The Synergistic Roles of Cholecystokinin B and Dopamine D5 Receptors on the Regulation of Renal Sodium Excretion

Renal dopamine D₁-like receptors (D₁R and D₅R) and the gastrin receptor (CCK_BR) are involved in the maintenance of sodium homeostasis. The D₁R has been found to interact synergistically with CCK_BR in renal proximal tubule (RPT) cells to promote natriuresis and diuresis. D₅R, which has a higher affinity for dopamine than D₁R, has some constitutive activity. Hence, we sought to investigate the interaction between D₅R and CCK_BR in the regulation of renal sodium excretion. In present study, we found D₅R and CCK_BR increase each other’s expression in a concentration- and time-dependent manner in the HK-2 cell, the specificity of which was verified in HEK293 cells heterologously expressing both human D₅R and CCK_BR and in RPT cells from a male normotensive human. The specificity of D₅R in the D₅R and CCK_BR interaction was verified further using a selective D₅R antagonist, LE-PM436. Also, D₅R and CCK_BR colocalize and co-immunoprecipitate in BALB/c mouse RPTs and human RPT cells. CCK_BR protein expression in plasma membrane-enriched fractions of renal cortex (PMFs) is greater in D₅R^-/- mice than D₅R^+/+ littermates and D₅R protein expression in PMFs is also greater in CCK_BR^-/- mice than CCK_BR^+/+ littermates. High salt diet, relative to normal salt diet, increased the expression of CCK_BR and D₅R proteins in PMFs. Disruption of CCK_BR in mice caused hypertension and decreased sodium excretion. The natriuresis in salt-loaded BALB/c mice was decreased by YF476, a CCK_BR antagonist and {"type":"entrez-protein","attrs":{"text":"Sch23390","term_id":"1052733334"}}Sch23390, a D₁R/D₅R antagonist. Furthermore, the natriuresis caused by gastrin was blocked by {"type":"entrez-protein","attrs":{"text":"Sch23390","term_id":"1052733334"}}Sch23390 while the natriuresis caused by fenoldopam, a D₁R/D₅R agonist, was blocked by YF476. Taken together, our findings indicate that CCK_BR and D₅R synergistically interact in the kidney, which may contribute to the maintenance of normal sodium balance following an increase in sodium intake.     

C-terminal di-leucine motif of dopamine D₁ receptor plays an important role in its plasma membrane trafficking

The dopamine D₁ receptor (D₁R), a G protein-coupled receptor, plays a critical role in regulating blood pressure through its actions on renal hemodynamics and epithelial ion transport, which are highly linked to its intracellular trafficking. In this study, we generated a series of C-terminal mutants of D₁R that were tagged with or without enhanced yellow fluorescent protein, and analyzed the consequences of these mutants on the plasma membrane trafficking of D₁R and cyclic AMP response to D₁R stimulation. D₁R with mutations within the endocytic recycling signal (amino acid residues 360–382) continued to be functional, albeit decreased relative to wild-type D₁R. Mutation of the palmitoylation site (347C>S) of D₁R did not impair its trafficking to the plasma membrane, but abolished its ability to increase cyclic AMP accumulation. In contrast, replacement of di-leucines (344–345L>A) by alanines resulted in the retention of D₁R in the early endosome, decreased its glycosylation, and prevented its targeting to the plasma membrane. Our studies suggest that di-L motif at the C-terminus of D₁R is critical for the glycosylation and cell surface targeting of D₁R.     

D5 Dopamine Receptor Knockout Mice and Hypertension

Abnormalities in dopamine production and receptor function have been described in human essential hypertension and rodent models of genetic hypertension. All of the five dopamine receptor genes (D₁, D₂, D₃, D₄, and D₅) expressed in mammals and some of their regulators are in loci linked to hypertension in humans and in rodents. Under normal conditions, D₁-like receptors (D₁ and D₅) inhibit sodium transport in the kidney and the intestine. However, in the Dahl salt-sensitive and spontaneously hypertensive rats, and humans with essential hypertension, the D₁-like receptor-mediated inhibition of sodium transport is impaired because of an uncoupling of the D₁-like receptor from its G protein/effector complex. The uncoupling is genetic, and receptor-, organ-, and nephron segment-specific. In human essential hypertension, the uncoupling of the D₁ receptor from its G protein/effector complex is caused by an agonist-independent serine phosphorylation/desensitization by constitutively active variants of the G protein-coupled receptor kinase type 4. The D₅ receptor is also important in blood pressure regulation. Disruption of the D₅ or the D₁ receptor gene in mice increases blood pressure. However, unlike the D₁ receptor, the hypertension in D₅ receptor null mice is caused by increased activity of the sympathetic nervous system, apparently due to activation of oxytocin, V₁ vasopressin, and non-N-methyl D-aspartate receptors in the central nervous system. The cause of the activation of these receptors remains to be determined.     

