D1A Receptor Gene Research Articles

Chemical hypoxia-induced increases in dopamine D1A receptor mRNA in renal epithelial cells are mediated by nitric oxide.

Nitric oxide (NO) and dopamine (DA) have similar effects on renal function, with both having natriuretic and diuretic effects mediated by vascular and tubular mechanisms. Renal ischaemia or hypoxia have been shown to influence the activity of both systems. However, it is not known whether there is any crosstalk between the NO and dopaminergic systems in the kidney. Here using the porcine proximal tubule-like renal epithelial LLC-PK1 cell line as a model system, we determined whether exposure of cells to chemical hypoxia altered the steady-state levels of D1A receptor mRNA and whether the changes involved the NO system. Exposure of LLC-PK1 cells to chemical hypoxia resulted in a marked increase in D1A receptor mRNA levels as measured by reverse transcription-polymerase chain reaction (RT-PCR). The increased levels of D1A receptor mRNA following hypoxia were blocked by the NO synthase inhibitors NG-nitro-L-arginine methylester (L-NAME) or NG-monomethyl-L-arginine (L-NMMA). Further evidence that the NO system exerted positive effects on D1A receptor gene expression came from finding that the NO donor sodium nitroprusside, the NO precursor L-arginine and the guanosine 3', 5'-cyclic monophosphate (cyclic GMP) analogue 8-Br-cGMP all increased D1A receptor mRNA levels in LLC-PK1 cells. These results indicate that expression of the D1A receptor in LLC-PK1 cells can be positively regulated by the NO system. Such an interaction between the renal NO and DA systems may contribute to the reported protective effects that NO and DA exert upon the kidney under conditions of ischaemia.

Regulation of Dopamine-1A (D1A) Receptor Gene Transcription

The D1A receptor is expressed primarily in the brain and kidney. The D1A receptor gene has been cloned from human, rat and pig and is organized similarly in each species. The 5′flanking region of the D1A receptor gene is high in GC content, is TATA box-less and contains multiple Sp1 binding sites. Comparison and alignment of the nucleotide sequences within the 5′ flanking and 5′ untranslated regions of each gene indicates that the highest sequence identity is in the area centered approximately 100 bases upstream from the transcription start site. There are numerous binding sites for transcription factors, including Sp1 and AP-2, in the 5′ flanking region. Approximately 200 bases upstream is a conserved cAMP regulatory element-like sequence. The conserved position of certain cis-acting elements in each gene suggests that the essential elements for regulated expression of the D1A receptor gene are contained within the first 300 bases of the 5′ flanking region.

Regulation of DI Receptor Function in Spontaneous Hypertension

Although multiple genes may interact to initiate hypertension, the kidney is a primary locus for the genesis and maintenance of hypertension by its ability to influence sodium balance and produce hormones that can increase or decrease blood pressure. Most of the salt transport in the kidney occurs in the proximal tubule, and several studies have reported abnormalities in the renal regulation of sodium transport at this site in human as well as in animal models of genetic hypertension. The production of dopamine by renal proximal tubules and its effect on sodium transport in this nephron segment is regulated by sodium intake. During moderately increased sodium intake, dopamine, via D1-like receptors, acts as an intrarenal natriuretic factor. This renal paracrine–autocrine effect is lost during states of negative sodium balance. This chapter surmises that a defect in the regulation of dopamine and its effect on renal sodium transport may be important in the pathogenesis of spontaneous hypertension. Decreased renal production of dopamine has been reported in human essential hypertension, especially in those individuals with the salt-sensitive type. Those patients have also been reported to have an exaggerated natriuretic response to exogenous dopamine, although this natriuretic response may be related exclusively to the renal hemodynamic effect of D1 receptors. However, the defect in spontaneous hypertension is not because of a mutation of the D1A receptor gene. The mechanism of the uncoupling of the renal D1-like receptor from its effector proteins remains to be determined. Although, the uncoupling is akin to homologous desensitization, it is not because of agonist-induced desensitization.

What we can learn from the selective manipulation of dopaminergic receptors about the pathogenesis and treatment of hypertension?

The natriuretic response that accompanies an acute or a chronic sodium load results, in a large part, from dopamine produced by the renal proximal tubules. This paracrine/ autocrine effect of dopamine, mediated by occupancy of renal tubular dopamine D1 receptors, is impaired in spontaneous hypertension. Disruption of the D1A receptor gene in mice increases blood pressure. The D3 receptor is also present in renal proximal tubules and juxtaglomerular cells, and it may be an inhibitor of renin release. Disruption of the D3 receptor gene in mice also increases blood pressure, but the ability to excrete a sodium chloride load is not impaired. Thus, selective manipulation of dopamine receptor subtypes might aid in studies of the pathogenesis of essential hypertension and the design of novel drugs to treat high blood pressure.

