Oxytocin receptor antagonists as a novel pharmacological agent for reducing smooth muscle tone in the human prostate

Pharmacotherapies for the treatment of Benign Prostatic Hyperplasia (BPH) are targeted at reducing cellular proliferation (static component) or reducing smooth muscle tone (dynamic component), but response is unpredictable and many patients fail to respond. An impediment to identifying novel pharmacotherapies is the incomplete understanding of paracrine signalling. Oxytocin has been highlighted as a potential paracrine mediator of BPH. To better understand oxytocin signalling, we investigated the effects of exogenous oxytocin on both stromal cell proliferation, and inherent spontaneous prostate contractions using primary models derived from human prostate tissue. We show that the Oxytocin Receptor (OXTR) is widely expressed in the human prostate, and co-localises to contractile cells within the prostate stroma. Exogenous oxytocin did not modulate prostatic fibroblast proliferation, but did significantly (p < 0.05) upregulate the frequency of spontaneous contractions in prostate tissue, indicating a role in generating smooth muscle tone. Application of atosiban, an OXTR antagonist, significantly (p < 0.05) reduced spontaneous contractions. Individual tissue responsiveness to both exogenous oxytocin (R2 = 0.697, p < 0.01) and atosiban (R2 = 0.472, p < 0.05) was greater in tissue collected from older men. Overall, our data suggest that oxytocin is a key regulator of inherent spontaneous prostate contractions, and targeting of the OXTR and associated downstream signalling is an attractive prospect in the development of novel BPH pharmacotherapies.

Functional constipation induces bladder overactivity associated with upregulations of Htr2 and Trpv2 pathways

Bladder and bowel dysfunction (BBD) is a common yet underdiagnosed paediatric entity that describes lower urinary tract symptoms (LUTS) accompanied by abnormal bowel patterns manifested as constipation and/or encopresis. LUTS usually manifest as urgency, urinary frequency, incontinence, and urinary tract infections (UTI). Despite increasing recognition of BBD as a risk factor for long-term urinary tract problems including recurrent UTI, vesicoureteral reflux, and renal scarring, the mechanisms underlying BBD have been unclear, and treatment remains empirical. We investigated how constipation affects the lower urinary tract function using a juvenile murine model of functional constipation. Following four days of functional constipation, animals developed LUTS including urinary frequency and detrusor overactivity evaluated by awake cystometry. Physiological examination of detrusor function in vitro using isolated bladder strips, demonstrated a significant increase in spontaneous contractions without affecting contractile force in response to electrical field stimulation, carbachol, and KCl. A significant upregulation of serotonin receptors, Htr2a and Htr2c, was observed in the bladders from mice with constipation, paralleled with augmented spontaneous contractions after pre-incubation of the bladder strips with 0.5 µM of serotonin. These results suggest that constipation induced detrusor overactivity and increased excitatory serotonin receptor activation in the urinary bladder, which contributes to the development of BBD.

Modulating the mechanokinetics of spontaneous contractions of the myometrium of rats using calix[4]arene C-90 – plasma membrane calcium ATPase inhibitor

Introduction. Plasma membrane calcium ATPase is a constitutive structure of cells that functions as a high affinity system of releasing Са2+ ions from the cytoplasm and ensures a long-term maintenance of the basal concentration of these cations in the state of dormancy. Currently, there are no satisfactory means for the pharmacological correction of plasma membrane calcium ATPase function. Thus, elaboration, synthesis, and study of substances with the targeted impact on plasma membrane calcium ATPase are topical issues. Previously, we determined the ability of this calix[4]arene in the concentration of 10 µM to inhibit the contractive activity and to slow down the relaxation of smooth muscle of the myometrium in the NO-dependent way, which considerably decreases the normalized maximal velocity of the relaxation phase. Materials and Methods. The tenzometric methods and mechanokinetic analysis were used to investigate the impact of the cumulative increase in the concentration of calix[4]arene С-90 (10 nM – 100 µM) on the spontaneous contractive activity of the myometrium of rats. The complete profile of spontaneous cycles of contractions-relaxa­tions was studied by the empirical multiparameter method of complex mechanokinetic analysis, elaborated by us (with the consideration of the parameters of time (τ0, τC and τR), force (Fmax, FC and FR), velocity (VC and VR), and impulse (Іmax, ІC and ІR). Results. Calix[4]arene C-90 evoked the dose-dependent inhibition of spontaneous contractive activity of the myometrium preparations. Its high concentrations caused a change in the structure of the contraction act, such as an increase in the duration of the contraction phase, while the duration of the relaxation phase did not show any changes. The multiparameter method of the complex mechanokinetic analysis demonstrated that in the whole range of the investigated concentrations, substance С-90 considerably decreases the indices of force parameters (Fmax, FC and FR) and the values of impulses of force Іmax, IC and IR of the spontaneous contractions of the myometrium. On the background of all the applied concentrations (10-7–10-4 М), calix[4]arene С-90 conditioned the slowing down of the relaxation of spontaneous contractions in uterine muscle prepa­rations of rats, which was reflected in a reliable decrease in the parameter for the maxi­mal velocity of the relaxation phase (VR). Conclusions. The results of the study demonstrate that calix[4]arene С-90 inhibits the processes of Са2+ extrusion from smooth muscle cells myoplasm, probably, impacting plasma membrane calcium ATPase molecules directly. It is noteworthy that C-90 is also likely to inhibit the processes of the intake of these cations to cells from the extracellular medium, causing a decrease in the velocity of force intensification during the contraction phase, and reducing the frequency and force of the spontaneous contractions in the myometrium.

