Repeated human leukocyte antigens eplets, importance of typing the partner.

INTRODUCTION: Antibody-mediated rejection (AMR) is the most common cause of immune-mediated allograft failure after kidney transplant and impacts allograft survival. Previous sensitization is a major risk factor for development of donor specific antibodies (DSA). AMR can have a wide range of clinical features such as impaired kidney function, proteinuria/hypertension or can be subclinical. HLA molecules have specific regions of antigens binding antibodies called epitopes and eplets are considered essential components responsible for immune recognition. We present a patient with subclinical AMR 1 week post transplantation. CASE REPORT: A 48-year-old, caucasian woman with end-stage kidney disease (ESKD) secondary to autosomal dominant polycystic kidney disease (ADPKD) on peritoneal dialysis was registered in deceased donor waitlist. She was a hypersensitized patient from 3 prior pregnancies with a calculated panel reactive antibody of 93,48%. She was transplanted through kidney paired exchange donation with no evidence of DSA pre transplantation. Surgery and post-op were unremarkable with excellent and immediate graft function. Per protocol DSA levels on the 5th day was DR1 of 3300 MFI, with an increase in MFI by day 13 with 7820 MFI and a new B41 1979MFI. Allograft kidney biopsy findings were diagnostic of AMR and she was treated with immunoglobulin and plasmapheresis. As early onset AMR post transplantation was observed an anamnestic response was hypothesized from a previous exposure to allo-HLA. We decided to type her husband, her son's father, which was presented with DSA. Mismatch eplet analysis revealed a shared 41 T and 67LQ eplets between the donor and husband, responsible for the reactivity and new HLA class I B41 and HLA class II DR1 DSA, respectively. DISCUSSION: Shared eplets between the patient husband and donor was responsible for the alloimmune response and early development of DSAs. This case highlights the importance of early monitoring DSA levels in highly sensitized patients after transplant in order to promptly address and lower inflammatory damage. Mismatch eplet analysis can provide a thorough and precise evaluation of immune compatibility providing a useful technique to immune risk stratification, donor selection and post-transplant immunosuppressive therapy and monitoring.

Terson Syndrome - Clinical Presentation, Management, and Visual Outcomes in a Tertiary Centre.

Purpose: The purpose of this study was to characterize the clinical presentation, management strategy and visual outcomes of patients diagnosed with Terson syndrome and followed in a tertiary centre in Portugal. Patients and Methods: A single-centre retrospective study was performed, based on the survey review of the medical records of every consecutive patient diagnosed with Terson syndrome and followed from January 2018 to August 2021. The change in best-corrected visual acuity (BCVA) from baseline to the final evaluation was the primary outcome. Results: Fifteen eyes from 8 patients (50% female) were included. The mean age at diagnosis was 55±7 years. The neurological event was traumatic brain injury in 37.5% (n=3) and subarachnoid haemorrhage in 62.5% of the patients (n=5). Bilateral intraocular haemorrhage occurred in 875% (n=7) of the patients. Vitreous and preretinal haemorrhages occurred each in 66.7% (n=10), intraretinal in 30% (n=3) and subretinal in 13.3% (n=2) of the eyes. In 40% of the eyes (n=6), spontaneous resolution of intraocular haemorrhage occurred, while PPV was performed in the remaining 60% (n=9). Ocular haemorrhage detection occurred 58.47 ± 40.94 days after the neurological event (range 11 to 121 days). Baseline BCVA was 1.11 ± 1.01 logMAR and improved to 0.32 ± 0.69 logMAR in the follow-up period (p=0.004). A positive correlation was found between initial and final BCVA (Spearman's rho = 0.643, p=0.01). Baseline BCVA of eyes undergoing PPV was lower than of those conservatively managed (1.84±0.72 vs 0.20±0.28 logMAR, p<0.001). However, there were no statistically significant differences in final BCVA after surgery or observation (0.56 ± 0.90 vs 0.04 ± 0.04 logMAR, p=0.149). Longer periods between the neurological and the ophthalmological diagnosis were correlated with worse final BCVA (Spearman's rho = 0.688, p=0.005). Conclusion: Terson syndrome is a potential cause of irreversible visual loss. Diagnosis delay may affect visual prognosis. PPV is indicated when intraocular haemorrhage is dense and does not resolve spontaneously or when visual acuity at presentation is low, allowing for good visual outcomes with minimal complications.

