has important therapeutic potential.

082 Testosterone replacement therapy may upregulate pro-erectile markers by modulating endothelial function In hypogonadal men with erectile dysfunction

Testosterone (T) deficiency (hypogonadism) is strongly associated with erectile dysfunction (ED). The relaxant response of T on non-genomic pathways on the corporal smooth muscle has been reported. However, there is no evidence on the in vitro mediating effects of T on human corpus cavernosum (HCC). We aimed to compare the mediating effects of different T concentrations on nitric oxide (NO)-dependent & -independent nitrergic relaxations in organ bath studies. HCC samples were obtained after consent from men undergoing penile prosthesis implantation (n = 9). After phenylephrine (Phe) contraction, electrical field stimulation (EFS), acetylcholine (ACh) and phosphodiesterase-5 (PDE-5) inhibitor (sildenafil) induced relaxation at 150, 400, and 600 ng/dL T incubations of HCC strips were performed using organ bath preparations. HCC measurements of endothelial nitric oxide synthase (eNOS), neuronal (nNOS) and PDE5 were evaluated through immunostaining, Western blotting, and cGMP and nitrite/nitrate assays. The relaxation responses to ACh and EFS in isolated HCC were significantly increased by all T levels, as compared to untreated tissues. However, sildenafil-induced relaxant responses were significantly increased at eugonadal T. The unaltered neurogenic contractions to EFS were observed. Eugonadal T levels may be accompanied by increased eNOS, nNOS and cGMP, along with lower PDE5 protein expressions. Tissue nitrate/nitrite concentration (NO production marker) was enhanced by eugonadal T levels (see Table).

Identification of a Rhythmic Firing Pattern in the Enteric Nervous System That Generates Rhythmic Electrical Activity in Smooth Muscle.

The enteric nervous system (ENS) contains millions of neurons essential for organization of motor behavior of the intestine. It is well established that the large intestine requires ENS activity to drive propulsive motor behaviors. However, the firing pattern of the ENS underlying propagating neurogenic contractions of the large intestine remains unknown. To identify this, we used high-resolution neuronal imaging with electrophysiology from neighboring smooth muscle. Myoelectric activity underlying propagating neurogenic contractions along murine large intestine [also referred to as colonic migrating motor complexes, (CMMCs)] consisted of prolonged bursts of rhythmic depolarizations at a frequency of ∼2 Hz. Temporal coordination of this activity in the smooth muscle over large spatial fields (∼7 mm, longitudinally) was dependent on the ENS. During quiescent periods between neurogenic contractions, recordings from large populations of enteric neurons, in mice of either sex, revealed ongoing activity. The onset of neurogenic contractions was characterized by the emergence of temporally synchronized activity across large populations of excitatory and inhibitory neurons. This neuronal firing pattern was rhythmic and temporally synchronized across large numbers of ganglia at ∼2 Hz. ENS activation preceded smooth muscle depolarization, indicating rhythmic depolarizations in smooth muscle were controlled by firing of enteric neurons. The cyclical emergence of temporally coordinated firing of large populations of enteric neurons represents a unique neural motor pattern outside the CNS. This is the first direct observation of rhythmic firing in the ENS underlying rhythmic electrical depolarizations in smooth muscle. The pattern of neuronal activity we identified underlies the generation of CMMCs.SIGNIFICANCE STATEMENT How the enteric nervous system (ENS) generates neurogenic contractions of smooth muscle in the gastrointestinal (GI) tract has been a long-standing mystery in vertebrates. It is well known that myogenic pacemaker cells exist in the GI tract [called interstitial cells of Cajal (ICCs)] that generate rhythmic myogenic contractions. However, the mechanisms underlying the generation of rhythmic neurogenic contractions of smooth muscle in the GI tract remains unknown. We developed a high-resolution neuronal imaging method with electrophysiology to address this issue. This technique revealed a novel pattern of rhythmic coordinated neuronal firing in the ENS that has never been identified. Rhythmic neuronal firing in the ENS was found to generate rhythmic neurogenic depolarizations in smooth muscle that underlie contraction of the GI tract.

