Intact rat superior mesenteric artery endothelium is an electrical syncytium and expresses strong inward rectifier K+ conductance

Background and purpose

Vascular endothelial and smooth muscle cell phenotypes may change dramatically after isolation and in cell cultures. This study was designed to investigate gap junctions coupling in an integrated intact preparation and to test if K_IR channels modulate resting membrane conductance in “in situ” endothelial cells (EC), and acetylcholine (ACh)-evoked relaxation of the rat superior mesenteric artery.

Experimental approach

Whole cell blind patch recordings of ionic currents from in situ EC, dye-coupling experiments, and functional studies were performed in rat superior mesenteric artery.

Key results

EC were dye-coupled through gap junctions. 18β-glycyrretinic acid (25 μM) decreased outward and inward currents, the 80% decay of time and time constant of the capacitative transients, capacitance, and increased input resistance. Barium chloride (30 μM) decreased resting and ACh-evoked inward currents, the sensitivity of ACh-evoked relaxation, and decreased both the sensitivity and the maximal relaxation to S-nitroso-N-acetyl penicillamine in arteries with, but not in arteries without endothelium.

Conclusions

The present results suggest that the EC layer of this large artery is electrically coupled, and that K_IR channels regulate resting inward conductance, hence suggesting that they are of importance for resting membrane potential in in situ EC. Moreover, EC K_IR channels are involved in ACh-evoked relaxation.     

Adenosine signaling in outer medullary descending vasa recta

We tested whether dilation of outer medullary descending vasa recta (OMDVR) is mediated by cAMP, nitric oxide (NO), and cyclooxygenase (COX). Adenosine (A; 10(-6) M)-induced vasodilation of ANG II (10(-9) M)-preconstricted OMDVR was mimicked by the cAMP analog 8-bromoadenosine 3',5'-cyclic monophosphate (10(-10) to 10(-4) M) and reversed by the adenylate cyclase inhibitor SQ-22536. Adenosine (10(-4) M) stimulated OMDVR cAMP production greater than threefold. NO synthase blockade with N(G)-nitro-L-arginine methyl ester and N(G)-monomethyl-L-arginine (10(-4) M) did not affect adenosine vasodilation. Adenosine induced endothelial cytoplasmic calcium transients that were small. Indomethacin (10(-6) M) reversed adenonsine-induced dilation of OMDVR preconstricted with ANG II, endothelin, 4-bromo-calcium ionophore A23187, or carbocyclic thromboxane A(2). In contrast, selective A(2)-receptor activation dilated endothelin-preconstricted OMDVR even in the presence of indomethacin. We conclude that OMDVR vasodilation by adenosine involves cAMP and COX but not NO. COX blockade does not fully inhibit selective A(2) receptor-mediated OMDVR dilation.     

Oxygen transport across vasa recta in the renal medulla

In this model of oxygen transport in the renal medullary microcirculation, we predicted that the net amount of oxygen reabsorbed from vasa recta into the interstitium is on the order of 10(-6) mmol/s, i.e., significantly lower than estimated medullary oxygen requirements based on active sodium reabsorption. Our simulations confirmed a number of experimental findings. Low medullary PO(2) results from the countercurrent arrangement of vessels and an elevated vasa recta permeability to oxygen, as well as high metabolic needs. Diffusional shunting of oxygen between descending vasa recta (DVR) and ascending vasa recta also explains why a 20-mmHg decrease in initial PO(2) at the corticomedullary junction only leads to a small drop in papillary tip PO(2) (<2 mmHg with baseline parameter values). Conversely, small changes in the consumption rate of DVR-supplied oxygen, in blood flow rate, in hematocrit, or in capillary permeability to oxygen, beyond certain values sharply reduce interstitial PO(2). Without erythrocytes, papillary tip PO(2) cannot be maintained above 10 mmHg, even when oxygen consumption is zero.     