Tli1, a resistance locus for carcinogen-induced T-lymphoma

Within 180 days after injection with N-methyl-N-nitrosourea (MNU), 83.5% of AKR/J mice and 37.5% of BALB/cJ mice developed T-lymphoma. The high tumor incidence was a dominant trait, as 93% of MNU-injected F₁ mice developed T-lymphoma. A genome screen of 285 MNU-injected F₂ mice identified a locus, designated T-lymphoma Induced 1 or Tli1, in a ∼10-cM interval on central Chr 1 between D1Mit87 and D1Mit423 with significant linkage to the incidence of MNU-induced T-lymphoma (P= 0.0004). Injection of BALB/cJ.AKR/J–Tli1 congenic mice with MNU confirmed the presence of Tli1 on central Chr 1. Mice homozygous for the BALB/cJ allele (Tli1 ^bb) were over-represented in the tumor-free F₂ mice, while the inheritance of parental alleles of Tli1 in tumor-bearing mice was close to expected. This suggests that the Tli1 ^b allele is recessive and suppresses MNU-induced T-lymphoma development in BALB/cJ mice and in Tli1 ^bb F₂ mice. Furthermore, the kinetics of lymphoma development in BALB/cJ and the Tli1 congenic mice suggests that Tli1 ^b acts to suppress lymphomas developing late after injection with MNU. Two known genes that map in the identified genomic interval on central Chr 1 are candidates for Tli1:IL10, encoding the lymphokine IL10, and Cmkar4, encoding the chemokine receptor CXCR4. Received: 2 November 1998 / Accepted 12 January 1999     

Overexpression of FAB1A-GFP recruits SNX2b on the endosome membrane in snx1–1 mutant in Arabidopsis

Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P₂] is one of the phosphoinositides that controls endosomal trafficking events in eukaryotes. PtdIns(3,5)P₂ is produced from PI(3)P by phosphatidylinositol 3-phosphate 5-kinase FAB1/PIKfyve. Recently, we reported that FAB1 predominantly localizes on the SNX1-residing late endosomes and a loss-of FAB1 function causes the release of late endosomal effector proteins, ARA7/RABF2b and SORTING NEXIN 1 from the endosome membrane, indicating that FAB1 or its product PtdIns(3,5)P₂ mediates the maturation process of the late endosomes. Intriguingly, the ectopic expression of FAB1A could complement the sucrose-dependent seedling growth phenotype of snx1–1 mutant. Here, we demonstrated that the depletion of SNX1 causes the release of SNX2b-mRFP from the endosomal membrane. However, overexpression of FAB1A-GFP reassembles SNX2b-mRFP on the endosomal membrane despite the absence of SNX1. From these results, we proposed that SNX2b homodimer or SNX2a/SNX2b heterodimer might function as functional Sorting Nexin complex instead of SNX1 to attach the endosomal membrane by binding of overproduced PI(3,5)P₂ in Arabidopsis.     

Striatal CB1 and D2 receptors regulate expression of each other,CRIP1A and delta opioid systems

