Transient depletion of macrophages alters local inflammatory response at the site of disc herniation in a transgenic mouse model.

OBJECTIVE: Macrophages are abundantly detected at sites of disc herniation, however, their function in the disease progression is unclear. We aim to investigate the functions of macrophages in acute disc herniation using a macrophage Fas-induced apoptosis (MaFIA) transgenic mouse strain. METHOD: To transiently deplete macrophages, a dimerizer, AP20187, or vehicle solution was administered via intraperitoneal injection to MaFIA mice immediately, day 1 and 2 after annular puncture induced disc herniation. Local infiltrated tissues at disc hernia and DRGs at corresponding levels were harvested to analyze immune cells and neuroinflammation on postoperative day (POD) 6 by flow cytometry and/or immunostaining. Mouse spines were harvested to analyze structures of degenerated discs and adjacent vertebrae and to assess osteoclast activity by histology and tartrate-resistant acid phosphatase (TRAP) staining on POD 6, 13, and 20, respectively. RESULTS: On POD 6, abundant macrophages were confirmed at disc hernia sites. Compared to vehicle control, AP20187 significantly reduced GFP+ cells in blood, spleen, and local inflammatory tissue. At disc hernia sites, AP20187 markedly reduced macrophages (CD11b+, F4/80+, GFP+CD11b+, CD11b+F4/80+) while increasing neutrophils and B cells. Transient macrophage depletion decreased ectopic bone formation and osteoclast activity in herniated discs and adjacent cortical bones for up to 20 days post herniation. Disc herniation elevated expressions of TNF-α, IL-6, substance P, calcitonin gene-related peptide, accompanied by increasing GFP+, CD11b+ and F4/80+ macrophages. Macrophage depletion did not attenuate these markers of neuroinflammation. CONCLUSIONS: Transient depletion of macrophages altered local inflammatory response at the site of disc herniation.

Common variants of the G protein-coupled receptor type 4 are associated with human essential hypertension and predict the blood pressure response to angiotensin receptor blockade.

Non-synonymous GRK4 variants, R65L, A142V and A486V, are associated with essential hypertension in diverse populations. This study replicated the association of GRK4 variants, including GRK4(142V), with human essential hypertension in a Japanese population (n=588; hypertensive, n=486 normotensive controls) and determined whether the presence of GRK4 variants predicted the blood pressure (BP) response to angiotensin receptor blockers (ARBs) in patients with essential hypertension. We analyzed 829 patients and compared the response to ARBs between individuals with no GRK4 variants (n=136) and those with variants at one or any of the three loci (n=693). Carriers of hGRK4(142V) had a greater decrease in systolic BP in response to ARBs than non-carrier hypertensive patients. By contrast, those with variants only at GRK4(486V) were less likely to achieve the BP goal in response to an ARB than those with no variants. These studies showed for the first time the association between GRK4(142V) and a larger decrease in BP with ARBs in hypertensive patients.

Defective dopamine-1 receptor adenylate cyclase coupling in the proximal convoluted tubule from the spontaneously hypertensive rat.

The natriuretic effect of DA-1 agonists is less in the spontaneously hypertensive rat (SHR) than its normotensive control, the Wistar-Kyoto rat (WKY). To determine a mechanism of the decreased effect of DA-1 agonists on sodium transport, DA-1 receptors in renal proximal convoluted tubule (PCT) were studied by radioligand binding and by adenylate cyclase (AC) determinations. Specific binding of 125I-SCH 23982 (defined by 10 microM SCH 23390, a DA-1 antagonist) was concentration dependent, saturable, and stereoselective. The dissociation constant, maximum receptor density, and DA-1 antagonist inhibition constant were similar in SHR and WKY. The apparent molecular weight of the DA-1 receptor determined by the photoaffinity D1 probe 125I-MAB was also similar in WKY and SHR. However, DA-1 agonists competed more effectively for specific 125I-SCH 23982 binding sites in WKY than in SHR. Basal as well as forskolin, parathyroid hormone, GTP and Gpp(NH)p-stimulated-AC activities were similar. In contrast DA-1 agonists (fenoldopam, SKF 38393, SND 911C12) stimulated AC activity to a lesser extent in SHR. GTP and Gpp(NH)p enhanced the ability of DA-1 agonists to stimulate AC activity in WKY but not in SHR. These data suggest a defect in the DA-1 receptor-second messenger coupling mechanism in the PCT of the SHR.

