QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

HOME  |   SEARCH  |   ARCHIVE  |   SUBSCRIBE  |   CONTACT  |   HELP

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (53) Citing Articles via Google Scholar Google Scholar Articles by Hollon, T. R. Articles by Sibley, D. R. Search for Related Content PubMed PubMed Citation Articles by Hollon, T. R. Articles by Sibley, D. R.

 Previous Article  |  Next Article 

The Journal of Neuroscience, December 15, 2002, 22(24):10801-10810

Mice Lacking D₅ Dopamine Receptors Have Increased Sympathetic Tone and Are Hypertensive

Tom R. Hollon^{1, *}, Martin J. Bek^{3, *}, Jean E. Lachowicz⁴, Marjorie A. Ariano⁵, Eva Mezey², Ramesh Ramachandran⁶, Scott R. Wersinger⁶, Patricio Soares-da-Silva⁷, Zhi Fang Liu¹, Alexander Grinberg⁸, John Drago⁸, W. Scott Young III⁶, Heiner Westphal⁸, Pedro A. Jose³, and David R. Sibley¹

¹ Molecular Neuropharmacology Section and ² Basic Neurosciences Program, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892-1406, ³ Department of Pediatrics, Georgetown University Medical Center, Washington, DC 20007, ⁴ CNS/Cardiovascular Research, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, ⁵ Department of Neuroscience, The Chicago Medical School, North Chicago, Illinois 60064, ⁶ Section on Neural Gene Expression, National Institute of Mental Health, Bethesda, Maryland 20892, ⁷ Institute of Pharmacology and Therapeutics, Faculty of Medicine of Porto, Porto, Portugal, and ⁸ Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Dopamine is an important transmitter in the CNS and PNS, critically regulating numerous neuropsychiatric and physiological functions. These actions of dopamine are mediated by five distinct receptor subtypes. Of these receptors, probably the least understood in terms of physiological functions is the D₅ receptor subtype. To better understand the role of the D₅ dopamine receptor (DAR) in normal physiology and behavior, we have now used gene-targeting technology to create mice that lack this receptor subtype. We find that the D₅ receptor-deficient mice are viable and fertile and appear to develop normally. No compensatory alterations in other dopamine receptor subtypes were observed. We find, however, that the mutant mice develop hypertension and exhibit significantly elevated blood pressure (BP) by 3 months of age. This hypertension appears to be caused by increased sympathetic tone, primarily attributable to a CNS defect. Our data further suggest that this defect involves an oxytocin-dependent sensitization of V₁ vasopressin and non-NMDA glutamatergic receptor-mediated pathways, potentially within the medulla, leading to increased sympathetic outflow. These results indicate that D₅ dopamine receptors modulate neuronal pathways regulating blood pressure responses and may provide new insights into mechanisms for some forms of essential hypertension in humans, a disease that afflicts up to 25% of the aged adult population in industrialized societies.

Key words: D₅ receptor; gene knock-out; hypertension; sympathetic tone; oxytocin; vasopressin

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Dopamine is an important neurotransmitter in the brain as well as the periphery and plays a critical role in regulating numerous locomotor, neuroendocrine, cognitive, and emotional functions. Dysregulation of dopaminergic systems has also been hypothesized to underlie several neuropsychiatric and endocrine disorders, including Parkinson's disease, schizophrenia, Tourette's syndrome, and hyperprolactinemia. The actions of dopamine are mediated by five distinct receptor subtypes that belong to the G-protein-coupled receptor super-family and are divided into two major subgroups, D₁-like and D₂-like, the basis of their structure and pharmacology (Neve and Neve, 1997). The D₁-like subfamily consists of the D₁ and D₅ subtypes (also called D_1A and D_1B, respectively), both of which transduce their signals by increasing intracellular cAMP levels. The D₂-like subfamily consists of the D₂, D₃, and D₄ receptors, all of which can diminish cAMP production as well as regulate the activity of various ion channels. Although the D₁-like and D₂-like subfamilies can be differentiated pharmacologically, it is difficult to discriminate between receptors within each subfamily using selective ligands. This has led to uncertainties in ascribing specific physiological and behavioral functions to individual receptor subtypes. Investigators have approached this issue, in part, by creating genetically altered animals that lack individual receptor subtypes. Thus far, mice lacking D₁, D₂, D₃, or D₄ receptors have been produced, all of which have exhibited informative phenotypes (Sibley, 1999; Glickstein and Schmauss, 2001). This has resulted in the elucidation of receptor functions that could not have been obtained through other means such as the predominant presynaptic and postsynaptic roles of the D_2S and D_2L receptor isoforms, respectively (Usiello et al., 2000).

The D₅ dopamine receptor (DAR) has generated significant interest because of its relatively high affinity for dopamine, compared with other DARs, and its purported constitutive activity (Sunahara et al., 1991; Tiberi and Caron, 1994). This has suggested that the D₅ DAR may be activated in the absence or presence of low concentrations of endogenous agonist. Although the D₅ DAR is functionally coupled to the activation of adenylate cyclase, recent studies suggest that the D₅ DAR may also modulate GABA_A receptor-mediated activity through both second messenger cascades (Yan and Surmeier, 1997) as well as through direct receptor-receptor interactions (Liu et al., 2000). Localization of the D₅ DAR in the brain has revealed a widespread distribution, with the highest expression in the cerebral cortex, hippocampus, and basal ganglia (Ariano et al., 1997; Ciliax et al., 2000). Interestingly, recent reports have suggested a possible association of the D₅ DAR gene with schizophrenia (Muir et al., 2001) or substance abuse (Vanyukov et al., 1998). D₅ DARs are also expressed in the hypothalamus, where they may regulate circadian rhythms (Rivkees and Lachowicz, 1997) and female sexual behaviors (Apostolakis et al., 1996a,b). Within the periphery, D₅ DARs have been found in adrenal tissue (Dahmer and Senogles, 1996), kidney (Sanada et al., 2000), and also the gastrointestinal tract, where they may exert a protective effect on the intestinal mucosa (Mezey et al., 1996). To further elucidate the physiological roles of the D₅ DAR, we have now used gene-targeting technology to generate mice lacking functional D₅ DARs.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Construction of targeting vector and gene targeting. The targeting construct "pD5KO" contained a 9.6 kb genomic fragment of the mouse D₅ receptor gene isolated from a 129/Sv genomic library. This genomic sequence was subcloned as an EcoRV-NheI fragment into the gene targeting vector pPNT (Tybulewicz et al., 1991). Through a number of intermediate subcloning steps, a neomycin resistance gene was ligated, in reverse orientation, at the unique SfiI site of the D₅ receptor gene, thus disrupting the reading frame within the coding region. The length of D₅ receptor genomic DNA flanking the neomycin gene in the targeting vector was 6.9 kb (3') and 2.7 kb (5'). The targeting vector pD5KO was subsequently electroporated into the J1 line (Li et al., 1992) of embryonic stem (ES) cells, and simultaneous G418 positive selection and gancyclovir negative selection were used to enrich for ES cell colonies with successful gene targeting. Southern hybridization analysis was used to examine DNA from ES colonies for gene-targeting events. Homologous recombination at the 5' end of the targeting construct was detected by digesting ES cell genomic DNA with NcoI and hybridizing the Southern blot with probe A (see Results). The normal allele was 6 kb in length, and the recombinant allele was 3.4 kb. Probe B (see Results) was hybridized to Southern blots of ES genomic DNA cut with KpnI to detect homologous recombination at the 3' end. The normal allele was 28 kb, and the length of the recombinant allele was 18 kb. A hybridization probe, derived from the Neo gene, detected a single 3.4 kb band on NcoI Southern blots for those ES colonies in which gene targeting was successful; bands different in length from 3.4 kb on these blots indicated random integration of one of more copies of the targeting vector into the ES cell genome.

