Basal Tone Research Articles

Physician-Directed Duplex Augments Distal Arteriovenous Fistula Creation

Best practice guidelines for arteriovenous access creation have emphasized selecting distal sites and autogenous tissue before using proximal sites and synthetic shunts. Preoperative vein mapping is a useful tool to evaluate the optimal access location. However, the vein size is often underestimated secondary to patient hydration, room temperature, and basal vascular tone. Infraclavicular brachial plexus blocks (BPBs) are routinely performed for anesthesia but also have known vasodilatory effects. Although many surgeons use both techniques, most do not repeat vein mapping after performance of the BPB to reevaluate the targets after BPB-mediated vasodilation. Therefore, we evaluated whether the role of physician-directed vein mapping after BPBs resulted in more favorable fistula creations. All patients who had undergone primary ipsilateral access creation with physician-directed post-BPB duplex ultrasound from 2017 to 2018 were evaluated. Vein mapping was reviewed for “theoretical access location” using the criterion of >25-mm vessels. Fistula priority was analogous to the current indications, with an order of preference given to wrist radiocephalic, forearm radiocephalic, brachiocephalic, and brachiobasilic, followed by a prosthetic graft. Forty-three patients met the inclusion criteria. Physician-directed vein mapping after initiation of a regional BPB resulted in creation of a more distal access than expected in 62.8% of the patients. In 34.9%, the access site was at the predicted level, with a more proximal graft used in 2.3%. Furthermore, no differences in the maturation rates between the access sites placed distally to the predicted sites compared with those placed at the predicted site (0.65 vs 0.63; P = .38). The overall revision rate for distal fistulas was 22.2%, of which 83.3% were revised to the theoretical location, and 17.7% had required a proximal revision or creation of a graft. Physician-directed ultrasound after BPB is able to identify larger targets for access creation compared with preoperative vein mapping. Of those fistulas created distally, no difference was found in the maturation rates compared with those created at the theoretical location from vein mapping.

Cardioluminescence in Transgenic Zebrafish Larvae: A Calcium Imaging Tool to Study Drug Effects and Pathological Modeling.

Zebrafish embryos and larvae have emerged as an excellent model in cardiovascular research and are amenable to live imaging with genetically encoded biosensors to study cardiac cell behaviours, including calcium dynamics. To monitor calcium ion levels in three to five days post-fertilization larvae, we have used bioluminescence. We generated a transgenic line expressing GFP-aequorin in the heart, Tg(myl7:GA), and optimized a reconstitution protocol to boost aequorin luminescence. The analogue diacetyl h-coelenterazine enhanced light output and signal-to-noise ratio. With this cardioluminescence model, we imaged the time-averaged calcium levels and beat-to-beat calcium oscillations continuously for hours. As a proof-of-concept of the transgenic line, changes in ventricular calcium levels were observed by Bay K8644, an L-type calcium channel activator and with the blocker nifedipine. The β-adrenergic blocker propranolol decreased calcium levels, heart rate, stroke volume, and cardiac output, suggesting that larvae have a basal adrenergic tone. Zebrafish larvae treated with terfenadine for 24 h have been proposed as a model of heart failure. Tg(myl7:GA) larvae treated with terfenadine showed bradycardia, 2:1 atrioventricular block, decreased time-averaged ventricular calcium levels but increased calcium transient amplitude, and reduced cardiac output. As alterations of calcium signalling are involved in the pathogenesis of heart failure and arrhythmia, the GFP-aequorin transgenic line provides a powerful platform for understanding calcium dynamics.

Arachnoid membrane as a source of sphingosine-1-phosphate that regulates mouse middle cerebral artery tone

Growing evidence indicates that perivascular tissue is critical to modulate vessel function. We hypothesized that the arachnoid membrane surrounding middle cerebral artery (MCA) regulates its function via sphingosine-1-phosphate (S1P)-induced vasoconstriction. The MCA from 3- to 9-month-old male and female wild-type (Oncine France 1 and C57BL/6) mice and sphingosine kinase 2 knockout (SphK2-/-) mice in the C57BL/6 background was mounted in pressure myographs with and without arachnoid membrane. Raman microspectroscopy and imaging were used for in situ detection of S1P. The presence of arachnoid tissue was associated with reduced external and lumen MCA diameters, and with an increase in basal tone regardless of sex and strain background. Strong S1P-positive signals were detected in the arachnoid surrounding the MCA wall in both mice models, as well as in a human post-mortem specimen. Selective S1P receptor 3 antagonist TY 52156 markedly reduced both MCA vasoconstriction induced by exogenous S1P and arachnoid-dependent basal tone increase. Compared to 3-month-old mice, the arachnoid-mediated contractile influence persisted in 9-month-old mice despite a decline in arachnoid S1P deposits. Genetic deletion of SphK2 decreased arachnoid S1P content and vasoconstriction. This is the first experimental evidence that arachnoid membrane regulates the MCA tone mediated by S1P.