Persistent defective coupling of dopamine-1 receptors to G proteins after solubilization from kidney proximal tubules of hypertensive rats.

The natriuretic effect of dopamine-1 (DA-1) agonists is reduced in spontaneously hypertensive rat (SHR), partly because of defective DA-1 receptor-adenylate cyclase (AC) coupling in renal proximal convoluted tubules. To investigate this defective coupling, DA-1 dopamine receptors from renal proximal tubules were solubilized and reconstituted into phospholipid vesicles. The binding of DA-1-selective ligand [125I]SCH 23982 was specific and saturable, with no differences in receptor density or Kd between SHR and normotensive rats (Wistar-Kyoto rats; WKY). Competition experiments of the reconstituted DA-1 dopamine receptors in WKY with a DA-1-selective agonist, SKF R-38393, revealed the presence of high- (Kh = 350 +/- 209 nM) and low-affinity (Kl = 70,500 +/- 39,500 nM) binding sites. 100 microM Gpp(NH)p abolished the agonist high-affinity sites, converting them to a low-affinity state (Ki = 33,650 +/- 10,850 nM). In SHR, one affinity site was noted (Ki = 13,800 +/- 500) and was not modulated by Gpp(NH)p (Ki = 11,505 +/- 2,295). The absence of guanine nucleotide-sensitive agonist high-affinity sites may explain the defective DA-1/AC coupling mechanism in the SHR.

Disruption of the dopamine D3 receptor gene produces renin-dependent hypertension.

Since dopamine receptors are important in the regulation of renal and cardiovascular function, we studied the cardiovascular consequences of the disruption of the D3 receptor, a member of the family of D2-like receptors, expressed in renal proximal tubules and juxtaglomerular cells. Systolic and diastolic blood pressures were higher (approximately 20 mmHg) in heterozygous and homozygous than in wild-type mice. An acute saline load increased urine flow rate and sodium excretion to a similar extent in wild-type and heterozygous mice but the increase was attenuated in homozygous mice. Renal renin activity was much greater in homozygous than in wild-type mice; values for heterozygous mice were intermediate. Blockade of angiotensin II subtype-1 receptors decreased systolic blood pressure for a longer duration in mutant than in wild-type mice. Thus, disruption of the D3 receptor increases renal renin production and produces renal sodium retention and renin-dependent hypertension.

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.

Dopaminergic Immunofluorescence Studies in Kidney Tissue.

The kidney is a highly integrated system of specialized differentiated cells that are responsible for fluid and electrolyte balance in the body. While much of today's research focuses on isolated nephron segments or cells from nephron segments grown in tissue culture, an often overlooked technique that can provide a unique view of many cell types in the kidney is slice culture. Here, we describe techniques that use freshly excised kidney tissue from rats to perform a variety of experiments shortly after isolating the tissue. By slicing the rat kidney in a "bread loaf" format, multiple studies can be performed on slices from the same tissue in parallel. Cryosectioning and staining of the tissue allow for the evaluation of physiological or biochemical responses in a wide variety of specific nephron segments. The procedures described within this chapter can also be extended to human or mouse kidney tissue.

Use of an anti-viral drug, Ribavirin, as an anti-glioblastoma therapeutic.

The median survival for glioblastoma patients is ~15 months despite aggressive surgery and radio-chemotherapy approaches. Thus, developing new therapeutics is necessary to improve the treatment of these invasive brain tumors, which are known to show high levels of the eukaryotic initiation factor, eIF4E, a potent oncogene. Ribavirin, the only clinically approved drug known to target eIF4E, is an anti-viral molecule currently used in hepatitis C treatment. Here, we report the effect of ribavirin on proliferation, cell cycle, cell death and migration of several human and murine glioma cell lines, as well as human glioblastoma stem-like cells, in vitro. In addition, we tested ribavirin efficacy in vivo, alone and in combination with temozolomide and radiation. Our work showed that ribavirin inhibits glioma cell growth and migration, and increases cell cycle arrest and cell death, potentially through modulation of the eIF4E, EZH2 and ERK pathways. We also demonstrate that ribavirin treatment in combination with temozolomide or irradiation increases cell death in glioma cells. Finally and most importantly, ribavirin treatment in vivo significantly enhances chemo-radiotherapy efficacy and improves survival of rats and mice orthotopically implanted with gliosarcoma tumors or glioma stem-like cells, respectively. On the basis of these results, we propose that ribavirin represents a new therapeutic option for glioblastoma patients as an enhancer of the cytotoxic effects of temozolomide and radiotherapy.