Although biochemical and physiological evidence suggests a strong interaction between striatal CB₁ cannabinoid (CB₁R) and D₂ dopamine (D₂R) receptors, the mechanisms are poorly understood. We targeted medium spiny neurons of the indirect pathway using shRNA to knockdown either CB₁R or D₂R. Chronic reduction in either receptor resulted in deficits in gene and protein expression for the alternative receptor and concomitantly increased expression of the cannabinoid receptor interacting protein 1a (CRIP1a), suggesting a novel role for CRIP1a in dopaminergic systems. Both CB₁R and D₂R knockdown reduced striatal dopaminergic‐stimulated [³⁵S]GTPγS binding, and D₂R knockdown reduced pallidal WIN55212‐2‐stimulated [³⁵S]GTPγS binding. Decreased D₂R and CB₁R activity was associated with decreased striatal phosphoERK. A decrease in mRNA for opioid peptide precursors pDYN and pENK accompanied knockdown of CB₁Rs or D₂Rs, and over‐expression of CRIP1a. Down‐regulation in opioid peptide mRNAs was followed in time by increased DOR1 but not MOR1 expression, leading to increased [D‐Pen2, D‐Pen5]‐enkephalin‐stimulated [³⁵S]GTPγS binding in the striatum. We conclude that mechanisms intrinsic to striatal medium spiny neurons or extrinsic via the indirect pathway adjust for changes in CB₁R or D₂R levels by modifying the expression and signaling capabilities of the alternative receptor as well as CRIP1a and the DELTA opioid system.     

Experimental allergic orchitis in mice. V. Resistance to actively induced disease in BALB/cJ substrain mice is mediated by CD4 + T cells

Previous studies have shown that differential susceptibility to actively induced experimental allergic orchitis (EAO) exists among various BALB/c substrains. Of 13 substrains studied, BALB/cJ mice consistently exhibit greater resistance to disease induction. Such resistance is associated with a single recessive genotypic difference in an immunoregulatory locus which is unlinked to any of the known alleles distinguishing the BALB/cJ substrain. In this study, gene complementation protocols were used to study the genetics of susceptibility and resistance to EAO. The results indicate that resistance in BALB/cJ mice is not due to a mutation in theH-2D ^d linked gene which governs the phenotypic expression of autoimmune orchitis. The mechanistic basis for disease resistance was examined using reciprocal bone marrow radiation chimeras generated between the disease-susceptible BALB/ cByJ (ByJ) substrain and BALB/cJ (Jax) mice. All constructs, including Jax - Jax and Jax - ByJ, developed severe EAO following inoculation with mouse testicular homogenate (MTH) and adjuvants whereas control chimeras immunized with adjuvants alone did not. These results suggest that an active immunoregulatory mechanism rather than a passive one, such as the lack of T cells and/or B cells with receptors for the aspermatogenic autoantigens relevant in the induction of EAO, is responsible for disease resistance in BALB/cJ mice. The role of immunoregulatory cells was examined by pretreating BALB/cJ mice with either cyclophosphamide (20 mg/kg) or low-dose whole body or total lymphoid irradiation (350 rads) 2 days prior to inoculation. BALB/cJ mice immunized with MTH plus adjuvants generate immunoregulatory spleen cells (SpCs) that, when transferred to naive BALB/cByJ recipients, significantly reduce the severity of autoimmune orchitis observed during actively induced EAO. Treatment of such cells with either cytotoxic monoclonal anti-Thy-1.2 or anti-CD4 plus C' before transfer abrogates the ability of BALB/cJ spleen cells to inhibit disease. In contrast, neither SpCs from adjuvantimmunized BALB/cJ nor MTH plus adjuvant-primed BALB/cByJ donors significantly influenced the severity of disease observed in recipients. Taken together, these results suggest that genetically controlled resistance to EAO in BALB/cJ mice is associated with a mutation in an immunoregulatory locus whose effects appear to be mediated through a cyclophosphamide and low-dose radiation-sensitive CD4⁺ T-cell population.     

Dopamine Reduction of GABA Currents in Striatal Medium-sized Spiny Neurons is Mediated Principally by the D<Subscript>1</Subscript> Receptor Subtype

Dopamine modulates voltage- and ligand-gated currents in striatal medium-sized neurons (MSNs) through the activation of D1- and D2-like family receptors. GABA_A receptor-mediated currents are reduced by D1 receptor agonists, but the relative contribution of D₁ or D₅ receptors in this attenuation has been elusive due to the lack of selective pharmacological agents. Here we examined GABA_A receptor-mediated currents and the effects of D1 agonists on MSNs from wildtype and D₁ or D₅ receptor knockout (KO) mice. Immunohistochemical and single-cell RT-PCR studies demonstrated a lack of compensatory effects after genetic deletion of D₁ or D₅ receptors. However, the expression of GABA_A receptor α1 subunits was reduced in D₅ KO mice. At the functional level, whole-cell patch clamp recordings in dissociated MSNs showed that GABA peak current amplitudes were smaller in cells from D₅ KO mice indicating that lack of this receptor subtype directly affected GABA_A-mediated currents. In striatal slices, addition of a D1 agonist reduced GABA currents significantly more in D₅ KO compared to D₁ KO mice. We conclude that D₁ receptors are the main D1-like receptor subtype involved in the modulation of GABA currents and that D₅ receptors contribute to the normal expression of these currents in the striatum. Special issue dedicated to Anthony Campagnoni.     