Mutant mouse generation and genotyping. C57BL/6 blastocysts were injected with ES cells from five different recombinant ES colonies and implanted into foster mothers. The 129/Sv-C57BL/6 chimeric offspring produced by blastocyst injection were bred with C57BL/6 mice to pass the recombinant D₅ receptor allele from the germline of these chimeras to an F1 generation. Southern blots of NcoI- or KpnI-digested genomic DNA from mouse tail biopsies, hybridized with probes A or B, respectively, were used to detect germline passage of the recombinant D₅ receptor allele in the F1 generation mice. Homozygous mutant (/) and wild-type (+/+) mice were generated from heterozygous mouse intermatings. In the later stages of this study, the mice were genotyped using a PCR-based method involving amplification from mouse genomic DNA isolated from tail biopsies. Oligonucleotide primers were designed to flank the SfiI restriction site into which the neomycin cassette was ligated. Primer 1 (5'-ACTCTCTTAATCGTCTGGACCTTG-3') and primer 2 (5'-TCGCAGGCTGGGGTCAGGTTCGCA-3') were used to amplify the wild-type allele, whereas primer 3 (5'-TGATCAACTAGTGCCCGGGCGGTA-3'), which was unique to the neomycin cassette, was used with primer 1 to amplify the recombinant allele. The PCR reaction used 0.2 µg of genomic tail DNA in a 50 µl reaction (50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl₂, 0.2 mM dNTP, 0.5 µM of each forward and reverse primer with 2.5 U TaqDNA polymerase). The initial cycle of amplification was as follows: denaturation at 94°C for 1 min, primer annealing at 55°C for 2 min, and extension at 72°C for 2 min. The reaction then was carried through 30 cycles consisting of 94°C for 45 sec, 55°C for 1 min, and 72°C for 1 min. The extension time at 72°C for the final cycle was 10 min.

Immunohistochemistry and in situ hybridization. The immunohistochemical analysis of the D₅ receptor protein was performed as described previously (Ariano et al., 1997). Adult male wild-type and homozygous mutant mice were killed, the brains were removed, and 10-µM-thick fresh-frozen brain sections were generated. Tissue sections from mutants and wild-type brains were processed simultaneously. In situ hybridization histochemistry was performed as described (Malik et al., 1996).

Receptor autoradiography. Brains from wild-type and D₅ mutant mice (n = 4 each) were removed after decapitation, frozen on dry ice, and stored at 80°C. Brain sections in the coronal plane were cut at 12 µm thickness and thaw-mounted on Superfrost plus slides (Fisher Scientific, Pittsburgh, PA). One set of slides from each animal that represented forebrain and midbrain regions was used for labeling the oxytocin receptor (OTR), whereas another set was used for labeling the vasopressin-1a receptor (VP). OTR autoradiography was performed using ¹²⁵I-labeled ornithine vasotocin analog [D(CH₂)₅-[Tyr(Me)², Thr⁴, Tyr-NH₂⁹], ¹²⁵I-OTA; specific activity, 2200 Ci/mmol; NEN Life Sciences Products, Boston, MA; NEX 254] as described previously (Insel and Shapiro, 1992). VP autoradiography was performed using ¹²⁵I-labeled linear VP ligand [HO-phenylacetyl1-D-Tyr(Me)²-Phe³-Gln⁴-Asn⁵-Arg⁶-Pro⁷-Arg⁸-NH₂; specific activity, 2200 Ci/mmol; NEN Life Sciences Products; NEX 310] as described previously (Young et al., 2000).

Receptor binding assays. Homogenate radioligand binding assays using striatal or kidney membrane preparations were performed as described previously for D₁-like receptors (Jiang and Sibley, 1999) and D₂-like receptors (Schetz et al., 2000). [³H]-SCH-23390 (DuPont/NEN; 71.3 Ci/mmol) was used to label D₁-like receptors, whereas [³H]-methylspiperone (DuPont/NEN; 84 Ci/mmol) was used to label D₂-like receptors. Adult male or female wild-type and homozygous mutant mice were killed, the brains were removed, and the corpus striatum was rapidly dissected and immediately frozen before subsequent membrane preparations and radioligand binding assays. Membrane protein concentrations were determined with the bicinchoninic acid protein reagent (Pierce, Rockford, IL) and a BSA standard curve.

Blood pressure studies. The mice were anesthetized with pentobarbital (50 mg/kg, i.p.), placed on heated board to maintain body temperature at 37°C, and tracheotomized (PE 90). Catheters were inserted into the femoral vessels and right jugular vein (PE 50 heat-stretched to 180 µm tip) for fluid administration, blood drawing, and blood pressure (BP) monitoring. After a 60 min stabilization period after the surgical procedures, the following agents were infused intravenously in random order: [1-(-mercapto-, -cyclopentamethylene propionic acid), 2-(O-methyl)tyrosine]-Arg⁸-vasopressin (Peninsula Laboratories, San Carlos, CA) at 10 µg/kg over 30 sec; BQ610 (Peninsula Laboratories) at 100 µg · kg¹ · min¹ for 10 min; BQ788 (Peninsula Laboratories) at 6.6 µg · kg¹ · min¹ for 15 min; phentolamine (RBI, Natick, MA) at 5 ng · kg¹ · min¹ for 30 min; losartan (Merck, Philadelphia, PA) at 3 mg/kg over 30 sec; GYKI 52466 (RBI) at 8 mg/kg over 30 sec; and CNQX (RBI) given at 1 mg/kg over 30 sec. The effects of these drugs, if any, on blood pressure and heart rate were monitored for 20-45 min. The blood pressure was allowed to stabilize at pre-infusion values for 30-60 min before the administration of subsequent drugs. In preliminary studies, the nonglutamatergic antagonists were shown to completely block the vasopressor effects of their respective agonists: arginine-vasopressin, phenylephrine, endothelin-1, and angiotensin II given over 30 sec at volumes of 40 µl (data not shown). In some mice, an oxytocin antagonist, D(CH2)₅[Tyr(Me)², Thr⁴, Thy-NH₂]OVT (Bachem AG, Torrance, CA) was administered intraperitoneally (0.3 µg/kg) 12 and 24 hr before blood pressure determination. The blood pressures were determined before oxytocin antagonist administration under a short-acting anesthetic agent, 2,2,2-tribromoethanol. The blood pressure effects of the V₁ vasopressin receptor antagonist and GYKI 52466 were subsequently tested under pentobarbital anesthesia as described above.

In some 2,2,2-tribromoethanol-anesthetized mice, blood pressures were measured during the placement of a femoral artery catheter, coated with 5% heparin complex, that was threaded upward and out of a 5 mm incision at the nape of the neck. Analgesia (buprenorphine) was given during the recovery period and continued on the first day after surgery. One-third ml of a sterile solution (1/2 mg plasmin and 1000 U heparin/ml of sterile saline) was used to flush the catheter immediately and every 2 d thereafter. Blood pressures were subsequently measured in freely moving, unanesthetized mice, 1-3 d after catheter placement.

Determination of catecholamine levels. The adrenal glands were homogenized with 0.1 M HClO₄ and centrifuged at 6000 × g for 20 min at 4°C, and catecholamine concentrations were determined by HPLC and electrochemical detection (Caramona and Soares-da-Silva, 1985).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Generation of D₅ DAR-deficient mice

A PCR-generated cDNA encoding the rat D₅ DAR was used to screen a 129/Sv mouse genomic library to isolate the mouse D₅ DAR gene. Several clones were isolated and characterized through partial sequencing and restriction mapping to confirm that they encoded the D₅ DAR gene. A restriction map of the mouse D₅ DAR gene is shown at the top of Figure 1A. To inactivate the D₅ DAR gene, a neomycin resistance gene was ligated, in reverse orientation, into a unique SfiI site of the D₅ receptor gene, thereby disrupting the reading frame within the coding region (Fig. 1A). A stop codon was engineered into the proximal neomycin gene linker such that the recombinant D₅ receptor would be prematurely truncated subsequent to Gly-190 in the second extracellular loop of the receptor. A total of 216 transfected ES cell lines were screened by Southern blotting (Fig. 1B), resulting in the identification of six cell lines exhibiting homologous recombination. ES cells amplified from five of these lines were used to generate chimeric male mice that were subsequently bred with C57/BL6 females. Only one line (Fig. 1B, 112) produced chimeras capable of transmitting the mutant allele to their offspring. Southern analysis of tail DNA from the progeny of heterozygous matings revealed the predicted restriction patterns for wild-type (+/+), heterozygote (+/), and mutant (/) genotypes (Fig. 1C).

