Arteriolar Cells Research Articles

PAR1-mediated Non-periodical Synchronized Calcium Oscillations in Human Mesangial Cells.

Mesangial cells offer structural support to the glomerular tuft and regulate glomerular capillary flow through their contractile capabilities. These cells undergo phenotypic changes, such as proliferation and mesangial expansion, resulting in abnormal glomerular tuft formation and reduced capillary loops. Such adaptation to the changing environment is commonly associated with various glomerular diseases, including diabetic nephropathy and glomerulonephritis. Thrombin-induced mesangial remodeling was found in diabetic patients, and expression of the corresponding protease-activated receptors (PARs) in the renal mesangium was reported. However, the functional PAR-mediated signaling in mesangial cells was not examined. This study investigated protease-activated mechanisms regulating mesangial cell calcium waves that may play an essential role in the mesangial proliferation or constriction of the arteriolar cells. Our results indicate that coagulation proteases such as thrombin induce synchronized oscillations in cytoplasmic Ca2+ concentration of mesangial cells. The oscillations required PAR1 G-protein coupled receptors-related activation, but not a PAR4, and were further mediated presumably through store-operated calcium entry and transient receptor potential canonical 3 (TRPC3) channel activity. Understanding thrombin signaling pathways and their relation to mesangial cells, contractile or synthetic (proliferative) phenotype may play a role in the development of chronic kidney disease and requires further investigation.

#6062 EFFECT OF KIDNEY TRANSPLANTATION ON ARTERIOLAR MOLECULAR PATHWAYS IN CHILDREN WITH PRECEDING PERITONEAL DIALYSIS

Abstract Background and Aims Patients on peritoneal dialysis suffer from progressive peritoneal membrane transformation and accelerated vascular disease, underlying molecular machineries have recently been described. Molecular effects of kidney transplantation (KTx) on vascular and peritoneal pathophysiology following PD are hardly understood. Method Fat surrounded omental arterioles (i.e. not directly exposed to PD fluids) from age-matched (median 6,5 years) children with chronic kidney disease (CKD5, n = 8), 5 children on PD [neutral pH, low glucose degradation product content, median PD duration 22 (5,50) months] and 6 children, who underwent successful KTx 4-5 weeks after a median 22 (12, 36) months of PD were analyzed by digital histomorphometry. Following microdissection from fat tissue, arterioles underwent transcriptome and proteome analyses, followed by Gene Set Enrichment Analysis (GSEA), weighted gene co-expression network analysis (WGCNA), gene ontology (GO) and protein interaction analysis (string-db.org). Key pathways were validated in independent, matched cohorts by quantitative immunohistochemistry (n = 15/group) and in vitro. Results 5000 most variable arteriolar transcripts (cut off p-value<0.05 and |r| >0.5) were grouped into 23 modules by WGCNA, of which 8 significantly differed between CKD5, PD and KTx. Seven were specifically related to KTx and one module to PD. Four out of seven KTx specific modules resulted in significant GO terms summarized as fatty acid biosynthesis, negative regulation of RNA metabolism, cell cycle arrest, and apoptosis. The PD specific module was associated with muscle cell proliferation, detoxification and complement activation (with thrombospondin as most interconnected gene). Multi-omics analysis of KTx vs. PD demonstrated concordant upregulation of lipid and fatty acid biosynthesis with the hub gene fatty acid synthase (FASN) and downregulation of positive cell cycle regulation. In independent, matched cohorts, arteriolar abundance of thrombospondin and terminal complement complex were higher in children on PD, arterioles from children after KTx had levels comparable to arterioles from children with CKD5. Key drivers of fibrotic process, arteriolar TGF-ß and pSMAD2/3, were also higher in PD arterioles compared to CKD5 and persistently high after KTx. Arteriolar cell cycle arrest marker p16 and apoptosis marker cleaved Casp3 were higher after KTx as compared to PD and CKD5. FASN was abundant in intima and media layers of all arterioles, and three-fold higher after KTx then in children with PD and CKD5, respectively. Neutral lipids (oil red staining) were present in all three groups, more abundant in the media than in the intima, and two-fold increased in the media of children after KTx compared to children on PD (p = 0.018) and with CKD5 (p = 0.002). In vitro, methylprednisolone and tacrolimus, but not mycophenolate mofetil increased FASN abundance and activity in human umbilical arterial endothelial and vascular smooth muscle cells compared to media controls. Conclusion We for the first time comprehensively describe molecular pathways activated after KTx in children following a standard immunosuppressive regime. After KTx, arteriolar fatty acid biosynthesis, apoptosis and cell cycle arrest markers are increased as compared to arterioles from children with PD and CKD5; profibrotic pathways induced by PD are persistently activated. Arteriolar hubgene FASN is three-fold more abundant after KTx and neutral lipid deposition is increased, presumably due to the induction of FASN activity by methylprednisolone and tacrolimus.

