scholarly journals Descending vasa recta endothelial cells and pericytes form mural syncytia

2014 ◽  
Vol 306 (7) ◽  
pp. F751-F763 ◽  
Author(s):  
Zhong Zhang ◽  
Hai Lin ◽  
Chunhua Cao ◽  
Kristie Payne ◽  
Thomas L. Pallone
Keyword(s):  
Endothelial Cells ◽  
Membrane Potential ◽  
Patch Clamp ◽  
Gap Junction ◽  
Lucifer Yellow ◽  
Sampling Rate ◽  
Input Resistance ◽  
Contralateral Side ◽  
Vasa Recta ◽  
Endothelial Response

Using patch clamp, we induced depolarization of descending vasa recta (DVR) pericytes or endothelia and tested whether it was conducted to distant cells. Membrane potential was measured with the fluorescent voltage dye di-8-ANEPPS or with a second patch-clamp electrode. Depolarization of an endothelial cell induced responses in other endothelia within a millisecond and was slowed by gap junction blockade with heptanol. Endothelial response to pericyte depolarization was poor, implying high-resistance myo-endothelial coupling. In contrast, dual patch clamp of neighboring pericytes revealed syncytial coupling. At high sampling rate, the spread of depolarization between pericytes and endothelia occurred in 9 ± 2 or 12 ± 2 μs, respectively. Heptanol (2 mM) increased the overall input resistance of the pericyte layer to current flow and prevented transmission of depolarization between neighboring cells. The fluorescent tracer Lucifer yellow (LY), when introduced through ruptured patches, spread between neighboring endothelia in 1 to 7 s, depending on location of the flanking cell. LY diffused to endothelial cells on the ipsilateral but not contralateral side of the DVR wall and minimally between pericytes. We conclude that both DVR pericytes and endothelia are part of individual syncytia. The rate of conduction of membrane potential exceeds that for diffusion of hydrophilic molecules by orders of magnitude. Gap junction coupling of adjacent endothelial cells may be spatially oriented to favor longitudinal transmission along the DVR axis.

scholarly journals Descending vasa recta endothelium is an electrical syncytium

2006 ◽  
Vol 291 (6) ◽  
pp. R1688-R1699 ◽  
Author(s):  
Qingli Zhang ◽  
Chunhua Cao ◽  
Michael Mangano ◽  
Zhong Zhang ◽  
Erik P. Silldorff ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Gap Junction ◽  
Lucifer Yellow ◽  
Smooth Muscle Actin ◽  
Muscle Actin ◽  
Endothelial Monolayer ◽  
Mechanical Perturbation ◽  
Vasa Recta ◽  
Gap Junction Coupling ◽  
Junction Coupling

We examined gap junction coupling of descending vasa recta (DVR). DVR endothelial cells or pericytes were depolarized to record the associated capacitance transients. Virtually all endothelia and some pericytes exhibited prolonged transients lasting 10–30 ms. Carbenoxolone (100 μM) and 18β-glycyrrhetinic acid (18βGRA; 100 μM) markedly shortened the endothelial transients. Carbenoxolone and heptanol (2 mM) reduced the pericyte capacitance transients when they were prolonged. Lucifer yellow (LY; 2 mM) was dialyzed into the cytoplasm of endothelial cells and pericytes. LY spread diffusely along the endothelial monolayer, whereas in most pericytes, it was confined to a single cell. In some pericytes, complex patterns of LY spreading were observed. DVR cells were depolarized by voltage clamp as fluorescence of bis(1,3-dibarbituric acid)-trimethine oxanol [DiBAC4( 3 )] was monitored ∼200 μm away. A 40-mV endothelial depolarization was accompanied by a 26.1 ± 5.5-mV change in DiBAC4( 3 ) fluorescence. DiBAC4( 3 ) fluorescence did not change after 18βGRA or when pericytes were depolarized. Similarly, propagated cytoplasmic Ca2+responses arising from mechanical perturbation of the DVR wall were attenuated by 18βGRA or heptanol. Connexin (Cx) immunostaining showed predominant linear Cx40 and Cx43 in endothelia, whereas Cx37 stained smooth muscle actin-positive pericytes. We conclude that the DVR endothelium is an electrical syncytium and that gap junction coupling in DVR pericytes exists but is less pronounced.

scholarly journals Mural propagation of descending vasa recta responses to mechanical stimulation

