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.     

Generation and pharmacological analysis of M2 and M4 muscarinic receptor knockout mice

Muscarinic acetylcholine receptors (M1-M5) play important roles in the modulation of many key functions of the central and peripheral nervous system. To explore the physiological roles of the two Gi-coupled muscarinic receptors, we disrupted the M2 and M4 receptor genes in mice by using a gene targeting strategy. Pharmacological and behavioral analysis of the resulting mutant mice showed that the M2 receptor subtype is critically involved in mediating three of the most striking central muscarinic effects, tremor, hypothermia, and analgesia. These studies also indicated that M4 receptors are not critically involved in these central muscarinic responses. However, M4 receptor-deficient mice showed an increase in basal locomotor activity and greatly enhanced locomotor responses following drug-induced activation of D1 dopamine receptors. This observation is consistent with the concept that M4 receptors exert inhibitory control over D1 receptor-mediated locomotor stimulation, probably at the level of striatal projection neurons where the two receptors are known to be coexpressed. These findings emphasize the usefulness of gene targeting approaches to shed light on the physiological and pathophysiological roles of the individual muscarinic receptor subtypes.     

Investigations into the physiological role of muscarinic M2 and M4 muscarinic and M4 receptor subtypes using receptor knockout mice

Determination of muscarinic agonist-induced parasympathomimetic effects in wild type and M2 and M4 muscarinic receptor knockout mice revealed that M2 receptors mediated tremor and hypothermia, but not salivation. The M4 receptors seem to play a modest role in salivation, but did not alter hypothermia and tremor. In the M2 knockout mice, agonist-induced bradycardia in isolated spontaneously beating atria was completely absent compared to their wild type litter mates, whereas agonist-induced bradycardia was similar in the M4 knockout and wild type mice. The potency of carbachol to stimulate contraction of isolated stomach fundus, urinary bladder and trachea was reduced by a factor of about 2 in the M2 knockout mice, but was unaltered in the M4 knockout mice. The binding of the muscarinic agonist, [3H]-oxotremorine-M, was reduced in cortical tissue from the M2 knockout mice and to a lesser extent from the M4 knockout mice, and was reduced over 90% in the brain stem of M2 knockout mice. The data demonstrate the usefulness of knockout mice in determining the physiological function of peripheral and central muscarinic receptors.     

Unaltered D1, D2, D4, and D5 dopamine receptor mRNA expression and distribution in the spinal cord of the D3 receptor knockout mouse

Dopamine (DA) acts through five receptor subtypes (D1–D5). We compared expression levels and distribution patterns of all DA mRNA receptors in the spinal cord of wild-type (WT) and loss of function D3 receptor knockout (D3KO) animals. D3 mRNA expression was increased in D3KO, but no D3 receptor protein was associated with cell membranes, supporting the previously reported lack of function. In contrast, mRNA expression levels and distribution patterns of D1, D2, D4, and D5 receptors were similar between WT and D3KO animals. We conclude that D3KO spinal neurons do not compensate for the loss of function of the D3 receptor with changes in the other DA receptor subtypes. This supports use of D3KO animals as a model to provide insight into D3 receptor dysfunction in the spinal cord.     

Role of dopamine D1-like receptors in methamphetamine locomotor responses of D2 receptor knockout mice

Behavioral sensitization to psychostimulants manifests as an increased locomotor response with repeated administration. Dopamine systems are accepted to play a fundamental role in sensitization, but the role of specific dopamine receptor subtypes has not been completely defined. This study used the combination of dopamine D2 receptor-deficient mice and a D1-like antagonist to examine dopamine D1 and D2 receptor involvement in acute and sensitized locomotor responses to methamphetamine. Absence of the dopamine D2 receptor resulted in attenuation of the acute stimulant effects of methamphetamine. Mutant and wild-type mice exhibited sensitization that lasted longer within the time period of the challenge test in the mutant animals. Pretreatment with the D1-like receptor antagonist SCH 23390 produced more potent reductions in the acute and sensitized locomotor responses to methamphetamine in D2 receptor-deficient mice than in wild-type mice; however, the expression of locomotor sensitization when challenged with methamphetamine alone was equivalently attenuated by previous treatment with SCH 23390. These data suggest that dopamine D2 receptors play a key role in the acute stimulant and sensitizing effects of methamphetamine and act in concert with D1-like receptors to influence the acquisition of methamphetamine-induced behavioral sensitization, traits that may influence continued methamphetamine use.     