The Development of Integrin Alpha-8 Deficient Lungs Shows Reduced and Altered Branching and a Correction of the Phenotype During Alveolarization

Lung development involves epithelial–mesenchymal interactions and integrins represent one of the key elements. These extracellular matrix receptors form hetero-dimers of alpha and beta subunits. The integrin α8β1 is highly expressed in mouse tissues, including lung. It forms a cellular receptor for fibronectin, vitronectin, osteopontin, nephronectin, and tenascin-C. This study aims to investigate the role of the integrin α8-subunit (α8) during lung development. Wild type and α8-deficient lungs were explanted at embryonic days 11.5/12.5. After 24–73 h in culture α8-deficient lung explants displayed reduced growth, reduced branching, enlarged endbuds, altered branching patterns, and faster spontaneous contractions of the airways as compared to wild type. Postnatally, a stereological investigation revealed that lung volume, alveolar surface area, and the length of the free septal edge were significantly reduced in α8-deficient lungs at postnatal days P4 and P7. An increased formation of new septa in α8-deficient lungs rescued the phenotype. At day P90 α8-deficient lungs were comparable to wild type. We conclude that α8β1 takes not only part in the control of branching, but also possesses a morphogenic effect on the pattern and size of the future airways. Furthermore, we conclude that the phenotype observed at day P4 is caused by reduced branching and is rescued by a pronounced formation of the new septa throughout alveolarization. More studies are needed to understand the mechanism responsible for the formation of new septa in the absence of α8β1 in order to be of potential therapeutic benefit for patients suffering from structural lung diseases.

Renal lymphatic vessel dynamics

Similar to other organs, renal lymphatics remove excess fluid, solutes, and macromolecules from the renal interstitium. Given the kidney’s unique role in maintaining body fluid homeostasis, renal lymphatics may be critical in this process. However, little is known regarding the pathways involved in renal lymphatic vessel function, and there are no studies on the effects of drugs targeting impaired interstitial clearance, such as diuretics. Using pressure myography, we showed that renal lymphatic collecting vessels are sensitive to changes in transmural pressure and have an optimal range of effective pumping. In addition, they are responsive to vasoactive factors known to regulate tone in other lymphatic vessels including prostaglandin E2 and nitric oxide, and their spontaneous contractility requires Ca2+ and Cl−. We also demonstrated that Na+-K+-2Cl− cotransporter Nkcc1, but not Nkcc2, is expressed in extrarenal lymphatic vessels. Furosemide, a loop diuretic that inhibits Na+-K+-2Cl− cotransporters, induced a dose-dependent dilation in lymphatic vessels and decreased the magnitude and frequency of spontaneous contractions, thereby reducing the ability of these vessels to propel lymph. Ethacrynic acid, another loop diuretic, had no effect on vessel tone. These data represent a significant step forward in our understanding of the mechanisms underlying renal lymphatic vessel function and highlight potential off-target effects of furosemide that may exacerbate fluid accumulation in edema-forming conditions.

