The dopamine D1 receptor has recently been identified in the rat heart and kidney. In the present study, using Western blot analysis and light microscopic immunohistochemistry, we examined D1 receptor protein expression in the human kidney and heart. Antipeptide polyclonal rabbit antiserum was raised against the third extracellular domain of the native receptor and affinity-purified using a protein-A column. Selectivity of the antiserum was validated by recognition of the D1 receptor expressed in stably transfected LTK- cells and Sf-9 cells. The immunohistochemical staining for D1 receptor protein was distributed throughout the atrium and ventricular myocardium and in the coronary vessels. In the kidney, positive immunoreactive signal was detected in the proximal and distal tubules, the collecting ducts, and the large intrarenal vasculature, whereas staining was absent in the juxtaglomerular (JG) cells and the glomeruli. D1 receptor antiserum preadsorbed against the immunizing peptide did not produce significant staining. In Western blot analysis, a single 55-kD band was detected for the D1 receptor in membranes from the D1 receptor transfected Sf-9 cells but not in nontransfected cells. In the heart and kidney, we detected a 55-kD band as well as an additional 40-kD band, which may reflect partial degradation of the receptor protein. These results provide the first evidence for the localization of the dopamine D1 receptor protein in the human heart and kidney. The similar distribution of this subtype receptor in the human heart and kidney to that in the rat supports the possible (patho)physiological significance of the peripheral dopamine system in humans.

Pharmacological, physiological, and autoradiographic studies have suggested the presence of dopamine receptors in the adrenal gland. Dopaminergic ligands have been shown to modulate adrenocortical aldosterone biosynthesis and secretion as well as adrenomedullary catecholamine production and release. Using a combination of Light microscopic immunochemistry and in situ amplification and hybridization, the present study sought to determine the site-specific expression of the recently cloned D-1A receptor subtype in rat adrenal gland.Light microscopic immunohistochemistry was conducted using polyclonal antisera raised to the putative rat D-1A receptor. Immunoreactive product was detected using an avidin-biotin immunoperoxidase method. D-1A receptor messenger RNA (mRNA) was detected using a transcription-based isothermal in situ amplification and hybridization approach using receptor-specific mRNA oligonucleotide probes. The amplified product was localized using an alkaline phosphatase 4-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolylphosphate technique. This combined experimental approach, using both receptor subtype-selective antibodies and oligonucleotide probes, allows for the site-specific localization of the D-1A receptor subtype, which would otherwise not be possible with the pharmacological methods currently available. The D-1A receptor protein and mRNA were expressed solely in the zona glomerulosa of the rat adrenal gland, with no signal evident in any of the other cortical layers or in the medulla. Such a distribution raises the possibility that the D-1A receptor subtype could modulate, at least in part, some of the known effects of dopamine on aldosterone secretion.

We performed the present studies to determine whether a proximal renal tubular dopamine D1-like receptor defect exists in human essential hypertension. Twenty-four subjects were studied (13 normotensive and 11 hypertensive) in a randomized, double-blind, vehicle-controlled study using fenoldopam, a selective D1-like receptor agonist. Subjects were studied in sodium metabolic balance at 300 mEq/d, after which the salt sensitivity of their blood pressure was determined. Fenoldopam at peak doses of 0.1 to 0.2 microgram/kg per minute decreased mean arterial pressure in hypertensive subjects but did not change mean pressure in normotensive subjects. Fenoldopam increased renal plasma flow to a greater extent in hypertensive than normotensive subjects. Fenoldopam increased both urinary and fractional sodium excretions in the hypertensive and normotensive groups. In normotensive but not hypertensive subjects, fenoldopam increased the fractional excretion of lithium and distal sodium delivery. In contrast, both distal fractional sodium reabsorption and sodium-potassium exchange fell significantly in hypertensive subjects. We conclude that human essential hypertension is associated with a reduction in the proximal tubular response to D1-like receptor stimulation compared with normotensive subjects. Hypertensive subjects appear to have a compensatory upregulation of renal vascular and distal tubular D1-like receptor function that offsets the proximal tubular defect, resulting in an enhanced natriuretic response to D1-like receptor stimulation.

The subtype 1A dopamine receptor (D1A) has recently been detected in the rat kidney. In the present study using light microscopic immunohistochemistry, electron microscopic immunocytochemistry, and in situ amplification of mRNA, we demonstrate the D1A receptor in Sprague-Dawley and Wistar Kyoto rat hearts. For immunohistochemistry and immunocytochemistry, anti-peptide polyclonal antibodies were directed toward amino acid sequences of the third extracellular and intracellular domains of the native receptor. Selectivity was validated by recognition of the D1A receptor expressed in stably transfected LTK- cells. D1A receptor mRNA was detected with a novel transcription-based isothermal in situ amplification system as well as with reverse transcription-polymerase chain reaction. D1A receptor protein was distributed throughout the atrium and ventricular myocardium. Preimmune and preabsorption controls were negative. Electron microscopic immunocytochemistry using the protein A gold method demonstrated the D1A receptor along the cellular membranes of coronary smooth muscle cells and ventricular myocytes and in the myosin thick filaments and M-lines. D1A receptor mRNA was present in coronary vessels and myocardium in amplified but not in unamplified sections. Western blot analysis showed specific D1A bands in transfected LTK- cells and the atrium but not in nontransfected LTK- cells and the ventricle. The selective D1-like receptor agonist SKF38393 stimulated adenylyl cyclase in ventricular myocardial plasma membranes in a dose-related fashion, and the response was abolished by the selective D1-like receptor antagonist SCH23390. These results demonstrate that the D1A receptor gene and protein are expressed in normal rat heart. The physiological and pathophysiological roles and predominant cell signaling mechanism or mechanisms of this receptor remain to be determined.

The dopamine D1A receptor subtype was identified in rat kidney with both light microscopic immunohistochemistry and electron microscopic immunocytochemistry. Antipeptide polyclonal antisera were directed to both extracellular and intracellular regions of the native receptor. The use of such receptor-subtype-selective antibodies allows for the identification of specific dopamine receptor subtype clones that are not distinguished by current pharmacological or receptor-ligand binding technology. Selectivity of the antipeptide antisera was validated by their ability to recognize native receptor protein expressed in permanently transfected mouse LTK- cells. In the rat kidney, D1A receptor protein was localized to the juxtaglomerular apparatus (JGA), proximal tubule, distal tubule, cortical collecting duct, and renal vasculature. In the JGA, the receptor was predominantly located in the arteriolar smooth muscle layer within cytoplasmic granules previously shown to contain renin. In the proximal tubules, staining was localized both on the brush-border and basolateral membranes. The D1A receptor, which is present in the central nervous system, is now identified in the rat kidney at those sites previously labeled as DA1 receptor sites on the basis of pharmacological binding studies. These results suggest that at least some of the renal dopamine DA1 receptors correspond structurally to the central dopamine D1A receptor.