Abstract P283: CCL5 Acts As A Vasoconstrictor On Penetrating Arterioles In The Cerebral Circulation By Enhancing 20-HETE Activity

20-HETE is synthesized from arachidonic acid by cytochrome P450 (CYP) enzymes 4A and 4F. Inactivating mutations in the CYP enzymes that produce 20-HETE are associated with hypertension and stroke in man. We previously revealed that inactivating variants of CYP4A/F enzymes are associated with dementia in the Atherosclerosis Risk in Communities Neurocognitive Study (ARIC-NS) population. 20-HETE is involved with sodium regulation in the kidney and is a powerful vasoconstrictor. It was recently discovered that CCL5 and 20-HETE share the same receptor, GPR75. We previously found that 20-HETE constricts and augments the myogenic response (MR) of the middle cerebral artery (MCA) and renal afferent arteriole. However, whether CCL5 has any effect on penetrating arterioles (PAs) and interacts with 20-HETE is unknown. We found that GPR75 is expressed in PAs and pericytes in the brain. CYP4A is also expressed in pericytes and is inversely proportional to levels of GPR75 in the brain. In the present study, we found that 20-HETE contributes to the basal myogenic tone of PAs in SD rats. Administration of HET0016, a 20-HETE synthesis inhibitor, dilated the PA by 34 ± 3% (n = 6) under 10 mmHg perfusion pressure. Administration of WIT003, a 20-HETE agonist, constricted the vessel by 23 ± 4% (n = 6) under the same perfusion pressure. We found that CCL5 also reduced PA diameter by 20 ± 4% (n = 7) in SD rats under 10 mmHg perfusion pressure. Moreover, we compared the response to CCL5 in SS rats that are 20-HETE deficient and SS.CYP4A1 transgenic rats in which 20-HETE production is restored. PAs isolated from SS rats treated with 0.1 nM CCL5 constricted by 9 ± 5% (n = 6) while those treated with 10 nM constricted by 12 ± 3% (n = 6). CCL5 had a greater response in PAs from the SS.CYP4A1 strain, and the diameter of the PAs constricted by 14 ± 2% (n = 5) and 24 ± 5% (n = 5) in response to 0.1 and 10 nM CCL5, respectively. These results demonstrate that CCL5 has a direct effect on PAs similar to 20-HETE that acts via the GPR75 receptor. However, further study is needed to determine how CCL5 and 20-HETE interact to promote vasoconstriction. These studies would help further understand the involvement of 20-HETE in disease and potentially identify novel drug targets.

Abstract MP17: Neuropilin-1 Is A Novel Regulator Of Vascular Tone And Blood Pressure

Introduction: Neuropilin-1 (NRP1) is a transmembrane receptor present in vascular smooth muscle cells (VSMC) that mediates the inhibition of Rho signaling by binding the Class 3 Semaphorin (SEMA) ligand SEMA3A. Hypothesis: We hypothesize that loss of NRP1 in VSMC mitigates SEMA3A-induced Rho inhibition, thereby increasing vascular tone and blood pressure in vivo . Methods: Male and female adult mice (8-12 weeks) with inducible, smooth muscle cell-specific deletion of NRP1 (SM22a-Cre ERT2 X Nrp1 flox/flox ) were examined. Following recombination using 4-hydroxy tamoxifen (SM- NRP1 KO), systolic blood pressure (SBP) was measured using a tail cuff and compared to age- and sex-matched mice that did not receive tamoxifen (control). Aortic vascular reactivity and expression of key proteins in the Rho signaling cascade were measured using ex vivo tension myography and western blotting, respectively. Results: SBP was significantly increased in SM- NRP1 KO mice following recombination compared to control mice (SBP: 136.5 ± 10.9 vs 112.9 ± 5.6 mmHg; p=0.0006). Contractile responses in aortas of SM- NRP1 KO mice to phenylephrine (p=0.025), KCl (p=0.012), and the thromboxane agonist U44619 (p=0.019) were significantly enhanced compared to controls. Expression of total myosin light chain and LIMK-2 proteins were increased in SM- NRP1 KO compared to control aortas. In vitro , treatment of murine primary VSMC expressing NRP1 with SEMA3A decreased angiotensin II-induced Rho-GTP activation. Additionally, control and SM- NRP1 KO mice (starting at 2 weeks post-recombination) were administered angiotensin II (490 ng/kg/day) for 4 weeks. While there was no significant difference in SBP at weeks 1 and 2, SM- NRP1 KO mice had significantly lower SBP at weeks 3 and 4 following angiotensin II infusion compared to controls (Week 4 SBP: 150 ± 1.4 vs 130.5 ± 2.5 mmHg; p=0.02), suggesting a low ejection fraction and cardiac dysfunction in these mice. In support of this observation, mRNA expression of atrial natriuretic peptide was increased in hearts of angiotensin II-infused SM- NRP1 KO mice. Conclusion: Our data suggest that VSMC NRP1 regulates basal tone and blood pressure, and that loss of NRP1 causes hypertension and exacerbates cardiac dysfunction.