Sleep,activity, temperature and arousal responses of mice deficient for muscarinic receptor M2 or M4

AimsThe type 2 muscarinic receptor (M2R) differs from the other G-protein-coupled muscarinic receptor (type 4, or M4R) in tissue distribution and physiologic effects. We studied the impact of these receptors on sleep and arousal by using M2R and M4R knock-out (KO) mice.Main methodsM2R and M4R KO and genetically intact mice were compared in terms of normal patterns of sleep, responses to sleep loss, infectious challenge and acoustic startle, and acoustic prepulse inhibition of startle (PPI).Key findingsUnder basal conditions, M2R and M4R KO mice do not differ from the background strain or each other in the amount or diurnal pattern of sleep, locomotor activity, and body temperature. After enforced sleep loss, M2R KO mice, in contrast to the other two strains, show no rebound in slow-wave sleep (SWS) time, although their SWS is consolidated, and they show a greater rebound in time spent in REMS (rapid-eye-movement sleep) and REMS consolidation. During influenza infection, M2R KO mice, as compared with the other strains, show marked hypothermia and a less robust increase in SWS. During Candida albicans infection, M2R KO mice show a greater increase in SWS and a greater inflammatory response than do the other strains. M2R KO mice also show greater acoustic startle amplitude than does the background strain, although PPI was not different across the 3 strains over a range of stimulus intensities.SignificanceTaken together, these findings support different roles for M2R and M4R in the modulation of sleep and arousal during homeostatic challenge.     

Effects of atypical antipsychotic drugs on body weight and food intake in dopamine D2 receptor knockout mice

Many atypical antipsychotic drugs cause weight gain, but the mechanism of this weight gain is unclear. To dissect the role of the dopamine D2 receptor (D2R), an important receptor in the pharmacology of antipsychotic drugs, we analyzed the effect of olanzapine, risperidone, and ziprasidone on changes in body weight and food intake in male wild-type (WT) and D2R knockout (D2R^−/−) mice. The oral delivery of atypical antipsychotics, olanzapine (5 and 10 mg/kg), risperidone (0.1 and 1.0 mg/kg) and ziprasidone (10 and 20 mg/kg) in both strains mice for 2 weeks suppressed body weight gain, except for olanzapine treatment in D2R^−/− mice. Olanzapine treatment suppressed body weight gain and decreased food intake in WT mice, but also reduced fat body mass and locomotor activity, whereas D2R^−/− mice did not show these changes. Ziprasidone and risperidone treatment produced similar responses in WT and D2R^−/− mice. These data suggest the involvement of D2R in the effect of olanzapine on metabolic regulation. Further studies are required to explore the implications of D2R activity in antipsychotic-mediated metabolic complications.     

Ciliary activity in the oviduct of cycling, pregnant, and muscarinic receptor knockout mice

The transport of the oocyte and the embryo in the oviduct is managed by ciliary beating and muscular contractions. Because nonneuronally produced acetylcholine influences ciliary beating in the trachea via the muscarinic receptors M2 and M3, we supposed that components of the cholinergic system may also modulate ciliary activity in the oviduct. To address this issue, we analyzed the expression profile of muscarinic receptors (CHRMs) in the murine oviduct by RT-PCR and assessed ciliary beat frequency (CBF) and cilia-driven particle transport speed (PTS) on the mucosal surface of opened oviductal segments in correlation with histomorphological investigations. RT-PCR of laser-assisted microdissected epithelium revealed expression of Chrm subtypes Chrm1 and Chrm3. In opened isthmic segments, particle transport was barely seen, correlating with a significantly lower number of ciliated cells compared to the ampulla. In the ampulla, basal PTS and CBF were high (71 μm/sec and 21 Hz, respectively) both in cycling and pregnant wild-type mice and in mice with targeted deletion of the Chrm genes Chrm1, Chrm3, Chrm4, and Chrm5. In contrast to the trachea, where basal ciliary activity was low and largely enhanced by muscarinic stimulation, muscarinic agonists and antagonists did not affect the high ampullar PTS. Our results imply that this high oviductal autonomous ciliary activity is independent from the intrinsic cholinergic system and serves to maintain optimal clearance of the tube throughout all stages of the estrous cycle and early pregnancy.     