Endothelial Ion Channels and Cell-Cell Communication in the Microcirculation

Endothelial cells in resistance arteries, arterioles, and capillaries express a diverse array of ion channels that contribute to Cell-Cell communication in the microcirculation. Endothelial cells are tightly electrically coupled to their neighboring endothelial cells by gap junctions allowing ion channel-induced changes in membrane potential to be conducted for considerable distances along the endothelial cell tube that lines arterioles and forms capillaries. In addition, endothelial cells may be electrically coupled to overlying smooth muscle cells in arterioles and to pericytes in capillaries via heterocellular gap junctions allowing electrical signals generated by endothelial cell ion channels to be transmitted to overlying mural cells to affect smooth muscle or pericyte contractile activity. Arteriolar endothelial cells express inositol 1,4,5 trisphosphate receptors (IP3Rs) and transient receptor vanilloid family member 4 (TRPV4) channels that contribute to agonist-induced endothelial Ca2+ signals. These Ca2+ signals then activate intermediate and small conductance Ca2+-activated K+ (IKCa and SKCa) channels causing vasodilator-induced endothelial hyperpolarization. This hyperpolarization can be conducted along the endothelium via homocellular gap junctions and transmitted to overlying smooth muscle cells through heterocellular gap junctions to control the activity of voltage-gated Ca2+ channels and smooth muscle or pericyte contraction. The IKCa- and SKCa-induced hyperpolarization may be amplified by activation of inward rectifier K+ (KIR) channels. Endothelial cell IP3R- and TRPV4-mediated Ca2+ signals also control the production of endothelial cell vasodilator autacoids, such as NO, PGI2, and epoxides of arachidonic acid contributing to control of overlying vascular smooth muscle contractile activity. Cerebral capillary endothelial cells lack IKCa and SKCa but express KIR channels, IP3R, TRPV4, and other Ca2+ permeable channels allowing capillary-to-arteriole signaling via hyperpolarization and Ca2+. This allows parenchymal cell signals to be detected in capillaries and signaled to upstream arterioles to control blood flow to capillaries by active parenchymal cells. Thus, endothelial cell ion channels importantly participate in several forms of Cell-Cell communication in the microcirculation that contribute to microcirculatory function and homeostasis.

Laser technologies in the diagnostics and care of patients with decompensated forms of chronic venous insufficiency in the lower extremities