View larger version (20K): [in this window] [in a new window]   Figure 1.   Generation of D5 receptor-deficient mice. A, The D5 receptor gene, targeting construct, and mutant loci of the D5 gene. The open box represents the coding region of the D5 gene, the solid box depicts the neomycin phosphotransferase gene, and the stippled box represents the herpes simplex thymidine kinase gene. Probes used in Southern hybridization analysis and predicted lengths of restriction fragments are also shown. A, AscI; E, EcoRV; K, KpnI; N, NcoI; Nh, NheI; S, SfiI, S/E, ligation junction of SrfI and EcoRV sites. B, Genomic analysis of targeted ES cell DNA. Recombination was detected at the 5' end by digesting ES cell DNA with NcoI and hybridizing the Southern blot with probe A. J1 represents the wild-type ES cell DNA, and 112 represents a targeted ES cell line exhibiting homologous recombination. C, Representative pedigree obtained from genotyping a litter of mice derived from crossing heterozygote parents. The genomic DNA was obtained from tail biopsies and digested with NcoI. The blot was subsequently hybridized with Probe A as described in B.

The D₅ DAR null mutant mice develop normally with no notable differences from wild-type littermates with respect to appearance, body weight, or home cage behaviors. The mutant allele appears to be inherited in a Mendelian manner, and the null mutants were fertile and capable of reproduction. Histological examinations revealed no abnormalities in major organ systems, and no obvious neurological or behavioral anomalies were noted. A more detailed behavioral characterization of these animals is presented elsewhere (Holmes et al., 2001).

Confirmation of D₅ DAR gene disruption

To confirm the disruption of the D₅ DAR gene, we took two different approaches. Because the D₁ and D₅ DARs are pharmacologically similar, there are no radioligands that selectively label the D₅ subtype, and because as the D₁ DAR is more prevalent than the D₅, binding assays with D₁-like ligands will predominantly label the D₁ DAR with little signal contributed by the D₅ subtype. Thus, we initially verified that the recombinant transcript was expressed by the mutant animals by performing RT-PCR analysis using RNA extracted from mouse brains. Figure 2A shows our strategy for the identification and detection of the wild-type and mutant transcripts by PCR. We designed three primers: 1 and 2 are unique to the coding sequence of the D₅ receptor and flank the SfiI site into which the neomycin resistance gene was inserted, whereas primer 3 is unique to the inserted neo gene sequence. Amplification with primers 1 and 2 should only result in a 702 bp fragment from the wild-type allele, whereas amplification with primers 1 and 3 should only result in a 468 bp fragment from the mutant allele. Although theoretically primers 1 and 2 could also give rise to a very large fragment from the mutant allele, the PCR conditions were not optimized for this to occur. Figure 2B shows an RT-PCR experiment using RNA extracted from the brains of homozygous wild-type (+/+), heterozygous (+/), and homozygous mutant (/) mice that had been genotyped via Southern blotting as shown in Figure 1C. As can be seen, all of the genotypes gave the predicted pattern of PCR fragments. Similar RT-PCR data were generated using RNA extracted from kidneys of wild-type and mutant genotypes (data not shown). These results indicate that the mutant mice are expressing the recombinant transcript as expected.

View larger version (32K): [in this window] [in a new window]   Figure 2.   Documentation that the D5 mutant allele is expressed in vivo. A, RT-PCR strategy for detection of the wild-type and mutant D5 receptor alleles in mouse tissues. Top, The coding region of the wild-type allele is shown with the unique SfiI restriction site. Primers 1 and 2 are derived from the coding region sequence. The predicted wild-type fragment is 702 bp in length. Bottom, The recombinant gene is shown with the neomycin resistance gene insert. Primer 3 is derived from the neomycin resistance gene. The predicted mutant fragment is 468 bp in length. B, RT-PCR analysis of RNA isolated from brain tissue. Wild-type (+/+), heterozygous (/+), and mutant (/) animals, identified by genotyping, were killed, brains were removed, and total RNA was isolated using the RNEasy Mini Kit (Qiagen, Hilden, Germany) and used for first-strand cDNA synthesis (Superscript II, Invitrogen, Gaithersburg, MD). An aliquot of this cDNA was subjected to amplification by PCR using the genotyping procedure described in Materials and Methods. The primers used in each amplification reaction are indicated at the top of each lane; the size of the fragments is indicated on the left.

Disruption of the D₅ DAR gene was further confirmed by directly examining the expression of the D₅ receptor protein using immunohistochemistry. We have previously described selective antisera for labeling the D₅ receptor protein in rat brain (Ariano et al., 1997). Two antisera were generated using peptides derived from the third extracellular and third intracellular loops of the D₅ DAR. Both of these epitopes are "downstream" of the truncation site in the recombinant D₅ DAR and should not be expressed in the mutant animals. Figure 3 shows immunofluorescence in fresh-frozen sections of frontal cortices from wild-type and mutant mouse brains. Both anti-D₅ DAR antisera detected neurons in the frontal cortices of wild-type animals (Fig. 3A,D), analogous to results in rat brain tissue (Ariano et al., 1997). In contrast, no staining was observed in brains from the D₅ mutant animals (Fig. 3B,E), and the fluorescence was at background levels (C, F). Taken together, the data in Figures 2 and 3 confirm that the D₅ DAR gene was disrupted in the mutant animals.

View larger version (46K): [in this window] [in a new window]   Figure 3.   Immunohistochemical detection of D5 receptor protein in wild-type and mutant mouse brains. Fresh-frozen mouse brains were mounted in the coronal plane and sectioned at 10 µm. Sections derived from frontal cortex were subsequently processed as described in Materials and Methods. A-C show results with antisera P14, which is directed to an extracellular epitope in the third external loop of the receptor. D-F show results with antisera C14, which is directed to an intracellular epitope in the third cytoplasmic loop of the receptor. C and F show wild-type tissue processed in the absence of primary antisera. Scale bar, 25 µm.

D₁- and D₂-like receptor binding is normal in the D₅ mutant mice

As part of our initial characterization of the D₅ mutant mice, we wished to assess whether there were any alterations in the expression of other dopamine receptor subtypes, perhaps arising as a consequence of developmental compensation. We thus performed radioligand binding assays using striatal membrane homogenates because the striatum is one of the regions of highest expression for the D₁ and D₂ receptor subtypes and also contains D₃ and D₄ receptors (Ariano, 1996). Figure 4A shows a saturation radioligand binding experiment using the D₁-like selective antagonist [³H]SCH-23390 in membranes prepared from wild-type and mutant mice. As can be seen, there were no differences between the genotypes. Similar results were obtained via D₁ receptor radioligand binding assays in kidney membranes derived from both genotypes (data not shown). Because only the D₁ receptor is being labeled in the D₅ mutant mice, these results indicated that there were no compensatory alterations in this receptor subtype and further illustrated the predominance of the D₁ DAR. In other studies, we also used D₁-selective antisera and immunohistochemical techniques to verify that the cellular staining of the D₁ receptor was unaltered in various brain regions of the D₅ DAR-deficient mice (data not shown).

View larger version (22K): [in this window] [in a new window]   Figure 4.   D1- and D2-like receptor binding assays in striatal membrane homogenates of D5 mutant mice. Striatal membranes were prepared from wild-type (WT) and mutant (KO) mice, and radioligand binding assays were performed as described in Materials and Methods with the indicated concentrations of ligands. Only specific binding is shown. The data represent the mean ± SEM values from three separate experiments using individual mice. A, Saturation binding analyses in striatal membranes using the D1-like selective antagonist [3H]SCH 23390. Computer analysis of the radioligand binding data resulted in the following parameters: wild-type, KD = 0.34 ± 0.01 nM, Bmax = 1.01 ± 0.18 pmol/mg protein; mutant, KD = 0.38 ± 0.03 nM, Bmax = 1.17 ± 0.28 pmol/mg protein. Using Student's t test, there was no significant difference between the Bmax values (p = 0.51). B, Saturation binding analyses in striatal membranes using the D2-like selective antagonist [3H]methylspiperone. Computer analysis of the radioligand binding data resulted in the following parameters: wild-type, KD = 0.30 ± 0.04 nM, Bmax = 430 ± 81 fmol/mg protein; mutant, KD = 0.28 ± 0.02 nM, Bmax = 460 ± 60 fmol/mg protein. Using Student's t test, there was no significant difference between the Bmax values (p = 0.67).