M(3)-receptor knockout mice: muscarinic receptor function in atria, stomach fundus, urinary bladder, and trachea

Negative chronotropic and smooth muscle contractile responses to the nonselective muscarinic agonist carbamylcholine were compared in isolated tissues from M(3)-muscarinic receptor knockout and wild-type mice. Carbamylcholine (10(-8)-3.0 x 10(-5) M) induced a concentration-dependent decrease in atrial rate that was similar in atria from M(3)-receptor knockout and wild-type mice, indicating that M(3) receptors were not involved in muscarinic receptor-mediated atrial rate decreases. In contrast, the M(3) receptor was a major muscarinic receptor involved in smooth muscle contraction of stomach fundus, urinary bladder, and trachea, although differences existed in the extent of M(3)-receptor involvement among the tissues. Contraction to carbamylcholine was virtually abolished in urinary bladder from M(3)-receptor knockout mice, suggesting that contraction was predominantly due to M(3)-receptor activation. However, approximately 50-60% maximal contraction to carbamylcholine occurred in stomach fundus and trachea from M(3)-receptor knockout mice, indicating that contraction in these tissues was also due to M(2)-receptor activation. High concentrations of carbamylcholine relaxed the stomach fundus from M(3)-receptor knockout mice by M(1)-receptor activation. Thus M(3)-receptor knockout mice provided unambiguous evidence that M(3) receptors 1) play no role in carbamylcholine-induced atrial rate reduction, 2) are the predominant receptor mediating carbamylcholine-induced urinary bladder contractility, and 3) share contractile responsibility with M(2) receptors in mouse stomach fundus and trachea.     

Gastric body cholinergic contractile signal transduction in M2 and M3 receptor knockout mice

Abstract

Although most smooth muscles express a greater density of M₂ than M₃ muscarinic receptors, based on the potency of subtype selective muscarinic receptor antagonists, the M₃ subtype predominantly mediates contraction. The effect of inhibitors of putative contractile signal transduction pathway enzymes on carbachol-induced contractions was determined in wild-type (WT) mice and mice lacking either the M₂ (M₂KO) or the M₃ (M₃KO) receptor subtype. Contractile responses to KCl, then increasing carbachol concentrations in the presence and absence of enzyme inhibitors was determined. The KCl-induced contraction was not different between strains. The carbachol response was unaffected in the M₂KO strain but decreased 42% in M₃KO mice (p?<?0.01). Darifenacin potency was high in both WT and M₂KO strains, indicating M₃-mediated contractions, and low in the M₃KO strain, suggesting M₂-mediated contractions. The phosphatidyl inositol-specific phospholipase C (Pi-PLC) inhibitor ET-18-OCH₃ had no effect. Inhibition of phosphatidyl choline-specific phospholipase C (PC-PLC) and sphingomyelin synthase with D609 decreased maximal contraction in all strains. M₃-mediated contractions in the M₂KO strain were decreased 54% by the protein kinase C (PKC) inhibitor chelerythrine. M₂-mediated contractions in the M₃KO and WT strains were decreased by the Rho kinase (ROCK) inhibitor Y27632 as well as the ROCK, PKA and PKG inhibitor H89. The M₃ subtype activates PKC and either PC-PLC or sphingomyelin synthase, while the M₂ subtype activates ROCK and either PC-PLC or sphingomyelin synthase. These studies suggest that multiple parallel pathways mediate cholinergic contractions in stomach body smooth muscle.     

Susceptibility of dopamine D5 receptor targeted mice to cysteamine

BACKGROUND: Recently we demonstrated that gastric mucosa of rats can synthesize, store and release dopamine. Out of five different subtypes, mRNA of D5 (=D1b) dopamine receptor is very abundant in the gastric epithelium. D1 receptor selective dopamine agonists have been shown to protect against experimental gastro-duodenal lesions. AIMS: To test the hypothesis that protective effects of dopamine involve D5 receptors, mucosal lesions were induced in D5 receptor deficient (KO) and wild-type (WT) mice using cysteamine. Morphology and gastric acid secretion of D5 KO mice were also studied. METHODS: Single doses of 600 mg/kg, 300 mg/kg cysteamine or vehicle were administered subcutaneously to fasted animals. After 24 h, number and severity of gastro-duodenal lesions were analyzed. Basal and histamine-induced maximal gastric acid output were measured by a stomach-sac wash-through method. RESULTS: All the KOs in the 600 mg/kg cysteamine group died within 4 h showing symptoms of toxicity while three out of four WTs survived (P<0.05). Mortality after 300 mg/kg cysteamine was significantly higher in KOs versus the WTs: 6/14 versus 2/11, P<0.05. Gastric lesion-index was also significantly higher in KOs (median, middle quartile): four (3-9) versus 0 (0-0), P<0.05. Duodenal lesions did not develop from this single dose of cysteamine in either genotype. Basal and histamine-induced maximal gastric acid output were comparable in the two genotypes. CONCLUSIONS: This study demonstrates that loss of D5 receptor causes mucosal vulnerability and increased toxicity of cysteamine in genetically manipulated mice. Thus, D5 receptor subtype is indeed likely to be involved in protective effects of dopamine in the stomach.     