Phosphodiestarase‐9 Inhibitor (BAY 73‐6691) Reduces the Cavernous Smooth Muscle Contractile Response and Improves the Effect of Chronic Treatment with Tadalafil in Heart Failure

INTRODUCTIONPatients with heart failure (HF) display erectile dysfunction (ED). Currently a pharmacological option for the ED treatment are the phosphodiesterase (PDE) 5 inhibitors, however, these has no complete satisfactory response in all patients. The role of other PDEs to treat the ED remains poorly investigated. PDE‐9 specifically hydrolyzes cyclic‐GMP, and was detected in mice and human corpus cavernosum (CC) moreover, the PDE9 inhibitor selective (BAY 73‐6691) amplifies the NO‐cGMP‐mediated cavernosal responses, may be of therapeutic value for ED treatment associated with HF.OBJECTIVEThe aims of the present study were to characterize the effect of BAY 73‐6691, both on the contractile response of rats CC submitted to aortocaval fistula (ACF) model, as in the CC contractile response of rats with HF submitted to chronic treatment with tadalafil.METHODSMale Sprague‐Dawley rats (250g) were submitted to the ACF. After 48 hours, the animals were divided in SHAM and ACF groups (acute protocol). After 8 weeks, to validation of HF, the animals underwent to echocardiogram and were divided into 4 groups: SHAM and HF groups (0.9% saline solution, daily), and TADALAFIL and HF TADALAFIL groups (treated with Tadalafil 5 mg/daily), the treatment was maintained for 4 weeks (chronic protocol). Echocardiogram were used to evaluate the final cardiac function. In all protocols, cumulative concentration‐response curve to phenylephrine (PE; 10 nM‐100μM) and neurogenic contractile responses induced by electrical‐field stimulation (EFS; 1‐32 Hz) were obtained in cavernous smooth muscle, both in the presence or absence of BAY 73‐6691.RESULTSAfter 48 hours, both the neurogenic contraction as well contractile response to PE were increased in the ACF group when compared to the Sham group (p <0.05). However, BAY 73‐6691 restored adrenergic hypercontractility in the ACF group (p <0.05). After 12 weeks, there was an increase in LV mass and a decrease in ejection fraction in HF group when compared to the Sham group (p <0.05). However, in the HF Tadalafil group, the treatment was able to restore the ventricular function and decrease LV mass (p <0.05). Moreover, the neurogenic contraction and contractile response to PE in CC were increased in the HF group when compared to the Sham group (p <0.05) and the treatment with tadalafil restored adrenergic hypercontractility of CC in the HF Tadalafil group (p <0.05). In addition, the CC hypercontractility, observed in the HF group, in response to EFS and PE were reduced with the addition of BAY 73‐6691 (P<0.05). Moreover, BAY 73‐6691 shown an additive in the reduction of contractile mechanism in the rats CC with HF treated with tadalafil (p <0.05).CONCLUSIONBAY 73‐6691 reduces the adrenergic hypercontractility in the CC of ACF rat. Treatment with Tadalafil improve the cardiac function and restore the increased of contractile response of rat CC with HF. Moreover, BAY 73‐6691 (in vitro) promote additive effect associated with treatment of tadalafil and restore the adrenergic hypercontractility in the CC of rats with HF.Support or Funding InformationFAPESP ‐ Grant number: 2011/21095‐4This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Participation of Toll‐like Receptor (TLR) 9 in Obesity‐Induced Benign Prostatic Hyperplasia (BPH) in Mice: Implication of Periprostatic Fat