Purpose. To assess the degree of disorders in regional microcirculation in patients with decompensated forms of chronic venous insufficiency of lower extremities and potentials for their correction with low-level laser therapy (LLLT).Material and methods. The authors analyzed results of examination and treatment of 66 patients with decompensated forms of chronic venous insufficiency in their lower extremities. 34 (51.5 %) patients, in addition to traditional therapy, had laser stimulation of microcirculation. For laser irradiation, infrared laser device “Mustang” (wavelength – 0.89 μm, frequency – 80 Hz, power – 10 W, exposure – 2 min per zone) was used. In laser course, there were 10 sessions. The state of microcirculation in tissues was examined with a computerized capillaroscope, a laser analyzer of capillary blood flow and a gas analyzer.Results. Microcirculation in patients with chronic venous insufficiency C6 has its specific features which, by findings of computerized capillaroscope, are manifested with deep suppression of tissue blood flow: namely, expressed polymorphism of capillary structures, violation of sizeratio of capillary sections, increased venous section, decreased density of the capillary network by 1.8 times, increased pericapillary zone by 1.47 times , decreased speed of capillary blood flow in the arterial section by 10.5 times and in the venous section by 11.5 times.Conclusion. Patients with decompensated forms of chronic venous insufficiency in lower extremities were observed to have significant morphological and functional changes in microcirculation, including structural changes in microvessels, decreased density of capillary network, increased prevascular zone, decreased speed of capillary blood flow. As a result, these changes cause disorders in tissue trophism, progressive decrease in TcpO2 and development of trophic ulcers. Low-level laser stimulation of microcirculation in the complex treatment of patients with decompensated forms of chronic venous insufficiency activates transcapillary metabolism, helps to restore structures and functions of microvasculature by increasing the myogenic activity of smooth muscle cells of arterioles and precapillaries as well as by normalizing arterio-venous relationships what accelerates tissue granulation and ulcer epithelialization.

Study of morphological changes in the kidney during modeling of ischemic-reperfusion injuries of the limb and massive blood loss

The article presents the features of morphological disorders of liver tissue on the background of limb ischemia-reperfusion and massive blood loss. The aim of the work was to establish the presence of structural changes and the severity of morphological disorders of internal organs, remote from the primary place of ischemia-reperfusion during modeling the pathological process. The experiment was performed on 33 adult nonlinear white rats weighing 200-250 g, which were on the standard vivarium diet. Experimental animals were simulated with ischemic-reperfusion syndrome of the lower extremity and massive blood loss. Collection of materials was performed 1 h after intervention or release from the tourniquet and on the 1st, 3rd, 7th and 14th days. All interventions were performed under thiopental-sodium anesthesia (40 mg/kg body weight). Mostly structural violations were expressed as disturbance of blood supply of little and middle caliber blood vessels and also in initial dystrophic changes. During comparison of groups, depending on the severity, it was shown that both the isolated use of a tourniquet and massive blood loss had long-term, systemic consequences, however, more expressed in EG-2; single vascular glomeruli were shrunked, endothelial cells of arterioles were slightly damaged. On the 7th and 14th days changes in the structure of EG-1 were mostly absent, although in EG-2 the epitheliocytes of the outer layer of the capsule remained flattened, retained full blood vessels of the venous bed in the interstitium, and the vast majority of epitheliocytes of the excretory tubules were at different stages of hydropic dystrophy with partly desquamation of the epithelium in the gaps of the tubules. Also, the basal membranes of the tubules were not completely visualized, which indicates deep damage in the structures caused by acute ischemia as a result of bleeding. Thus, as combat trauma (blood loss) is in itself a life-threatening factor, the use of a tourniquet due to the development of ischemic-reperfusion process can complicate the course of the primary affection. Knowledge of the periodization of traumatic disease on the background of this pathology is important for the development of sanogenic effects in order to minimize this pathogenic factor.

Laboratory findings in hematology, clinical chemistry, and urinalysis for patients with thrombotic thrombocytopenic purpura

Laboratory diagnosis of thrombotic thrombocytopenic purpura (TTP) often begins with routine laboratory tests; a complete blood count (CBC), clinical chemistry panel, and urinalysis. The classical findings may include anemia with schistocytes, thrombocytopenia, reticulocytosis or polychromasia, bilirubinemia, dark urine, and hemoglobinuria without red blood cells in the sediment. Additional findings including decreased haptoglobin can identify fragmentation as the cause for the hemolysis. The hemolysis in TTP arises from increased shear stress on red blood cells in arterioles and capillaries narrowed by microthrombi. Hemoglobinemia and schistocytes may generate spurious results in hematology analyzers that require correction before results can be released to the patient chart.

