Ambulatory blood pressure monitoring is more useful for the diagnosis of various forms of hypertension including white coat and morning surge as well as other conditions that are associated with increased morbidity and mortality such as sleep apnea. Nocturnal changes in blood pressure (BP) can also aid in the diagnosis of various manifestations of blood pressure independent of hypertension known as salt sensitivity (SS) and inverse salt sensitivity (ISS) of blood pressure. SS individuals experience an increase in BP on a high salt diet while ISS individuals experience a paradoxical increase in BP on a low salt diet. SS and ISS phenotypes affect approximately 18% and 15% of normotensives, respectively, which may result in significant morbidity and mortality similar to untreated hypertension. Consuming a personally appropriate salt diet can result in a circadian drop in BP during sleep, and failure to “dip” can lead to significant cardiovascular diseases. Nocturnal dipping is usually recorded using an ambulatory blood pressure monitoring (ABPM) device. Arterial pulse wave measurements using oscillatory photoplethysmography (OP) are minimally invasive when compared to an inflatable cuff, which can disturb sleep and raise blood pressure. We measured nocturnal blood pressure using a ring based photoplethysmograph (SensoGram Technologies, Plano Texas). A UVA Salt Study participant wore their device upon going to bed and then uploaded nocturnal data to the internet each morning. Systolic and diastolic BP is capable of being measured 30 times a minute for approximately 9 hours and providing over 5000 data points each night. Three representative dipping profiles for an individual yielded a dipping reduction of 24% ± 3% for systolic and 40% ± 10% for diastolic (1 - mid-sleeping lowest value/start of evening highest value). This additional data afforded by nocturnal BP measurement is anticipated to improve diagnostic opportunities in the measurement of SS and ISS phenotypes and provide the user with reassurance that nocturnal BP dipping is occurring.

The renal dopaminergic D 1 receptor (D 1 R) regulates sodium excretion which is terminated by phosphorylation by G protein-coupled receptor kinases 4 (GRK4). GRK4 gene variants are associated with increased GRK4 activity and reduced sodium excretion resulting in hypertension. Breast cancer incidence is higher in hypertensive women. We found that GRK4 is a potential molecule linking these two diseases. We hypothesized that GRK4 inhibitors would be beneficial to patients with hypertension and breast cancer. Three potential GRK4 inhibitors (compounds A, B, and C) were tested for their effect on the growth of breast cancer cells MDA-MB-468, MCF-7, and benign mammary epithelial cells MCF-10A controls. These cell lines had high, low, and no GRK4 expression respectively. Growth of MDA-MB-468 and MCF-7 cells was effectively inhibited by compound C with IC 50 16.8±1.9 nM (n=2). MCF-10A cells were relatively resistant to compound C with IC 50 49.3±4.0 nM (n=3) that is significantly higher than the cancer cells (p<0.001). Compound B was the least effective inhibitor in all three cell lines (IC 50 was 1.5-2.3 μM). Growth inhibition of compound A was similar in MCF-7 and MCF-10A cells but less effective in MDA-MB-468 cells indicating GRK4 inhibition may not be the only target for growth inhibition of this compound. It has been reported that D 1 R agonists inhibit growth of breast cancer cells. We hypothesized that blockade of GRK4 would increase sensitivity of breast cancer cells to the inhibitory effect of a D 1 R agonist. In MDA-MB-468 cells, SKF38393 (SKF) at 20 μM caused a 36% reduction in cell number (from 276.7±0.47E4 to 177.7±4.33E4). Compound C alone reduced cell number by 37% (172.4±0.04E4, 5 nM) and 51% (136.3±7.87E4, 10 nM) respectively. Combination treatment induced more reduction in cell number, 63% (100.4±5.54E4, 5 nM) and 84% (44.1±12.7E4, 10 nM). Similarly, Compound A also enhanced the inhibitory effect of SKF. A left-shift of the SKF dose-response curve in GKR4 knock-down MDA-MB-468 cells confirmed that inhibition of GRK4 increases sensitivity of breast cancer cells to SKF. Our preliminary results suggest that targeting GRK4 with compound C and a dopaminergic agonist could be a novel strategy for breast cancer therapy especially for the patients with hypertension.