Figure 4B shows a saturation radioligand binding assay using the D₂-like selective antagonist [³H]methylspiperone. This ligand will label the D₂, D₃, and D₄ receptors with approximately equal affinity. Within the striatum, however, ~90-95% of the D₂-like receptors are composed of the D₂ subtype, with the rest consisting of the lower abundant D₃ and D₄ receptors. As can be seen, there are no significant differences in [³H]methylspiperone binding between the two mouse genotypes. Although the D₃ and D₄ receptors remain to be analyzed in greater detail, these results suggested that there were no alterations in D₂ receptor expression in the D₅ mutant animals.

The D₅ receptor mutant mice are hypertensive and have increased sympathetic tone

Several physiological parameters were examined in adult D₅ mutant mice (/), and comparisons were made with wild-type littermates (+/+) as well as with the parental C57BL/6 and 129/Sv mouse strains (Table 1). Although there were no differences in body or kidney weights between the D₅ +/+ and D₅ / genotypes, the D₅ mutant mice exhibited significantly elevated heart weights as well as elevated systolic, diastolic, and mean blood pressures. Blood pressures were also significantly elevated in the mutant animals even in the absence of anesthesia: wild-type (n = 4) systolic BP = 119 ± 4, mean BP = 103 ± 3, diastolic BP = 93 ± 4; mutant (n = 5) systolic BP = 154 ± 6, mean BP = 127 ± 8, diastolic BP = 115 ± 8; p < 0.05 for all wild-type versus mutant values; Student's t test. No gender differences were noted.

View this table: [in this window] [in a new window]   Table 1.   Physiological measurements in adult D5 mutant (/), wild-type littermates (+/+), and parental strains of mice (3-9 months old)

Because the 129 parental strain also showed significantly higher heart weights (Table 1), the elevated heart weights in the D₅ / mice could be attributable to 129-linked genes. Alternatively, the increased heart size in the mutants may represent a compensatory response to the elevation in blood pressure. To address this issue, we evaluated the cardiovascular parameters in young mice not older than 2 months of age. Table 2 shows these results. No differences were observed in the genotypes for the parameters obtained using the young mice. The blood pressures were slightly but not significantly elevated at this age, and there were no differences in the heart weights. These results indicate that the hypertension exhibited by the D₅ / mice was age dependent and suggest that the cardiac hypertrophy observed in the adult mutants was a result of the elevated blood pressure.

View this table: [in this window] [in a new window]   Table 2.   Physiological measurements in young (2 months old) D5 mutant (/) and wild-type (+/+) mice

Because D₅ receptors inhibit catecholamine release from adrenal chromaffin cells (Dahmer and Senogles, 1996), we tested whether elevated adrenal catecholamines might contribute to the increased blood pressure in the null mutants. Adrenal norepinephrine and epinephrine levels are presented in Table 3. No significant differences in the absolute levels of norepinephrine or epinephrine were found between genotypes, but there was a significant elevation in the epinephrine/norepinephrine ratio in the mutant animals. Furthermore, acute adrenalectomy resulted in a greater reduction in mean arterial pressure in the D₅ mutant animals (110 ± 8 to 62 ± 6 mmHg; n = 7) compared with wild-type mice (88 ± 0.4 to 56 ± 5 mmHg; n = 6). Given these results, we wondered whether sympathetic blockade would normalize the blood pressure in the mutant mice relative to wild-type animals. Infusion of the -adrenergic antagonist phentolamine resulted in a greater and more rapid reduction of blood pressure in the mutant mice compared with wild-type animals (Fig. 5A). Taken together, these experiments suggested that activation of the sympathetic nervous system occurred after disruption of the D₅ receptor gene.

View this table: [in this window] [in a new window]   Table 3.   Catecholamine levels in adrenal glands from D5 mutant (/) and wild-type (+/+) mice

View larger version (33K): [in this window] [in a new window]   Figure 5.   Mean arterial blood pressure responses in mice resulting from infusion of various pharmacological agents. Infusions were performed as described in Materials and Methods. The data represent the means ± SEM. In some cases, the error bars are smaller than the data points. A, The -adrenergic antagonist phentolamine was infused into wild-type (n = 7) and mutant (n = 14). B, The peripherally restricted (CNQX) and centrally acting (GYKI 52466) glutamatergic antagonists were infused into wild-type and mutant mice: CNQX: wild-type (n = 6), mutant (n = 6); GYKI 52466: wild-type (n = 9), mutant (n = 9). C, The V1 vasopressin antagonist, [1-(-mercapto-, -cyclopentamethylene propionic acid), 2-(O-methyl)tyrosine]-Arg8-vasopressin was infused into wild-type (n = 8) and mutant (n = 14) mice. D, The angiotensin II AT1 receptor antagonist, losartan, and the endothelin A and B receptor antagonists, BQ610 and BQ788, respectively, were infused into wild-type and mutant mice (n = 6-14 per group).

Because of the low-level expression of D₅ receptors in the adrenal medulla, we thought that the increased sympathetic tone in the D₅-deficient mice might be explained more readily by a defect within the CNS. Because central sympathetic nerve responses originating in the pons and medulla are regulated by non-NMDA glutamatergic pathways (Butcher and Cechetto, 1998), we evaluated the effect of glutamatergic blockade on the blood pressure responses. Figure 5B shows the results of infusing the AMPA/kainate glutamatergic receptor antagonists CNQX and GYKI 52466 (Yoshiyama et al., 1995) into wild-type and mutant mice. CNQX does not cross the blood-brain barrier and had no effect on the mean arterial blood pressure in either wild-type or mutant mice. In contrast, GYK1 52466, which does cross the blood-brain barrier, reduced blood pressure in the mutant mice but had no effect on the wild-type animals. These results suggested that central non-NMDA glutamatergic pathways were abnormally activated in the null mutants, resulting in an elevation in blood pressure.

To further explore the central mechanisms underlying the hypertension in the D₅ DAR mutant mice, we investigated the role of the neuropeptide vasopressin. Arginine vasopressin (AVP) and V₁ vasopressin receptors regulate cardiovascular function and blood pressure and may be involved in human essential hypertension (Bakris et al., 1997). AVP also increases arterial blood pressure via activation of V₁ receptors in the area postrema, which projects to the nucleus tractus solitarius in the dorsomedial medulla (Migita et al., 1998). Interestingly, both NMDA and non-NMDA glutamate receptors are known to regulate the synaptic pathway between the area postrema and nucleus tractus solitarius (Aylwin et al., 1998; Migita et al., 1998). Figure 5C shows that infusion of the centrally acting V₁ receptor antagonist, [1-(-mercapto-, -cyclopentamethylene propionic acid), 2-(O-methyl)tyrosine]-Arg⁸-vasopressin (Bealer and Abell, 1995) reduced the mean arterial pressure in the mutant mice to normal levels yet did not significantly affect blood pressure in the wild-type animals. These results suggested that increased central V₁ receptor activity contributed to the elevated blood pressure in the D₅ mutant mice. Importantly, co-administration of the V₁ receptor antagonist and the glutamate antagonist GYKI 52466 to the mutant mice did not result in an additive reduction of blood pressure (data not shown), suggesting a common mechanism or pathway of action.

Because endothelins and angiotensin II are pressor agents that have been implicated in the pathogenesis of hypertension, we examined the cardiovascular effects of antagonizing these receptor systems (Fig. 5D). The endothelin A and B receptor antagonists, BQ610 (Beyer et al., 1999) and BQ788 (Allcock et al., 1995), respectively, did not affect the mean arterial blood pressure in either genotype. In contrast, the angiotensin II AT₁ receptor antagonist losartan (Asico et al., 1998) reduced the mean arterial pressure to the same extent in both genotypes. These results indicated that deletion of the D₅ receptor did not alter these peptide/receptor systems and demonstrated that the mutant mice were not selectively sensitive to depressor agents in general.