Implications of Systemic Hematocrit on the Radial Distribution of Red Cells in Arterioles

It is well‐established that the tube hemacocrit (HctT) in blood vessels and glass tubes with an internal diameter less than 200‐microns is significantly lower than feed or discharge Hct (the Fahraeus Effect). Microscopic imaging of the flow stream reveals a decrease in Hct in the vicinity of the wall (the plasma layer, PL) but the mechanism of the effect is unclear. To address this question, we recorded high speed videos (3000 fps) of blood flow in 35‐micron diameter arterioles of the rat cremaster muscle at systemic Hct (HctS) levels of 20% to 72%. We found that the PL is an annulus bordering the wall that occupies 12% to 50% of the flow stream cross section, and that it is inversely related to HctS. In addition, results indicate that the Hct in the core region of the flow stream is not significantly different from HctS. The position of the PL coincides with the region of maximal shear stress in the flow stream, which may strip away the red cell monolayer adjacent to the wall, creating the PL while the monolayer moves toward the faster‐flowing center of the flow stream. In conclusion, while volumetric flow is related to the cross section of the flow stream, the PL is related to the circumference, thus reducing HctT as radius decreases.Support or Funding InformationThis work was supported by the NIH Heart Lung and Blood Institute under Grants R01HL126945 and R01HL138116.

Expression of troponin subunits in the rat renal afferent arteriole.

Vascular smooth muscle cells of the renal afferent arteriole are unusual in that they must be able to contract very rapidly in response to a sudden increase in systemic blood pressure in order to protect the downstream glomerular capillaries from catastrophic damage. We showed that this could be accounted for, in part, by exclusive expression, at the protein level, of the "fast" (B) isoforms of smooth muscle myosin II heavy chains in the afferent arteriole, in contrast to other vascular smooth muscle cells such as the rat aorta and efferent arteriole which express exclusively the "slow" (A) isoforms (Shiraishi et al. (2003) FASEB. J. 17, 2284-2286). As contraction of the more rapidly contracting striated (skeletal and cardiac) muscles is regulated by the thin filament-associated troponin (Tn) system, we hypothesized that Tn or a Tn-like system may exist in afferent arteriolar cells and contribute to the unusually rapid contraction of this tissue in response to increased intraluminal pressure. We examined the expression of TnC (Ca2+ -binding subunit), TnI (inhibitory subunit), and TnT (tropomyosin-binding subunit) in vascular smooth muscle cells of the rat renal afferent arteriole at the mRNA level. Fast-twitch skeletal muscle and slow-twitch skeletal muscle/cardiac TnC isoforms and slow-twitch skeletal muscle and cardiac TnI isoforms were detected by reverse transcription-polymerase chain reaction (RT-PCR) and confirmed by cDNA sequencing. Furthermore, cardiac and slow-twitch skeletal muscle TnI isoforms, but not fast-twitch skeletal muscle TnI, were detected in isolated afferent arterioles at the protein level by proximity ligation assay. Finally, striated muscle myosin II heavy chain expression was identified in isolated rat afferent arterioles by RT-PCR. We conclude that, in addition to Ca2+ -mediated phosphorylation of myosin II regulatory light chains, contraction of the afferent arteriole may be regulated by a mechanism normally associated with the much more rapidly contracting cardiac and skeletal muscles, which involves Ca2+ binding to TnC, leading to alleviation of inhibition of the actomyosin MgATPase by TnI and tropomyosin and rapid contraction of the vessel.

Role of Pericytes in theInitiation and Propagation of Spontaneous Activity in theMicrovasculature.

The microvasculature is composed of arterioles, capillaries and venules. Spontaneous arteriolar constrictions reduce effective vascular resistance to enhance tissue perfusion, while spontaneous venular constrictions facilitate the drainage of tissue metabolites by pumping blood. In the venules of visceral organs, mural cells, i.e. smooth muscle cells (SMCs) or pericytes, periodically generate spontaneous phasic constrictions, Ca2+ transients and transient depolarisations. These events arise from spontaneous Ca2+ release from the sarco-endoplasmic reticulum (SR/ER) and the subsequent opening of Ca2+-activated chloride channels (CaCCs). CaCC-dependent depolarisation further activates L-type voltage-dependent Ca2+ channels (LVDCCs) that play a critical role in maintaining the synchrony amongst mural cells. Mural cells in arterioles or capillaries are also capable of developing spontaneous activity. Non-contractile capillary pericytes generate spontaneous Ca2+ transients primarily relying on SR/ER Ca2+ release. Synchrony amongst capillary pericytes depends on gap junction-mediated spread of depolarisations resulting from the opening of either CaCCs or T-type VDCCs (TVDCCs) in a microvascular bed-dependent manner. The propagation of capillary Ca2+ transients into arterioles requires the opening of either L- or TVDCCs again depending on the microvascular bed. Since the blockade of gap junctions or CaCCs prevents spontaneous Ca2+ transients in arterioles and venules but not capillaries, capillary pericytes appear to play a primary role in generating spontaneous activity of the microvasculature unit. Pericytes in capillaries where the interchange of substances between tissues and the circulation takes place may provide the fundamental drive for upstream arterioles and downstream venules so that the microvasculature network functions as an integrated unit.

