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Project 3

Sympathetic Neurotransmitters & Ouabain Hypertension

Project Leader

Dr. Wier
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

calcium transients
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

myograph
 

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. 

Dr. Wier's Lab

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 

working in the lab

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. http://ajpheart.physiology.org/cgi/reprint/292/3/H1523