Renal proximal tubule (RPT) dopamine D 1 -like receptors (D 1 R) and angiotensin II type-2 receptor (AT 2 R) inhibit sodium reabsorption and counter regulate the renin angiotensin systems AT 1 R which stimulates sodium reabsorption. Salt sensitivity of blood pressure (SS) is defined as a ≥7-mmHg rise in blood pressure following a week of daily consumption of 350 mM sodium chloride (NaCl). Inverse salt sensitivity (ISS) is defined as a ≥7-mm Hg increase in blood pressure (BP) after a week of 10 mM NaCl/day. Salt resistant controls were defined as < 7mM Hg change in BP whether on 10 or 350mmHg NaCl/day for one week. Previously, we demonstrated that D 1 R RPT membrane recruitment was inversely proportional to an individual’s degree of BP increase on a 350 mM diet. We hypothesize that the degree of salt sensitivity of blood pressure would be inversely correlated with the recruitment of the AT 2 R to the plasma membrane induced by NaCl. Immunostaining shows that D 1 R was distributed in a fine granular manner throughout the whole plasma membrane, while AT 2 R shows a punctate pattern in both urine-derived SR and ISS RPTCs. There was no difference of basal D 1 R or AT 2 R expression. Increasing cell NaCl (monensin ionophore 10 μM, 1 hour) resulted in a significantly more AT 2 R and D 1 R(control) recruitment to cell surface in ISS cells than in SR cells (D 1 R: MON/VEH: SR, 1.032 ± 0.056, n=4; ISS, 1.537 ± 0.097, n=4; t-test, p<0.01; AT 2 R :MON/VEH: SR, 0.923 ± 0.063, n=3; ISS, 1.28 ± 0.106, n=3; t-test, p<0.05). Because ISS individuals present to the medical system with elevated blood pressure while on a low salt diet, they are often misdiagnosed as hypertensive. As our studies were conducted on RPT cells isolated from individual's urine, the D 1 R and AT 2 R response may contribute to the diagnosis of ISS individuals with elevated blood pressure while on a 10 mM salt diet, and provide better understanding on the etiology of ISS.

G protein coupled kinase type 4 (GRK4) reduces renal sodium excretion by deactivating the renal dopamine type 1 receptor (D 1 R) through serine phosphorylation. At least 3 polymorphisms in GRK4 have been shown to be associated with the expression of hypertension and/or salt sensitivity in humans and in mice. GRK4 is an ideal therapeutic target for treating hypertension since putting human GRK4 variants in mice results in hypertension which can be reversed by reducing the expression of GRK4. Inhibitors of GRK4 could serve as potent and selective agents to promote sodium excretion in salt sensitive and/or hypertensive individuals. Therefore, we developed an automated high throughput homogeneous time resolved fluorescent resonance energy transfer assay (TR-FRET) to identify small molecule inhibitors of GRK4 isoenzymes. The assay relies on the close proximity (90 A) transfer of 337 nm energy from a europium anti-phosphoserine antibody (Eu-pSer) to an allophycocyanin labeled streptavidin acceptor (streptavidin-APC), producing FRET at 665 nm. The assay was optimized through serial dilutions of streptavidin-APC, Eu-pSer, GRK4, and a peptide consisting of serine sites in the D 1 R, serving as a substrate. To validate the assay and determine its suitability for a large scale high throughput screen, three inhibitors, GRKA, GRKB, and GRKC were selected and concentration response assays were performed to determine IC50 values and variability analysis. The IC50 and (R 2 ) values of GRKA, GRKB, and GRKC were 4.99 μM (0.99), 26.52 μM (0.99), and 179.8 μM (0.82), respectively. GRKA is the most efficient GRK4 inhibitor, while GRKC is the least. This assay will be used to test congeners of compounds A, B, and C to identify more selective candidates, as well as for screening available small molecule compound libraries.