Cellular mechanisms underlying nitric oxide-induced vasodilation of descending vasa recta

It has been observed that vasoactivity of explanted descending vasa recta (DVR) is modulated by intrinsic nitric oxide (NO) and superoxide (O(2)(-)) production (Cao C, Edwards A, Sendeski M, Lee-Kwon W, Cui L, Cai CY, Patzak A, Pallone TL. Am J Physiol Renal Physiol 299: F1056-F1064, 2010). To elucidate the cellular mechanisms by which NO, O(2)(-) and hydrogen peroxide (H(2)O(2)) modulate DVR pericyte cytosolic Ca(2+) concentration ([Ca](cyt)) and vasoactivity, we expanded our mathematical model of Ca(2+) signaling in pericytes. We incorporated simulations of the pathways that translate an increase in [Ca](cyt) to the activation of myosin light chain (MLC) kinase and cell contraction, as well as the kinetics of NO and reactive oxygen species formation and their effects on [Ca](cyt) and MLC phosphorylation. The model reproduced experimentally observed trends of DVR vasoactivity that accompany exposure to N(ω)-nitro-L-arginine methyl ester, 8-Br-cGMP, Tempol, and H(2)O(2). Our results suggest that under resting conditions, NO-induced activation of cGMP maintains low levels of [Ca](cyt) and MLC phosphorylation to minimize basal tone. This results from stimulation of Ca(2+) uptake from the cytosol into the SR via SERCA pumps, Ca(2+) efflux into the extracellular space via plasma membrane Ca(2+) pumps, and MLC phosphatase (MLCP) activity. We predict that basal concentrations of O(2)(-) and H(2)O(2) have negligible effects on Ca(2+) signaling and MLC phosphorylation. At concentrations above 1 nM, O(2)(-) is predicted to modulate [Ca(cyt)] and MCLP activity mostly by reducing NO bioavailability. The DVR vasoconstriction that is induced by high concentrations of H(2)O(2) can be explained by H(2)O(2)-mediated downregulation of MLCP and SERCA activity. We conclude that intrinsic generation of NO by the DVR wall may be sufficient to inhibit vasoconstriction by maintaining suppression of MLC phosphorylation.     

Membrane potential controls calcium entry into descending vasa recta pericytes

We tested the hypothesis that constriction of descending vasa recta (DVR) is mediated by voltage-gated calcium entry. K(+) channel blockade with BaCl(2) (1 mM) or TEACl (30 mM) depolarized DVR smooth muscle/pericytes and constricted in vitro-perfused vessels. Pericyte depolarization by 100 mM extracellular KCl constricted DVR and increased pericyte intracellular Ca(2+) ([Ca(2+)](i)). The K(ATP) channel opener pinacidil (10(-7)-10(-4) M) hyperpolarized resting pericytes, repolarized pericytes previously depolarized by ANG II (10(-8) M), and vasodilated DVR. The DVR vasodilator bradykinin (10(-7) M) also reversed ANG II depolarization. The L-type Ca(2+) channel blocker diltiazem vasodilated ANG II (10(-8) M)- or KCl (100 mM)-preconstricted DVR, and the L-type agonist BayK 8644 constricted DVR. The plateau phase of the pericyte [Ca(2+)](i) response to ANG II was inhibited by diltiazem. These data support the conclusion that DVR vasoreactivity is controlled through variation of membrane potential and voltage-gated Ca(2+) entry into the pericyte cytoplasm.     

Role of chloride in constriction of descending vasa recta by angiotensin II

We investigated the dependence of ANG II (10(-8) M)-induced constriction of outer medullary descending vasa recta (OMDVR) on membrane potential (Psim) and chloride ion. ANG II depolarized OMDVR, as measured by fully loading them with the voltage-sensitive dye bis[1,3-dibutylbarbituric acid-(5)] trimethineoxonol [DiBAC(4)(3)] or selectively loading their pericytes. ANG II was also observed to depolarize pericytes from a resting value of -55.6 +/- 2.6 to -26.2 +/- 5.4 mV when measured with gramicidin D-perforated patches. When measured with DiBAC(4)(3) in unstimulated vessels, neither changing extracellular Cl(-) concentration ([Cl(-)]) nor exposure to the chloride channel blocker indanyloxyacetic acid 94 (IAA-94; 30 microM) affected Psim. In contrast, IAA-94 repolarized OMDVR pretreated with ANG II. Neither IAA-94 (30 microM) nor niflumic acid (30 microM, 1 mM) affected the vasoactivity of unstimulated OMDVR, whereas both dilated ANG II-preconstricted vessels. Reduction of extracellular [Cl(-)] from 150 to 30 meq/l enhanced ANG II-induced constriction. Finally, we identified a Cl(-) channel in OMDVR pericytes that is activated by ANG II or by excision into extracellular buffer. We conclude that constriction of OMDVR by ANG II involves pericyte depolarization due, in part, to increased activity of chloride channels.     