The Leptin,Dopamine and Serotonin Receptors in Hypothalamic POMC-Neurons of Normal and Obese Rodents

The pro-opiomelanocortin (POMC)-expressing neurons of the hypothalamic arcuate nucleus (ARC) are involved in the control of food intake and metabolic processes. It is assumed that, in addition to leptin, the activity of these neurons is regulated by serotonin and dopamine, but only subtype 2C serotonin receptors (5-HT_2CR) was identified earlier on the POMC-neurons. The aim of this work was a comparative study of the localization and number of leptin receptors (LepR), types 1 and 2 dopamine receptors (D₁R, D₂R), 5-HT_1BR and 5-HT_2CR on the POMC-neurons and the expression of the genes encoding them in the ARC of the normal and diet-induced obese (DIO) rodents and the agouti mice (A ^y /a) with the melanocortin obesity. As shown by immunohistochemistry (IHC), all the studied receptors were located on the POMC-immunopositive neurons, and their IHC-content was in agreement with the expression of their genes. In DIO rats the number of D₁R and D₂R in the POMC-neurons and their expression in the ARC were reduced. In DIO mice the number of D₁R and D₂R did not change, while the number of LepR and 5-HT_2CR was increased, although to a small extent. In the POMC-neurons of agouti mice the number of LepR, D₂R, 5-HT_1BR and 5-HT_2CR was increased, and the D₁R number was reduced. Thus, our data demonstrates for the first time the localization of different types of the serotonin and dopamine receptors on the POMC-neurons and a specific pattern of the changes of their number and expression in the DIO and melanocortin obesity.     

Dopamine and G protein-coupled receptor kinase 4 in the kidney: Role in blood pressure regulation

Complex interactions between genes and environment result in a sodium-induced elevation in blood pressure (salt sensitivity) and/or hypertension that lead to significant morbidity and mortality affecting up to 25% of the middle-aged adult population worldwide. Determining the etiology of genetic and/or environmentally-induced high blood pressure has been difficult because of the many interacting systems involved. Two main pathways have been implicated as principal determinants of blood pressure since they are located in the kidney (the key organ responsible for blood pressure regulation), and have profound effects on sodium balance: the dopaminergic and renin–angiotensin systems. These systems counteract or modulate each other, in concert with a host of intracellular second messenger pathways to regulate sodium and water balance. In particular, the G protein-coupled receptor kinase type 4 (GRK4) appears to play a key role in regulating dopaminergic-mediated natriuresis. Constitutively activated GRK4 gene variants (R65L, A142V, and A486V), by themselves or by their interaction with other genes involved in blood pressure regulation, are associated with essential hypertension and/or salt-sensitive hypertension in several ethnic groups. GRK4γ ?142V?transgenic mice are hypertensive on normal salt intake while GRK4γ? 486V? transgenic mice develop hypertension only with an increase in salt intake. GRK4 gene variants have been shown to hyperphosphorylate, desensitize, and internalize two members of the dopamine receptor family, the D₁ (D₁R) and D₃ (D₃R) dopamine receptors, but also increase the expression of a key receptor of the renin–angiotensin system, the angiotensin type 1 receptor (AT₁R). Knowledge of the numerous blood pressure regulatory pathways involving angiotensin and dopamine may provide new therapeutic approaches to the pharmacological regulation of sodium excretion and ultimately blood pressure control.     