Ischemia Changes the Contribution of KATP-Channels to Basal Tone and Dilation of Rat Pial Arteries

Ischemia Changes the Contribution of KATP-Channels to Basal Tone and Dilation of Rat Pial Arteries

Heart rate variability and heart rate turbulence in patients with vasovagal syncope.

Aim The autonomic nervous system plays an important role in the pathogenesis of vasovagal syncope, but studies on the effect of basal autonomic tone have found confusing results. The aim of this study was to investigate the effect of basal autonomic functions, as assessed by heart rate variability (HRV) and heart rate turbulence (HRT), in patients with vasovagal syncope.Material and methods Patients who underwent head-up tilt test (HUTT) due to unexplained syncope and who had a 24 hr Holter ECG recording in the same period were retrospectively analyzed. Patients with diabetes, a history of myocardial infarction, heart failure, orthostatic hypotension, atrial fibrillation, or use of vasoactive drugs, such as beta blockers, were excluded from the study. 161 patients who met these criteria were included in the study. Time domain HRV parameters from Holter ECG recordings and HRT parameters from patients with sufficient number of ventricular premature contractions were measured.Results The age of the patients varied from 16 to 75 yrs (mean: 44.8±18.5 yrs). HUTT results of 60 (37.2 %) patients were evaluated as positive. There were no significant differences in the basal demographic, clinical, or laboratory findings of the tilt-positive and tilt-negative patient groups. Likewise, there were no significant differences between the time domain HRV parameters and HRT parameters of both groups.Conclusion HRV and HRT parameters reflecting basal autonomic function were not different between HUTT positive and HUTT negative patient groups. These findings suggest that basal autonomic functions have no effect on vasovagal syncope pathogenesis.

Age-related differences of γ-aminobutyric acid (GABA)ergic transmission in human colonic smooth muscle.

BackgroundEnteric neurons undergo to functional changes during aging. We investigated the possible age‐associated differences in enteric γ‐aminobutyric acid (GABA)ergic transmission evaluating function and distribution of GABAergic receptors in human colon.MethodsMechanical responses to GABA and GABA receptor agonists on slow phasic contractions were examined in vitro as changes in isometric tension in colonic muscle strips from young (<65 years old) and aged patients (>65 years old). GABAergic receptor expression was assessed by quantitative RT‐PCR.Key ResultsIn both preparations GABA induced an excitatory effect, consisting in an increase in the basal tone, antagonized by the GABAA receptor antagonist, bicuculline, and potentiated by phaclofen, GABAB receptor antagonist.Tetrodotoxin (TTX) and atropine‐sensitive contractile responses to GABA and GABAA receptor agonist, muscimol, were more pronounced in old compared to young subjects. Baclofen, GABAB receptor agonist, induced a TTX‐sensitive reduction of the amplitude of the spontaneous. Nω–nitro‐l‐arginine methyl ester (L‐NAME), nitric oxide (NO) synthase inhibitor abolished the inhibitory responses in old preparations, but a residual responses persisted in young preparations, which in turn was abolished by suramin, purinergic receptor antagonist. α3–GABAA receptor subunit expression tends to change in an age‐dependent manner.Conclusions and inferencesOur results reveal age‐related differences in GABAergic transmission in human colon. At all the age tested GABA regulates muscular contractility modulating the activity of the intrinsic neurons. Activation of GABAA receptor, through acetylcholine release, induces contraction, which increases in amplitude with age. GABAB receptor activation leads to neural release of NO and purines, being a loss of purinergic‐component in aged group.

Study of the constrictor properties of the neobladder under the influence of new chemical compounds in vivo experiment

The orthotopic ileocystoplasty is the most preferred urine derivation method for patients after removal of the bladder. The best quality of life, self-esteem, cosmetic appearance of one’s own body, and the possibility of physiological urination characterize the artificial bladder as the “gold standard” of urine derivation. There are a greater number of postoperative complications associated with the functionality of the neobladder, considering the complexity of orthotopic derivation of urine. The autocaturation is required in some patients day or night urinary incontinence, and in the other group, periodic urinary retention. Urinary retention is more common in women up to 43 % compared with 20 % in men, who need intermittent catheterization.&#x0D; The aim of investigation was to evaluate the possibility of pharmacological correction of contractile activity of mini-pigs neobladder in vivo, under the influence of new chemical compounds and the anticholinesterase drug nivalin.&#x0D; Materials and methods Experimental studies were performed on 21 female mini-pigs, with an average age of 6 to 14 months. The duration of the experiment was 12 months. Performing of enterocystometry in experimental animals is possible only under anesthesia and, accordingly, only part of the urodynamic parameters can be obtained.&#x0D; Results. The obtained results of in vivo experimental studies demonstrated the constrictor activity of the studied new chemical compounds. Compound I in comparison with the control group statistically significantly increased basal tone by 62.09 %, the amplitude of contractions by 37.34 %, and the duration of contractions by 35.71 %. The new chemical compound II, in comparison with the control group, showed less pronounced results of constrictor activity, so the basal tone statistically significantly increased by 38.86 %, and the amplitude of contractions by 15.0 %.&#x0D; Conclusion. Thus, under the influence of new chemical compounds in the conditions of an experimental artificial bladder, constrictor properties are proved, which require further research and study. In turn, cholinomimetic nivalin has prospects for use in urology, in particular in patients with hypoactive forms of artificial and neurogenic bladder.