2013 ◽  
Vol 305 (3) ◽  
pp. F286-F294 ◽  
Author(s):  
Zhong Zhang ◽  
Kristie Payne ◽  
Chunhua Cao ◽  
Thomas L. Pallone
Keyword(s):  
Membrane Potential ◽  
Patch Clamp ◽  
Mechanical Stimulation ◽  
Channel Blockade ◽  
Patch Clamp Recording ◽  
Automated Method ◽  
Whole Cell Patch Clamp ◽  
Vasa Recta ◽  
Gated Channel ◽  
Phosphoinositide 3 Kinase

To investigate the responses of descending vasa recta (DVR) to deformation of the abluminal surface, we devised an automated method that controls duration and frequency of stimulation by utilizing a stream of buffer from a micropipette. During stimulation at one end of the vessel, fluorescent responses from fluo4 or bis[1,3-dibutylbarbituric acid-(5)] trimethineoxonol [DiBAC4(3)], indicating cytoplasmic calcium ([Ca2+]CYT) or membrane potential, respectively, were recorded from distant cells. Alternately, membrane potential was recorded from DVR pericytes by nystatin whole cell patch-clamp. Mechanical stimulation elicited reversible [Ca2+]CYT responses that increased with frequency. Individual pericyte responses along the vessel were initiated within a fraction of a second of one another. Those responses were inhibited by gap junction blockade with 18 β-glycyrrhetinic acid (100 μM) or phosphoinositide 3 kinase inhibition with 2-morpholin-4-yl-8-phenylchromen-4-one (50 μM). [Ca2+]CYT responses were blocked by removal of extracellular Ca2+ or L-type voltage-gated channel blockade with nifedipine (10 μM). At concentrations selective for the T-type channel blockade, mibefradil (100 nM) was ineffective. During mechanostimulation, pericytes rapidly depolarized, as documented with either DiBAC4(3) fluorescence or patch-clamp recording. Single stimuli yielded depolarizations of 22.5 ± 2.2 mV while repetitive stimuli at 0.1 Hz depolarized pericytes by 44.2 ± 4.0 mV. We conclude that DVR are mechanosensitive and that rapid transmission of signals along the vessel axis requires participation of gap junctions, L-type Ca2+ channels, and pericyte depolarization.

Intracellular cyclic AMP concentration modulates gap junction permeability in parturient rat myometrium

1992 ◽  
Vol 70 (3) ◽  
pp. 358-364 ◽  
Author(s):  
Nobuyoshi Sakai ◽  
Michael G. Blennerhassett ◽  
Robert E. Garfield
Keyword(s):  
Gap Junction ◽  
Lucifer Yellow ◽  
Contractile Activity ◽  
Input Resistance ◽  
Uterine Wall ◽  
Resting Membrane Potential ◽  
Cell Coupling ◽  
Cyclic Monophosphate ◽  
Myometrial Cells ◽  
High Concentrations

We investigated whether cell-to-cell coupling between myometrial cells of parturient rats is influenced by intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) concentration. To evaluate the coupling, we measured input resistance (R0) and injected Lucifer Yellow (LY) using microelectrode techniques. The intercellular spread of the dye was then observed. Longitudinal muscle strips from rat myometrium were exposed to isoproterenol, forskolin, or dibutyryl cAMP (DB-cAMP) to elevate cAMP. Isoproterenol (10−11–10−6 M) and DB-cAMP (10−5–10−3 M) hyperpolarized the resting membrane potential (Em) and increased R0 in a dose-dependent fashion. Forskolin (10−6 M) also hyperpolarized Em and increased R0. When LY was injected into a single cell, LY spread rapidly and extensively to neighboring cells in parturient control tissues, while LY transfer was completely blocked by any of the three agents at high concentrations. The increased R0 and blocked transfer of LY owing to these agents indicate that the cell-to-cell coupling was decreased both electrically and metabolically. Myometrial cells of parturient rats show increased number and size of gap junctions (GJs). The rapid and reversible decrease in coupling is interpreted to reflect the reduced permeability of GJs between the muscle cells because of an elevation of cAMP. Control of GJ permeability by this second messenger may be important for the physiological regulation of intercellular coupling and the extent of synchronizing and coordinating electrical, metabolic, and contractile activity in the uterine wall during pregnancy and parturition.Key words: adenosine 3′,5′-cyclic monophosphate, cell-to-cell coupling, gap junction permeability, rat myometrium.