IntroductionClinical and experimental studies indicate a strong correlation between obesity and benign prostatic hyperplasia (BPH)[1]. Recently, prostatic inflammation and innate immune system have been shown to play a pivotal role in BPH pathogenesis/progression [2]. In the present study we explored the presence and participation of TLR9 signaling pathway on obesity‐induced BPH in mice.MethodsUsing prostate from C57BL6/JUnib mice, we performed immunofluorescence analysis for TLR9 localization. We also evaluated the effects of the in vitro TLR9 activation by ODN 2395 on functional (concentration‐response curves) and molecular (MYD88, TRAF6 and COX2 ‐ TLR9 pathway protein expression) protocols. Besides, male C57BL6/JUnib (Wild type) and TLR9 mutant mice were fed for 12 weeks with either a standard chow or high‐fat diet (HFD) to induce obesity. Fasting glucose levels and body, epididymal and periprostatic fat weight were evaluated. Concentration‐response curves to phenylephrine (PE; α1‐adrenoceptor agonist) and potassium chloride (KCl; depolarizing agent), as well as neurogenic contractions produced by electrical‐field stimulation (EFS; 1–32 Hz, 50 V, 10 s) were also obtained.ResultsThe immunofluorescence assay revealed the presence of TLR9 in epithelial layer of the mouse prostate. The in vitro TLR9 activation with ODN 2395 (TLR9 agonist; 5 μM) led to a significant decrease (P<0.05) in prostate smooth muscle (PSM) contractions to PE and EFS along with increases in MYD88, TRAF6 and COX2 protein expression in prostate from C57BL6/JUnib mice. After 12 weeks of HFD, the TLR9 mutant obese mice exhibited the same body weight compared with WT obese group, however, fasting glucose (FG) and epididymal fat (EF) were reduced on TLR9 mutant obese mice. Despite improvements in FG and EF, TLR9 mutant obese group showed increases in prostate weight and PSM reactivity to PE, KCl and EFS compared with WT and TLR9 mutant lean group. Interestingly, the periprostatic fat (PF) showed augmented (P<0.001) in TLR9 mutant lean group compared with WT lean group. Under obesity conditions, an increase in PF was seen in WT obese group, which was further increased in TLR9 mutant obese group.ConclusionDespite amelioration in fasting glucose and reduction in epididymal fat, TLR9 mutant obese mice still exhibit BPH, which can be, at least in part, due to augmented periprostatic fat.Support or Funding InformationFinancial support: FAPESP (2016/20592‐8); NIH (NHLBI 001961)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acute radiation impacts contractility of guinea-pig bladder strips affecting mucosal-detrusor interactions.

Radiation-induced bladder toxicity is associated with radiation therapy for pelvic malignancies, arising from unavoidable irradiation of neighbouring normal bladder tissue. This study aimed to investigate the acute impact of ionizing radiation on the contractility of bladder strips and identify the radiation-sensitivity of the mucosa vs the detrusor. Guinea-pig bladder strips (intact or mucosa-free) received ex vivo sham or 20Gy irradiation and were studied with in vitro myography, electrical field stimulation and Ca2+-fluorescence imaging. Frequency-dependent, neurogenic contractions in intact strips were reduced by irradiation across the force-frequency graph. The radiation-difference persisted in atropine (1μM); subsequent addition of PPADs (100μM) blocked the radiation effect at higher stimulation frequencies and decreased the force-frequency plot. Conversely, neurogenic contractions in mucosa-free strips were radiation-insensitive. Radiation did not affect agonist-evoked contractions (1μM carbachol, 5mM ATP) in intact or mucosa-free strips. Interestingly, agonist-evoked contractions were larger in irradiated mucosa-free strips vs irradiated intact strips suggesting that radiation may have unmasked an inhibitory mucosal element. Spontaneous activity was larger in control intact vs mucosa-free preparations; this difference was absent in irradiated strips. Spontaneous Ca2+-transients in smooth muscle cells within tissue preparations were reduced by radiation. Radiation affected neurogenic and agonist-evoked bladder contractions and also reduced Ca2+-signalling events in smooth muscle cells when the mucosal layer was present. Radiation eliminated a positive modulatory effect on spontaneous activity by the mucosa layer. Overall, the findings suggest that radiation impairs contractility via mucosal regulatory mechanisms independent of the development of radiation cystitis.