Chronic ANG II infusion increases NO generation by rat descending vasa recta

We tested whether chronic ANG II infusion into rats affects descending vasa recta (DVR) contractility, synthesis of superoxide, or synthesis of nitric oxide (NO). Rats were infused with ANG II at 250 ng.kg(-1).min(-1) for 11-13 days. DVR were loaded with dihydroethidium (DHE) to measure superoxide and 3-amino-4-aminomethyl-2',7'-difluorofluorescein (DAFFM) to measure NO. Acute constriction of DVR by ANG II (0.1, 1, and 10 nM) was diminished, and NO generation rate was raised by chronic ANG II infusion. DHE oxidation by DVR from ANG II-infused rats was similar to controls and was significantly higher when NO synthesis was prevented with N(omega)-nitro-L-arginine methyl ester (L-NAME). The superoxide dismutase mimetic Tempol (1 mM) increased NO generation compared with controls. The increased synthesis of NO by chronic ANG II-treated vessels persisted in the presence of Tempol. DVR endothelial cytoplasmic Ca(2+) response to ACh was diminished by chronic ANG II treatment, but the capacity of ACh to increase NO generation was unaltered. We conclude that DVR generation of superoxide is not affected by chronic ANG II exposure but that basal NO synthesis is increased. DVR superoxide is unlikely to be an important mediator of chronic ANG II slow pressor hypertension in rats.     

Inhibition of calcium signaling in descending vasa recta endothelia by ANG II

The intracellular calcium ([Ca(2+)](i)) response of outer medullary descending vasa recta (OMDVR) endothelia to ANG II was examined in fura 2-loaded vessels. Abluminal ANG II (10(-8) M) caused [Ca(2+)](i) to fall in proportion to the resting [Ca(2+)](i) (r = 0. 82) of the endothelium. ANG II (10(-8) M) also inhibited both phases of the [Ca(2+)](i) response generated by bradykinin (BK, 10(-7) M), 835 +/- 201 versus 159 +/- 30 nM (peak phase) and 169 +/- 26 versus 103 +/- 14 nM (plateau phase) (means +/- SE). Luminal ANG II reduced BK (10(-7) M)-stimulated plateau [Ca(2+)](i) from 180 +/- 40 to 134 +/- 22 nM without causing vasoconstriction. Abluminal ANG II added to the bath after luminal application further reduced [Ca(2+)](i) to 113 +/- 9 nM and constricted the vessels. After thapsigargin (TG) pretreatment, ANG II (10(-8) M) caused [Ca(2+)](i) to fall from 352 +/- 149 to 105 +/- 37 nM. This effect occurred at a threshold ANG II concentration of 10(-10) M and was maximal at 10(-8) M. ANG II inhibited both the rate of Ca(2+) entry into [Ca(2+)](i)-depleted endothelia and the rate of Mn(2+) entry into [Ca(2+)](i)-replete endothelia. In contrast, ANG II raised [Ca(2+)](i) in the medullary thick ascending limb and outer medullary collecting duct, increasing [Ca(2+)](i) from baselines of 99 +/- 33 and 53 +/- 11 to peaks of 200 +/- 47 and 65 +/- 11 nM, respectively. We conclude that OMDVR endothelia are unlikely to be the source of ANG II-stimulated NO production in the medulla but that interbundle nephrons might release Ca(2+)-dependent vasodilators to modulate vasomotor tone in vascular bundles.     