Notice of Retraction: Acharya P, Chen J-J, Correia MA (2010) Hepatic heme-regulated inhibitor (HRI) eukaryotic initiation factor 2α kinase: a protagonist of heme-mediated translational control of CYP2B enzymes and a modulator of basal endoplasmic reticulum stress tone. Mol Pharmacol 77(4):575-592; doi:10.1124/mol.109.061259.

At the request of the corresponding author and investigator at the University of California, San Francisco, and in consultation with Molecular Pharmacology , this paper has been retracted. It was found there was improper manipulation, reuse, and analyses represented by Figs. 1A, 1B, 1D, 2C, 3A, 3B,

Caffeic Acid Phenethyl Ester (CAPE), Active Phenolic Compound of Propolis Attenuates Endothelin, Prostaglandin F2α and U46619 Elicited Contractions of Isolated Human Umbilical Artery

Background: Propolis is a product of honeybees that contains a variety of different compounds, including caffeic acid phenethyl ester (CAPE). Propolis and its bioactive compounds are widely used in folk medicine and as a dietary supplement. Previously it has been shown that CAPE has antioxidant, anti-inflammatory, antimicrobial, antiviral, immunomodulatory, and anticancer activity. Objective: This in vitro study was designed to investigate the vasoactive effects of CAPE on quiescent and precontracted human umbilical arteries. Methods: Umbilical artery strips were suspended in aerated organ baths containing a buffer solution. The strips were randomly allocated to study groups (n=8). Via a transducer and computer, changes in isometric tension were recorded. The effects of cumulative CAPE (10-8-10-4M) on basal tone of the artery, and in different groups of strips, the vasodilatory effect of cumulative CAPE on the constriction elicited by endothelin (ET-1), prostaglandin F2α (PGF2α) and U46619, and the effect of incubation with NO synthase inhibitor L-NAME, were recorded. Conclusion: CAPE elicits concentration-dependent relaxation on precontracted human umbilical artery strips depending on the constrictor agent. NO plays significant role in CAPE’s vasorelaxant effect.

Differential effects of β-methylphenylethylamine and octopamine on contractile parameters of the rat gastrointestinal tract

This study tested the effects of β-methylphenylethylamine (β-MPEA) and octopamine on contractile parameters of the gastrointestinal tract in rats. We hypothesized that some of their effects result from interactions with trace amine (TA)-associated receptors or serotoninergic 5-hydroxytryptamine (5-HT) receptors. β-MPEA-induced contractions in rat gastric fundus strips under resting tonus conditions, but induced relaxation in preparations that were previously contracted with carbachol. Octopamine relaxed gastric fundus strips maintained at resting tonus or contracted with carbachol. The contractile effect of β-MPEA was reduced by cyproheptadine and methiothepin, antagonists of excitatory 5-HT receptors. The relaxing effect of β-MPEA on gastric fundus was insensitive to pretreatment with N-(3-ethoxyphenyl)-4-(1-pyrrolidinyl)-3-(trifluoromethyl)benzamide (EPPTB) and tropisetron, antagonists of TA1 and 5-HT4 receptors, respectively. Both EPPTB and tropisetron inhibited the relaxant effects of octopamine on carbachol-contracted preparations. Contrarily, EPPTB did not reduce the relaxant effects of RO5263397 (TA1 agonist) or zacopride (5-HT4 agonist). Octopamine, but not β-MPEA, delayed the gastrointestinal transit of a liquid test meal in awaken rats. In isolated preparations of the small intestine under resting conditions, β-MPEA did not alter the basal tonus, but octopamine relaxed it. Intestinal preparations previously contracted with carbachol relaxed after the addition of octopamine and decreased the magnitude of their spontaneous rhythmic contractions in a tropisetron-dependent manner. Thus, β-MPEA and octopamine exerted pharmacological actions on the rat gastrointestinal tract. The excitatory effects of β-MPEA involved 5-HT receptors. Octopamine inhibited the rat gut contractility through the likely involvement of 5-HT4 and TA receptors. Overall, octopamine effectively inhibited rat gastrointestinal transit.