Social stress-induced blood pressure increase in borderline hypertensive rats is associated with endothelial dysfunction in the resistant arteries

ObjectivesSeveral studies have observed that altered endothelial function is involved in the development of stress-induced hypertension. The aim of this study was to investigate the effects of chronic social stress (crowding) on endothelium-dependent relaxation (EDR) of the superior mesenteric artery (SMA) and of small resistant mesenteric arteries (MA) as well as on neurogenic contractions of SMA in adult borderline hypertensive rats (BHR).MethodsTwelve-week-old BHR (offspring of spontaneously hypertensive dams and Wistar-Kyoto sires) males were exposed to crowding (living space: 200 cm2/rat) for eight weeks. Control BHR were kept in the groups of four rats per cage (living space: 480 cm2/rat). Systolic blood pressure (sBP) was determined by the tail-cuff method.Vascular function was investigated in the isolated arteries at isometric conditions.EDR was assessed using acetylcholine test.ResultsCrowding significantly increased sBP of BHR to the hypertensive values. Results showed that stress did not affect total acetylcholine-induced relaxation and its nitric oxide (NO)-dependent and NO-independent components in the SMA. In the resistant MA, stress reduced total acetylcholine-induced relaxation by reducing NO-independent component, without the alterations of its NO-dependent component.However, stress failed to affect significantly neurogenic contractions of SMA elicited by electrical stimulation of perivascular sympathetic nerves and vasoconstriction induced by exogenous noradrenaline in SMA.ConclusionIn conclusion, chronic social stress can accelerate the development of hypertension in BHR, which seems to be associated with NO-independent endothelial dysfunction in small resistant arteries.Supported by the grants VEGA No. 2/0190/17 and APVV-16-0263.

Development of contractile properties in the fetal porcine urinary bladder.

BackgroundIn early fetal life, the bladder is merely a conduit allowing urine to pass through freely into the amniotic cavity. As the striated external urethral sphincter evolves, the bladder acquires its reservoir and voiding functions. We characterized the myogenic and neurogenic contractions of the normal fetal porcine bladder from midterm until close to full-term gestation.MethodsContractile responses were measured in vitro using bladder strips from fetuses at 60 (N=23) and 100 days (N=21) of gestation. Spontaneous activity, and the responses to potassium chloride (KCl) solution, electrical field stimulation (EFS), and receptor activation were recorded. The smooth muscle content was evaluated histologically.ResultsHistological studies revealed that the fractional content of smooth muscle doubled between the two time points, and passive tension was adjusted to take that into account. Spontaneous activity was regular at 60 days, changing toward an irregular pattern at 100 days. Contractile force elicited by KCl and carbachol increased significantly with gestational age, while contractions to the purinergic agonist, α-β-methylene adenosine 5'-triphosphate did not. The responses to EFS were almost completely blocked by atropine.ConclusionSpontaneous myogenic contractions become irregular and contractile responses to muscarinic receptor stimulation increase during gestation, as the bladder reservoir and voiding functions develop.

Inhibition of smooth muscle contraction and ARF6 activity by the inhibitor for cytohesin GEFs, secinH3, in the human prostate.

