Project 3: Sympathetic Neurotransmitters and Ouabain Hypertension

Project Leader: Withrow G. Wier, PhD

Ouabain-induced hypertension is characterized by hyperactivity of the sympathetic nervous system and increased contraction of vascular smooth muscle. It may also involve changes in sympathetic neuromuscular transmission in small arteries.

The proposed research aims first to determine certain basic mechanisms of sympathetic transmitter release in small arteries, and then, to determine how these are affected by ouabain. Basic premises of the research are i) that sympathetic neuromuscular transmission and arterial contraction importantly involve the two co-transmitters, ATP and nor-epinephrine (NE) and, ii) that these two neurotransmitters are differentially released by Ca²⁺ dependent mechanisms, possibly involving different synaptic vesicles, ‘residual [Ca²⁺]’ and stored Ca²⁺. 

An overall hypothesis on mechanisms of ouabain actions is that inhibition of nerve terminal Na⁺ pumps increases ‘residual’ [Ca²⁺] and/or stored Ca²⁺ (thereby increasing NE and ATP release) and that the increases in terminal [Ca²⁺] are mediated by Na/Ca exchange. To test this hypothesis we are determining the probabilities, at individual sympathetic nerve varicosities, of ATP and NE release and measuring the sizes of ATP and NE transmitter packets (‘quanta’).  

We are also testing the hypotheses that differential release of NE and ATP results from the release of different types of synaptic vesicles and that acute, low-dose, ouabain inhibits the α₃-isoform of the Na⁺/K⁺ATPase in sympathetic varicosities and changes the probability of release, but not quantal size. We are determining whether release probability or quantal size is altered in ouabain hypertensive rats. 

Figure 1.  Junctional Ca²⁺ transients in pressurized small artery. (A) Images of perivascular nerves and smooth muscle cells (SMC). The image is an average
of 30 frames (1 s total), from a set of 600 images obtained with a real-time
confocal microscope during EFS.   (B) A virtual line-scan image was derived
from this data, by taking a single line of pixels, extending through the image
vertically, between the two black arrowheads in A,  from each of the 600 frames.
The chosen line crosses prominent nerve fibers at the top of the image, bisects
a smooth muscle cell longitudinally, and crosses a region (white arrow in A) in
which a small nerve fiber crosses the muscle cell.  In the line-scan image, nerve
fiber Ca²⁺ transients in the large fibers at the top of are seen as periodic (0.5 s^-1)
increases in fluorescence occurring at the times of the stimulus pulses (black arrowheads).  A jCaT is seen in the SMC at 4.8 s in the line-scan image. (C) jCaTs
often arise at streaks in line-scan images. (D) A region where a jCat occurred was
scanned repeatedly. In (a) a jCat occurred. In (b) no jCaT occurred, but a
fluorescence transient occurred in the streak. We identify such transients as nerve
fiber Ca²⁺ transients. These images demonstrate that jCaTs can arise precisely in the region of an electrically excitable nerve fiber (from Lamont & Wier, 2002, References). 

Rat and mouse mesenteric small arteries are loaded with fluorescent Ca2+ indicators and studied in a myograph that permits simultaneous confocal fluorescence imaging, electrical stimulation/recording, and recording of
isometric force development.

Figure 2. The effects of prazosin on neurogenic constrictions of WT and KO arteries. A: two  superimposed constrictions of a WT artery before (black) and after (red) application of 1 _M prazosin. B: two superimposed constrictions of a P2X1-null artery before (black) and after (red) application of 1 _M prazosin. The constrictions were all elicited by 1 min of EFS (16 Hz) at the
times indicated by the black bars under the traces.

Mice with genetically altered  Na/Ca exchangers or ATP receptors are used. Junctional Ca²⁺ transients (jCaTs) are used to measure neurally released ATP. Carbon fiber microelectrodes and amperometry are used to measure NE release as ‘NE oxidation currents’ (NEOCs). The research measures NE and ATP release together for the first time and thereby determines some of the basic mechanisms that control neurogenic contractions of arteries. It will elucidate the mechanisms by which sympathetic nerves contribute to ouabain-induced hypertension.

References

Lamont, C., Vial, C., Evans, R.J. and W.G. Wier P2X1 receptors mediate sympathetic post-junctional Ca²⁺ transients (jCaTs) in mesenteric small arteries. Am J Physiol Heart Circ Physiol. 2006 Aug 18; [Epub ahead of print] PMID: 16920810 [PubMed - as supplied by publisher]. http://ajpheart.physiology.org/cgi/reprint/00466.2006v1

Lamont, C., and W.G. Wier. Different roles of ryanodine receptors and inositol (1,4,5)-trisphosphate receptors in adregeneric stimulated contractions of small artieres. Am J Physiol Heart Circ Physiol. 287:H617-H625, 2004. http://ajpheart.physiology.org/cgi/reprint/287/2/H617

Lamont, C., Vainorius, E., and W.G. Wier.  Purinergic and adrenergic Ca²⁺ transients during neurogenic contractions of rat mesenteric small arteries. J Physiol. 549(Pt 3):801-808, 2003. http://jp.physoc.org/cgi/reprint/549/3/801

Lamont, C., and W.G. Wier. Evoked and spontaneous purinergic junctional Ca²⁺ transients (jCaTs) in rat small arteries. Circ Res. 91:454-456, 2002. http://circres.ahajournals.org/cgi/reprint/91/6/454

Wier, W.G., Zang, W.J., Lamont. C., and H. Raina.  Sympathetic Neurogenic Ca²⁺ Signaling in Arteries: ATP, Noradrenaline and NPY. Exp Physiol. 2008 Oct 17. [Epub ahead of print]

Li, M., Zacharia, J., Sun, Xx, and W.G. Wier.  Effects of siRNA knock-down of TRPC6 and InsP(3)R1 in vasopressin-induced Ca ²⁺ oscillations of A7r5 vascular smooth muscle cells.  Pharmacol Res. 2008 Sep 13. [Epub ahead of print] http://www.sciencedirect.com/science

Blaustein, M.P. and W.G. Local sodium, global reach: filling the gap between salt and hypertension.  Circ Res. 101:959-961, 2007. http://circres.ahajournals.org/cgi/reprint/101/10/959

Zacharia, J., Zhang, J., and W.G. Wier. Ca ²⁺ signaling in mouse mesenteric small arteries: myogenic tone and adrenergic vasoconstriction.  Am J Physiol Heart Circ Physiol. 292:H1523-32, 2007. 2007http://ajpheart.physiology.org/cgi/reprint/292/3/H1523

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