ADP-ribosylation factor-dependent phospholipase D activation by the M3 muscarinic receptor

G protein-coupled receptors can potentially activate phospholipase D (PLD) by a number of routes. We show here that the native M3 muscarinic receptor in 1321N1 cells and an epitope-tagged M3 receptor expressed in COS7 cells substantially utilize an ADP-ribosylation factor (ARF)-dependent route of PLD activation. This pathway is activated at the plasma membrane but appears to be largely independent of G, phospholipase C, Ca2+ q/11, protein kinase C, tyrosine kinases, and phosphatidyl inositol 3-kinase. We report instead that it involves physical association of ARF with the M3 receptor as demonstrated by co-immunoprecipitation and by in vitro interaction with a glutathione S-transferase fusion protein of the receptor's third intracellular loop domain. Experiments with mutant constructs of ARF1/6 and PLD1/2 indicate that the M3 receptor displays a major ARF1-dependent route of PLD1 activation with an additional ARF6-dependent pathway to PLD1 or PLD2. Examples of other G protein-coupled receptors assessed in comparison display alternative pathways of protein kinase C- or ARF6-dependent activation of PLD2.     

Cross-talk between M2 muscarinic and D1 dopamine receptors in the cat adrenal medulla.

In the study reported here we have reached two conclusions. First, the cat adrenal medulla chromaffin cell possesses a dopamine D1 receptor that seems to be coupled to an adenylyl cyclase. Second, this receptor regulates the muscarinic-mediated catecholamine release response through a negative feed-back loop which uses cyclic AMP as a second messenger. These conclusions are supported by the following findings: (i) SKF38393 (a selective D1 receptor agonist), but not quinpirole (a selective D2 agonist), inhibits the methacholine-mediated catecholamine release responses in a concentration-dependent manner (IC50 of around 1-2 microM). (ii) SCH23390 (a selective D1 antagonist), but not sulpiride (a selective D2 antagonist), reversed by 70% the inhibitory effects of SKF38393. (iii) Dibutyril cyclic AMP (500 microM) inhibited by 80% the secretory effects of methacholine.     

Maternal care of heterozygous dopamine receptor D4 knockout mice: Differential susceptibility to early‐life rearing conditions

The differential susceptibility hypothesis proposes that individuals who are more susceptible to the negative effects of adverse rearing conditions may also benefit more from enriched environments. Evidence derived from human experiments suggests the lower efficacy dopamine receptor D4 (DRD4) 7‐repeat as a main factor in exhibiting these for better and for worse characteristics. However, human studies lack the genetic and environmental control offered by animal experiments, complicating assessment of causal relations. To study differential susceptibility in an animal model, we exposed Drd4^+/? mice and control litter mates to a limited nesting/bedding (LN), standard nesting (SN) or communal nesting (CN) rearing environment from postnatal day (P) 2‐14. Puberty onset was examined from P24 to P36 and adult females were assessed on maternal care towards their own offspring. In both males and females, LN reared mice showed a delay in puberty onset that was partly mediated by a reduction in body weight at weaning, irrespective of Drd4 genotype. During adulthood, LN reared females exhibited characteristics of poor maternal care, whereas dams reared in CN environments showed lower rates of unpredictability towards their own offspring. Differential susceptibility was observed only for licking/grooming levels of female offspring towards their litter; LN reared Drd4^+/? mice exhibited the lowest and CN reared Drd4^+/? mice the highest levels of licking/grooming. These results indicate that both genetic and early‐environmental factors play an important role in shaping maternal care of the offspring for better and for worse.     

D1-like dopamine receptor-mediated function in congenic mutants with D1 vs. D5 receptor "knockout"

Current understanding of the functional roles of individual dopamine D1-like [D1, D5] and D2-like [D2L/s, D3, D4] receptor subtypes remains incomplete. In particular, the lack of pharmacological agonists and antagonists able to distinguish between D1 and D5 receptors means that any differential roles in the regulation of behavior are poorly understood. Mutant mice with targeted gene deletion ("knockout") of individual dopamine receptor subtypes offer an important alternative approach to resolving these functional roles. In congenic D1 mutants examined ethologically, progressive increases in specific topographies of behavior over wildtypes were considerably greater than those in D1 mutants on a mixed genetic background; D1 knockout appears to influence the neuronal substrate(s) of habituation to disrupt sculpture of the changing topography of behavior from initial exploration through to quiescence. Similarly, the D1 receptor appears to regulate specific topographies of orofacial movement in the mouse as these are "sculpted" in a time-dependent manner. Although the well-recognized role of the D1-like family in regulating several aspects of behavioral topography has been assumed to involve primarily D1 receptors, this presumption may require modification to accommodate a subtle but not negligible role for their D5 counterparts as evidenced in the phenotype of congenic D5 mutants.     