MO640LASER DOPPLER FLOWMETRY IN STUDYING OF MICROVASCULAR DISORDERS IN PATIENTS WITH DIABETIC NEPHROPATHY

Abstract Background and Aims According to various research, vascular complications of type 2 diabetes mellitus are the main reason for patients' mortality. The most specific one, observed in patients with diabetes only, is diabetic microangiopathy, especially diabetic nephropathy. This complication accounts for more than 20% of cases of chronic kidney disease. So, the development of non-invasive methods for the diagnosis of vascular complications of type 2 diabetes mellitus is critically important. There is a perspective method for this problem - Laser Doppler flowmetry (LDF). It is currently used in the diagnosis of diabetic microangiopathy, but the limiting factor is the lack of a unified algorithmic approach to the data interpretation. This work aimed to analyze changes in the amplitude indicators of the low-frequency part of the LDF signal spectrum in patients with chronic kidney disease and type 2 diabetes and to identify their correlations with the glomerular filtration rate. Method The study included 42 patients (20 men and 22 women) with type 2 diabetes mellitus chronic kidney disease (stage C3-C4). The age of patients was 58-77 years (66 years on average). The duration of diabetes was more than 5 years (on average 7 years). All patients had diabetic nephropathy with a decrease in glomerular filtration rate, chronic kidney disease stage C3-C4. Laser Doppler flowmetry was done using the "LAZMA MC-1" system ("Lazma", Russia). Each patient had a 10-minute LDF registration. The sensor was placed on the skin of the rear of the foot. After recording the LDF curve, the special software has calculated amplitudes of endothelial, myogenic, neurogenic, respiratory, and pulse flux motions. Then we assessed the amplitude contribution of every frequency range to the total power of the local flux motion region. The next step was a correlation analysis with the estimated glomerular filtration rate. For statistical analysis, we used the GraphPad Prism 8 (GraphPad Software, USA). Results All examined patients had amplitude peaks in the neurogenic, myogenic, respiratory, and pulse ranges. There were no significant correlations between the glomerular filtration rate and the amplitudes of myogenic and neurogenic flux motions (p&amp;gt;0.05) (Fig. 1). However, there was a significant positive correlation between the contribution of myogenic flux motions to the low-frequency range and glomerular filtration rate (p&amp;lt;0.01), and a negative one – for the contribution of neurogenic flux motions (p&amp;lt;0.01) (Fig. 2). In six observations there was a tendency to a decrease in the contribution of endothelial flux motions as the glomerular filtration rate decreased. Conclusion The results of this study showed that laser Doppler flowmetry has the potential to diagnosis the nature of the dysfunction of individual microcirculation modulation mechanisms. In patients with chronic kidney disease of the C3-C4 stage decreasing the glomerular filtration rate correlated with decreasing the contribution of myogenic flux motions and increasing the contribution of neurogenic flux motions to the total power of the low-frequency part of the LDF signal amplitude-frequency spectrum. These changes can be explained within the framework of the existing understanding of the pathogenesis of diabetic microangiopathy, namely, damage to the smooth muscle layer of the wall of arterioles and venules with damage to myocyte pacemakers and changes of basal vascular tone pattern. It causes an increase in the role of neurogenic modulation of the micro-vascular bloodstream. These data can be an additional argument in favor of the further development of improving laser Doppler flowmetry using for the tasks of early (preclinical) non-invasive diagnosis of microvascular disorders in patients with type 2 diabetes mellitus, as well as for monitoring the effectiveness of the therapy. The reported study was funded by RFBR, project number 19-315-90080.

On the Three-Dimensional Correlation Between Myofibroblast Shape and Contraction.

Myofibroblasts are responsible for wound healing and tissue repair across all organ systems. In periods of growth and disease, myofibroblasts can undergo a phenotypic transition characterized by an increase in extracellular matrix (ECM) deposition rate, changes in various protein expression (e.g., alpha-smooth muscle actin (αSMA)), and elevated contractility. Cell shape is known to correlate closely with stress-fiber geometry and function and is thus a critical feature of cell biophysical state. However, the relationship between myofibroblast shape and contraction is complex, even as well in regards to steady-state contractile level (basal tonus). At present, the relationship between myofibroblast shape and basal tonus in three-dimensional (3D) environments is poorly understood. Herein, we utilize the aortic valve interstitial cell (AVIC) as a representative myofibroblast to investigate the relationship between basal tonus and overall cell shape. AVICs were embedded within 3D poly(ethylene glycol) (PEG) hydrogels containing degradable peptide crosslinkers, adhesive peptide sequences, and submicron fluorescent microspheres to track the local displacement field. We then developed a methodology to evaluate the correlation between overall AVIC shape and basal tonus induced contraction. We computed a volume averaged stretch tensor ⟨U⟩ for the volume occupied by the AVIC, which had three distinct eigenvalues (λ1,2,3=1.08,0.99, and 0.89), suggesting that AVIC shape is a result of anisotropic contraction. Furthermore, the direction of maximum contraction correlated closely with the longest axis of a bounding ellipsoid enclosing the AVIC. As gel-imbedded AVICs are known to be in a stable state by 3 days of incubation used herein, this finding suggests that the overall quiescent AVIC shape is driven by the underlying stress-fiber directional structure and potentially contraction level.