Renal dopamine receptors in health and hypertension.

During the past decade, it has become evident that dopamine plays an important role in the regulation of renal function and blood pressure. Dopamine exerts its actions via a class of cell-surface receptors coupled to G-proteins that belong to the rhodopsin family. Dopamine receptors have been classified into two families based on pharmacologic and molecular cloning studies. In mammals, two D1-like receptors that have been cloned, the D1 and D5 receptors (known as D1A and D1B, respectively, in rodents), are linked to stimulation of adenylyl cyclase. Three D2-like receptors that have been cloned (D2, D3, and D4) are linked to inhibition of adenylyl cyclase and Ca2+ channels and stimulation of K+ channels. All the mammalian dopamine receptors, initially cloned from the brain, have been found to be expressed outside the central nervous system, in such sites as the adrenal gland, blood vessels, carotid body, intestines, heart, parathyroid gland, and the kidney and urinary tract. Dopamine receptor subtypes are differentially expressed along the nephron, where they regulate renal hemodynamics and electrolyte and water transport, as well as renin secretion. The ability of renal proximal tubules to produce dopamine and the presence of receptors in these tubules suggest that dopamine can act in an autocrine or paracrine fashion; this action becomes most evident during extracellular fluid volume expansion. This renal autocrine/paracrine function is lost in essential hypertension and in some animal models of genetic hypertension; disruption of the D1 or D3 receptor produces hypertension in mice. In humans with essential hypertension, renal dopamine production in response to sodium loading is often impaired and may contribute to the hypertension. The molecular basis for the dopaminergic dysfunction in hypertension is not known, but may involve an abnormal post-translational modification of the dopamine receptor.

D-2 dopaminergic agonists and adenosine 3',5'-monophosphate directly regulate the synthesis of alpha-melanocyte-stimulating hormone-like peptides by cultured rat melanotrophs.

When placed in short term (2-day) tissue culture, the melanotrophs from the intermediate lobe of the rat pituitary gland synthesize a proopiomelanocortin-like material (POMC-LM). Exposure of these cells to bromocriptine (CB 154), an agonist upon their D-2 dopamine receptor, reduces the synthesis of POMC-LM; spiroperidol, an antagonist of the D-2 receptor, prevents this effect of CB 154. Cultured melanotrophs secrete an alpha MSH-like material. The amount of this alpha MSH-like material, either stored intracellularly or secreted into the culture medium, can be quantified in a specific RIA; the material identified in this manner is designated immunoreactive alpha MSH (IR-alpha MSH). CB 154 inhibits the secretion of IR-alpha MSH from these cells. Either spiroperidol or 8-bromo-cAMP prevent this inhibitory effect of CB 154. The capacity of these cells to synthesize alpha MSH-like molecules and release them into the culture medium can be assessed by incubation in the presence of [3H]tyrosine, followed by immunoprecipitation with an antibody directed against alpha MSH. This newly synthesized immunoprecipitable material is designated immunoprecipitable alpha MSH (IP-alpha MSH) and should be distinguished from IR-alpha MSH. Both CB 154 and quinpirole, a selective D-2 agonist (but not SKF 38393, a selective D-1 agonist), inhibit the synthesis and secretion of IP-alpha MSH. YM-09151-2, a selective D-2 antagonist (but not SCH 23390, a selective D-1 antagonist), blocks the inhibitory effects of quinpirole. Several compounds affecting cAMP metabolism (cholera toxin, forskolin, 8-bromo-cAMP, and 3-isobutyl-1-methylxanthine) can also prevent the inhibitory effect of CB 154 on the synthesis of IP-alpha MSH. We conclude the following. The D-2 receptor in the intermediate lobe directly regulates the synthesis and secretion of IP-alpha MSH. cAMP can regulate either the synthesis of POMC-LM or the processing of this substance into alpha MSH-like peptides.