Prostate smooth muscle contraction is critical for etiology and treatment of male lower urinary tract symptoms (LUTS) and is promoted by small monomeric GTPases (RhoA and Rac). GTPases may be activated by guanosine nucleotide exchange factors (GEFs). GEFs of the cytohesin family may indirectly activate Rac, or ADP ribosylation factor (ARF) GTPases directly. Here we investigated the expression of cytohesin family GEFs and effects of the cytohesin inhibitor Sec7 inhibitor H3 (secinH3) on smooth muscle contraction and GTPase activities in human prostate tissues. Of all four cytohesin isoforms, cytohesin-1 and -2 showed the highest expression in real-time PCR. Western blot and fluorescence staining suggested that cytohesin-2 may be the predominant isoform in prostate smooth muscle cells. Contractions induced by norepinephrine, the α1-adrenoceptor agonist phenylephrine, the thromboxane A2 analog U-46619 , and endothelin-1 and -3, as well as neurogenic contractions induced by electric field stimulation (EFS), were reduced by secinH3 (30 µM). Inhibition of EFS-induced contractions appeared to have efficacy similar to that of inhibition by the α1-adrenoceptor antagonist tamsulosin (300 nM). Combined application of secinH3 plus tamsulosin caused larger inhibition of EFS-induced contractions than tamsulosin alone. Pull-down assays demonstrated inhibition of the small monomeric GTPase ARF6 by secinH3, but no inhibition of RhoA or Rac1. In conclusion, we suggest that a cytohesin-ARF6 pathway takes part in smooth muscle contraction. This may open attractive new possibilities in medical treatment of male LUTS.

Ammonia modifies enteric neuromuscular transmission through glial γ-aminobutyric acid signaling.

Impaired gut motility may contribute, at least in part, to the development of systemic hyperammonemia and systemic neurological disorders in inherited metabolic disorders, or in severe liver and renal disease. It is not known whether enteric neurotransmission regulates intestinal luminal and hence systemic ammonia levels by induced changes in motility. Here, we propose and test the hypothesis that ammonia acts through specific enteric circuits to influence gut motility. We tested our hypothesis by recording the effects of ammonia on neuromuscular transmission in tissue samples from mice, pigs, and humans and investigated specific mechanisms using novel mutant mice, selective drugs, cellular imaging, and enzyme-linked immunosorbent assays. Exogenous ammonia increased neurogenic contractions and decreased neurogenic relaxations in segments of mouse, pig, and human intestine. Enteric glial cells responded to ammonia with intracellular Ca2+ responses. Inhibition of glutamine synthetase and the deletion of glial connexin-43 channels in hGFAP::CreERT2+/-/connexin43f/f mice potentiated the effects of ammonia on neuromuscular transmission. The effects of ammonia on neuromuscular transmission were blocked by GABAA receptor antagonists, and ammonia drove substantive GABA release as did the selective pharmacological activation of enteric glia in GFAP::hM3Dq transgenic mice. We propose a novel mechanism whereby local ammonia is operational through GABAergic glial signaling to influence enteric neuromuscular circuits that regulate intestinal motility. Therapeutic manipulation of these mechanisms may benefit a number of neurological, hepatic, and renal disorders manifesting hyperammonemia.NEW & NOTEWORTHY We propose that local circuits in the enteric nervous system sense and regulate intestinal ammonia. We show that ammonia modifies enteric neuromuscular transmission to increase motility in human, pig, and mouse intestine model systems. The mechanisms underlying the effects of ammonia on enteric neurotransmission include GABAergic pathways that are regulated by enteric glial cells. Our new data suggest that myenteric glial cells sense local ammonia and directly modify neurotransmission by releasing GABA.

Imaging sympathetic neurogenic Ca2+ signaling in blood vessels.

We review the information that has been provided by optical imaging experiments directed at understanding the role and effects of sympathetic nerve activity (SNA) in the functioning of blood vessels. Earlier studies utilized electric field stimulation of nerve terminals (EFS) in isolated arteries and vascular tissues (ex vivo) to elicit SNA, but more recently, imaging studies have been conducted in vivo, enabling the study of SNA in truly physiological conditions. Ex vivo: In vascular smooth muscle cells (VSMC) of isolated arteries, the three sympathetic neurotransmitters, norepinephrine (NE), ATP and neuropeptide Y (NPY), elicit or modulate distinct patterns of Ca2+ signaling, as revealed by confocal imaging of exogenous fluorescent Ca2+ indicators. Purinergic junctional Ca2+ transients (jCaTs) arise from Ca2+ influx during excitatory junction potentials (eJPs), and are associated with the initial neurogenic contraction. Adrenergic Ca2+ waves and oscillations cause contraction while SNA-induced endothelial Ca2+ 'pulsars' cause relaxation. In vivo: optical biosensor mice, which express genetically encoded Ca2+ indicators (GECI's) specifically in smooth muscle, combined with non-invasive imaging techniques has enabled imaging SNA-induced Ca2+ signaling and arterial diameter in vivo. SNA induces Ca2+ oscillations in intact arteries. [Ca2+] of arterial smooth muscle cells increased in hypertension, in association with increased SNA. High resolution imaging has revealed local sympathetic, neurogenic Ca2+ signaling within smooth muscle and endothelial cells of the vasculature. The ongoing development of in vivo imaging together with an expanding availability of different biosensor animals promises to enable the further assessment of SNA and its effects in the vasculature of living animals.