Because pretreatment of rats with the peptide oxytocin sensitizes the V₁ receptor-mediated pressor response to vasopressin (Poulin et al., 1994), we evaluated the role of oxytocin pathways in the elevated blood pressure in the mutant mice. Figure 6 shows the results from an experiment in which a centrally acting oxytocin receptor antagonist, D(CH2)₅[Tyr(Me)², Thr⁴, Thy-NH₂]OVT (Boccia et al., 1998), was administered 12 and 24 hr before the blood pressure measurements. Interestingly, pretreatment with the oxytocin antagonist alone was sufficient to reduce the systolic blood pressure to normal levels in the mutant mice, whereas there was no effect on the wild-type animals. Moreover, pretreatment with the oxytocin receptor antagonist negated any further effect on blood pressure by subsequent infusion of the glutamatergic antagonist GYKI 52466 or the V₁ receptor antagonist. These results suggested that oxytocin elevated blood pressure in the D₅ DAR-deficient mice by increasing the activity of central glutamatergic and vasopressin pathways potentially involved in regulating sympathetic outflow.

View larger version (15K): [in this window] [in a new window]   Figure 6.   Blood pressure responses in mice pretreated with a centrally acting oxytocin receptor antagonist. The blood pressures were initially determined (BASAL) followed by two injections of the oxytocin receptor antagonist D(CH2)5[Tyr(Me)2, Thr4, Thy-NH2]OVT, as described in Materials and Methods. Systolic blood pressures were then evaluated alone (OTR) or subsequent to the infusion of GYKI 52466 (OTR + GYKI) or [1-(-mercapto-, -cyclopentamethylene propionic acid), 2-(O-methyl)tyrosine]-Arg8-vasopressin (OTR + V1) as described in Figure 5, B and C. The data represent the mean ± SEM values from five to nine animals per group. In some cases, the error bars are smaller than the data points. *p < 0.05 versus other groups; ANOVA; Newman-Keuls test. Similar results were observed for diastolic blood pressures (data not shown).

Because dopamine, acting partially via D₁-like DARs, stimulates the synthesis of vasopressin and oxytocin in the brain and pituitary (Cornish and van den Buuse, 1995; Mathiasen et al., 1996; Galfi et al., 2001), we examined their corresponding mRNA levels in the D₅ DAR-deficient mice. In situ hybridization histochemical analysis of vasopressin and oxytocin mRNA in the hypothalami of wild-type and mutant mice showed abundant expression of both mRNAs in the paraventricular and supraoptic nuclei (Fig. 7). Surprisingly, there was a significant decrease in the expression of vasopressin mRNA in D₅ DAR-deficient mice but no alteration in the mRNA levels for oxytocin. We also observed decreased plasma levels of AVP in the mutant mice, although this did not achieve statistical significance (wild type: 0.070 ± 0.023 ng/ml, n = 12; mutant: 0.027 ± 0.007 ng/ml, n = 12; p = 0.15).

View larger version (44K): [in this window] [in a new window]   Figure 7.   In situ hybridization histochemical (ISHH) analyses of vasopressin and oxytocin mRNA in the hypothalami of wild-type (n = 8) and mutant mice (n = 9). ISHH was performed as described in Materials and Methods, with representative images shown at the bottom of the figure. Phosphorimage analysis was performed by choosing a level that went through the middle of the paraventricular and supraoptic nuclei in each brain. The y-axis represents arbitrary optical density (OD) values assigned by the Image Gauge program of the Fuji phosphor imager. The vasopressin mRNA levels were significantly decreased in the mutant brains: OD mean ± SEM values: wild-type, 9.0 ± 0.73; mutant, 5.1 ± 0.73; p < 0.05; Student's t test.

Given the results in Figure 7, we thought it would be of interest to examine the expression of oxytocin and vasopressin V₁ receptors in the D₅ mutant brains. Receptor autoradiography was performed throughout multiple serial brain sections using radioiodinated ligands for both V₁ vasopressin and oxytocin receptors. Figure 8 shows representative coronal sections through the hypothalami of wild-type and mutant mice. No consistent differences were noted between the genotypes at any level examined. Deletion of the D₅ receptor therefore did not appear to affect the expression levels of these receptors.

View larger version (120K): [in this window] [in a new window]   Figure 8.   Receptor autoradiography of V1 vasopressin and oxytocin receptors in brain sections of wild-type (n = 3) and mutant mice (n = 3). Representative autoradiograms of sections from wild-type (A, C) and mutant (B, D) mice through the hypothalamus show binding of the oxytocin (A, B) and vasopressin (C, D) receptor ligands. No consistent differences were noted between the wild-type and knock-out mice at any level examined. IV, Cortical layer 4; En, endopiriform nucleus; HF, hippocampal formation; LH, lateral hypothalamus; PL, posterolateral cortical amygdaloid nucleus; PV, thalamic paraventricular nucleus; VM, ventromedial hypothalamic nucleus. Three sets of wild-type and D5 mutant brains were examined with similar results.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Since its initial discovery and characterization, the exact physiological and behavioral roles of the D₅ receptor have been difficult to clarify with certainty. This has been attributable, in large part, to the fact that the D₁ and D₅ DARs are pharmacologically indistinguishable. There are few, if any, ligands that exhibit >10-fold selectivity for either subtype (Neve and Neve, 1997). Interestingly, dopamine is one of the most selective agents demonstrating ~10-fold higher affinity at the D₅ DAR compared with the D₁. This lack of selective ligands has made it virtually impossible to selectively activate or block D₁ or D₅ receptors in vivo. Genetic approaches to this problem have been used by investigators using antisense technologies to downregulate D₁ or D₅ DAR expression as well as the creation of D₁ DAR-deficient mice (Sibley, 1999; Glickstein and Schmauss, 2001). These studies have demonstrated a predominant role of the D₁ receptor in regulating various locomotor, cognitive, and other behaviors. Reports of antisense "knock-down" of D₅ receptor expression have been sparse but have suggested a role for the D₅ DAR in regulating female sexual behaviors (Apostolakis et al., 1996a,b) and locomotor responses to dopaminergic agonists (Dziewczapolski et al., 1998).

In the present study, we have used gene targeting technology to generate mice completely lacking functional D₅ DARs. Documenting the inactivation of the D₅ DAR gene presented a challenge because there are no radioligands that can be used to selectively label the D₅ subtype without simultaneously labeling the D₁ DAR, and because as the D₁ receptor is more abundant relative to the D₅, radioligand binding assays with D₁-like ligands will predominantly label the D₁ DAR with very little signal being contributed by the D₅ subtype. This has been well demonstrated in the recent publication of Montague et al. (2001), who performed radioligand binding assays in mice lacking the D₁ receptor subtype. Using [³H]SCH-23390, which labels both D₁ and D₅ receptors, almost all D₁-like binding was found to be ablated in the brains of these animals. There were, however, demonstrable levels of [³H]SCH-23390 binding in the hippocampi of the D₁ knock-out mice. This was presumed to represent binding to the D₅ receptor because the hippocampus is a brain region of (relatively) high expression for this subtype. Although the use of radioiodinated ligands could probably detect more D₅ receptor binding sites in the D₁ DAR-deficient mice, it is clear that the D₁ receptor is more abundant than the D₅, and approaches other than radioligand binding are needed to demonstrate the absence of a functional D₅ receptor. We thus used two complementary approaches. First, we used RT-PCR analyses to show that the D₅ mutant mice were expressing the recombinant transcript as expected. Second, we used selective antisera to the D₅ DAR and showed that the D₅-deficient mice lacked specific immunohistochemical staining that was observed in wild-type mice. Taken together, these results indicate that the D₅ DAR gene was inactivated as planned.

The D₅ DAR-deficient mice were viable, appeared to develop normally, and were fertile and capable of reproduction. This latter observation was especially interesting given the antisense studies (Apostolakis et al., 1996a,b) that described suppression of lordosis behavior in receptive females after D₅ DAR knock-down in the ventromedial nucleus of the hypothalamus. Despite their ability to reproduce, it will be interesting to determine whether the sexual behaviors of these animals are abnormal in any way. In general, the home cage behaviors of the D₅ DAR-deficient mice appeared normal. Casual observation could not distinguish between mutant and wild-type animals. As is reported elsewhere, however, the D₅ mutant mice did show some altered behavioral responses to dopaminergic agonist stimulation (Holmes et al., 2001). Additional experimentation using the D₅ DAR-deficient mice is currently underway to elucidate additional behavioral roles of this receptor subtype.