ANG II signaling in vasa recta pericytes by PKC and reactive oxygen species

ANG II constricts descending vasa recta (DVR) through Ca(2+) signaling in pericytes. We examined the role of PKC DVR pericytes isolated from the rat renal outer medulla. The PKC blocker staurosporine (10 microM) eliminated ANG II (10 nM)-induced vasoconstriction, inhibited pericyte cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)) elevation, and blocked Mn(2+) influx into the cytoplasm. Activation of PKC by either 1,2-dioctanoyl-sn-glycerol (10 microM) or phorbol 12,13-dibutyrate (PDBu; 1 microM) induced both vasoconstriction and pericyte [Ca(2+)](cyt) elevation. Diltiazem (10 microM) blocked the ability of PDBu to increase pericyte [Ca(2+)](cyt) and enhance Mn(2+) influx. Both ANG II- and PDBu-induced PKC stimulated DVR generation of reactive oxygen species (ROS), measured by oxidation of dihydroethidium (DHE). The effect of ANG II was only significant when ANG II AT(2) receptors were blocked with PD-123319 (10 nM). PDBu augmentation of DHE oxidation was blocked by either TEMPOL (1 mM) or diphenylene iodonium (10 microM). We conclude that ANG II and PKC activation increases DVR pericyte [Ca(2+)](cyt), divalent ion conductance into the cytoplasm, and ROS generation.     

Expression of TRPC4 channel protein that interacts with NHERF-2 in rat descending vasa recta

The PDZ domain adaptor protein Na⁺/H⁺ exchanger regulatory factor (NHERF)-2 is expressed in renal medullary descending vasa recta (DVR), although its function has not been defined. Transient receptor potential channels (TRPC) TRPC4 and TRPC5, nonselective cation channels that transport Ca²⁺, were recently demonstrated to complex with the NHERF proteins. We investigated whether TRPC4 and/or TRPC5 are associated with NHERF-2 in DVR. RT-PCR revealed mRNA for TRPC4 and NHERF-2, but not for TRPC5 or NHERF-1, in microdissected DVR. Immunohistochemical studies demonstrated expression of TRPC4 and NHERF-2 proteins in both the endothelial cells and pericytes. These proteins colocalized in some cells of the DVR. TRPC4 coimmunoprecipitated with NHERF-2 from renal medullary lysates, and NHERF-2 coimmunoprecipitated with TRPC4. TRPC5 was not detected in DVR with the use of immunohistochemistry or in NHERF-2 immunoprecipitates. We conclude that DVR pericytes and endothelia coexpress TRPC4 and NHERF-2 mRNA and protein and that these proteins colocalize and coimmunoprecipitate, indicating a possible physical association. These findings suggest that TRPC4 and NHERF-2 may play a role in interactions related to Ca²⁺ signaling. PDZ proteins; calcium channels; medulla; pericytes; endothelium; microcirculation     

Measurements on the kidneys and vasa recta of various mammals in relation to urine concentrating capacity.

Maximum urine-concentrating capacity (UCC) differs widely among mammals of different species, being very high in some desert species (e.g. kangaroo rats) and very low in freshwater acquatic species (e.g. beaver). In this study, kidneys of 21 species of mammals from widely different habitats were studied in histological sections to determine whether differences in UCC can be attributed to differences in kidney structure. Parameters studied included the ratio of medullary to cortical thickness, the proportional subdivision of the medulla into inner and outer zones, and the dimensions of the vasa recta expressed in terms of the total area and the number of lumens within the vascular bundles. Determinations were made at a level where the size of individual vasa recta bundles has reached a constant maximum size, i.e. in the distal half of the outer zone. A positive correlation was found between the UCC and the ratio of medullary length to cortical thickness. No clear correlation existed between the proportion of the medullary length comprised of outer or inner zones and the UCC, although a trend to higher UCC in animals with relatively longer inner zones was apparent. Thus, it appears that the relative length of the entire medullary region is a major factor determining UCC, but the length of individual medullary zones is of lesser importance. A correlation was also found between the density of vasa recta per cubic millimeter of medullary tissue (the number of lumens regardless of identify in bundles, based on the number counted at the level sampled) and the UCC of the species. Data reported here support the view that UCC can be correlated with two parameters of kidney structure - the length of medulla relative to that of cortex and the density of vasa recta within the outer zone. It is proposed that the anatomical characteristics of the vascular supply to the medulla - that is, the vasa recta - are equally as important for the concentration of urine as is the primary mechanism determined by the characteristics of the loop of Henle and collecting ducts.     