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 Ca2+-activated Cl- currents (I ClCa) 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, CACCinh-A01 and T16Ainh-A01, on I ClCa 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. Single-cell RT-PCR experiments revealed that only TMEM16A was expressed in freshly isolated RUICC. Depolarization-evoked I ClCa in isolated RUICC, recorded using voltage clamp, were inhibited by CACCinh-A01 and T16Ainh-A01 with IC50 values of 1.2 and 3.4μM, respectively. Similarly, spontaneous transient inward currents (STICs) recorded from RUICC voltage clamped at -60mV and spontaneous transient depolarizations (STDs), recorded in current clamp, were also inhibited by CACCinh-A01 and T16Ainh-A01. In contrast, spontaneous Ca2+ waves in isolated RUICC were only partially reduced by CACCinh-A01 and T16Ainh-A01. Finally, neurogenic contractions of strips of rabbit urethra smooth muscle (RUSM), evoked by electric field stimulation (EFS), were also significantly reduced by CACCinh-A01 and T16Ainh-A01. These data are consistent with the idea that TMEM16A is involved with CACCs in RUICC and in contraction of rabbit urethral smooth muscle.

Altered detrusor contractility in MPTP-treated common marmosets with bladder hyperreflexia

Bladder hyperreflexia is a common non-motor feature of Parkinson’s disease. We now report on the contractility of the isolated primate detrusor strips devoid of nerve input and show that following MPTP, the amplitude and frequency of spontaneous contraction was increased. These responses were unaffected by dopamine D1 and D2 receptor agonists A77636 and ropinirole respectively. Contractions by exogenous carbachol, histamine or ATP were similar and no differences in the magnitude of noradrenaline-induced relaxation were seen in detrusor strip obtained from normal and MPTP-treated common marmosets (Callithrix jacchus). However, the neurogenic contractions following electrical field stimulation of the intrinsic nerves (EFS) were markedly greater in strips obtained from MPTP treated animals. EFS evoked non-cholinergic contractions following atropine were also greater but the contribution of the cholinergic innervation as a proportion of the overall contraction was smaller in the detrusor strips of MPTP treated animals, suggesting a preferential enhancement of the non-cholinergic transmission. Although dopaminergic mechanism has been proposed to underlie bladder hyperreflexia in MPTP-treated animals with intact bladder, the present data indicates that the increased neurogenically mediated contractions where no extrinsic innervation exists might be due to long-term adaptive changes locally as a result of the loss of the nigrostriatal output.

Spatiotemporal Mapping Techniques Show Clozapine Impairs Neurogenic and Myogenic Patterns of Activity in the Colon of the Rabbit in a Dose-Dependent Manner