Role of the D1A dopamine receptor in the pathogenesis of genetic hypertension.

Since dopamine produced by the kidney is an intrarenal regulator of sodium transport, an abnormality of the dopaminergic system may be important in the pathogenesis of hypertension. In the spontaneously hypertensive rat (SHR), in spite of normal renal production of dopamine and receptor density, there is defective transduction of the D1 receptor signal in renal proximal tubules, resulting in decreased inhibition of sodium transport (Na+/H+ exchanger [NHE] and Na+/K+ATPase activity) by dopamine. To determine if impaired D1 receptor regulation of NHE in proximal tubules is related to hypertension, studies were performed in a F2 generation from female Wistar Kyoto (WKY) and male SHR crosses. A D1 agonist, SKF 81297, inhibited (37.6 +/- 4.7%) NHE activity in brush border membranes of normotensive F2s (systolic blood pressure < 140 mm Hg, n = 7) but not in hypertensive F2s (n = 21). Furthermore, a D1 agonist, SKF 38393, when infused into the renal artery, dose dependently increased sodium excretion in normotensive F2s (n = 3) without altering renal blood flow but was inactive in hypertensive F2s (n = 21). Since the major D1 receptor gene expressed in renal proximal tubules is the D1A subtype, we determined the importance of this gene in the control of blood pressure in mice lacking functional D1A receptors. Systolic blood pressure was greater in homozygous (n = 6) and heterozygous (n = 5) mice compared to normal sex matched litter mate controls (n = 12); moreover, the mice lacking one or both D1A alleles developed diastolic hypertension. The cosegregation with hypertension of an impaired D1 receptor regulation of renal sodium transport and the development of elevated systolic and diastolic pressure in mice lacking one or both D1A alleles suggest a causal relationship of the D1A receptor gene with hypertension.

INCREASED SERINE-PHOSPHORYLATION OF THE D1A RECEPTOR IN RENAL PROXIMAL TUBULE CELLS IN SPONTANEOUS HYPERTENSION. • 2218

We have suggested that a defect in the coupling of the D1 receptor to G-protein/enzyme complex in renal proximal tubules is involved in the pathogenesis of spontaneous hypertension. The D1A receptor subtype may be involved since hypertension develops in mice mutant for the D1A receptor allele. However, we have found no mutation of the D1A receptor gene in the spontaneously hypertensive rat (SHR) or Dahl salt-sensitive rat suggesting that the D1A receptor defect may be due to post-translational modification. Since the renal D1 receptor resembles the phenomenon of desensitization, and desensitization occurs as a result of receptor phosphorylation, we studied the phosphorylation of the D1A receptor in renal proximal tubules from SHR and its normotensive control, the Wistar-Kyoto rat (WKY). Vehicle alone or fenoldopam, a D1 agonist, 1μg/kg/min, was infused for 5-10′ into the renal artery via the right suprarenal artery of anesthetized rats. Renal cortical homogenates (n=5) were then immunoprecipitated with anti-phosphoserine antibodies and immunoblotted with anti D1A antibodies. Basal serine-phosphorylated D1A receptor was greater in SHR (21.0±1.5 density units, DU) than in WKY(7.4±2.9 DU) (P<0.01 t-test); the D1 agonist increased serine-phosphorylated D1A receptor in WKY (55.0±2.8 DU) but not in SHR (17.0±6.3 DU) (P<0.01 t-test) (n=5/group). The“hyper” phosphorylation of the D1A receptor in the SHR was not the result of homologous desensitization because similar studies were obtained when the rats (n=3) were injected with carbidopa (40 μmol/kg) 24, 18, and 1 hr prior to study to decrease renal dopamine production. Moreover, the decreased ability of D1 agonist to stimulate adenylyl cyclase and phospholipase C (n=5-8) and the “hyper” phosphorylation (n=3) of the D1A receptor persist in immortalized proximal tubular cells from SHR (dopamine synthesis does not occur because of the absence of DOPA, the substrate for renal dopamine synthesis). The mechanism of “hyper” phosphorylation of the D1A receptor in renal proximal tubules from SHR is not known. However, we have recently found that protein phosphatase 2A activity in cytosol but not in membranes of renal proximal tubules is decreased in the SHR compared to the WKY; protein phosphatases are important in the resensitization process. We conclude that a defect in the post-translational modification of the D1A receptor is important in the pathogenesis of spontaneous hypertension.

Expression of the subtype 1A dopamine receptor in the rat heart.

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 human D1A dopamine receptor gene promoter directs expression of a reporter gene to the central nervous system in transgenic mice.