Troponin Subunits are Expressed in the Rat Afferent Arteriole

Vascular smooth muscle cells of the renal afferent arteriole are unusual: they must be able to contract very rapidly in response to a sudden increase in systemic blood pressure, thereby protecting the downstream glomerular capillaries from catastrophic damage. We showed that this could be accounted for in part by exclusive expression, at the protein level, of the “fast” (B) isoforms of smooth muscle myosin II heavy chains in the afferent arteriole, in contrast to other vascular smooth muscle cells such as the rat aorta and efferent arteriole, which express exclusively the “slow” (A) isoforms (Shiraishi M et al (2003) FASEB J 17: 2284–2286). Since contraction of the more rapidly‐contracting striated (skeletal and cardiac) muscles is regulated by the thin filament‐associated troponin (Tn) system, we hypothesized that Tn or a Tn‐like system may exist in afferent arteriolar cells and contribute to the unusually rapid contraction of this tissue in response to increased intraluminal pressure. We examined the expression of TnC (Ca2+‐binding subunit), TnI (inhibitory subunit) and TnT (tropomyosin‐binding subunit) in vascular smooth muscle cells of the rat renal afferent arteriole at the mRNA level. Fast skeletal muscle and slow skeletal muscle/cardiac TnC isoforms and slow skeletal muscle and cardiac TnI isoforms were detected by RT‐PCR and confirmed by cDNA sequencing. Furthermore, cardiac and slow skeletal muscle TnI isoforms, but not fast skeletal muscle TnI were detected in isolated afferent arterioles at the protein level by proximity ligation assay. We conclude that, in addition to Ca2+‐mediated phosphorylation of myosin regulatory light chains, contraction of the afferent arteriole may be regulated by a mechanism normally associated with the much more rapidly contracting cardiac and skeletal muscles, which involves Ca2+ binding to TnC, leading to alleviation of inhibition of the actomyosin MgATPase by TnI and tropomyosin and rapid contraction of the vessel.Support or Funding InformationSupported by the Canadian Institutes of Health ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Properties of synchronous spontaneous Ca2+ transients in the mural cells of rat rectal arterioles.

Synchrony of spontaneous Ca2+ transients among venular mural cells (smooth muscle cells and pericytes) in visceral organs relies on the intercellular spread of L-type voltage-dependent Ca2+ channel (LVDCC)-dependent depolarisations. However, the mechanisms underlying the synchrony of spontaneous Ca2+ transients between arteriolar mural cells are less understood. The spontaneous intracellular Ca2+ dynamics of arteriolar mural cells in the rat rectal submucosa were visualised by Cal-520 Ca2+ imaging to analyse their synchrony. The mural cells in fine arterioles that had a rounded cell body with several extended processes developed spontaneous 'synchronous' Ca2+ transients arising from Ca2+ released from sarcoendoplasmic reticulum Ca2+ stores. Gap junction blockers (3μM carbenoxolone, 10μM 18β-glycyrrhetinic acid), a Ca2+-activated Cl- channel (CaCC) blocker (100μM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) or lowering extracellular Cl- concentration (from 134.4 to 12.4mM) disrupted the synchrony of Ca2+ transients between arteriolar mural cells. Blockers of T-type voltage-dependent Ca2+ channels (TVDCCs, 1μM mibefradil or ML218) or LVDCCs (1μM nifedipine) reduced the Ca2+ transient frequency or their area under curve (AUC), respectively. However, neither TVDCC nor LVDCC blockers disrupted the synchrony of Ca2+ transients among arteriolar mural cells. This is in contrast with rectal venules in which nifedipine disrupted the synchrony of spontaneous Ca2+ transients. Thus, spontaneous transient depolarisations arising from the opening of CaCCs may effectively spread to neighbouring arteriolar mural cells via gap junctions to maintain the Ca2+ transient synchrony. Activation of TVDCCs appears to accelerate spontaneous Ca2+ transients, while LVDCCs predominantly contribute to the duration of Ca2+ transients.