IgA allotype suppression in mice: A cellular implication for the IgA preponderance in the gut

Paternal IgA allotype suppressions were induced in (A/HeJ x SJL/J)F₁, (BALB/cJ x CB-20) F₁, and (BALB/cJ x SJL/J)F₁ mice by perinatal exposure of the neonates to antibody against paternal IgA allotype (Ig-2b). All the Ig-2b allotype suppressions were temporary and were prominently seen at the age of 4–10 weeks. These transient Ig-2b allotype suppressions were manifested by a suppression of Ig-2b allotype in the serum and a suppression of Ig-2b allotype-producing cells in the gut lamina propria. During the suppression, no replacement of the Ig-2b allotype by IgM and IgG plasma cells was seen in the gut lamina propria. The transient IgA allotype suppression was primarily due to a depletion of IgA allotype precursors in Peyer's patches; suppressor T cells were not actively involved. These results suggest that IgA preponderance in the gut is due to the migration of the patch IgA precursors and their accumulation and maturation in the gut lamina propria.     

Effects of long-term ouabain treatment on blood pressure, sodium excretion, and renal dopamine D1 receptor levels in rats

To examine the effects of chronic ouabain treatment on blood pressure (BP), sodium excretion, and renal dopamine D₁ receptor level, male Sprague-Dawley (SD) rats were treated with ouabain (27.8 μg kg⁻¹ d⁻¹) intraperitoneally for 5 weeks, and systolic blood pressure (SBP) were recorded weekly. After 5 weeks, sodium excretion and dopamine D₁ receptor agonist fenoldopam-mediated natriuresis were measured, and the expression and phosphorylation levels of the renal cortical dopamine D₁ receptor were confirmed by Western blot analysis. The effects of ouabain on fenoldopam-mediated inhibition of Na⁺-K⁺-ATPase activity were determined by colorimetric assays in human proximal tubular epithelial cells (HK-2 cells). After 5 weeks, the SBP in ouabain group was significantly higher than that in the control group (P < 0.01), but the sodium excretion and renal cortical D₁ receptor expression levels were reduced, and D₁ receptor phosphorylation levels were increased after ouabain treatment. Intravenous administration of fenoldopam caused an increased sodium excretion in control rats, but failed to induce natriuresis in ouabain-treated rats. In addition, fenoldopam induced a dose–respone (10⁻⁹ to 10⁻⁶ M) inhibition of Na⁺-K⁺-ATPase activity in HK-2 cells,but these effects were significantly diminished in HK-2 cells pretreated with nanomolar concentration of ouabain for 5 days (P < 0.01). We propose that the ouabain-induced reduction of the renal dopamine D₁ receptor function serves as a mechanism responsible for sodium retention, and this contributes to the hypertension induced by chronic ouabain treatment.     

Regulation of P2Y1 Receptor Traffic by Sorting Nexin 1 is Retromer Independent

The activity and traffic of G‐protein coupled receptors (GPCRs) is tightly controlled. Recent work from our laboratory has shown that P2Y₁ and P2Y₁₂ responsiveness is rapidly and reversibly modulated in human platelets and that the underlying mechanism requires receptor trafficking as an essential part of this process. However, little is known about the molecular mechanisms underlying P2Y receptor traffic. Sorting nexin 1 (SNX1) has been shown to regulate the endosomal sorting of cell surface receptors either to lysosomes where they are downregulated or back to the cell surface. These functions may in part be due to interactions of SNX1 with the mammalian retromer complex. In this study, we investigated the role of SNX1 in P2Y receptor trafficking. We show that P2Y₁ receptors recycle via a slow recycling pathway that is regulated by SNX1, whereas P2Y₁₂ receptors return to the cell surface via a rapid route that is SNX1 independent. SNX1 inhibition caused a dramatic increase in the rate of P2Y₁ receptor recycling, whereas inhibition of Vps26 and Vps35 known to be present in retromer had no effect, indicating that SNX1 regulation of P2Y₁ receptor recycling is retromer independent. In addition, inhibition of SNX4, 6 and 17 proteins did not affect P2Y₁ receptor recycling. SNX1 has also been implicated in GPCR degradation; however, we provide evidence that P2Y receptor degradation is SNX1 independent. These data describe a novel function of SNX1 in the regulation of P2Y₁ receptor recycling and suggest that SNX1 plays multiple roles in endocytic trafficking of GPCRs.     