Depolarization-mediated inhibition of Ca2+ entry in endothelial cells

1999 ◽  
Vol 277 (4) ◽  
pp. H1498-H1504 ◽  
Author(s):  
Xiaodong Wang ◽  
Cornelis van Breemen
Keyword(s):  
Endothelial Cells ◽  
Membrane Potential ◽  
Patch Clamp ◽  
Benzoic Acid ◽  
Plateau Phase ◽  
Free Medium ◽  
Current Clamp ◽  
Aortic Endothelial Cells ◽  
Induced Membrane ◽  
Patch Clamp Electrophysiology

The effect of extracellular Cl− in regulating ACh-induced Ca2+ entry into freshly isolated rabbit aortic endothelial cells was studied using Ca2+-sensitive fluorescence microscopy and patch-clamp electrophysiology. After ACh caused transient Ca2+ release in Ca2+-free medium, readdition of 3 mM Ca2+ to the bath maintained Ca2+ entry. Removal of extracellular Cl− abolished the plateau phase in Ca2+ signal and inhibited divalent cation entry. However, in the presence of the K+ ionophore valinomycin, removal of Cl− had no effect on the Ca2+ plateau. Under current-clamp conditions, substitution of gluconate for Cl− induced membrane depolarization. Under voltage clamp, with CsCl in the pipette, ACh activated a slowly developing Cl− current, which was blocked by SITS and 5-nitro-2-(3-phenylpropylamino)benzoic acid. Varying the membrane potential by utilizing different extracellular K+ concentrations in the presence of 5 μM valinomycin demonstrated that depolarization blocked ACh-stimulated Mn2+ entry. These data suggest that ACh-induced Ca2+entry in freshly isolated endothelial cells requires the presence of extracellular Cl− to maintain a polarized membrane potential.

Effect of HUVEC on human osteoprogenitor cell differentiation needs heterotypic gap junction communication

2002 ◽  
Vol 282 (4) ◽  
pp. C775-C785 ◽  
Author(s):  
F. Villars ◽  
B. Guillotin ◽  
T. Amédée ◽  
S. Dutoya ◽  
L. Bordenave ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Differentiation ◽  
Gap Junction ◽  
Type I Collagen ◽  
Lucifer Yellow ◽  
Umbilical Vein ◽  
Functional Coupling ◽  
Type I ◽  
Osteoprogenitor Cell ◽  
Junction Protein

Bone development and remodeling depend on complex interactions between bone-forming osteoblasts and other cells present within the bone microenvironment, particularly vascular endothelial cells that may be pivotal members of a complex interactive communication network in bone. Our aim was to investigate the interaction between human umbilical vein endothelial cells (HUVEC) and human bone marrow stromal cells (HBMSC). Cell differentiation analysis performed with different cell culture models revealed that alkaline phosphatase activity and type I collagen synthesis were increased only by the direct contact of HUVEC with HBMSC. This “juxtacrine signaling” could involve a number of different heterotypic connexions that require adhesion molecules or gap junctions. A dye coupling assay with Lucifer yellow demonstrated a functional coupling between HUVEC and HBMSC. Immunocytochemistry revealed that connexin43 (Cx43), a specific gap junction protein, is expressed not only in HBMSC but also in the endothelial cell network and that these two cell types can communicate via a gap junctional channel constituted at least by Cx43. Moreover, functional inhibition of the gap junction by 18α-glycyrrhetinic acid treatment or inhibition of Cx43 synthesis with oligodeoxyribonucleotide antisense decreased the effect of HUVEC cocultures on HBMSC differentiation. This stimulation could be mediated by the intercellular diffusion of signaling molecules that permeate the junctional channel.

Effect of gap junction number and permeability on intercellular coupling in rat myometrium

1991 ◽  
Vol 261 (6) ◽  
pp. C1001-C1009 ◽  
Author(s):  
M. G. Blennerhassett ◽  
R. E. Garfield
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Lucifer Yellow ◽  
Input Resistance ◽  
Cell Coupling ◽  
Intercellular Coupling ◽  
Myometrial Cells ◽  
Intercellular Spread