Selective D2 dopamine receptor agonists prevent catalepsy induced by SCH 23390, a selective D1 antagonist

SCH 23390, an apparently selective antagonist of central D1 dopamine receptors, produced profound catalepsy at low doses (0.1 mg/kg, s.c.). Pretreatment with the selective D2 receptor agonists LY 141865, RU 24213 or LY 171555, the active (-) enantiomer of LY 141865, elicited a dose-dependent inhibition of the cataleptic response. Pergolide and apomorphine were also effective. This effect was not due to altered disposition or penetration of SCH 23390 into the brain since pretreatment with a dose of LY 171555 which completely blocked catalepsy had no effect on the ID50 of SCH 23390 to inhibit 3H-cis-piflutixol binding to D1 receptors measured ex vivo. Alternative mechanisms are considered to explain the results, which offer new insights into striatal dopaminergic regulation of motor activity.     

G protein-coupled receptor kinase 5 regulates airway responses induced by muscarinic receptor activation

G protein-coupled receptors (GPCRs) transduce extracellular signals into intracellular events. The waning responsiveness of GPCRs in the face of persistent agonist stimulation, or desensitization, is a necessary event that ensures physiological homeostasis. GPCR kinases (GRKs) are important regulators of GPCR desensitization. GRK5, one member of the GRK family, desensitizes central M(2) muscarinic receptors in mice. We questioned whether GRK5 might also be an important regulator of peripheral muscarinic receptor responsiveness in the cardiopulmonary system. Specifically, we wanted to determine the role of GRK5 in regulating muscarinic receptor-mediated control of airway smooth muscle tone or regulation of cholinergic-induced bradycardia. Tracheal pressure, heart rate, and tracheal smooth muscle tension were measured in mice having a targeted deletion of the GRK5 gene (GRK5(-/-)) and littermate wild-type (WT) control mice. Both in vivo and in vitro results showed that the airway contractile response to a muscarinic receptor agonist was not different between GRK5(-/-) and WT mice. However, the relaxation component of bilateral vagal stimulation and the airway smooth muscle relaxation resulting from beta(2)-adrenergic receptor activation were diminished in GRK5(-/-) mice. These data suggest that M(2) muscarinic receptor-mediated opposition of airway smooth muscle relaxation is regulated by GRK5 and is, therefore, excessive in GRK5(-/-) mice. In addition, this study shows that GRK5 regulates pulmonary responses in a tissue- and receptor-specific manner but does not regulate peripheral cardiac muscarinic receptors. GRK5 regulation of airway responses may have implications in obstructive airway diseases such as asthma or chronic obstructive pulmonary disease.     

Amplification of the rat M2 muscarinic receptor gene by the polymerase chain reaction: functional expression of the M2 muscarinic receptor

A selective amplification of the coding sequence of the rat M2 muscarinic receptor gene was achieved by the polymerase chain reaction. The error rate of this amplification system under conditions specified was 1 nucleotide substitution in 841 base pairs. In vitro expression of this gene in murine fibroblasts (B82) via the eukaryotic expression vector, pH beta APr-1-neo, resulted in high level expression of specific [3H] (-)MQNB binding in transfected B82 cell lines. One of these clones, M2LKB2-2, showed a stable expression of [3H] (-)MQNB binding with a Kd value of 265 pM and a Bmax value of 411 +/- 50 fmol/10(6) cells. Cardiac selective muscarinic antagonists such as himbacine and AF-DX 116 show high affinities for this binding site in the M2LKB2-2 cells. The rank order of potency of several antagonists in inhibiting [3H] (-)MQNB binding in these cells conformed to the characteristics of an M2 type muscarinic receptor. Carbachol showed a single affinity state for the receptors in the M2LKB2-2 cells with a Ki value of 2.0 microM. This receptor appeared to be inversely coupled to adenylate cyclase via a pertussis toxin sensitive G-protein. Carbachol also had a slight stimulatory effect on the hydrolysis of inositol lipids. The polymerase chain reaction proves highly effective in cloning genes from genomic material, as demonstrated by the first in vitro functional expression of the rat M2 type muscarinic receptor.