Intravitreal Fluocinolone Acetonide Implant (FAc, 0.19 mg, ILUVIEN®) in the Treatment of Patients with Recurrent Cystoid Macular Edema After Pars Plana Vitrectomy.

INTRODUCTION: Postoperative cystoid macular edema (PCME) is a complication of several ocular procedures, including pars plana vitrectomy (PPV), due to the activation of the inflammatory cascade. The purpose of this case series is to evaluate the effectiveness and safety of fluocinolone acetonide intravitreal implant (FAc, 0.2 µg/day; ILUVIEN®) in the treatment of refractory PCME after successful PPV. METHODS: This retrospective observational case series includes consecutive eyes of patients with recurrent PCME after PPV and treated with a single FAc implant at Centro Hospitalar Universitário de São João, Porto, Portugal. Previous treatments, best-corrected visual acuity (BCVA, ETDRS letters), central macular thickness (CMT, µm), intraocular pressure (IOP, mmHg), and IOP-lowering medication needed were recorded at baseline and during follow-up. Total macular edema resolution was defined as CMT less than 300 µm or a reduction of greater than 20%, and partial macular edema resolution was defined as a reduction of greater than 10%. RESULTS: Nine eyes from nine patients were included. Before FAc implant, all eyes received intravitreal short-action corticosteroids (triamcinolone and dexamethasone implant), with a good response but relapse 1-5 months later. At baseline, BCVA was 55.0 ± 10.6 letters, CMT was 514.9 ± 165.6 µm, and IOP was 15.4 ± 2.4 mmHg with four eyes under IOP-lowering medication. After FAc implant, all eyes achieved edema resolution (eight total and one partial) with a peak gain of 17.2 letters and a maximum decrease of 208.2 µm in CMT. During follow-up (44.0 ± 14.8 months), 66.7% of the eyes kept their macula dry and three showed recurrence after 11, 14, and 28 months, respectively. The maximum IOP registered was 17.0 ± 6.0 mmHg. IOP-lowering regimen was increased in one eye and two additional eyes started hypotensive drops. CONCLUSION: FAc implant can be considered a therapeutic alternative in PCME refractory to other therapies in vitrectomized eyes, reducing the need for repeated treatments.

Coronal brain atlas in stereotaxic coordinates of the African spiny mouse, Acomys cahirinus.

The African spiny mouse (Acomys cahirinus) is an emerging model of mammalian epimorphic regeneration that has aroused the interest of the scientific community in the last decade. To date, studies on brain repair have been hindered by the lack of knowledge on the neuroanatomy of this species. Here, we present a coronal brain atlas in stereotaxic coordinates, which allows for three-dimensional identification and localization of the brain structures of this species. The brain of 12-week-old spiny mice was mapped in stereotaxic coordinates using cresyl violet-stained brain sections obtained from coronal cryosectioning of the brain after transcardial perfusion with fixative. The atlas is presented in 42 plates representing sections spaced 240 µm apart. Stereotaxic coordinates were validated using both a model of Parkinsonian lesion of the striatum with 6-hydroxydopamine and labeling of the corticospinal tract in the spiny mouse spinal cord using AAV1/2-GFP intracortical injections. This work presents a new tool in A. cahirinus neurobiology and opens new avenues of research for the investigation of the regenerative ability of A. cahirinus in models of brain disorders.

Avaliação de resíduos de agrotóxicos em amostras de farinha de trigo por espectrometria de massas / Evaluation of pesticide residues in wheat flour by mass spectrometry