Gap junctions (GJ) increase between myometrial cells immediately before labor. To provide evidence of their role in cell-to-cell coupling, we evaluated input resistance (Ro) and intercellular spread of Lucifer yellow (LY) in intact preparations of rat longitudinal myometrium of preterm, term, and antiprogesterone-treated preterm delivering animals. LY injected into cells from either term or preterm delivering rats (many GJ) spread rapidly to neighboring cells by 60 s, but in preparations from nondelivering controls required 4-6 min to become detectable in adjacent cells. Ro of cells in preterm nondelivering preparations was 24.1 +/- 0.8 (SE) M omega, but dropped to 12.0 +/- 0.4 M omega (P less than 0.05) at delivery, similar to preterm delivering tissues at 13.8 +/- 0.6 M omega. The putative GJ uncoupling agent octanol reversibly increased Ro of term- and preterm-delivering tissues fourfold (P less than 0.01) within 60 s, and Ro of preterm-nondelivering tissue was further increased so that Ro values were similar among the three classes. These increased Ro values are interpreted as decreased coupling. Both K+ depolarization and oxytocin (10(-8) to 10(-6) M) increased Ro of delivering tissues (P less than 0.05), suggesting that high levels of contractile agonists may lead to reduced cell-to-cell coupling. Therefore, myometrial coupling can be modulated over seconds via GJ permeability as well as over hours by GJ number.

Descending vasa recta endothelia express inward rectifier potassium channels

2007 ◽  
Vol 293 (4) ◽  
pp. F1248-F1255 ◽  
Author(s):  
Chunhua Cao ◽  
Whaseon Lee-Kwon ◽  
Kristie Payne ◽  
Aurélie Edwards ◽  
Thomas L. Pallone
Keyword(s):  
Endothelial Cells ◽  
Membrane Potential ◽  
Potassium Channels ◽  
Inward Rectifier ◽  
Equilibrium Potential ◽  
Dependent Manner ◽  
Membrane Hyperpolarization ◽  
Inward Rectifier Potassium Channels ◽  
Vasa Recta ◽  
Inwardly Rectifying

Descending vasa recta (DVR) are capillary-sized microvessels that supply blood flow to the renal medulla. They are composed of contractile pericytes and endothelial cells. In this study, we used the whole cell patch-clamp method to determine whether inward rectifier potassium channels (KIR) exist in the endothelia, affect membrane potential, and modulate intracellular Ca2+ concentration ([Ca2+]cyt). The endothelium was accessed for electrophysiology by removing abluminal pericytes from collagenase-digested vessels. KIR currents were recorded using symmetrical 140 mM K+ solutions that served to maximize currents and eliminate cell-to-cell coupling by closing gap junctions. Large, inwardly rectifying currents were observed at membrane potentials below the equilibrium potential for K+. Ba2+ potently inhibited those currents in a voltage-dependent manner, with affinity k = 0.18, 0.33, 0.60, and 1.20 μM at −160, −120, −80, and −40 mV, respectively. Cs+ also blocked those currents with k = 20, 48, 253, and 1,856 μM at −160, −120, −80, and −40 mV, respectively. In the presence of 1 mM ouabain, increasing extracellular K+ concentration from 5 to 10 mM hyperpolarized endothelial membrane potential by 15 mV and raised endothelial [Ca2+]cyt. Both the K+-induced membrane hyperpolarization and the [Ca2+]cyt elevation were reversed by Ba2+. Immunochemical staining verified that both pericytes and endothelial cells of DVR express KIR2.1, KIR2.2, and KIR2.3 subunits. We conclude that strong, inwardly rectifying KIR2.x isoforms are expressed in DVR and mediate K+-induced hyperpolarization of the endothelium.

scholarly journals Syncytial communication in descending vasa recta includes myoendothelial coupling

2014 ◽  
Vol 307 (1) ◽  
pp. F41-F52 ◽  
Author(s):  
Zhong Zhang ◽  
Kristie Payne ◽  
Thomas L. Pallone
Keyword(s):  
Endothelial Cells ◽  
Membrane Potential ◽  
Endothelial Cell ◽  
Cell Communication ◽  
Membrane Potentials ◽  
Current Injection ◽  
Patch Clamp Recording ◽  
Vasa Recta ◽  
Highly Correlated ◽  
Heterologous Cell

