Expression of Ryanodine Receptor 3 and Trp Channels in Endothelium of Human Mesenteric Artery: A Single-Cell Rt-Pcr and Patch-Clamp Analysis in Situ .

Hypertension ◽  
2000 ◽  
Vol 36 (suppl_1) ◽  
pp. 719-719
Author(s):  
Joachim Hoyer ◽  
Meike Kuehn ◽  
Christiane Degenhardt ◽  
Norbert Runkel ◽  
Martin Paul ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Endothelial Function ◽  
Single Cell ◽  
Blood Vessels ◽  
Human Blood ◽  
Mesenteric Artery ◽  
Trp Channels ◽  
Rt Pcr ◽  
Store Depletion

P146 Ca 2+ mobilization plays an important role in endothelial function by stimulating Ca 2+ -dependent synthesis of vasodilating factors. In addition to InsP 3 - mediated Ca 2+ store depletion, Ca 2+ release from ryanodine-sensitive pools and Ca 2+ -influx through cation channels of the TRP-gene family have been suggested to be involved in endothelial Ca 2+ signaling. In cultured endothelial cells (EC) the function and expression of ryanodine-receptors (RyR) and TRP channels might differ substantially from those in native endothelium of human blood vessels. We, therefore, characterized expression and function of RyR and TRP channels in EC of small human mesenteric artery (MA) by use of single-cell RT-PCR and patch-clamp techniques in situ. MA were isolated from colon specimens of patients subjected to hemicolectomy. For single-cell RT-PCR and PC experiments single human mesenteric artery EC (HMAEC) were harvested directly from the luminal vessel with the patch pipette. Expression of the RyR subtype 3, but not of the RyR1 and RyR2 subtpyes was detected in 25% of HMAEC samples. Correspondingly, in PC experiments in HMAEC, application of caffeine (0.5 mM) induced Ca 2+ -release from ryanodine-sensitive stores and subsequently activation of Ca 2+ -activated K + channels leading to a sustained endothelial hyperpolarization from a resting potential of 28 ± 3 mV to -46 ± 4 mV. Single HMAEC expressed the TRP subtypes, TRP1 and TRP3, but not TRP 4 and 6. The TRP1 was the predominantly expressed TRP subtype as expression was detected in 16% of HMAEC. TRP3 expression was detected in only 3% of HMAEC. In single-channel and whole-cell patch clamp experiments in HMAEC, InsP 3 -mediated Ca 2+ -store depletion in response to bradykinin (100 nM) activated TRP related nonselective cation currents leading to Ca 2+ entry. In conclusion, Ca 2+ release from ryanodine-sensitive stores mediated by RyR3 and Ca 2+ entry through TRP1 might represent important components of endothelial Ca 2+ signaling and thereby of endothelial function in intact human blood vessels.

The Distribution of Vasculokinase in Human Blood Vessels

1962 ◽  
Vol 08 (01) ◽  
pp. 096-100
Author(s):  
Marvin Murray ◽  
Robert Johnson
Keyword(s):  
Inferior Vena Cava ◽  
Superior Mesenteric Artery ◽  
Blood Vessels ◽  
Human Blood ◽  
Iliac Artery ◽  
Popliteal Artery ◽  
Mesenteric Artery ◽  
Inferior Vena ◽  
Vena Cava ◽  
High Concentrations

Summary133 blood vessels were evaluated for vasculokinase concentration in the freshly morbid state. High concentrations of activity were found in the aorta, iliac artery, superior mesenteric artery and popliteal artery. Activity was occasionally found in the inferior vena cava and common iliacs veins. Other vessels evaluated had no activity. Evaluation of the data with respect to vas-culokinase activity and atherosclerosis suggests higher levels of vasculokinase in those vessels having atherosclerosis.

scholarly journals Combined single cell RT-PCR analysis with patch clamp recording in acute brain slices

2000 ◽  
Vol 82 ◽  
pp. 12
Author(s):  
Keisuke Tsuzuki ◽  
Bertrand Lambolez ◽  
Etienne Audinat ◽  
James T. Porter ◽  
Bruno Cauli ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Single Cell ◽  
Brain Slices ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Patch Clamp Recording

Heterogeneous distribution of chloride channels along the distal convoluted tubule probed by single-cell RT-PCR and patch clamp

2004 ◽  
Vol 287 (6) ◽  
pp. F1233-F1243 ◽  
Author(s):  
Antoine Nissant ◽  
Stéphane Lourdel ◽  
Sophie Baillet ◽  
Marc Paulais ◽  
Pedro Marvao ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Single Cell ◽  
Distal Convoluted Tubule ◽  
Transport Systems ◽  
Rt Pcr ◽  
Heterogeneous Distribution ◽  
Anion Selectivity ◽  
Basolateral Side