ANG II AT2 receptor modulates AT1 receptor-mediated descending vasa recta endothelial Ca2+ signaling

We tested whether the respective angiotensin type 1 (AT(1)) and 2 (AT(2)) receptor subtype antagonists losartan and PD-123319 could block the descending vasa recta (DVR) endothelial intracellular calcium concentration ([Ca(2+)](i)) suppression induced by ANG II. ANG II partially reversed the increase in [Ca(2+)](i) generated by cyclopiazonic acid (CPA; 10(-5) M), acetylcholine (ACh; 10(-5) M), or bradykinin (BK; 10(-7) M). Losartan (10(-5) M) blocked that effect. When vessels were treated with ANG II before stimulation with BK and ACh, concomitant AT(2) receptor blockade with PD-123319 (10(-8) M) augmented the suppression of endothelial [Ca(2+)](i) responses. Similarly, preactivation with the AT(2) receptor agonist CGP-42112A (10(-8) M) prevented AT(1) receptor stimulation with ANG II + PD-123319 from suppressing endothelial [Ca(2+)](i). In contrast to endothelial [Ca(2+)](i) suppression by ANG II, pericyte [Ca(2+)](i) exhibited typical peak and plateau [Ca(2+)](i) responses that were blocked by losartan but not PD-123319. DVR vasoconstriction by ANG II was augmented when AT(2) receptors were blocked with PD-123319. Similarly, AT(2) receptor stimulation with CGP-42112A delayed the onset of ANG II-induced constriction. PD-123319 alone (10(-5) M) showed no AT(1)-like action to constrict microperfused DVR or increase pericyte [Ca(2+)](i). We conclude that ANG II suppression of endothelial [Ca(2+)](i) and stimulation of pericyte [Ca(2+)](i) is mediated by AT(1) or AT(1)-like receptors. Furthermore, AT(2) receptor activation opposes ANG II-induced endothelial [Ca(2+)](i) suppression and abrogates ANG II-induced DVR vasoconstriction.     

Mouse LYVE-1 is an endocytic receptor for hyaluronan in lymphatic endothelium

The glycosaminoglycan hyaluronan is a key substrate for cell migration in tissues during inflammation, wound healing, and neoplasia. Unlike other matrix components, hyaluronan (HA) is turned over rapidly, yet most degradation occurs not locally but within distant lymph nodes, through mechanisms that are not yet understood. While it is not clear which receptors are involved in binding and uptake of hyaluronan within the lymphatics, one likely candidate is the lymphatic endothelial hyaluronan receptor LYVE-1 recently described in our laboratory (Banerji, S., Ni, J., Wang, S., Clasper, S., Su, J., Tammi, R., Jones, M., and Jackson, D.G. (1999) J. Cell Biol. 144, 789-801). Here we present evidence that LYVE-1 is involved in the uptake of hyaluronan by lymphatic endothelial cells using a new murine LYVE-1 orthologue identified from the EST data base. We show that mouse LYVE-1 both binds and internalizes hyaluronan in transfected 293T fibroblasts in vitro and demonstrate using immunoelectron microscopy that it is distributed equally among the luminal and abluminal surfaces of lymphatic vessels in vivo. In addition, we show by means of specific antisera that expression of mouse LYVE-1 remains restricted to the lymphatics in homozygous knockout mice lacking a functional gene for CD44, the closest homologue of LYVE-1 and the only other Link superfamily HA receptor known to date. Together these results suggest a role for LYVE-1 in the transport of HA from tissue to lymph and imply that further novel hyaluronan receptors must exist that can compensate for the loss of CD44 function.     

3HOH-osmotic water fluxes and ultrastructure of an epithelial syncytium

Summary Ultrastructural changes associated with osmotically-induced water transport and water permeability were examined in two flatworm species,Schistosoma mansoni andHymenolepis diminuta. The structure of the surface layer of these parasites is unusual in that it is a syncytial epithelial layer that lacks tight junctions and lateral extracellular spaces. The permeability coefficients observed in this study are therefore necessarily associated only with the transcellular route of transepithelial transport. The ultrastructural changes associated with volume transport across the epithelial syncytium were also unusual in that the basally located channels extending distally from the inward-facing membrane into the syncytial layer remained open regardless of the direction of water flow.Despite the structural differences, most of the features of diffusive (P _d ) and osmotic (P _osm ) water fluxes across the syncytium resembled those observed in other epithelia: (i) Low water permeability with maximum values of 4.1×10^–5 forP _d and 9.6×10^–5 forP _osm.(ii)P _osm>P _d by 2.0- to 3.2-fold. (iii) Outward water permeability less than inward water permeability. This asymmetry could not be attributed to collapsing channels when net volume transport was directed outward since channels in the syncytium remained open regardless of the direction of water flow. The asymmetry could be explained by tissue contraction or swelling when bathed in anisotonic fluids. (iv)P _osm values were not significantly altered by tissue unstirred layers but bothP _osm andP _d values were underestimated when the bulk fluid was not vigorously stirred.The lower permeability inS. mansoni relative toH. diminuta may be attributed to the membranous surface coat of the former species.     