Using dual cell patch-clamp recording, we examined pericyte, endothelial, and myoendothelial cell-to-cell communication in descending vasa recta. Graded current injections into pericytes or endothelia yielded input resistances of 220 ± 21 and 128 ± 20 MΩ, respectively ( P < 0.05). Injection of positive or negative current into an endothelial cell depolarized and hyperpolarized adjacent endothelial cells, respectively. Similarly, current injection into a pericyte depolarized and hyperpolarized adjacent pericytes. During myoendothelial studies, current injection into a pericyte or an endothelial cell yielded small, variable, but significant change of membrane potential in heterologous cells. Membrane potentials of paired pericytes or paired endothelia were highly correlated and identical. Paired measurements of resting potentials in heterologous cells were also correlated, but with slight hyperpolarization of the endothelium relative to the pericyte, −55.2 ± 1.8 vs. −52.9 ± 2.2 mV ( P < 0.05). During dual recordings, angiotensin II or bradykinin stimulated temporally identical variations of pericyte and endothelial membrane potential. Similarly, voltage clamp depolarization of pericytes or endothelial cells induced parallel changes of membrane potential in the heterologous cell type. We conclude that the descending vasa recta endothelial syncytium is of lower resistance than the pericyte syncytium and that high-resistance myoendothelial coupling also exists. The myoendothelial communication between pericytes and endothelium maintains near identity of membrane potentials at rest and during agonist stimulation. Finally, endothelia membrane potential lies slightly below pericyte membrane potential, suggesting a tonic role for the former to hyperpolarize the latter and provide a brake on vasoconstriction.

Measurement of spontaneous neuronal membrane activity by time lapse confocal microscopy

1995 ◽  
Vol 53 ◽  
pp. 972-973
Author(s):  
R H. Selinfreund ◽  
A. H. Cornell-Bell
Keyword(s):  
Membrane Potential ◽  
Confocal Microscopy ◽  
Patch Clamp ◽  
Electrical Activity ◽  
Time Lapse ◽  
Neuronal Firing ◽  
Neuronal Membrane ◽  
Pituitary Cells ◽  
Patch Clamp Recording ◽  
Spontaneous Electrical Activity

Cellular electrophysiological properties are normally monitored by standard patch clamp techniques . The combination of membrane potential dyes with time-lapse laser confocal microscopy provides a more direct, least destructive rapid method for monitoring changes in neuronal electrical activity. Using membrane potential dyes we found that spontaneous action potential firing can be detected using time-lapse confocal microscopy. Initially, patch clamp recording techniques were used to verify spontaneous electrical activity in GH4\C1 pituitary cells. It was found that serum depleted cells had reduced spontaneous electrical activity. Brief exposure to the serum derived growth factor, IGF-1, reconstituted electrical activity. We have examined the possibility of developing a rapid fluorescent assay to measure neuronal activity using membrane potential dyes. This neuronal regeneration assay has been adapted to run on a confocal microscope. Quantitative fluorescence is then used to measure a compounds ability to regenerate neuronal firing.The membrane potential dye di-8-ANEPPS was selected for these experiments. Di-8- ANEPPS is internalized slowly, has a high signal to noise ratio (40:1), has a linear fluorescent response to change in voltage.

Effects of ouabain on background and voltage-dependent currents in canine colonic myocytes

1990 ◽  
Vol 259 (3) ◽  
pp. C402-C408 ◽  
Author(s):  
E. P. Burke ◽  
K. M. Sanders
Keyword(s):  
Membrane Potential ◽  
Potential Gradient ◽  
Circular Muscle ◽  
Input Resistance ◽  
Pump Current ◽  
Muscle Layer ◽  
Membrane Properties ◽  
Resting Potential ◽  
Voltage Dependent ◽  
In Cells

Previous studies have suggested that the membrane potential gradient across the circular muscle layer of the canine proximal colon is due to a gradient in the contribution of the Na(+)-K(+)-ATPase. Cells at the submucosal border generate approximately 35 mV of pump potential, whereas at the myenteric border the pump contributes very little to resting potential. Results from experiments in intact muscles in which the pump is blocked are somewhat difficult to interpret because of possible effects of pump inhibitors on membrane conductances. Therefore, we studied isolated colonic myocytes to test the effects of ouabain on passive membrane properties and voltage-dependent currents. Ouabain (10(-5) M) depolarized cells and decreased input resistance from 0.487 +/- 0.060 to 0.292 +/- 0.040 G omega. The decrease in resistance was attributed to an increase in K+ conductance. Studies were also performed to measure the ouabain-dependent current. At 37 degrees C, in cells dialyzed with 19 mM intracellular Na+ concentration [( Na+]i), ouabain caused an inward current averaging 71.06 +/- 7.49 pA, which was attributed to blockade of pump current. At 24 degrees C or in cells dialyzed with low [Na+]i (11 mM), ouabain caused little change in holding current. With the input resistance of colonic cells, pump current appears capable of generating at least 35 mV. Thus an electrogenic Na+ pump could contribute significantly to membrane potential.

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