The pathomorphology features of kidneys lesion in the metabolic syndrome (evidences of autopsy and experiment)

The global increase in the number of people with chronic kidney disease requires new approaches to their prevention and treatment. Aim: To investigate the pathomorphology features of kidneys lesion in the metabolic syndrome (evidences of autopsy and experiment) Methods and results. The pathomorphology features of kidneys lesion on autopsy of kidneys of 124 deceased patients with the metabolic syndrome and in 40 experimental animals (rats) were identified by light and electron microscopy method. All the changes of kidney, which we observed, were typical for common conditions – diabetes mellitus type 2, hypertension, obesity and dyslipidemia. We established that the damage of kidney's components was characterized by heterogeneity and heterochronicity. Conclusion. The pathomorphology features were characterized by dystrophy in smooth muscle cells of arterioles, thickening of the basement membrane, mesangium proliferation, hyalinosis and focal-segmental sclerosis.

Ca(2+) signaling in arterioles and small arteries of conscious, restrained, optical biosensor mice.

Two-photon fluorescence microscopy and conscious, restrained optical biosensor mice were used to study smooth muscle Ca2+ signaling in ear arterioles. Conscious mice were used in order to preserve normal mean arterial blood pressure (MAP) and sympathetic nerve activity (SNA). ExMLCK mice, which express a genetically-encoded smooth muscle-specific FRET-based Ca2+ indicator, were equipped with blood pressure telemetry and immobilized for imaging. MAP was 101 ± 4 mmHg in conscious restrained mice, similar to the freely mobile state (107 ± 3 mmHg). Oscillatory vasomotion or irregular contractions were observed in most arterioles (71%), with the greatest oscillatory frequency observed at 0.25 s−1. In a typical arteriole with an average diameter of ~35 μm, oscillatory vasomotion of a 5–6 μm magnitude was accompanied by nearly uniform [Ca2+] oscillations from ~0.1 to 0.5 μM, with maximum [Ca2+] occurring immediately before the rapid decrease in diameter. Very rapid, spatially uniform “Ca2+ flashes” were also observed but not asynchronous propagating Ca2+ waves. In contrast, vasomotion and dynamic Ca2+ signals were rarely observed in ear arterioles of anesthetized exMLCK biosensor mice. Hexamethonium (30 μg/g BW, i.p.) caused a fall in MAP to 74 ± 4 mmHg, arteriolar vasodilation, and abolition of vasomotion and synchronous Ca2+ transients.Summary: MAP and heart rate (HR) were normal during high-resolution Ca2+ imaging of conscious, restrained mice. SNA induced continuous vasomotion and irregular vasoconstrictions via spatially uniform Ca2+ signaling within the arterial wall. FRET-based biosensor mice and two-photon imaging provided the first measurements of [Ca2+] in vascular smooth muscle cells in arterioles of conscious animals.