Background: Clozapine, an antipsychotic used in treatment-resistant schizophrenia, has adverse gastrointestinal effects with significant associated morbidity and mortality. However, its effects on defined patterns of colonic contractile activity have not been assessed.Method: We used novel radial and longitudinal spatiotemporal mapping techniques, combined with and monitoring of ambient lumen pressure, in ex vivo preparations of triply and of singly haustrated portions of rabbit colon. We identified the contractile patterns of mass peristalses, fast phasic, and ripple contractions and directly qualified the effects of clozapine, at concentrations of 10 μmol/L, 20 μmol/L, and 30 μmol/L, and of norclozapine, the main metabolite of clozapine, on contractile patterns. The effects of carbachol, serotonin and naloxone on clozapine-exposed preparations were also determined. Tetradotoxin was used to distinguish neurogenic from myogenic contractions.Results: At 10 μmol/L, clozapine temporarily abolished the longitudinal contractile components of mass peristalsis, which on return were significantly reduced in number and amplitude, as was maximal mass peristaltic pressure. These effects were reversed by carbachol (1 μmol/L) and to some extent by serotonin (15 μmol/L). At 10 μmol/L, myogenic ripple contractions were not affected. At 20 μmol/L, clozapine had a similar but more marked effect on mass peristalses with both longitudinal and radial components and corresponding maximal pressure greatly reduced. At 30 μmol/L, clozapine suppressed the radial and longitudinal components of mass peristalses for over 30 min, as well as ripple contractions. Similar dose-related effects were observed on addition of clozapine to the mid colon. At 20 μmol/L, norclozapine had opposite effects to those of clozapine, causing an increase in the frequency of mass peristalsis with slight increases in basal tone. These slightly augmented contractions were abolished on addition of clozapine. Concentrations of norclozapine below 20 μmol/L had no discernible effects.Conclusion: Clozapine, but not norclozapine, has potent effects on the motility of the rabbit colon, inhibiting neurogenic contractions at lower concentrations and myogenic contractions at higher concentrations. This is the likely mechanism for the serious and life-threatening gastrointestinal complications seen in human clozapine-users. These effects appear to be mediated by cholinergic and serotonergic mechanisms. Spatiotemporal mapping is useful in directly assessing the effects of pharmaceuticals on particular patterns of gastrointestinal motility.

Obesity‐Induced Mouse Prostatic Dysfunction is Improved by Chronic Treatment with Resveratrol: Implication of Oxidative Stress and Neuronal Growth Factor

IntroductionThe worldwide incidence of obesity has increased dramatically in the past 25 years. Clinical studies indicate a strong correlation between obesity and genitourinary tract diseases, being benign prostatic hyperplasia (BPH) one of the most common prostatic disorders affecting obese individuals1,2. Recently, increased oxidative stress has been shown to play a pivotal role in BPH pathogenesis/progression3. In the present study we explored the effects of the polyphenol resveratrol, an antioxidant drug, on obesity‐induced prostatic dysfunctions.MethodsMale C57BL6/JUnib mice, fed for 12 weeks with either a standard chow or high‐fat diet to induce obesity were treated daily with resveratrol (100 mg/kg/day, gavage) from weeks 10–12 of diet. Concentration‐response curves to phenylephrine (PE; α1‐adrenoceptor agonist) and potassium chloride (KCl; depolarizing agent) as well as neurogenic contractions produced by electrical‐field stimulation (EFS; 1–32 Hz, 50 V, 10 s) were obtained. Stereological analysis, reactive‐oxygen species (ROS) levels, RT‐PCR for neuronal growth factor (NGF) and NADPH subunit gp91phox were all performed in the prostatic tissues in all groups.ResultsA significant increase (P<0.01) in the prostate smooth muscle contractions (PSM) to PE and KCl were observed in obese group compared with control animals. Likewise, EFS‐induced PSM contractions were higher in obese animals in all frequencies employed. Resveratrol fully restored the enhanced PSM contractions to PE, KCl and EFS in obese group. ROS levels were 87% higher (P<0.001) in prostate from obese mice. Moreover, mRNA expression for gp91phox was 57% higher (P<0.05) in prostate of obese mice. Resveratrol restored the augmented ROS production and gp91phox mRNA in obese mice, without affecting these parameters in the control group. In prostate from obese mice, the stereological analysis showed epithelial polyps formation and reduced lumen area whereas stromal and smooth muscle cells layer remained unchanged. A significant increase by about of 70% (P<0.01) in mRNA expression for NGF was also found in prostate of obese mice. Resveratrol significantly restored the NGF mRNA expression, and attenuated the epithelial alterations, without affecting the reduced luminal area.ConclusionA two‐week treatment with resveratrol is capable to reverse the prostatic dysfunctions in obesity mice. Resveratrol may be a good pharmacological option to prevent obesity‐associated prostatic dysfunctions.Support or Funding InformationFundação de Amparo à Pesquisa do Estado de São Paulo ‐ FAPESP (2016/01178‐6)