PEPTIDOGLYCAN OF STAPHYLOCOCCUS AUREUS ALTERS THE MYOMETRIAL CONTRACTILITY OF NON-PREGNANT RATS TROUGH INCREASING INTRACELLULAR CALCIUM LEVELS

The work is devoted to the study of the effect of Staphylococcus aureus peptidoglycan on the main parameters of myometrial contractions and the mechanism of this influence. The research was carried out by tensometry and calcimetry methods. The results of the experiments demonstrated that peptidoglycan modulates all the main parameters of contractions of the non-pregnant rat uterus, including increasing their amplitude and duration by 10 and 30%, respectively, mainly by prolonging the relaxation phase (56.7 ± 1.6%): myometrial band relative to quickly shrunk and relaxed for a long time. Application of peptidoglycan on freshly isolated uterine myocytes for 5 min led to a periodic increase in their intracellular content of calcium ions. Peptidoglycan enhanced myometrial contractions caused by hyperpotassium solution at a concentration of 60 mmol. Administration of 1 μmol of nifedipine, a blocker of potential-directed L-type calcium channels, suppressed spontaneous contractions enhanced by peptidoglycan. These changes in myometrium contractility of on the background of peptidoglycan action occur not only due to increased transmembrane calcium intake, but also its mobilization from the sarcoplasmic reticulum.

Alterations in detrusor contractility in rat model of bladder cancer

Urinary incontinence of idiopathic nature is a common complication of bladder cancer, yet, the mechanisms underlying changes in bladder contractility associated with cancer are not known. Here by using tensiometry on detrusor smooth muscle (DSM) strips from normal rats and rats with bladder cancer induced by known urothelial carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), we show that bladder cancer is associated with considerable changes in DSM contractility. These changes include: (1) decrease in the amplitude and frequency of spontaneous contractions, consistent with the decline of luminal pressures during filling, and detrusor underactivity; (2) diminution of parasympathetic DSM stimulation mainly at the expense of m-cholinergic excitatory transmission, suggestive of difficulty in bladder emptying and weakening of urine stream; (3) strengthening of TRPV1-dependent afferent limb of micturition reflex and TRPV1-mediated local contractility, promoting urge incontinence; (4) attenuation of stretch-dependent, TRPV4-mediated spontaneous contractility leading to overflow incontinence. These changes are consistent with the symptomatic of bladder dysfunction in bladder cancer patients. Considering that BBN-induced urothelial lesions in rodents largely resemble human urothelial lesions at least in their morphology, our studies establish for the first time underlying reasons for bladder dysfunction in bladder cancer.

Multiple nerve rings coordinate Hydra mechanosensory behavior

Hydra vulgaris is an emerging model organism for neuroscience due to its small size, transparency, genetic tractability, and regenerative nervous system; however, fundamental properties of its sensorimotor behaviors remain unknown. Here, we use microfluidic devices combined with fluorescent calcium imaging and surgical resectioning to study how the nervous system coordinates Hydra’s mechanosensory response. We find that mechanical stimuli cause animals to contract, and this response relies on both the oral and aboral nerve rings. We also find that these nerve rings work together to coordinate spontaneous contractions suggesting that spontaneous behavior and sensorimotor responses converge on to a common neural circuit. These findings improve our understanding of how Hydra’s diffuse nervous system supports sensorimotor behaviors, which is needed to increase the utility of Hydra as a model organism for neuroscience.

A Microtissue Platform to Simultaneously Actuate and Detect Mechanical Forces via Non-Contact Magnetic Approach

Mechanical control is essential for adaptive regulation in biological systems. This work presents a magnetic, non-contact approach to simultaneous detection and actuation in a microscale tissue testbed. The platform builds upon previously developed passive mechanical platforms, where tissues self-assemble on flexible pillars. Standard detection is typically derived from microscope images and actuation is often conducted using invasive approaches. In the presented platform, actuation and detection, both derived from magnetic fields, are demonstrated with high spatial, temporal, and force resolution resulting in a detectable 0.6 µm step-size in air. The forced deflection range is +/-125 microns (+/-10% strain), and an arbitrary magnitude step-and-settle actuation is achieved in less than 1 ms. Engineered human cardiac microtissue is used to demonstrate the capabilities of the system, as cardiac tissues generate contractions and adapt to external forces. Spontaneous contractions are monitored for an hour by a built-in sensor with a signal-to-noise of 2. Cyclic actuation at 1 Hz using a 1 mT/mm magnetic field is demonstrated, where max tissue strain is 0.3%. All this is achieved in a moderate-throughput, compact device, which is easily integrated into the typical flow of biological experimentation. Simultaneous control of actuation and detection enables decisions to be made on a sample-specific basis, and in future developments, will enable arbitrary design of the mechanical environment for 3D tissue conditioning, maturation, and control.