MOLECULAR DISSECTION OF DOPAMINE D5 RECEPTOR ACTIONS

In addition to its importance as a neurotransmitter, dopamine has
profound effects on heart rate, blood pressure, renal blood flow, sodium
absorption and water retention. In the clinical setting dopamine is used
to treat patients suffering from shock or impaired renal function. At
low doses the renal effects of dopamine predominate and are mediated by
two receptor subtypes: DA1 and DA2. Little is known about renal DA2
receptor physiology. In contrast, DA1 dopamine receptors mediate
natriuresis and diuresis in the tubules of the renal cortex and
vasodilation of the mesenteric and renal vascular beds. DA1 receptors
in proximal convoluted tubule (PCT) epithelial cells couple to the
stimulation of cAMP and diacylglycerol (DAG) production. Increases in
the concentration of these two second messengers activate protein kinases
A and C, respectively. Two targets of these kinases are the luminal
Na+/H+ exchanger (NHE) and the basolateral Na+-K+ATPase. Both the NHE
and the ATPase are involved in sodium transport but it is the
phosphorylation and subsequent inhibition of Na+/H+ exchanger activity
that results in sodium excretion (natriuresis). Our results suggest that
the dopamine D5 receptor that we recently cloned is identical to the
renal PCT DA1 receptor subtype. We propose that dopamine D5 receptors
in the renal PCT epithelia couple to second messenger systems that
participate in the regulation of Na+/H+ exchanger activity. Therefore,
any interference with the ability of D5/Da1 receptor's to regulate sodium
transport may play an important role in the etiology of certain forms of
essential hypertension. We propose to test several aspects of this
hypothesis. Recently we demonstrated that activated dopamine D5
receptors stimulate cAMP production and the secretion of H+. This latter
effect is sensitive to amiloride suggesting that a Na+/H+ exchanger (NHE)
activity is involved. We propose to pursue the in vitro characterization
of D5's coupling to adenylyl cyclase and phospholipase C. In addition,
we have the opportunity to develop a very powerful in vitro system in
which to dissect D5's regulation of NHE activity. Cell lines expressing
each of the three recently cloned NHEs and the D5 receptor will be
evaluated with respect to dopaminergic regulation of sodium transport.
We also have evidence to suggest that recombinant vaccinia virus vectors
can be used to generate polyclonal anti-receptor antiserum. We will use
this technology to produce anti-D5 antiserum, a valuable reagent for the
analysis of dopamine D5 receptor expression and post translational
modification in normal and diseased renal cortex tissue. Abnormal renal
responses to dopamine have also been reported in two well-documented rat
models of inherited hypertension and we are now in a unique position to
determine whether the genetic defect lies within the dopamine D5 receptor
gene. finally we intend to generate transgenic mice that lack functional
dopamine D5 receptors. The production of these "knockout" mice will
provide a new and powerful mouse model system in which the role of
dopamine D5 receptors in renal physiology in general, and sodium
transport in particular, can be evaluated.