The distal convoluted tubule (DCT) is a heterogeneous segment subdivided into early (DCT1) and late (DCT2) parts, depending on the distribution of various transport systems. We do not have an exhaustive picture of the Cl− channels on the basolateral side: the presence of ClC-K2 channels is generally accepted, whereas that of ClC-K1 remains controversial. We used here single-cell RT-PCR and patch clamp to probe Cl− channel heterogeneity in microdissected mouse DCT at the molecular and functional levels. Our findings show that 63% of the DCT cells express ClC-K2 mRNA, either alone (type 1 cells: 47 and 23% in DCT1 and DCT2, respectively), or combined with ClC-K1, mostly in DCT2 (type 2 cells: 33%), but 37% of DCT1 and DCT2 cells do not express any ClC-K. Patch-clamp experiments revealed that a Cl− channel, with 9-pS conductance and Cl− > NO3− = Br− anion selectivity sequence, is present in the DCT1 and DCT2 basolateral membranes (87 and 71% of the patches, respectively). This dominant channel is likely to be ClC-K2 in type 1 cells. In type 2 cells, it could be ClC-K2 and/or ClC-K1 homodimers, but also ClC-K1/ClC-K2 heterodimers, or a mixture of all combinations. A second, distinct Cl− channel (13% of DCT1 patches, 29% of DCT2 patches) also displayed 9-pS conductance but had a completely different anion selectivity (I− > NO3− > Br− > Cl−), which was not compatible with that of the ClC-Ks. This indicates that a Cl− channel that is unlikely to belong to the ClC family may also be involved in Cl− absorption in the DCT2.

Single cell RT-PCR analysis of ClC-2 mRNA expression in ureteric bud tip

1998 ◽  
Vol 274 (5) ◽  
pp. F951-F957 ◽  
Author(s):  
Stephan Huber ◽  
Bernd Schröppel ◽  
Matthias Kretzler ◽  
Detlef Schlöndorff ◽  
Michael Horster
Keyword(s):  
Patch Clamp ◽  
Single Cell ◽  
Collecting Duct ◽  
Pcr Analysis ◽  
Ureteric Bud ◽  
Rt Pcr ◽  
Outer Cortex ◽  
Mrna Isoforms ◽  
Pcr Products ◽  
Pcr Techniques

Embryonic epithelia at the tip of the ureteric bud (UB) face the interspace between epithelial and mesenchymal cells and are fundamentally involved in reciprocal signaling during early nephrogenesis. To characterize their membrane conductive proteins, patch-clamp and single cell RT-PCR techniques were applied to embryonic rat UBs [embryonic day 17( day E17)] microdissected from the outer cortex. Cells at the UB tip had a high whole cell conductance (14 ± 2 nS/10 pF, n = 8). The main fractional conductance resembled that of Ca-activated Cl channels in nonepithelial cells, with its time-dependent activation at depolarizing and inactivation at hyperpolarizing voltages. A second Cl-selective current fraction, by contrast, activated slowly during strong hyperpolarization, suggestive of a ClC-2-mediated conductance. To determine the origin of this current, cytoplasm was harvested into the patch pipette, RNA was reverse transcribed, and cDNA encoding the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeper gene or the ClC-2 Cl channel was amplified by polymerase chain reaction (PCR). GAPDH and ClC-2 PCR products were identified in 23 and 8 (out of a total of 57) single cell cDNA samples, respectively. ClC-2 PCR products with two different lengths were obtained, which might be due to two alternatively spliced ClC-2 mRNA isoforms. This first and combined approach by patch-clamp and single cell RT-PCR techniques to embryonic epithelia indicates that 1) cells at the UB tip express a phenotype remarkably different from that of postembryonic collecting duct principal cells and that 2) ClC-2 is likely to have a key role in early nephrogenesis.

scholarly journals Regenerative Translation of Human Blood-Vessel-Derived MSC Precursors

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
William C. W. Chen ◽  
Bruno Péault ◽  
Johnny Huard
Keyword(s):  
Blood Vessel ◽  
Blood Vessels ◽  
Human Blood ◽  
Current Status ◽  
Tissue Repair And Regeneration ◽  
Isolation And Characterization ◽  
Adventitial Cells ◽  
Preclinical Animal Models

Mesenchymal stem/stromal cells (MSCs) represent a promising adult progenitor cell source for tissue repair and regeneration. Their mysterious identityin situhas gradually been unveiled by the accumulating evidence indicating an association between adult multipotent stem/progenitor cells and vascular/perivascular niches. Using immunohistochemistry and fluorescence-activated cell sorting, we and other groups have prospectively identified and purified subpopulations of multipotent precursor cells associated with the blood vessels within multiple human organs. The three precursor subsets, myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are located, respectively, in the three structural tiers of typical blood vessels: intima, media, and adventitia. MECs, PCs, and ACs have been extensively characterized in prior studies and are currently under investigation for their therapeutic potentials in preclinical animal models. In this review, we will briefly discuss the identification, isolation, and characterization of these human blood-vessel-derived stem cells (hBVSCs) and summarize the current status of regenerative applications of hBVSC subsets.

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