Because functional deletion of the D₅ DAR might result in compensatory upregulation of other dopamine receptor subtypes, especially the D₁ DAR, we examined the expression of D₁-like and D₂-like receptors in the striatum, a brain region that expresses all DAR subtypes (Ariano, 1996). Using radioligands, which label either D₁-like or D₂-like receptors, we found no differences in the receptor binding activities when comparing mutant and wild-type animals. Similarly, no alterations in D₁ receptor expression were noted using immunohistochemical methods. These results indicate that there are no compensatory alterations in the expression of the D₁ DAR and suggest that the D₂-like receptors (D₂, D₃, and D₄) are similarly unaffected.

As part of our initial characterization of the D₅ mutant animals, we discovered that they were hypertensive, exhibiting significantly elevated blood pressures. The elevation in the epinephrine/norepinephrine ratio and the greater reduction in mean arterial pressure after adrenalectomy, or with -adrenergic blockade, in the mutant mice compared with wild-types, suggested that the hypertension was caused by increased sympathetic activity. However, because the percentage decrease in systolic blood pressure after adrenalectomy was similar in both mutant and wild-type mice, we sought to determine whether there were CNS mechanisms that may have contributed to the increase in blood pressure in the D₅ mutant mice.

Dopamine receptors, including the D₅ DAR, are present in the prefrontal cortex (Ariano et al., 1997; Ciliax et al., 2000), which projects to several brain areas involved with cardiovascular regulation (Verbene and Owens, 1998). Sympathetic responses from the prefrontal cortex are mediated within the lateral hypothalamic area (LHA) and ventrolateral medulla (VLM). Moreover, sympathetic responses originating in the prefrontal cortex and LHA are mediated by non-NMDA glutamate receptors in the VLM (Butcher and Cechetto, 1998). Indeed, CNS stimulation of non-NMDA glutamate receptors, specifically in the VLM, increases blood pressure (Chen et al., 1994; Araujo et al., 1999). Our studies suggest that the increased blood pressure in the D₅ DAR-deficient mice may be caused by activation of a sympathetic/non-NMDA glutamatergic axis because only a centrally acting non-NMDA glutamatergic antagonist decreased blood pressure in D₅ mutant mice.

The D₅ receptor may also negatively interact with oxytocin and vasopressin pathways in the prefrontal cortex and other brain areas associated with autonomic control (Ariano et al., 1997; Hermes et al., 1998; Buijs and Van Eden, 2000; Ciliax et al., 2000). Thus, V₁ vasopressin (Bealer and Abell, 1995) and oxytocin (Boccia et al., 1998) antagonists that cross the blood-brain barrier were found to decrease the blood pressure in the D₅ mutant but not wild-type mice. Interestingly, the hypotensive effect of the oxytocin antagonist occurred only 24 hr after its administration and negated any further reduction in blood pressure by vasopressin or glutamatergic blockade. These results are consistent with the observation that oxytocin has been shown to sensitize V₁ vasopressin receptors (Poulin et al., 1994) and further suggests that the decrease in blood pressure in the mutant mice engendered by these various antagonists occurs via a common output pathway.

In summary, we have found that functional deletion of the D₅ DAR gene produces hypertension in mice. The elevated blood pressure appears to be attributable to increased sympathetic tone with an involvement of adrenal catecholamines. The exact defect leading to the increase in sympathetic tone is unclear, although it appears to be primarily central in origin. Our current results suggest that D₅ receptor deletion results in an oxytocin-dependent sensitization of V₁ vasopressin and non-NMDA glutamatergic receptor-mediated pathways, potentially within the medulla, leading to increased sympathetic outflow in the mutant mice. This change is not associated with increased synthesis of either oxytocin or vasopressin, and in fact, vasopressin synthesis appears reduced in the D₅ DAR-deficient mice. Furthermore, there is no increase in oxytocin or V₁ vasopressin receptor numbers. This suggests that the increased sensitivity must occur at the level of receptor signaling, possibly via enhanced G-protein interactions, or other regulatory mechanisms, and/or downstream intracellular signaling pathways. The physiological events described here resulting from D₅ DAR deletion may provide new insights into mechanisms for some forms of essential hypertension in humans and may lead to new therapeutic approaches for its treatment.

	FOOTNOTES

Received May 31, 2002; revised Sept. 19, 2002; accepted Sept. 20, 2002.

^* T.R.H. and M.J.B. contributed equally to this work.

This work was partially supported by Department of Defense Grant 17-99-1-9542 to M.A.A. We thank Dr. Dong Jiang, Dr. Laureano D. Asico, David Cabrera, Sing Ping Huang, and Binu Tharakan for their assistance.

Correspondence should be addressed to Dr. David R. Sibley, Molecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke/National Institutes of Health, Building 10, Room 5C108, 10 Center Drive, MSC 1406, Bethesda, MD 20892-1406. E-mail: sibley{at}helix.nih.gov.

J. Drago's present address: Neurosciences Group, Monash University Department of Medicine, Monash Medical Centre, Clayton, Victoria, 3168, Australia.

M. J. Bek's present address: Department of Internal Medicine, Freiburg University Medical School, Freiburg, Germany.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