Premature aging in vitamin D receptor mutant mice

Hypervitaminosis vitamin D₃ has been recently implicated in premature aging through the regulation of 1alpha hydroxylase expression by klotho and fibroblast growth factor-23 (Fgf-23). Here we examined whether the lack of hormonal function of vitamin D₃ in mice is linked to aging phenomena. For this, we used vitamin D₃ receptor (VDR) “Tokyo” knockout (KO) mice (fed with a special rescue diet) and analyzed their growth, skin and cerebellar morphology, as well as overall motor performance. We also studied the expression of aging-related genes, such as Fgf-23, nuclear factor kappaB (NF-kappaB), p53, insulin like growth factor 1 (IGF1) and IGF1 receptor (IGF1R), in liver, as well as klotho in liver, kidney and prostate tissues. Overall, VDR KO mice showed several aging related phenotypes, including poorer survival, early alopecia, thickened skin, enlarged sebaceous glands and development of epidermal cysts. There was no difference either in the structure of cerebellum or in the number of Purkinje cells. Unlike the wildtype controls, VDR KO mice lose their ability to swim after 6 months of age. Expression of all the genes was lower in old VDR KO mice, but only NF-kappaB, Fgf-23, p53 and IGF1R were significantly lower. Since the phenotype of aged VDR knockout mice is similar to mouse models with hypervitaminosis D₃, our study suggests that VDR genetic ablation promotes premature aging in mice, and that vitamin D₃ homeostasis regulates physiological aging.     

Effect of dietary calcium and 1,25-(OH)2D3 on the expression of calcium transport genes in calbindin-D9k and -D28k double knockout mice

The phenotypes of calbindin-D9k (CaBP-9k) and -28k (CaBP-28k) single knockout (KO) mice are similar to wild-type (WT) mice due to the compensatory action of other calcium transport proteins. In this study, we generated CaBP-9k/CaBP-28k double knockout (DKO) mice in order to investigate the importance of CaBP-9k and CaBP-28k in active calcium processing. Under normal dietary conditions, DKO mice did not exhibit any changes in phenotype or the expression of active calcium transport genes as compared to WT or CaBP-28k KO mice. Under calcium-deficient dietary conditions, the phenotype and expression of calcium transport genes in CaBP-28k KO mice were similar to WT, whereas in DKO mice, serum calcium levels and bone length were decreased. The intestinal and renal expression of transient receptor potential vanilloid member 6 (TRPV6) mRNA was significantly decreased in DKO mice fed a calcium-deficient diet as compared to CaBP-28k KO or WT mice, and DKO mice died after 4 weeks on a calcium-deficient diet. Body weight, bone mineral density (BMD) and bone length were significantly reduced in all mice fed a calcium and 1,25-(OH)₂D₃-deficient diet, as compared to a normal diet, and none of the mice survived more than 4 weeks. These results indicate that deletion of CaBP-28k alone does not affect body calcium homeostasis, but that deletion of CaBP-9k and CaBP-28k has a significant effect on calcium processing under calcium-deficient conditions, confirming the importance of dietary calcium and 1,25-(OH)₂D₃ during growth and development.     

Tyrosine‐protein phosphatase non‐receptor type 2 inhibits alveolar bone resorption in diabetic periodontitis via dephosphorylating CSF1 receptor

Tyrosine‐protein phosphatase non‐receptor type 2 (PTPN2) is an important protection factor for diabetes and periodontitis, but the underlying mechanism remains elusive. This study aimed to identify the substrate of PTPN2 in mediating beneficial effects of 25‐Hydroxyvitamin D₃ (25(OH)2D₃) on diabetic periodontitis. 25(OH)2D₃ photo‐affinity probe was synthesized with the minimalist linker and its efficacy to inhibit alveolar bone loss, and inflammation was evaluated in diabetic periodontitis mice. The probe was used to pull down the lysates of primary gingival fibroblasts. We identified PTPN2 as a direct target of 25(OH)2D₃, which effectively inhibited inflammation and bone resorption in diabetic periodontitis mice. In addition, we found that colony‐stimulating factor 1 receptor (CSF1R) rather than JAK/STAT was the substrate of PTPN2 to regulate bone resorption. PTPN2 direct interacted with CSF1R and dephosphorylated Tyr807 residue. In conclusion, PTPN2 dephosphorylates CSF1R at Y807 site and inhibits alveolar bone resorption in diabetic periodontitis mice. PTPN2 and CSF1R are potential targets for the therapy of diabetic periodontitis or other bone loss‐related diseases.