Inhibition of agonist-induced smooth muscle contraction by picotamide in the male human lower urinary tract outflow region

Male lower urinary tract symptoms (LUTS) due to bladder outlet obstruction are characterized by abnormal smooth muscle contractions in the lower urinary tract. Alpha1-adrenoceptor antagonists may induce smooth muscle relaxation in the outflow region and represent the current gold standard of medical treatment. However, results may be unsatisfactory or inadequate. Apart from α1-adrenoceptor agonists, smooth muscle contraction in the outflow region may be induced by thromboxane A2 (TXA2), endothelins, or muscarinic receptor agonists. Here, we studied effects of the thromboxane A2 receptor (TP receptor) antagonist picotamide on contraction in the human male bladder trigone and prostate. Carbachol, the α1-adrenoceptor agonist phenylephrine, the thromboxane A2 analog U46619, and electric field stimulation (EFS) induced concentration- or frequency-dependent contractions of trigone tissues in an organ bath. Picotamide (300µM) inhibited carbachol-, phenylephrine-, U46619-, and EFS-induced contractions. Endothelins 1-3 induced concentration-dependent contractions of prostate tissues, which were inhibited by picotamide. Analyses using real time polymerase chain reaction and antibodies suggested expression of thromboxane A2 receptors and synthase in trigone smooth muscle cells. Thromboxane B2 (the stable metabolite of thromboxane A2) was detectable by enzyme immune assay in trigone samples, with most values ranging between 50 and 150pg/mg trigone protein. Picotamide inhibits contractions induced by different stimuli in the human lower urinary tract, including cholinergic, adrenergic, thromboxane A2- and endothelin-induced, and neurogenic contractions in different locations of the outflow region. This distinguishes picotamide from current medical treatments for LUTS, and suggests that picotamide may induce urodynamic effects in vivo.

Botulinum Neurotoxins Serotypes A and B induce paralysis of mouse striated and smooth muscles with different potencies.

To address the scarcity of direct comparison of botulinum neurotoxin serotypes activity on smooth versus striatal muscle, we have studied the action of BoNT/A1 and BoNT/B1 on ex vivo preparations of both muscle types. We have set up and characterized a model of neurogenic contractions in the isolated mouse bladder, and used this model to explore the effects of the two serotypes on contractions evoked by electrical field stimulation. Both toxins were also tested in the mouse phrenic nerve hemidiaphragm assay, to compare their potency in smooth versus striated muscle. The characterization of the model of neurogenic contractions in the isolated mouse bladder indicates that about half of the activity is driven by purinergic signaling, and about half by cholinergic signaling. Furthermore, we find that BoNT/B1 is more potent than BoNT/A1 in inhibiting activity in the mouse detrusor smooth muscle preparation, but that both toxins have comparable potency on the striated muscle activity of the phrenic nerve hemidiaphragm model. We also show that these findings are mouse strain independent. In conclusion, the established mouse bladder detrusor smooth muscle model is able to discriminate between different botulinum neurotoxin serotypes and could be a useful preclinical tool to explore the pathophysiology of bladder overactivity, as well as the effects of new therapeutic candidates. It is interesting to note that the high proportion of purinergic transmission driving detrusor contractions in this model is similar to that seen in neurodetrusor overactivity disease, making this model relevant with regard to pathophysiological interest.