• Allcock GH, Warner TD, Vane JR (1995) Roles of endothelin receptors in the regional and systemic vascular responses to ET-1 in the anaesthetized ganglion-blocked rat: use of selective antagonists. Br J Pharmacol 116:2482-2486[Web of Science][Medline].
• Apostolakis EM, Garai J, Fox C, Smith CL, Watson SJ, Clark JH, O'Malley BW (1996a) Dopaminergic regulation of progesterone receptors: brain D₅ dopamine receptors mediate induction of lordosis by D₁-like agonists in rats. J Neurosci 16:4823-4834[Abstract/Free Full Text].
• Apostolakis EM, Garai J, Clark JH, O'Malley BW (1996b) In vivo regulation of central nervous system progesterone receptors: cocaine induces steroid-dependent behavior through dopamine transporter modulation of D5 receptors in rats. Mol Endocrinol 10:1595-1604[Abstract/Free Full Text].
• Araujo GC, Lopes OU, Campos RR (1999) Importance of glycinergic and glutamatergic synapses within the rostral ventrolateral medulla for blood pressure regulation in conscious rats. Hypertension 34:752-755[Abstract/Free Full Text].
• Ariano MA (1996) In: Dopamine receptor localization. The dopamine receptors (Neve KA, Neve RL, eds), pp 77-104. Totowa, NJ: Humana.
• Ariano MA, Wang J, Noblett KL, Larson ER, Sibley DR (1997) Cellular distribution of the rat D_1B receptor in central nervous system using anti-receptor antisera. Brain Res 746:141-150[Web of Science][Medline].
• Asico LD, Ladines C, Fuchs S, Accili D, Carey RM, Semeraro C, Pocchiari F, Felder RA, Eisner GM, Jose PA (1998) Disruption of the dopamine D₃ receptor gene produces renin-dependent hypertension. J Clin Invest 102:493-498[Web of Science][Medline].
• Aylwin ML, Horowitz JM, Bonham AC (1998) Non-NMDA and NMDA receptors in the synaptic pathway between area postrema and nucleus tractus solitarius. Am J Physiol 275:H1236-H1246[Medline].
• Bakris G, Bursztyn M, Gavras I, Bresnahan M, Gavras H (1997) Role of vasopressin in essential hypertension: racial differences. J Hypertens 15:545-550[Web of Science][Medline].
• Bealer SL, Abell SO (1995) Paraventricular nucleus histamine increases blood pressure by adrenoceptor stimulation of vasopressin release. Am J Physiol 269:H80-H85[Web of Science][Medline].
• Beyer ME, Slesak G, Hovelborn T, Kazmaier S, Nerz S, Hoffmeister HM (1999) Inotropic effects of endothelin-1: interaction with molsidomine and with BQ 610. Hypertension 33:145-152[Abstract/Free Full Text].
• Boccia MM, Kopf SR, Baratti CM (1998) Effects of a single administration of oxytocin or vasopressin and their interactions with two selective receptor antagonists on memory storage in mice. Neurobiol Learn Mem 69:136-146[Web of Science][Medline].
• Buijs RM, Van Eden CG (2000) The integration of stress by the hypothalamus, amygdala and prefrontal cortex: balance between the autonomic nervous system and the neuroendocrine system. Prog Brain Res 126:117-132[Medline].
• Butcher KS, Cechetto DF (1998) Neurotransmission in the medulla mediating insular cortical and lateral hypothalamic sympathetic responses. Can J Physiol Pharmacol 76:737-746[Medline].
• Caramona MM, Soares-da-Silva P (1985) The effects of chemical sympathectomy on dopamine, noradrenaline and adrenaline content in some peripheral tissues. Br J Pharmacol 86:351-356[Web of Science][Medline].
• Chen K, Hernandez YM, Dretchen KL, Gillis RA (1994) Intravenous NBQX inhibits spontaneously occurring sympathetic nerve activity and reduces blood pressure in cats. Eur J Pharmacol 252:155-160[Web of Science][Medline].
• Ciliax BJ, Nash N, Heilman C, Sunahara R, Hartney A, Tiberi M, Rye DB, Caron MG, Niznik HB, Levey AI (2000) Dopamine D₅ receptor immunolocalization in rat and monkey brain. Synapse 37:125-145[Web of Science][Medline].
• Cornish JL, van den Buuse M (1995) Stimulation of the rat mesolimbic dopaminergic system produces a pressor response which is mediated by dopamine D-1 and D-2 receptor activation and the release of vasopressin. Brain Res 701:28-38[Web of Science][Medline].
• Dahmer MK, Senogles SE (1996) Dopaminergic inhibition of catecholamine secretion from chromaffin cells: evidence that inhibition is mediated by D₄ and D₅ dopamine receptors. J Neurochem 66:222-232[Medline].
• Dziewczapolski G, Menalled LB, Garcia MC, Mora MA, Gershanik OS, Rubinstein M (1998) Opposite roles of D₁ and D₅ dopamine receptors in locomotion revealed by selective antisense oligonucleotides. NeuroReport 9:1-5[Web of Science][Medline].
• Galfi M, Janaky T, Toth R, Prohaszka G, Juhasz A, Varga C, Laszlo FA (2001) Effects of dopamine and dopamine-active compounds on oxytocin and vasopressin production in rat neurohypophyseal tissue cultures. Regul Pept 98:49-54[Medline].
• Glickstein SB, Schmauss C (2001) Dopamine receptor function: lessons from knockout mice. Pharmacol Ther 91:63-83[Web of Science][Medline].
• Hermes ML, Buijs RM, Masson-Pevet M, Pevet P (1998) Oxytocinergic innervation of the brain of the garden dormouse (Eliomys quercinus L.). J Comp Neurol 273:252-262.
• Holmes A, Hollon TR, Gleason TC, Liu Z, Dreiling J, Sibley DR, Crawley JN (2001) Behavioral characterization of dopamine D₅ receptor null mutant mice. Behav Neurosci 115:1129-1144[Web of Science][Medline].
• Insel TR, Shapiro LE (1992) Oxytocin receptor distribution reflects social organization in monogamous and polygamous voles. Proc Natl Acad Sci USA 89:5981-5985[Abstract/Free Full Text].
• Jiang D, Sibley DR (1999) Agonist-induced desensitization of D₁ dopamine receptors with mutations of cyclic AMP-dependent protein kinase phosphorylation sites: attenuation of the rate of agonist-induced desensitization. Mol Pharmacol 56:675-683[Abstract/Free Full Text].
• Li E, Bestor TH, Jaenisch R (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69:915-926[Web of Science][Medline].
• Liu F, Wan Q, Pristupa ZB, Yu XM, Wang YT, Niznik HB (2000) Direct protein-protein coupling enables cross-talk between dopamine D5 and -aminobutyric acid A receptors. Nature 403:274-280[Medline].
• Malik KF, Kim J, Hartman AL, Kim P, Young III WS (1996) Binding preferences of the POU domain protein Brain-4: implications for autoregulation. Mol Brain Res 38:209-221[Medline].
• Mathiasen JR, Larson ER, Ariano MA, Sladek CD (1996) Neurophysin expression is stimulated by dopamine D₁ agonist in dispersed hypothalamic cultures. Am J Physiol 39:R404-R412.
• Mezey E, Eisenhofer G, Harta G, Hansson S, Gould L, Hunyady B, Hoffman BJ (1996) A novel nonneuronal catecholaminergic system: exocrine pancreas synthesizes and releases dopamine. Proc Natl Acad Sci USA 93:10377-10382[Abstract/Free Full Text].
• Migita K, Hori N, Manako J, Saito R, Takano Y, Kamiya H (1998) Effects of arginine-vasopressin on neuronal interaction from the area postrema to the nucleus tractus solitari in rat brain slices. Neurosci Lett 256:45-48[Medline].
• Montague DM, Striplin CD, Overcash JS, Drago J, Lawler CP, Mailman RB (2001) Quantification of D_1B (D₅) receptors in dopamine D_1A receptor-deficient mice. Synapse 39:319-322[Web of Science][Medline].
• Muir WJ, Thomson ML, McKeon P, Mynett-Johnson L, Whitton C, Evans KL, Porteous DJ, Blackwood DH (2001) Markers close to the dopamine D5 receptor gene (DRD5) show significant association with schizophrenia but not bipolar disorder. Am J Med Genet 105:152-158[Web of Science][Medline].
• Neve KA, Neve RL (1997) In: The dopamine receptors. Totowa, NJ: Humana.
• Poulin P, Komulainen A, Takahashi Y, Pittman (1994) QJ enhanced pressor responses to ICV vasopressin after pretreatment with oxytocin. Am J Physiol 266:R592-R598[Medline].
• Rivkees S, Lachowicz JE (1997) Functional D₁ and D₅ dopamine receptors are expressed in the suprachiasmatic, supraoptic and paraventricular nuclei of primates. Synapse 26:1-10[Web of Science][Medline].
• Sanada H, Xu J, Watanabe H, Jose PA, Felder RA (2000) Differential expression and regulation of dopamine-1 (D₁) and dopamine-5 (D₅) receptor function in human kidney. Am J Hypertens 13:156A.
• Schetz JA, Benjamin PS, Sibley DR (2000) Non-conserved residues in the second transmembrane-spanning domain of the D₄ dopamine receptor are molecular determinants of D₄-selective pharmacology. Mol Pharmacol 57:144-152[Abstract/Free Full Text].
• Sibley DR (1999) New insights into dopaminergic receptor function using antisense and genetically altered animals. Annu Rev Pharmacol Toxicol 39:313-341[Web of Science][Medline].
• Sunahara RK, Guan HC, O'Dowd BF, Seeman P, Laurier LG, Ng G, George SR, Torchia J, Van Tol HH, Niznik HB (1991) Cloning of the gene for a human dopamine D₅ receptor with higher affinity for dopamine than D₁. Nature 350:614-619[Medline].
• Tiberi M, Caron MG (1994) High agonist-independent activity is a distinguishing feature of the dopamine D_1B receptor subtype. J Biol Chem 269:27925-27931[Abstract/Free Full Text].
• Tybulewicz VLJ, Crawford CE, Jackson PK, Bronson RT, Mulligan RC (1991) Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl protooncogene. Cell 65:1153-1163[Web of Science][Medline].
• Usiello A, Baik JH, Rouge-Pont F, Picetti R, Dierich A, LeMeur M, Piazza PV, Borrelli E (2000) Distinct functions of the two isoforms of the dopamine D₂ receptors. Nature 408:199-203[Medline].
• Vanyukov MM, Moss HB, Gioio AE, Hughes HB, Kaplan BB, Tarter RE (1998) An association between a microsatellite polymorphism at the DRD5 gene and the liability to substance abuse: pilot study. Behav Genet 28:75-82[Medline].
• Verbene AJ, Owens NC (1998) Cortical modulation of the cardiovascular system. Prog Neurobiol 54:149-168[Web of Science][Medline].
• Yan Z, Surmeier DJ (1997) D₅ dopamine receptors regulate Zn²⁺-sensitive GABA_A currents in striatal cholinergic interneurons through a PKA/PP1 cascade. Neuron 19:1115-1126[Web of Science][Medline].
• Yoshiyama M, Roppolo JR, de Groat WC (1995) Effects of GYKI 52466 and CNQX, AMPA/kainate receptor antagonists, on the micturition reflex in the rat. Brain Res 691:185-194[Web of Science][Medline].
• Young LJ, Wang Z, Cooper TT, Albers HE (2000) Vasopressin (V1a) receptor binding, mRNA expression and transcriptional regulation by androgen in the Syrian hamster brain. J Neuroendocrinol 12:1179-1185[Web of Science][Medline].