Os cereais são uma importante fonte alimentícia e econômica, dentre eles se destaca o trigo e sua farinha. Durante o cultivo, visando o controle de pragas, frequentemente são utilizados os agrotóxicos, porém seu uso abusivo pode acarretar danos à saúde humana e ao meio ambiente. O trabalho teve como objetivo avaliar a presença de resíduos de agrotóxicos em amostras de farinha de trigo comercializadas no estado de São Paulo, utilizando o método QuEChERS (Rápido, Fácil, Barato, Efetivo, Robusto e Seguro) modificado, seguido de análise por CG-EM/EM e CLUE-EMAR para identificação e quantificação. No total, 124 ingredientes ativos (i.a.) estavam dentro dos critérios de aceitação para linearidade, limites de detecção e quantificação, exatidão e precisão (intervalo de confiança de 95%; k = 2). Os resultados de 25 amostras indicaram a presença de bifentrina, fenitrotiona, clorpirifós, deltametrina, lambda-cialotrina e pirimifós-metílico em farinha de trigo comum e os quatro últimos i.a. também foram detectados em farinha de trigo orgânica. O pirimifósmetílico foi detectado em 92% das amostras. Os i.a. encontrados nas amostras estavam abaixo do Limite Máximo de Resíduos (LMR) estabelecidos pela Agência Nacional de Vigilância Sanitária (Anvisa) para farinha de trigo e as amostras foram consideradas próprias para o consumo (AU).

Cereals are an important economic factor and food source, which wheat and its flour stand out. Pesticides are often employed to control harmful organisms, but their abusive use can cause damage to human health and the environment. This study aimed to evaluate the presence of pesticide residues in wheat flour samples commercialized in the state of São Paulo ­ Brazil, using the modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method, followed by GC-MS/MS and UHPLC-HRMS analysis for identification and quantification. Thus, 124 active ingredients (a.i.) were within acceptable criteria for linearity, limits of detection and quantification, accuracy (trueness), and precision (95% confidence level; k = 2). The results for 25 wheat flour samples showed the presence of bifenthrin, fenitrothion, chlorpyrifos, deltamethrin, lambda-cyhalothrin, and pirimiphos-methyl, and the last four a.i. were also detected in organic wheat flour. Pirimiphos-methyl was detected in 92% of all samples. All compounds were found below the Maximum Residue Levels (MRL) established by Brazilian Health Regulatory Agency (Anvisa) for wheat flour and the samples were considered suitable for consumption (AU).

In vitro inhibition of phosphodiesterase type 4 enhances rat corpus cavernosum nerve-mediated relaxation induced by gasotransmitters.

AIMS: Nitric oxide (NO) and hydrogen sulfide (H2S) are involved in nerve-mediated corpus cavernosum (CC) relaxation. Expression of phosphodiesterase type 5 (PDE5) and type 4 (PDE4), cyclic guanosine monophosphate (cGMP)- and cyclic adenosine monophosphate (cAMP)-specific, respectively, has been described and PDE5- and PDE4-inhibitors induce cavernous smooth muscle relaxation. Whereas the NO/cGMP signaling pathway is well established in penile erection, the cAMP-mediated mechanism is not fully elucidated. The aim of this study is to investigate the localization and the functional significance of PDE4 in rat CC tone regulation. MAIN METHODS: We performed immunohistochemistry for the detection of the PDE4A isoenzyme. Isometric tension recordings for roflumilast and tadalafil, PDE4 and PDE5 inhibitors, respectively, electrical field stimulation (EFS) and ß-adrenoceptor agonist isoproterenol and endogenous H2S production measurement. KEY FINDINGS: A marked PDE4A expression was detected mainly localized in the nerve cells of the cavernous smooth muscle. Furthermore, roflumilast and tadalafil exhibited strong corpus cavernous relaxations. Endogenous H2S production was decreased by NO and H2S synthase inhibitors and increased by roflumilast. Isoproterenol- and EFS-induced relaxations were increased by roflumilast. SIGNIFICANCE: These results indicate that PDE4A is mainly expressed within the nerves cells of the rat CC, where roflumilast induces a potent corpus cavernous relaxation per se and potentiates the response induced by ß-adrenoceptor activation. The fact that roflumilast enhances H2S production, as well as EFS-elicited responses suggests that PDE4 inhibitors modulate, in a positive feedback fashion, nerve-mediated relaxation induced by gasotransmitters, thus indicating a key role for neuronal PDE4 in penile erection.

Scleral Fixation of Akreos AO60 Intraocular Lens Using Gore-Tex Suture: An Eye on Visual Outcomes and Postoperative Complications.