Finger constrictor and thermoperceptual responsiveness to localised cooling following 5 weeks of intermittent regional exposures to moderately augmented transmural vascular pressure

PurposeTo examine the effects of prolonged intermittent exposures to moderately increased transmural pressure on finger vasoreactivity and thermoperception to localised cooling. MethodsEleven men completed a 5-week regimen (3 sessions·week−1; 55 min·session−1), during which the vasculature in one arm (EXP) was exposed intermittently (10-min exposure: 5-min pause) to increased transmural pressure (from +65 mmHg week-1 to +105 mmHg week-5). Before and after the regimen, finger cutaneous vascular conductance (CVC), temperature (Tavg), and thermoperception (thermal sensation, discomfort and pain) were monitored during a 30-min hand cold (8 °C water) provocation trial. The responses of the non-trained hand were examined during an additional cold trial. ResultsAfter the regimen, baseline finger CVC and Tavg were higher in both hands (p ≤ 0.01). During cooling, neither finger CVC nor Tavg were modified (p > 0.05). Yet the magnitude of the cold-induced drop of CVC was augmented in both hands, and to a similar extent (p ≤ 0.02). The regimen alleviated thermal pain in both hands (p ≤ 0.02); the sensation of coldness and thermal discomfort were attenuated mainly in the EXP hand (p = 0.02). ConclusionsPresent findings indicate that iterative local exposures to augmented intravascular pressure do not alter finger vasoreactivity to localised cooling. The pressure training, however, might impair finger basal vasomotor tone, and aggravate the magnitude of constrictor responsiveness to cooling. The pressure training also elicits thermoperceptual desensitisation to noxious thermal stimulus. To large extent, these vascular and perceptual adjustments seem to be transferred to the cutaneous vasculature of the non-trained limb.

Endogenous Endothelin‐1 Contributes to Enhanced Basal Pulmonary Arterial Tone Following Chronic Hypoxia: Role of Mitochondria‐Derived Reactive Oxygen Species

Previous studies from our laboratory demonstrate the importance of mitochondria-derived reactive oxygen species (mitoROS) in the development of pulmonary hypertension after chronic hypoxia (CH) exposure. Furthermore, we have identified a major role for the endothelium to augment mitoROS-dependent pulmonary vascular tone following CH. However, the mechanism of this response remains unclear. We hypothesized that the endogenous vasoconstrictor peptide, endothelin-1 (ET-1), facilitates mitoROS-dependent increases in basal tone after CH exposure. To test our hypothesis, we measured basal tone by videomicroscopy in isolated, pressurized, small pulmonary arteries [~150 μm inner diameter (i.d.)] from normoxic and CH (4 wk, 0.5 atm) rats with and without the ETA receptor and ETB receptor inhibitors, BQ123 and BQ788 (10 μM each), respectively. Parallel protocols were conducted in the presence of the mitochondria-targeted antioxidant, MitoQ (1 μM). All experiments were performed in the presence of the nitric oxide (NO) synthase inhibitor, Nω-nitro-L-arginine (300 μM), to limit the influence of endogenous NO. Pulmonary arterial tone was calculated as the percent difference in i.d. between Ca2+-free and Ca2+-containing conditions. To evaluate the contribution of mitoROS to ET-1-mediated vasoconstriction, we further assessed vasoconstrictor responses (percentage of baseline i.d.) to serial concentrations of exogenous ET-1 (10-11 to 10-7 M) in the presence or absence of MitoQ in endothelium-disrupted pulmonary arteries from each group. Additional experiments employed confocal microscopy to measure ET-1 (1 nM)-induced mitoROS production in primary cultures of pulmonary arterial smooth muscle cells (PASMCs) from control and CH rats using the fluorescent mitochondrial superoxide indicator MitoSOX (10 μM). Antimycin A (10 µM) treatment, which stimulates mitoROS production from complex III of electron transport chain, was used as a positive control. Both MitoQ and the combined administration of ETA and ETB receptor blockers abolished the effect of CH to increase basal tone (P<0.05). Arteries from CH rats also exhibited greater constriction to exogenous ET-1 (at 1 nM) compared to vessels from normoxic controls, which was prevented by MitoQ (P<0.05). Consistent with these findings, application of ET-1 significantly increased mitoROS production in PASMCs from CH rats (P<0.05), but not control animals. Application of antimycin A led to mitoROS generation in both control and CH groups (P<0.05). We conclude that endogenous ET-1 promotes pulmonary arterial constriction through mitoROS signaling in PASMCs following CH. These findings advance our understanding of the mechanistic basis for the vasoconstrictor component of CH-induced pulmonary hypertension.