Propagation of electrical activity in the nerve cord and muscle syncytium of the nematodeAscaris lumbricoides

Summary The functional muscle syncytium overlying the nerve cord inAscaris lumbricoides is preferentially excited by anodal stimulation with an extracellular electrode. Cathodal stimulation preferentially excites the nerve cord, allowing determination of separate conduction velocities for the nerve cord and syncytium. The propagation velocity of the nerve cord is 16.2±1.2 cm/s; that of the syncytium varies with Ca²⁺ concentration, being 21.6±1.3 cm/s under normal conditions (Figs. 5, 6). Both values are too high to account for the propagation of contractile waves in the intact animal.Modulation of spontaneous electrical activity on a behaviorally significant time scale is observed. The modulation shows dorsal-ventral coordination if the right lateral line is intact, in accord with known nervous system asymmetry (Fig. 10).We thank Mac McGlaughlin for help in obtainingAscaris, Professor Felix Strumwasser for the loan of the CAT, and Dr. Lou Byerly for assistance in statistical analysis and in the design of the trigger circuit. This work was supported by a Sloan Foundation Grant in Neuroscience and a U.S. Public Health Service Grant (NS 09654) to R.L.R., and by a N.I.H. Traineeship on Grant BCH 5 Tol GM 01262-12 to D.A.W.     

Aligned human cardiac syncytium for in vitro analysis of electrical,structural, and mechanical readouts

Human pluripotent stem cell‐derived cardiomyocytes (hPSC‐CMs) have emerged as an exciting new tool for cardiac research and can serve as a preclinical platform for drug development and disease modeling studies. However, these aspirations are limited by current culture methods in which hPSC‐CMs resemble fetal human cardiomyocytes in terms of structure and function. Herein we provide a novel in vitro platform that includes patterned extracellular matrix with physiological substrate stiffness and is amenable to both mechanical and electrical analysis. Micropatterned lanes promote the cellular and myofibril alignment of hPSC‐CMs while the addition of micropatterned bridges enable formation of a functional cardiac syncytium that beats synchronously over a large two‐dimensional area. We investigated the electrophysiological properties of the patterned cardiac constructs and showed they have anisotropic electrical impulse propagation, as occurs in the native myocardium, with speeds 2x faster in the primary direction of the pattern as compared to the transverse direction. Lastly, we interrogated the mechanical function of the pattern constructs and demonstrated the utility of this platform in recording the strength of cardiomyocyte contractions. This biomimetic platform with electrical and mechanical readout capabilities will enable the study of cardiac disease and the influence of pharmaceuticals and toxins on cardiomyocyte function. The platform also holds potential for high throughput evaluation of drug safety and efficacy, thus furthering our understanding of cardiovascular disease and increasing the translational use of hPSC‐CMs.     

Hypoxic pulmonary vasoconstriction is not endothelium dependent.

Feline intrapulmonary arteries (mean diameter, 0.9 mm) were equilibrated in Earle's solution at constant tension in a chamber bubbled with an hyperoxic gas mixture (30% oxygen, 5% carbon dioxide, balance nitrogen). The endothelium was removed from half the vessels by gentle rubbing. The isometric response to the addition of acetylcholine (1*10(-6) M) was dilator in the vessels with endothelium and constrictor in those without endothelium. Intermittent exposure to a hypoxic gas mixture (0% oxygen, 5% carbon dioxide, balance nitrogen) for 20 min with five repetitions demonstrated sustained constrictor responses in the presence or absence of endothelium. Endothelial cells are, therefore, not required for the mediation of hypoxic pulmonary vasoconstriction.