PURPOSE: "In-the-bag" placement of an IOL is the Holy Grail for any cataract surgeon. However, in the absence of capsular integrity, alternative surgical options to place the IOL must be sought. We aim to report the clinical outcomes and safety profile of scleral-fixated Akreos AO60 intraocular lens implantation using Gore-Tex suture, combined with pars plana vitrectomy. METHODS: This is a single-center, retrospective case series descriptive study. Electronic clinical records of all patients subjected to scleral fixation of a Bausch and Lomb Akreos AO60 IOL combined with pars plana vitrectomy, between April 1, 2017, and August 1, 2021, were reviewed. Data concerning age, sex, laterality, past ophthalmological history, pre- and postoperative best-available visual acuity, surgical indication, and intra- and postoperative complications were collected. Measured outcomes were the differences in best-available visual acuity and frequency of postoperative complications. RESULTS: A total of 37 eyes (20 right eyes and 17 left eyes) from 36 patients (16 females and 20 males) were included in the statistical analysis. The mean age at time of surgery was 72.0 ± 12.4 years. The mean follow-up period was 548.9 days (range 39-1564 days). Globally, the mean best-available logMAR visual acuity improved from 1.61 preoperatively (0.025 decimal equivalent) to 0.57 postoperatively (0.3 decimal equivalent), this difference being statistically significant (P < 0.001). Indications for surgery included aphakia due to complicated cataract surgery (24.3%; n = 9); subluxated IOL due to closed trauma (21.6%; n = 8); PEX-related subluxated IOL (16.2%; n = 6); non-traumatic, non-PEX-related subluxated IOL (18.9%; n = 7); subluxated crystalline lens due to closed trauma (8.1%; n = 3); aphakia due to open-globe injury (5.4%; n = 2); silicone-induced IOL opacification (2.7%; n = 1); and aphakia post-endophthalmitis (2.7%; n = 1). Postoperative complications included transient ocular hypertension (27.0%; n = 10), transient corneal edema (18.9%; n = 7), cystoid macular edema (18.9%, n = 7), self-limited hypotension (5.4%, n = 2), self-limited vitreous hemorrhage (2.7%, n = 1), central retinal vein occlusion (2.7%, n = 1), late retinal detachment (2.7%, n = 1), and Akreos IOL opacification (2.7%, n = 1). No suture-related complications were observed. CONCLUSION: There was a statistically significant improvement in visual acuity after scleral fixation of Akreos AO60 intraocular lens using Gore-Tex suture, with no suture-related problems recorded. This procedure seems to be a valuable alternative for posterior chamber IOL placement when secondary IOL implantation is required.

Percutaneous nephrolithotomy: Three-needle technique on two planes. Cury's technique.

INTRODUCTION: Percutaneous nephrolithotomy is the main type of surgery indicated for kidney stones larger than 2 cm. The present study describes a three-needle technique for percutaneous nephrolithotomy on two planes. SURGICAL TECHNIQUE: The patient is first placed in the lithotomy position for cystoscopy, which guides the ureteral and urethral catheter. Next, the patient is placed in ventral decubitus for the three-needle technique. With the aid of the nephroscope, the first needle is positioned in the projection of the renal pelvis, the second needle is placed in the most posterior of the inferior calyces and the third needle is aligned with the other two at the most depressible point determined by the surgeon's index finger. After alignment in the topography of the third needle, an incision is made with the scalpel 1-2 cm perpendicular to the aponeurosis of the latissimus dorsi muscle. The second needle is replaced with a peridural needle, which is used to confirm the location of the inferior renal calyx and limit the depth of the Chiba needle inserted in the topography of the third needle, forming a 90° angle with the peridural needle. The Chiba needle enables the passage of the guidewire and subsequent dilatation until the 30F caliber for the passage of the Amplatz dilator, initiating the conventional procedure. COMMENTS: The technique described has been used at our service for 15 years and has the advantages of less morbidity, fewer complications and less use of the nephrostomy tube. The technique is also easy to learn and highly reproducible.

Bladder Dysfunction in an Obese Zucker Rat: The Role of TRPA1 Channels, Oxidative Stress, and Hydrogen Sulfide.