Renal dopamine-1 receptors in hypertensive inbred rat strains with and without hyperactivity.

Renal dopamine-1 (DA-1) receptors are involved in the regulation of sodium transport in several nephron segments, including the proximal convoluted tubule (PCT). DA-1 receptors in the PCT and cortical collecting duct of normotensive rats are linked to the stimulation of adenylyl cyclase (AC). We have reported a defect in the DA-1 receptor/AC coupling in the PCT of the spontaneously hypertensive rat (SHR) of the Okamoto-Aoki strain. Hyperactivity and hypertension are both expressed in the SHR. To determine if the DA-1 receptor coupling defect is associated with hyperactivity or hypertension, we studied the DA-1 receptor in the PCT of two new inbred rat strains derived from the SHR: the hyperactive WKHA and the hypertensive WKHT rat. Tail-cuff blood pressures taken at 4 weeks indicated that WKHT rats were not hypertensive (86 +/- 3 mm Hg, n = 6), whereas at 12 weeks systolic pressures in both SHR and WKHT rats exceeded 150 mm Hg. Hyperactivity, however, was noted in WKHA rats even at this early age. Basal AC activity was similar in WKHA and WKHT PCT in either age group. In the older rats, the DA-1 agonist fenoldopam (10(-7) mol/L) stimulated AC activity in WKHA (70.6 +/- 16.1 fmol per 3 mm PCT per 20 minutes, n = 3) but not in WKHT PCT (43.3 +/- 5.3 fmol per 3 mm PCT per 20 minutes, n = 4). Gpp(NH)p (10(-5) mol/L), a nonhydrolyzable GTP analogue, stimulated AC activity to a similar extent in WKHA and WKHT PCT.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective inhibition of the renal dopamine subtype D1A receptor induces antinatriuresis in conscious rats.

Both dopamine D1-like (D1A and D1B) and D2-like (D2, D3, and D4) receptor subfamilies are present in the kidney. Blockade of the intrarenal D1-like receptor family is associated with natriuresis and diuresis. Because the D1A and D1B receptor subtypes are not distinguishable by currently available dopaminergic agents, their functional role remains undefined. In the present study, the effect of selective inhibition of the renal D1A receptor with phosphorothioated antisense oligodeoxynucleotide (AS-ODN) was investigated in conscious uninephrectomized rats. After renal interstitial administration of Texas red-labeled D1A receptor AS-ODN, intense fluorescent signal was localized in the renal tubular epithelium and vasculature. In rats on normal salt intake, AS-ODN injected interstitially into the kidney reduced daily urinary sodium excretion (1.4+/-0.04 versus 0.8+/-0.2 mEq/d, n=5, P<0.05) and urine output (16.9+/-3.8 versus 12.5+/-3.6 mL/d, n=5, P<0.05). In rats on high sodium intake, continuous renal interstitial administration of D1A receptor AS-ODN transiently decreased daily urinary sodium excretion (5.4+/-0.5 versus 4.2+/-0.3 mEq/d, n=7, P<0.01) and urine output (27.6+/-4.5 versus 18.1+/-1.8 mL/d, n=7, P<0.01). Neither vehicle nor sense oligodeoxynucleotide had significant effects. Systolic blood pressure remained unchanged. The renal D1A receptor protein was significantly decreased by 35% and 46% at the end of the study in AS-ODN-treated rats on normal and high salt intake, respectively, whereas the D1B receptor and beta-actin were not affected. These results provide the first direct evidence that the renal D1A receptor subtype plays an important role in the control of sodium excretion.

Differential human renal tubular responses to dopamine type 1 receptor stimulation are determined by blood pressure status.

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.

Intrarenal dopamine production and distribution in the rat. Physiological control of sodium excretion.