A paradigm shift for local blood flow regulation

a remarkable example of physiological regulation is the coordination between metabolic rate and local blood flow in microscopic volumes of an organ. In skeletal muscle an increase in oxygen consumption (Vo2) over a wide range evokes a proportional (∼5 to 6 × Vo2) increase in blood flow ([29][

Fenamates block gap junction coupling and potentiate BKCa channels in guinea pig arteriolar cells

We determined the actions of the fenamates, flufenamic acid (FFA) and niflumic acid (NFA), on gap junction-mediated intercellular coupling between vascular smooth muscle cells (VSMC) in situ of acutely isolated arteriole segments from the three vascular beds: the spiral modiolar artery (SMA), anterior inferior cerebellar artery (AICA) and mesenteric artery (MA), and on non-junctional membrane channels in dispersed VSMCs. Conventional whole-cell recording methods were used. FFA reversibly suppressed the input conductance (Ginput) or increased the input resistance (Rinput) in a concentration dependent manner, with slightly different IC50s for the SMA, AICA and MA segments (26, 33 and 56μM respectively, P>0.05). Complete electrical isolation of the recorded VSMC was normally reached at ≥300μM. NFA had a similar effect on gap junction among VSMCs with an IC50 of 40, 48 and 62μM in SMA, AICA and MA segments, respectively. In dispersed VSMCs, FFA and NFA increased outward rectifier K+-current mediated by the big conductance calcium-activated potassium channel (BKCa) in a concentration-dependent manner, with a similar EC50 of ∼300μM for both FFA and NFA in the three vessels. Iberiotoxin, a selective blocker of the BKCa, suppressed the enhancement of the BKCa by FFA and NFA. The KV blocker 4-AP had no effect on the fenamates-induced K+-current enhancement. We conclude that FFA and NFA blocked the vascular gap junction mediated electrical couplings uniformly in arterioles of the three vascular beds, and complete electrical isolation of the recorded VSMC is obtained at ≧300μM; FFA and NFA also activate BKCa channels in the arteriolar smooth muscle cells in addition to their known inhibitory effects on chloride channels.

Distribution of Adenylyl Cyclases in the Rat Ovary by Immunofluorescence Microscopy

In the mammalian ovary both gonadotropins and local cytokines, acting through G-protein coupled receptors, govern the physiology of the ovary in part by regulating the cyclic adenosine 3',5'-monophosphate via adenylyl cyclases. The nine transmembrane adenylyl cyclases and a soluble adenylyl cyclase are regulated by a diversity of ligands. In this study we have examined the rat ovaries, prior to and subsequent to gonadotropin treatment, for the presence of different transmembrane adenylyl cyclases by indirect immunofluorescence microscopy. Adenylyl cyclase I immunoreactivity was observed in the nuclei of oocytes in preantral and antral follicles along with some staining in granulosa cells. Equine chorionic gonadotropin injection increased adenylyl cyclase I staining in granulosa cells. Adenylyl cyclase I staining was also observed in luteal and endothelial cells. Adenylyl cyclase II was observed throughout the ovary, including granulosa cells and the ovarian surface epithelium. Adenylyl cyclase II staining was also found to increase in granulosa cells after equine chorionic gonadotropin injection. Adenylyl cyclase III was distributed primarily in theca and smooth muscle cells of arterioles, with faint staining in the oocytes of equine chorionic gonadotropin-injected ovaries. Adenylyl cyclase IV staining was present throughout the ovary, including the nuclei of oocytes. Adenylyl cyclase VIII staining in granulosa cells increased subsequent to equine chorionic gonadotropin injection and remained in luteal cells. Our study reveals the redundancy of adenylyl cyclases present in the rat ovary and, therefore, implies potential regulation of follicular and corpus luteum physiology by cyclic adenosine 3',5'-monophosphate generated through distinct adenylyl cyclases.

Treatment of atypical uraemic syndrome in the era of eculizumab.

Treatment of atypical uraemic syndrome in the era of eculizumab.

Facilitated diffusion of angiotensin II from perivascular interstitium to AT1 receptors of the arteriole. A regulating step in vasoconstriction