PURPOSE: This study investigates whether functionality and/or expression changes of transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) channels, oxidative stress, and hydrogen sulfide (H2S) are involved in the bladder dysfunction from an insulin-resistant obese Zucker rat (OZR). MATERIALS AND METHODS: Detrusor smooth muscle (DSM) samples from the OZR and their respective controls, a lean Zucker rat (LZR), were processed for immunohistochemistry for studying the expression of TRPA1 and TRPV1 and the H2S synthase cystathionine beta-synthase (CBS) and cysthathionine-γ-lyase (CSE). Isometric force recordings to assess the effects of TRPA1 agonists and antagonists on DSM contractility and measurement of oxidative stress and H2S production were also performed. RESULTS: Neuronal TRPA1 expression was increased in the OZR bladder. Electrical field stimulation- (EFS-) elicited contraction was reduced in the OZR bladder. In both LZR and OZR, TRPA1 activation failed to modify DSM basal tension but enhanced EFS contraction; this response is inhibited by the TRPA1 blockade. In the OZR bladder, reactive oxygen species, malondialdehyde, and protein carbonyl contents were increased and antioxidant enzyme activities (superoxide dismutase, catalase, GR, and GPx) were diminished. CSE expression and CSE-generated H2S production were also reduced in the OZR. Both TRPV1 and CBS expressions were not changed in the OZR. CONCLUSIONS: These results suggest that an increased expression and functionality of TRPA1, an augmented oxidative stress, and a downregulation of the CSE/H2S pathway are involved in the impairment of nerve-evoked DSM contraction from the OZR.

Mantle cell lymphoma of the cecum.

The authors have read with great interest the recently published article "Colon lymphomas: an analysis of our experience over the last 23 years" by Martín Domínguez V et al., a single center retrospective review of 29 patients diagnosed with colon lymphoma. The present report describes a case of mantle cell lymphoma (MCL) of the cecum that aims to improve the knowledge regarding this unusual clinical and endoscopic entity.

Intracellular replication of Streptococcus pneumoniae inside splenic macrophages serves as a reservoir for septicaemia.

Bacterial septicaemia is a major cause of mortality, but its pathogenesis remains poorly understood. In experimental pneumococcal murine intravenous infection, an initial reduction of bacteria in the blood is followed hours later by a fatal septicaemia. These events represent a population bottleneck driven by efficient clearance of pneumococci by splenic macrophages and neutrophils, but as we show in this study, accompanied by occasional intracellular replication of bacteria that are taken up by a subset of CD169+ splenic macrophages. In this model, proliferation of these sequestered bacteria provides a reservoir for dissemination of pneumococci into the bloodstream, as demonstrated by its prevention using an anti-CD169 monoclonal antibody treatment. Intracellular replication of pneumococci within CD169+ splenic macrophages was also observed in an ex vivo porcine spleen, where the microanatomy is comparable with humans. We also showed that macrolides, which effectively penetrate macrophages, prevented septicaemia, whereas beta-lactams, with inefficient intracellular penetration, failed to prevent dissemination to the blood. Our findings define a shift in our understanding of the pneumococcus from an exclusively extracellular pathogen to one with an intracellular phase. These findings open the door to the development of treatments that target this early, previously unrecognized intracellular phase of bacterial sepsis.

Aflibercept in diabetic macular edema refractory to previous bevacizumab: outcomes and predictors of success.

PURPOSE: To evaluate functional and anatomical outcomes after aflibercept in patients with diabetic macular edema (DME) with poor response to bevacizumab. METHODS: We retrospectively reviewed patients with DME recalcitrant to bevacizumab who were switched to aflibercept between January and December 2015. All patients had a minimal follow-up of three months before the conversion and underwent at least three injections of bevacizumab. Functional outcome consisted in best corrected visual acuity (VA). Anatomical outcomes were demonstrated through central macular thickness (CMT) measured by optical coherence tomography. RESULTS: Forty-nine eyes of 34 subjects were reviewed. Mean VA improved from 0.55 ± 0.32 logMAR to 0.46 ± 0.33 logMAR (p = 0.038). Mean CMT decreased from 473 ± 146 µm to 349 ± 85 µm (p < 0.001). Twelve eyes (24%) demonstrated absence of macular edema after aflibercept. Previous bevacizumab exposure did not correlate with different outcomes. The variation of VA in response to aflibercept was significantly superior in the group with poorer VA before the switch (mean variation of -0.097 ± 0.21 logMAR) when compared to eyes with VA < 0.4 logMAR (mean variation of +0.019 ± 0.090 logMAR; p = 0.036). The same scenario was verified for anatomical outcomes as eyes with poor vision before the switch (≥0.4 logMAR) achieved superior reduction in CMT in response to aflibercept (mean CMT variation of -157 ± 171 µm versus -49.5 ± 39.9 µm; p < 0.01). Pre-switch CMT was a predictor of CMT reduction after switching (B = -0.945; confidence interval 95% -1.1; -0.76; p < 0.001). CONCLUSIONS: Conversion to aflibercept for persistent DME resulted in functional and anatomical improvements and these outcomes were not influenced by previous bevacizumab exposure. Pre-switch CMT was a predictor of anatomical changes after aflibercept.

