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Project 4

Ouabain & Descending Vasa Recta Ca2+ Signaling

Project Leader

Dr. Pallone
Thomas L. Pallone, MD 

The microcirculation of the renal medulla traps NaCl and urea deposited to the interstitium by the loops of Henle and collecting ducts and distributes blood flow to a hypoxic region of the kidney. Evidence links medullary perfusion to regulation of salt and water excretion, hypertension and genesis of acute renal failure. 

Descending vasa recta (DVR) are 15 micron arteriolar microvessels through which blood flow reaches the renal medulla. DVR are transporting, capillary sized vessels that express the AQP1 water channel and UTB facilitated urea transporter. DVR vasoactivity is controlled by contractile pericytes and adjacent endothelia (Figure 1) (Zhang Q et al in press, 2006). 

We have established methods to study Ca2+ signaling and channel architecture in those cells (Zhang Z et al, 2005; Lee-Kwon, W et al, 2006). Past studies reveal that endothelial Ca2+ signaling is stimulated by vasodilators and inhibited by angiotensin II. Maneuvers affecting cellular Na+ (Na+/K+ ATPase inhibition and extracellular Na+ reduction) strongly modulate DVR endothelial Ca2+  (Figures 2, 3) (Pittner J et al., 2006). We are testing the hypothesis that high ouabain affinity Na+ pump isoforms and Na+/Ca2+ exchange (NCX) modulate DVR endothelial Ca2+ signaling, participate in AngII signaling and become deranged in the chronic ouabain hypertensive rat.

  • In Aim 1 of the Project 4, we are testing whether Na+K+ATPase high affinity isoforms (α2/α3) and NCX are confined to subplasmalemmal microdomains that affect Ca2+ signaling. We are examining colocalization of signaling proteins with SR/ER using immunofluorescence. We are determining whether ouabain inhibition, α2 Na+ pump deficiency, reduction of extracellular Na+ ([Na+]e) and NCX blockade modulate DVR endothelial [Ca2+]CYT responses to vasodilators (Pittner et al., 2006).
  • In Aim 2, we are using low affinity fluorescent Ca2+ probes (furaFF, furaptra) to determine the effect of the maneuvers in Aim 1 on store Ca2+ sequestration. 
  • woman at microscope
  • In Aim 3, we are investigating the mechanisms responsible for AngII inhibition of DVR endothelial [Ca2+]CYT responses to sarcoplasmic / endoplasmic release Ca2+ (SERCA) pump inhibition and vasodilators. We are characterizing Ca2+ entry pathways in DVR endothelia and testing whether AngII inhibits them.  We are testing for roles of NCX and Ca2+ store sequestration in the AngII induced reduction of endothelial [Ca2+]CYT.
  • In Aim 4 we are following up our observation that DVR endothelial dysfunction accompanies chronic ouabain hypertension (OH) in the rat. We are testing whether endothelial Ca2+ responses and NO release are altered in OH rats and whether Na+ pump isoforms are down-regulated. We are testing whether DVR contractions to norepinephrine, AngII and KCl are increased, measuring NO production and assessing effects of OH on endothelial and pericyte Ca2+ signaling.

Figure 1

immunostaining
Immunostaining of Cx40.  Panels show immunostaining with antibody directed against Cx40 (left, green), α-smooth muscle actin (SMA, red) and a corresponding white light image (right). Cx40 shows linear staining confined to the endothelium with very little SMA colocalization.  Abluminal pericytes are identified by the red SMA.  Bar = 10 microns.
 

Figure 2

fluorescence ratio 
DVR endothelial [Ca2+]CYT changes evoked by ouabain. Vessels exposed to log-molar increasing concentrations of ouabain from 10-10 to 10-6 M (n = 9). Panel A: representative recording shows background subtracted fluorescent ratios from fura-2 excitation. Panel B: Peak (solid bars) and plateau (open bars, at 5 minutes) [Ca2+]CYT expressed as % elevations from baseline.
 

Figure 3

Ca2 timing 
Effect of ouabain on DVR endothelial [Ca2+]CYT elevation evoked by bradykinin. Panel A: DVR exposed to BK for 5 minutes to elicit a baseline response.  Subsquently vehicle (n = 6), or ouabain (500 nM, n = 9) was introduced for 5 minutes and BK applied a second time. The second BK induced [Ca2+]CYT response was higher after ouabain (, P < 0.05).  Panel B: Summary of area under the curve comparisons of [Ca2+]CYT during the first and second exposures to BK (mean ± SE, , P < 0.05). Ouabain enhanced the peak-phase BK induced [Ca2+]CYT response.
 

References

researchers in lab  

Zhang Z, Cao C, Lee-Kwon W, Pallone TL. Voltage gated sodium conductance in descending vasa recta pericytes. J. Physiol. 567:445-457, 2005.

Pittner J, Rhinehart K, Pallone TL. Ouabain modulation of endothelial calcium signaling in descending vasa recta.  Am J Physiol Renal Physiol 291:F761-F769, 2006.

Zhang Q, Cao C, Mangano M, Zhang Z, Silldorff EP, Lee-Kwon W, Pallone TL. Descending vasa recta endothelium is an electrical syncytium. Am J Physiol Regulatory Integrative Physiol 291(6):R1688-99, 2006.

Lee-Kwon W, Goo JH, Zhang Z, Silldorff EP, Pallone TL. Vasa recta voltage gated Na+ channel NaV1.3 is regulated by calmodulin. Am J Physiol Renal Physiol 292 (1):F404-F414, 2007.

Cao, C, Payne, K, Lee-Kwon, W, Zhang, Z, Lim, SW, Hamlyn, JM, Blaustein, MP, Kwon, HM, and Pallone, TL. Chronic ouabain treatment induces vasa recta endothelial dysfunction in the rat.  Am J Physiol Renal Physiol (2008 in press).