Dopamine (DA), produced by the renal proximal tubule, has been demonstrated as an intrarenal paracrine hormone mediating diuresis and natriuresis. The precise mechanism by which DA exerts its cell-to-cell action is not fully understood. In the present study, renal interstitial fluid (RIF) DA (by in vivo microdialysis) and urinary DA excretion (UDAV) were compared in anesthetized rats on either normal (0.28% NaCI, NS) or high (4.0% NaCI, HS) sodium balance and in response to acute gamma-L-glutamyl-L-dopa (gludopa) administration. Urine flow (UV) and sodium excretion (UNaV) in HS were greater than in NS rats. UDAV was increased in HS compared with NS rats. RIF DA was significantly lower in HS than NS rats. Gludopa at 3, 5, and 7.5 nmol/kg (IV bolus) produced a larger increase in UDAV than RIF DA. Only the highest dose of gludopa (7.5 nmol/kg), which resulted in a 7.3-fold increase in UDAV and 1.7-fold increase in RIF DA, was associated with significant diuresis and natriuresis. Cortical and medullary blood flow remained unchanged after gludopa (7.5 nmol/kg) administration, while angiotensin II (100 ng.kg-1.min-1) induced significant reduction in cortical and medullary blood flow. Prior bilateral renal denervation did not have a significant effect on basal DA levels (RIF DA and UDAV) or gludopa-induced DA production or natriuresis and diuresis. These data demonstrated that both chronic sodium loading and acute gludopa administration stimulated renal DA production and release predominantly into the tubule lumen, where DA had a direct tubule action in the control of UNaV. Renal DA production and its renal effects were not significantly regulated by renal sympathetic nerve activity.

Attenuated renal response to dopaminergic drugs in spontaneously hypertensive rats.

Activation of renal dopamine-1 receptors decreases sodium transport. However, the spontaneously hypertensive rat retains sodium despite increased renal dopamine concentration. We tested the hypothesis that the abnormal sodium handling in spontaneously hypertensive rats (Okamoto-Aoki strain) is related to a decreased dopaminergic response by studying the effects of the intrarenal infusion of the dopamine-1 agonist SKF-38393 and the dopamine-1 antagonist SCH-23390 in hypertensive and in normotensive Wistar-Kyoto rats. Rats (9-16 weeks old) were studied with renal nerves intact under pentobarbital anesthesia (n = 5-6 in each group). Specificity of dopamine-1 effects of SKF-38393 was verified because its natriuretic effect was blocked in a dose-related manner by the dopamine-1 antagonist SCH-23390 (n = 5). Intrarenal but resulted in a dose-related natriuresis and diuresis in normotensive but not in hypertensive rats. Intrarenal arterial infusion of the dopamine-1 antagonist SCH-23390 alone induced an antinatriuresis, without affecting glomerular filtration rate, in normotensive but not in hypertensive rats. Addition of the dopamine-2 antagonist YM-09151 to the dopamine-1 antagonist infusion did not enhance the effect of the dopamine-1 antagonist. The lack of response to the dopamine-1 agonist or antagonist in hypertensive rats was not due to differences in renal dopamine-1 receptor density (1.3 +/- 0.3 pmol/mg protein for spontaneously hypertensive rats, n = 4; 1 +/- 0.2 for Wistar-Kyoto rats, n = 4) or affinity; distribution determined by autoradiography was also similar. The abnormal renal sodium handling in 9-16-week-old spontaneously hypertensive rats is in part due to decreased response distal to the dopamine-1 receptor.

Diuresis and natriuresis during continuous dopamine-1 receptor stimulation.

Stimulation of renal dopamine-1 (DA1) receptors for 3 hours produces an increase in renal plasma flow and sustained natriuresis. The present study was designed to assess the response of renal hemodynamic and tubular function to long-term DA1 receptor stimulation. Fenoldopam, a selective DA1 receptor agonist, was infused intravenously for 24 hours in 10 normal male subjects in metabolic balance at 150 mEq sodium and 60 mEq potassium intake in a single-blind, vehicle-controlled protocol. During DA1 receptor activation, urine flow rate and fractional excretion of sodium increased for the first 5 hours, 16.9 +/- 0.9 ml/min compared with a vehicle control value of 12.4 +/- 0.5 ml/min (p less than 0.001) and 2.0 +/- 0.1% compared with a vehicle control value of 1.1 +/- 0.1% (p less than 0.005), respectively. Urinary sodium excretion rose at 5 hours, 0.27 +/- 0.02 mEq/min compared with a vehicle control value of 0.14 +/- 0.01 mEq/min (p less than 0.01). Renal plasma flow increased during fenoldopam at 5 hours, 505 +/- 47 ml/min compared with a vehicle control value of 397 +/- 25 ml/min (p less than 0.01), and was sustained for 24 hours, 523 +/- 40 ml/min compared with 432 +/- 31 ml/min (p less than 0.05). The distal sodium load increased and the percentage of distal sodium reabsorption decreased during fenoldopam. Glomerular filtration rate, blood pressure, heart rate, plasma aldosterone concentration, plasma renin activity, and fractional excretion of potassium were unchanged. Selective DA1 receptor activation produced sustained 5-hour diuresis and 11-hour natriuresis without kaliuresis or a systemic hemodynamic effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of the dopamine D1 receptor protein in the human heart and kidney.