Paraesophageal hernia and gastric volvulus: an uncommon etiology of vomiting and upper gastrointestinal bleeding.

The authors report the case of a 75 year-old woman admitted to the emergency room with abdominal pain and coffee ground vomiting. Marked epigastric distension with tenderness and signs of severe dehydration were present. Upper GI endoscopy showed a black esophagus covered by a large amount of dark fluid, diffuse hyperaemia and superficial erosions. Marked distortion of gastric anatomy caused by stomach rotation and a large paraesophageal hernia was observed and the pylorus was not identified. Chest X-ray and CT scan confirmed the presence of an organoaxial gastric volvulus with antero superior rotation and incarceration of the gastric antrum, which was located above the diaphragm. Immediate surgery repaired the diaphragmatic hernia, obtained volvulus reduction and a Nissen fundoplication was performed to prevent recurrence. The patient was discharged without further complications. Acute gastric volvulus is a rare entity that may manifest with vomiting due to gastric outlet obstruction and gastrointestinal bleeding associated with mucosal ischemia and sloughing. Emergency surgery wass required to resolve symptoms and prevent complications.

Air pollution alters Staphylococcus aureus and Streptococcus pneumoniae biofilms, antibiotic tolerance and colonisation.

Air pollution is the world's largest single environmental health risk (WHO). Particulate matter such as black carbon is one of the main components of air pollution. The effects of particulate matter on human health are well established however the effects on bacteria, organisms central to ecosystems in humans and in the natural environment, are poorly understood. We report here for the first time that black carbon drastically changes the development of bacterial biofilms, key aspects of bacterial colonisation and survival. Our data show that exposure to black carbon induces structural, compositional and functional changes in the biofilms of both S. pneumoniae and S. aureus. Importantly, the tolerance of the biofilms to multiple antibiotics and proteolytic degradation is significantly affected. Additionally, our results show that black carbon impacts bacterial colonisation in vivo. In a mouse nasopharyngeal colonisation model, black carbon caused S. pneumoniae to spread from the nasopharynx to the lungs, which is essential for subsequent infection. Therefore our study highlights that air pollution has a significant effect on bacteria that has been largely overlooked. Consequently these findings have important implications concerning the impact of air pollution on human health and bacterial ecosystems worldwide.

Role of endogenous hydrogen sulfide in nerve-evoked relaxation of pig terminal bronchioles.

Hydrogen sulfide (H2S) is a gasotransmitter employed for intra- and inter-cellular communication in almost all organ systems. This study investigates the role of endogenous H2S in nerve-evoked relaxation of pig terminal bronchioles with 260 µm medium internal lumen diameter. High expression of the H2S synthesis enzyme cystathionine γ-lyase (CSE) in the bronchiolar muscle layer and strong CSE-immunoreactivity within nerve fibers distributed along smooth muscle bundles were observed. Further, endogenous H2S generated in bronchiolar membranes was reduced by CSE inhibition. In contrast, cystathionine ß-synthase expression, another H2S synthesis enzyme, however was not consistently detected in the bronchiolar smooth muscle layer. Electrical field stimulation (EFS) and the H2S donor P-(4-methoxyphenyl)-P-4-morpholinylphosphinodithioic acid (GYY4137) evoked smooth muscle relaxation. Inhibition of CSE, nitric oxide (NO) synthase, soluble guanylyl cyclase (sGC) and of ATP-dependent K+, transient receptor potential A1 (TRPA1) and transient receptor potential vanilloid 1 (TRPV1) channels reduced the EFS relaxation but failed to modify the GYY4137 response. Raising extracellular K+ concentration inhibited the GYY4137 relaxation. Large conductance Ca2+-activated K+ channel blockade reduced both EFS and GYY4137 responses. GYY4137 inhibited the contractions induced by histamine and reduced to a lesser extent the histamine-induced increases in intracellular [Ca2+]. These results suggest that relaxation induced by EFS in the pig terminal bronchioles partly involves the H2S/CSE pathway. H2S response is produced via NO/sGC-independent mechanisms involving K+ channels and intracellular Ca2+ desensitization-dependent pathways. Thus, based on our current results H2S donors might be useful as bronchodilator agents for the treatment of lung diseases with persistent airflow limitation, such as asthma and chronic obstructive lung disease.