A kinetic model for the binding of angiotensin (Ang) II to AT1 receptors (AT1R) in arterioles in vivo did suggest a novel mechanism of stimulus amplification. To further clarify the role of this mechanism in the functioning of the local renin-angiotensin systems, as opposed to circulating Ang II. The model was refined in order to account for geometric characteristics of the vascular smooth muscle (VSM) cells in arterioles with a single VSM cell layer. Results show that, unlike experiments in vitro, the graph of AT1R occupancy, that is, [Rec(occ)]/[Rec(total)] where [Rec(total)]=[Rec(occ)]+[Rec(free)], as a function of log [Ang II], is shifted to the left at higher [Rec(total)]. This leads to the concept of association rate amplification (ASRA) and facilitated Ang II diffusion. Considering that abluminal Ang II has to cross a diffusion fluid-barrier 1-10 times the glycocalyx to reach VSM AT1R, it appears that the ASRA factor is 1500 to 150 respectively, whereas more than 90% of Ang II is captured, at 10% occupancy, and with [Ang II] as low as 10(-15)-10(-14) mol/ml. Due to the presence of endothelium, intraluminal [Ang II] needs to be 20-30 times higher. ASRA favors a low [Ang II] threshold for AT1R stimulation, but it also favors a flat stimulus/response curve by promoting receptor-mediated endocytosis and receptor downregulation. The model predicts that, in small resistance vessels, abluminal rather than intraluminal Ang II is important for maintaining vasoconstrictor tone. ASRA minimizes the overflow of de-novo generated tissue Ang II into the circulation. It explains why Ang II acts at levels far below K(D), why AT1R blockers are effective in hypertension even when [Ang II] is low, and why the constrictor action of Ang II appears so much suppressed by sodium depletion.

Immunohistochemical localization of d-aspartate oxidase in porcine peripheral tissues

D-Aspartate (D-Asp) is an endogenous substance in mammals. Degradation of D-Asp is carried out only by D-aspartate oxidase (DDO). We measured DDO activity in porcine tissues, and produced an anti-porcine DDO antibody to examine the cellular localization of DDO. All the tissues examined showed DDO activities, whereas the substrate D-Asp was not detected in kidney cortex, liver, heart, and gastric mucosa. In the kidney, intensive immunohistochemical staining for DDO was found in the epithelial cells of the proximal tubules. In the liver, the epithelial cells of interlobular bile ducts, liver sinusoid-lining cells with cytoplasmic processes, and the smooth muscle cells of arterioles were strongly stained for DDO. In the heart, cardiomyocytes and the smooth muscle cells of arterioles showed DDO-immunoreactivity. In the gastric mucosa, only the chief cells were DDO-positive. These newly identified DDO-positive cells seem to actively degrade D-Asp to prevent an excess of D-Asp from exerting harmful effects on the respective functions of porcine tissues.

Renin secretion and total body sodium: Pathways of integrative control

1. Herein, we review mechanisms of sodium balance operating at constant mean arterial blood pressure (MABP); that is, under conditions where MABP does not provide the primary signal to the kidney. 2. Relative constancy of body fluids requires accurate regulation of total body sodium (TBS). Normally, plenty of sodium is ingested and balance is achieved by control of renal excretion driven by multiple central nervous, cardiovascular, endocrine and renal tubular mechanisms. Subtle changes in sodium balance are associated with parallel changes in extracellular volume (due to fast and precise osmoregulation), but not necessarily in MABP. Therefore, signals other than MABP seem to be the primary link between TBS and kidney function. 3. Renal functions involved in sodium homeostasis include: (i) the rate of glomerular filtration (GFR) determined by renal haemodynamics, including tubuloglomerular feedback (TGF); (ii) proximal tubular reabsorption involving glomerulotubular balance (GTB) and neurohumoral control; (iii) macula densa mechanisms influencing TGF and renin secretion; and (iv) distal tubular reabsorption dominated by the renin-angiotensin-aldosterone system (RAAS). 4. The present review focuses on the interactive, homeostatic operation of TBS, MABP, GTB, TGF and the RAAS. Regulation of sodium balance involves neurohumoral control of tubular sodium reabsorption, including proximal reabsorption. Central nervous system-mediated regulation of the latter modulates renin secretion. Homeostatically, the RAAS-TGF interaction seems analogous to a spring-shock absorber set-up: non-adaptive RAAS functions determine the new steady state position, whereas TGF controls the rate of change. Recruitment of renin-secreting cells during sustained stimulation may be essential for chronic adaptation, although details of this afferent arteriolar cell plasticity are unclear at present.