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.

Expression of the dopamine D3 receptor protein in the rat kidney.

The dopamine D3 receptor subtype was identified in rat kidney using both light microscopic immunohistochemistry and electron microscopic immunocytochemistry. Antipeptide polyclonal antisera were directed to both extracellular and intracellular regions of the native D3 receptor. Selectivity of the antipeptide antisera was validated by their ability to recognize native receptor protein expressed in permanently transfected mouse LTK- cells or Spodoptera fragiperda (Sf9) cell membranes. Light microscopic immunohistochemical staining for the D3 receptor was observed only in the cortex. Specific staining was present in proximal and distal tubules, cortical collecting ducts, glomeruli, and renal vasculature. Immunostaining was observed predominantly in the apical portion of both the proximal and distal tubules. Renal arterial staining was prominent in the medial and adventitial layers. Electron microscopic immunocytochemistry revealed immunogold particles in arteriolar smooth muscle cells of the renal vasculature. In proximal and distal tubules and cortical collecting duct, immunogold staining was localized to apical portions of tubule cells. D3 receptor immunogold staining in the glomeruli was clearly present in podocytes. Western blot analysis demonstrated a single D3 receptor band in infected Sf9 cell membranes, in transfected LTK- cells, and in kidney and brain but not in noninfected Sf9 cell membranes or in D2 or D3 receptor transfected or nontransfected LTK- cells. The use of receptor subtype-selective antibodies allows for the tissue localization of specific dopamine receptors that are not distinguished by current pharmacological or ligand-binding technology. The rat kidney expresses the D3 receptor at sites previously deemed to have D2-like receptors.

Dopamine-1 receptor coupling defect in renal proximal tubule cells in hypertension.

The ability of the dopamine-1 (D1)-like receptor to stimulate adenylyl cyclase (AC) and phospholipase C (PLC), inhibit sodium transport in the renal proximal tubule (RPT), and produce natriuresis is attenuated in several rat models of hypertension. Since the inhibitory effect of D1-like receptors on RPT sodium transport is also reduced in some patients with essential hypertension, we measured D1-like receptor coupling to AC and PLC in cultures of human RPT cells from normotensive (NT) and hypertensive (HT) subjects. Basal cAMP concentrations were the same in NT (n=6) and HT (n=4). However, the D1-like receptor agonist fenoldopam increased cAMP production to a greater extent in NT (maximum response=67+/-1%) than in HT (maximum response=17+/-5%), with a potency ratio of 105. Dopamine also increased cAMP production to a greater extent in NT (32+/-3%) than in HT (14+/-3%). The fenoldopam-mediated increase in cAMP production was blocked by SCH23390 (a D1-like receptor antagonist) and by antisense D1 oligonucleotides in both HT and NT, indicating action at the D1 receptor. The stimulatory effects of forskolin and parathyroid hormone-related protein of cAMP accumulation were not statistically different in NT and HT, indicating receptor specificity and an intact G-protein/AC pathway. The fenoldopam-stimulated PLC activity was not impaired in HT, and the primary sequence and expression of the D1 receptor were the same in NT and HT. However, D1 receptor serine phosphorylation in the basal state was greater in HT than in NT and was not responsive to fenoldopam stimulation in HT. These studies demonstrate the expression of D1 receptors in human RPT cells in culture. The uncoupling of the D1 receptor in both rats (previously described) and humans (described here) suggests that this mechanism may be involved in the pathogenesis of hypertension; the uncoupling may be due to ligand-independent phosphorylation of the D1 receptor in 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.