Dopamine receptors are involved in many aspects of dopaminergic neurotransmission including regulation of motor control, cognition, affect and neuroendocrine function. The D1A receptor is the most widely distributed dopamine receptor in the brain and is expressed at high levels in the striatum and nucleus accumbens, but is also found throughout cortical, limbic, hypothalamic and thalamic brain regions. We have cloned a 6.4 kb fragment 5' of the human D1A dopamine receptor gene and shown that this region activates transcription of the chloramphenicol acetyltransferase (CAT) gene in a cell-specific manner. To study the expression of these sequences in vivo we analyzed the expression of the E. coli lac Z gene under the regulation of the 6.4 kb fragment in transgenic mice. Expression of the transgene was primarily detected in the brain, with only low levels detected in peripheral tissues. The 5' flanking sequences were able to direct the tissue-specific expression of lac Z in three different lines of transgenic mice, to a number of brain regions including the caudate-putamen, thalamus, amygdala, cerebral cortex, hippocampus and hypothalamus. Greatest expression of the lac Z gene was detected in areas of the thalamus and amygdaloid complex. In the striatum, beta-galactosidase activity was restricted to neurons within the matrix and was not detected within striosomes. Results of this study demonstrate that the 6.4 kb region upstream of the human D1A receptor gene is sufficient to confer tissue-specific expression in the CNS of transgenic mice. Furthermore, expression of the transgene to neurons within the matrix of the striatum, but not the striosomes suggests that expression of the D1A receptor may be regulated differently within these areas.

Cloning of the dopamine-1A (D1A) receptor gene expressed in porcine renal epithelial cells.

We sought to determine the molecular identify of the dopamine-1 (D1) receptor expressed in the porcine renal epithelial cell line LLC-PK1. We first isolated a partial cDNA by the reverse transcription-polymerase chain reaction procedure and then used the partial cDNA to isolate positive overlapping clones from a porcine genomic DNA library. Sequence analysis of the gene revealed that the longest open-reading frame encoded a 446 amino acid protein that was 95% identical to the human D1A receptor. Expression studies in mammalian cells were also consistent with the clones encoding a D1 receptor. Northern blot hybridizations with LLC-PK1 poly (A+) RNA were strongly positive. The porcine D1A gene has two exons and a short intron in the 5' untranslated region. The 5' flanking region lacks a TATA and CAAT box but is high in GC content (68%) and contains multiple Sp1 binding sites. The 5' flanking region also contains numerous other cis-acting elements for transcription factors. These results indicate that the D1A receptor is the major D1 receptor expressed in LLC-PK1 cells and further suggest that LLC-PK1 cells may be a useful model to study the regulation of renal D1A receptor gene transcription.

Identification of Dopamine1A Receptors in the Rat Small Intestine

Dopamine receptors have been localized to several tissues outside the central nervous system including the kidney and mesenteric vessels. To determine if there are dopamine1 receptors within the small intestine, homogenates of the antimesenteric halves of the entire jejunum and ileum of adult rats were prepared and competitive inhibition studies and Scatchard analysis were performed at room temperature using 125I-SCH 23982 and SCH 23390. The specific binding of 125I-SCH 23982 to the intestinal tissue homogenates was rapid, saturable with ligand concentration, and reversible. Analysis of the Scatchard plots revealed a single class of receptors with an apparent dissociation constant of 10.77 ± 2.32 nM and maximum receptor density of 1.37 ± 0.34 fmole/mg protein. Emulsion autoradiography performed using 125I-SCH 23982 on antimesenteric sections of the rat small intestine revealed that the dopamine1 receptors are located on cells at the base of the intestinal crypts. Two dopamine1 subtypes (D1A and D1B) have been identified by molecular biological techniques. Using a ribonuclease protection assay we found expression of the D1A receptor gene in the small intestinal tissue. These studies are the first to identify, characterize, and localize receptors for the endogenous catecholamine, dopamine, within the rat small intestine and to confirm the expression of the D1A receptor gene.

Expression of dopamine D1A receptor gene in proximal tubule of rat kidneys.

The existence of dopamine receptor subtypes outside the central nervous system has been suggested by pharmacological and biochemical techniques. Whether the renal dopamine receptors are distinct from those cloned from the brain remains to be determined. Indeed, the expression of any of these receptor genes in the kidney has not been demonstrated definitively. In this study, we amplified D1A receptor cDNA from microdissected proximal convoluted tubules of the rat kidney by reverse transcription-polymerase chain reaction; primers were based on the sequence of rat D1A cDNA cloned from the brain and corresponded to the third cytoplasmic loop of the receptor. Specificity of the amplified products was verified by restriction analysis, Southern blots, and sequencing. Furthermore, solution hybridization indicated the presence of a single ribonuclease-protected RNA species corresponding to the D1A receptor mRNA in proximal tubules. These studies report for the first time the presence of D1A receptor message in the kidney.