Constitutively active PKA regulates neuronal acetylcholine release and contractility of guinea pig urinary bladder smooth muscle.

Autonomic and somatic motor neurons that innervate the urinary bladder and urethra control the highly coordinated functions of the lower urinary tract, the storage, and the emptying of urine. ACh is the primary excitatory neurotransmitter in the bladder. Here, we aimed to determine whether PKA regulates neuronal ACh release and related nerve-evoked detrusor smooth muscle (DSM) contractions in the guinea pig urinary bladder. Isometric DSM tension recordings were used to measure spontaneous phasic and electrical field stimulation (EFS)- and carbachol-induced DSM contractions with a combination of pharmacological tools. The colorimetric method was used to measure ACh released by the parasympathetic nerves in DSM isolated strips. The pharmacological inhibition of PKA with H-89 (10 µM) increased the spontaneous phasic contractions, whereas it attenuated the EFS-induced DSM contractions. Intriguingly, H-89 (10 µM) attenuated the (primary) cholinergic component, whereas it simultaneously increased the (secondary) purinergic component of the nerve-evoked contractions in DSM isolated strips. The acetylcholinesterase inhibitor, eserine (10 µM), increased EFS-induced DSM contractions, and the subsequent addition of H-89 attenuated the contractions. H-89 (10 µM) significantly increased DSM phasic contractions induced by the cholinergic agonist carbachol. The inhibition of PKA decreased the neuronal release of ACh in DSM tissues. This study revealed that PKA-mediated signaling pathways differentially regulate nerve-evoked and spontaneous phasic contractions of guinea pig DSM. Constitutively active PKA in the bladder nerves controls synaptic ACh release, thus regulating the nerve-evoked DSM contractions, whereas PKA in DSM cells controls the spontaneous phasic contractility.

BK channel regulation by phosphodiesterase type 1: a novel signaling pathway controlling human detrusor smooth muscle function.

Large-conductance Ca(2+)-activated K(+) (BK) channels are critical regulators of detrusor smooth muscle (DSM) function. We aimed to investigate phosphodiesterase type 1 (PDE1) interactions with BK channels in human DSM to determine the mechanism by which PDE1 regulates human urinary bladder physiology. A combined electrophysiological, functional, and pharmacological approach was applied using human DSM specimens obtained from open bladder surgeries. The perforated whole cell patch-clamp technique was used to record transient BK currents (TBKCs) and the cell membrane potential in freshly isolated human DSM cells in combination with the selective PDE1 inhibitor, 8-methoxymethyl-3-isobutyl-1-methylxanthine (8MM-IBMX). Isometric DSM tension recordings were used to measure spontaneous phasic and electrical field stimulation-induced contractions in human DSM isolated strips. Selective pharmacological inhibition of PDE1 with 8MM-IBMX (10 µM) increased TBKC activity in human DSM cells, which was abolished by subsequent inhibition of protein kinase A (PKA) with H-89 (10 µM). The stimulatory effect of 8MM-IBMX on TBKCs was reversed upon activation of muscarinic acetylcholine receptors with carbachol (1 µM). 8MM-IBMX (10 µM) hyperpolarized the DSM cell membrane potential, an effect blocked by PKA inhibition. 8MM-IBMX significantly decreased spontaneous phasic and nerve-evoked contractions of human DSM isolated strips. The results reveal a novel mechanism that pharmacological inhibition of PDE1 attenuates human DSM excitability and contractility by activating BK channels via a PKA-dependent mechanism. The data also suggest interactions between PDE1 and muscarinic signaling pathways in human DSM. Inhibition of PDE1 can be a novel therapeutic approach for the treatment of overactive bladder associated with detrusor overactivity.