Bi‐directional cAMP signaling crosstalk between dopamine D2 receptors and the constitutively active orphan receptor GPR52

G protein-coupled receptors (GPCRs) are valuable drug targets with ~35% of FDA approved medicines targeting these receptors. However, nearly 120 GPCRs remain orphan receptors, whose endogenous ligands, G protein signaling, and therapeutic potential are unknown. Dopamine D2 receptor (D2R) antagonists are effective antipsychotics while D2R agonists correct motor dysfunction in Parkinson's disease. The novel human orphan receptor GPR52 was recently identified and determined to activate cAMP signaling cascades. Notably, GPR52 is primarily co-expressed in the striatum of the brain with dopamine D2 receptors. Here we elucidate G protein signaling mechanisms responsible for both constitutive and agonist-induced GPR52 cAMP signaling and examine signaling crosstalk between GPR52 and the D2R. Expression of human GPR52 in wildtype HEK293 cells profoundly elevated basal cAMP levels by over 100 fold (Glosensor assay), indicating high constitutive receptor activity. CRISPR/Cas9 stable knockout of Gs/olf G proteins in HEK293 cells eliminated the GPR52 constitutive activity. Subsequent addback of Gs to Gs/olf knockout cells rescued GPR52 constitutive activity to near wildtype levels of basal cAMP, while addback of Golf did not significantly elevate the constitutive activity. However, when treated with novel GPR52 agonist PW0787, both Gs and Golf showed strong agonist-induced responses with unchanged potency. These findings indicate high constitutive activity of GPR52 is mediated via Gs signaling, while agonist-induced GPR52 signaling occurs via either Gs or Golf. We hypothesized the high GPR52 constitutive signaling may set the basal tone of cAMP in cells to afford more robust effects from adenylyl cyclase inhibition by Gi/o-coupled receptors, such as the D2R. Expression of D2R alone in HEK293 cells produced low basal cAMP levels (~4000 light counts/sec, Glosensor assay), and treatments with agonist quinpirole and antagonist haloperidol both yielded minimal changes to cAMP levels (~1000-2000 light counts/sec). Notably, co-expression of GPR52 and D2R substantially increased basal cAMP levels (~500,000 light counts/sec). This elevated basal cAMP also allowed for larger windows of agonism and inverse agonism by D2R ligands quinpirole (decrease of ~500,000 light counts/sec) and haloperidol (increase of ~150,000 light counts/sec). When the cells were treated concurrently with the GPR52 agonist PW0787 to further elevate basal cAMP, the efficacies of quinpirole (decrease of ~1,500,000 light counts/sec) and haloperidol (increase of ~500,000 light counts/sec) grew even more significantly. These studies indicate that striatal orphan receptor GPR52 may set the tone of basal cAMP levels through constitutive activity to allow more dynamic changes in cAMP signaling by the D2R. These findings also suggest potential for GPR52 as a drug target by modulating D2R cAMP signaling in the striatum, with therapeutic possibilities for psychosis, Parkinson's disease, and substance use disorders.

Chlorogenic Acid Relaxes Rat Ileal Smooth Muscles by Mechanisms Independent of Rho Kinase Mediated Effects on Myosin Light Chain Phosphorylation

Chlorogenic acid (CGA) is a polyphenolic compound found in fruits and vegetables and generally known to have antioxidant, anti-inflammatory and antithrombotic effects. Studies have shown that CGA induces nitric oxide-sGC-PKG pathway to cause relaxation of the vascular smooth muscles. But the effects of CGA on ileal smooth muscles are still unknown. In our previous experiments, we have shown that CGA increases basal tone with no effect on relaxation of rat ileal smooth muscles when they are depolarized by 140 mM potassium kreb's buffer. The present study is designed to evaluate if CGA acts through the rho-kinase mediated pathway in relaxing rat ileal smooth muscles when challenged with acetylcholine. Effects of CGA (10 μM) on basal tension and maximum contractile tension induced by acetylcholine (0.5μM) in isolated rat ileal rings (3mm) were determined using tissue bath apparatus. CGA effects on myosin light chain phosphorylation at maximum contractile tension in response to acetylcholine (0.5 μM) were also measured by freezing the ileal rings and extracting the protein and running Western blots. The role of rho-kinase in regulating smooth muscle relaxation is tested by incubating ileal rings with rho-kinase inhibitor Y-27632 (1 μM) and measuring the basal tension, maximum contractile tension and myosin light chain phosphorylation as described above. The results show that basal tension increases from 100% (control group) to 130.8 ± 19.0% and 121.7 ± 30.6 % when treated with CGA alone and the combination of CGA and Y-27632 respectively. The peak contractile tension in response acetylcholine changes from 100% (control group) to 50.4 ± 12.13 % and 55 ± 4.05 % when treated with CGA alone and the combination of CGA and Y-27632 respectively. The results from Western blots show that myosin light chain phosphorylation didn't change significantly between CGA (41.8 ± 5.5%) and the combination of CGA and Y-27632 (37.7 ± 3.7%) treatments. These results suggest that CGA alone and in combination with rho-kinase inhibitor is involved in increasing the basal tone of ileal smooth muscles when compared with the control group. In contrast, CGA significantly decreased the acetylcholine induced peak contractile tension both in presence and absence of rho-kinase inhibitor with out any corresponding changes in myosin light chain phosphorylation. Therefore, we conclude that CGA relaxes ileal smooth muscles by mechanisms independent of rho-kinase mediated effects on myosin light chain phosphorylation. Further studies on effects of CGA on activity of soluble guanylate cyclase and phosphodiesterase activity may provide some insights on mechanism of action of CGA on smooth muscles.