---

Copyright © 2002 Society for Neuroscience  0270-6474/02/222410801-10$05.00/0

This article has been cited by other articles:

				F.-W. Zhou, Y. Jin, S. G. Matta, M. Xu, and F.-M. Zhou An Ultra-Short Dopamine Pathway Regulates Basal Ganglia Output J. Neurosci., August 19, 2009; 29(33): 10424 - 10435. [Abstract] [Full Text] [PDF]

				H.-H. Hsu, K. Duning, H. H. Meyer, M. Stolting, T. Weide, S. Kreusser, T. van Le, C. Gerard, R. Telgmann, S.-M. Brand-Herrmann, et al. Hypertension in mice lacking the CXCR3 chemokine receptor Am J Physiol Renal Physiol, April 1, 2009; 296(4): F780 - F789. [Abstract] [Full Text] [PDF]

				A. Sahu, K. R. Tyeryar, H. O. Vongtau, D. R. Sibley, and A. S. Undieh D5 Dopamine Receptors are Required for Dopaminergic Activation of Phospholipase C Mol. Pharmacol., March 1, 2009; 75(3): 447 - 453. [Abstract] [Full Text] [PDF]

				C. S. Wilcox and A. Pearlman Chemistry and Antihypertensive Effects of Tempol and Other Nitroxides Pharmacol. Rev., December 1, 2008; 60(4): 418 - 469. [Abstract] [Full Text] [PDF]

				E. Argilli, D. R. Sibley, R. C. Malenka, P. M. England, and A. Bonci Mechanism and Time Course of Cocaine-Induced Long-Term Potentiation in the Ventral Tegmental Area J. Neurosci., September 10, 2008; 28(37): 9092 - 9100. [Abstract] [Full Text] [PDF]

				R. Howden, E. Liu, L. Miller-DeGraff, H. L. Keener, C. Walker, J. A. Clark, P. H. Myers, D. C. Rouse, T. Wiltshire, and S. R. Kleeberger The genetic contribution to heart rate and heart rate variability in quiescent mice Am J Physiol Heart Circ Physiol, July 1, 2008; 295(1): H59 - H68. [Abstract] [Full Text] [PDF]

				C. Zeng, I. Armando, Y. Luo, G. M. Eisner, R. A. Felder, and P. A. Jose Dysregulation of dopamine-dependent mechanisms as a determinant of hypertension: studies in dopamine receptor knockout mice Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H551 - H569. [Abstract] [Full Text] [PDF]

				J. J. Gildea, X. Wang, P. A. Jose, and R. A. Felder Differential D1 and D5 Receptor Regulation and Degradation of the Angiotensin Type 1 Receptor Hypertension, February 1, 2008; 51(2): 360 - 366. [Abstract] [Full Text] [PDF]

				Z. Wang, I. Armando, L. D. Asico, C. Escano, X. Wang, Q. Lu, R. A. Felder, C. G. Schnackenberg, D. R. Sibley, G. M. Eisner, et al. The elevated blood pressure of human GRK4{gamma} A142V transgenic mice is not associated with increased ROS production Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2083 - H2092. [Abstract] [Full Text] [PDF]

				L. J. Mullins, M. A. Bailey, and J. J. Mullins Hypertension, Kidney, and Transgenics: A Fresh Perspective Physiol Rev, April 1, 2006; 86(2): 709 - 746. [Abstract] [Full Text] [PDF]

				M. J. Bek, X. Wang, L. D. Asico, J. E. Jones, S. Zheng, X. Li, G. M. Eisner, D. K. Grandy, R. M. Carey, P. Soares-da-Silva, et al. Angiotensin-II Type 1 Receptor-Mediated Hypertension in D4 Dopamine Receptor-Deficient Mice Hypertension, February 1, 2006; 47(2): 288 - 295. [Abstract] [Full Text] [PDF]

				Z. Yang, L. D. Asico, P. Yu, Z. Wang, J. E. Jones, C. S. Escano, X. Wang, M. T. Quinn, D. R. Sibley, G. G. Romero, et al. D5 dopamine receptor regulation of reactive oxygen species production, NADPH oxidase, and blood pressure Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2006; 290(1): R96 - R104. [Abstract] [Full Text] [PDF]

				C. J. Zeiss Neuroanatomical Phenotyping in the Mouse: The Dopaminergic System Vet. Pathol., November 1, 2005; 42(6): 753 - 773. [Abstract] [Full Text] [PDF]

				M. Sjoquist, S.-L. Lee, and P. Hansell CNS-induced natriuresis, neurohypophyseal peptides and renal dopamine and noradrenaline excretion in prehypertensive salt-sensitive Dahl rats Exp Physiol, November 1, 2005; 90(6): 847 - 853. [Abstract] [Full Text] [PDF]

				C. S. Wilcox Oxidative stress and nitric oxide deficiency in the kidney: a critical link to hypertension? Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2005; 289(4): R913 - R935. [Abstract] [Full Text] [PDF]

				C. Zeng, Z. Yang, Z. Wang, J. Jones, X. Wang, J. Altea, A. J. Mangrum, U. Hopfer, D. R. Sibley, G. M. Eisner, et al. Interaction of Angiotensin II Type 1 and D5 Dopamine Receptors in Renal Proximal Tubule Cells Hypertension, April 1, 2005; 45(4): 804 - 810. [Abstract] [Full Text] [PDF]

				Z. Yang, L. D. Asico, P. Yu, Z. Wang, J. E. Jones, R.-k. Bai, D. R. Sibley, R. A. Felder, and P. A. Jose D5 dopamine receptor regulation of phospholipase D Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H55 - H61. [Abstract] [Full Text] [PDF]

				C. Zeng, H. Sanada, H. Watanabe, G. M. Eisner, R. A. Felder, and P. A. Jose Functional genomics of the dopaminergic system in hypertension Physiol Genomics, November 17, 2004; 19(3): 233 - 246. [Abstract] [Full Text] [PDF]

				M. Kobayashi, C. Iaccarino, A. Saiardi, V. Heidt, Y. Bozzi, R. Picetti, C. Vitale, H. Westphal, J. Drago, and E. Borrelli Simultaneous absence of dopamine D1 and D2 receptor-mediated signaling is lethal in mice PNAS, August 3, 2004; 101(31): 11465 - 11470. [Abstract] [Full Text] [PDF]

				K. J. Pettersson-Fernholm, C. M. Forsblom, M. Perola, J. A. Fagerudd, and P.-H. Groop Dopamine D3 receptor gene polymorphisms, blood pressure and nephropathy in type 1 diabetic patients Nephrol. Dial. Transplant., June 1, 2004; 19(6): 1432 - 1436. [Abstract] [Full Text] [PDF]

				C. Zeng, Y. Luo, L. D. Asico, U. Hopfer, G. M. Eisner, R. A. Felder, and P. A. Jose Perturbation of D1 Dopamine and AT1 Receptor Interaction in Spontaneously Hypertensive Rats Hypertension, October 1, 2003; 42(4): 787 - 792. [Abstract] [Full Text] [PDF]

				D. Centonze, C. Grande, E. Saulle, A. B. Martin, P. Gubellini, N. Pavon, A. Pisani, G. Bernardi, R. Moratalla, and P. Calabresi Distinct Roles of D1 and D5 Dopamine Receptors in Motor Activity and Striatal Synaptic Plasticity J. Neurosci., September 17, 2003; 23(24): 8506 - 8512. [Abstract] [Full Text] [PDF]

				D. Centonze, C. Grande, A. Usiello, P. Gubellini, E. Erbs, A. B. Martin, A. Pisani, N. Tognazzi, G. Bernardi, R. Moratalla, et al. Receptor Subtypes Involved in the Presynaptic and Postsynaptic Actions of Dopamine on Striatal Interneurons J. Neurosci., July 16, 2003; 23(15): 6245 - 6254. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (53) Citing Articles via Google Scholar Google Scholar Articles by Hollon, T. R. Articles by Sibley, D. R. Search for Related Content PubMed PubMed Citation Articles by Hollon, T. R. Articles by Sibley, D. R.

Home  |   Search  |   Archive  |   Subscribe  |   Contact  |   Help