Exercise Training Rescues Impaired H 2 O 2 ‐Mediated Vasodilation in Collateral‐Dependent Coronary Arterioles Through Enhanced K + Channel Activation

The objective of this study was to determine downstream cellular signaling pathways by which exercise training promotes H2O2-mediated dilation in arterioles distal to chronic coronary artery occlusion. We tested the hypothesis that exercise training would correct impaired H2O2-mediated dilation in collateral-dependent arterioles through enhanced coupling of H2O2 with voltage-gated K+ (Kv) and large-conductance, calcium-dependent K+ (BKCa) channels. Yucatan miniature swine were surgically instrumented with an Ameroid constrictor around the proximal left circumflex coronary artery, which gradually induced occlusion and created a collateral-dependent vascular bed. Arterioles from the left anterior descending artery region served as nonoccluded control vessels. Pigs were separated into two groups: one that underwent a progressive exercise training program (treadmill run; 5 days/week for 14 weeks) and another that remained sedentary and was limited to normal pen activity. Collateral-dependent arterioles from sedentary pigs were significantly less sensitive to H2O2-induced dilation compared with nonoccluded arterioles (EC50: -4.55 ± 0.15 vs. -4.91 ± 0.15 log M, respectively; P<0.05), whereas this difference was eliminated after completion of the exercise training regimen when collateral-dependent and nonoccluded arterioles responded similarly to H2O2 (EC50: -4.86 ± 0.16 vs. -4.95 ± 0.18 log M, respectively). H2O2-mediated dilation in nonoccluded or collateral-dependent arterioles from sedentary animals was not altered by inhibitors of Kv (4-aminopyridine; 1 mM) or BKCa (iberiotoxin; 100 nM) channels. In contrast, after exercise training, Kv channels contributed significantly to dilation in nonoccluded arterioles and both Kv and BKCa channels played a significant role in dilation of collateral-dependent arterioles. Interestingly, the contribution of Kv and BKCa channels to basal tone was not altered by occlusion or exercise training, suggesting similar availability of these channels in nonoccluded and collateral-dependent arterioles of sedentary and exercise-trained pigs. Immunoblot analysis was performed to assess levels of PKG dimerization, a potential mediator between H2O2 and K+ channels. These studies revealed that H2O2 treatment significantly increased PKG dimerization in all artery treatment groups, and this effect was significantly greater in nonoccluded compared with collateral-dependent arteries from sedentary pigs, but not different after exercise training. Taken together, our studies suggest that impaired H2O2-mediated vasodilation may be in part attributable to attenuated PKG dimerization in collateral-dependent arterioles of sedentary pigs. However, while exercise training appeared to correct impaired H2O2-mediated dilation via enhanced coupling to K+ channel activation, exercise-induced changes in PKG dimerization did not appear to play a role.

Impaired Colonic Contractility and Intestinal Permeability in Symptomatic Uncomplicated Diverticular Disease

Background/AimsImpaired intestinal motility seems to play a crucial role in symptomatic uncomplicated diverticular disease (SUDD), although the mechanism is not clear. The aim of the present study is to explore the contractility patterns of colonic smooth muscle strips (MS) and smooth muscle cells (SMCs) and to assess mucosal integrity in SUDD patients.MethodsMS or SMCs were isolated from specimens of human distal colon of 18 patients undergoing surgery for non-obstructive colonic cancer, among them 9 with SUDD. Spontaneous phasic contractions on strips and morpho-functional parameters on cells were evaluated in basal conditions and in response to acetylcholine (ACh). Mucosal integrity of SUDD colonic biopsies was evaluated by the Ussing Chamber system. Immunohistochemical staining for tight junction protein complex and for Toll-like receptor 4 (TLR4) was performed.ResultsColonic MS of SUDD group showed a significant reduced basal tone and ACh-elicited contraction, compared to the control group (9.5 g and 47.0% in the SUDD group; 14.16 g and 69.0% in the control group; P < 0.05). SMCs of SUDD group showed a maximal contractile response to ACh significantly reduced compared to control group (8.8% vs 16.5%, P < 0.05). SUDD patients displayed lower transepithelial electrical resistance and increased paracellular permeability compared to control group. Immunohistochemical expression of TLR4 was not different in both groups, while tight junction protein complex expression was lower in SUDD patients compared to control group patients.ConclusionIt could be hypothesized that in SUDD, in absence of severe inflammation, an increased intestinal mucosal permeability is related to altered colonic motility probably responsible for symptoms genesis.