Molecular characterization and distribution of motilin and motilin receptor in the Japanese medaka Oryzias latipes.

Motilin (MLN) is a peptide hormone originally isolated from the mucosa of the porcine intestine. Its orthologs have been identified in various vertebrates. Although MLN regulates gastrointestinal motility in tetrapods from amphibians to mammals, recent studies indicate that MLN is not involved in the regulation of isolated intestinal motility in zebrafish, at least in vitro. To determine the unknown function of MLN in teleosts, we examined the expression of MLN and the MLN receptor (MLNR) at the cellular level in Japanese medaka (Oryzias latipes). Quantitative PCR revealed that mln mRNA was limitedly expressed in the gut, whereas mlnr mRNA was not detected in the gut but was expressed in the brain and kidney. By in situ hybridization and immunohistochemistry, mlnr mRNA was detected in the dopaminergic neurons of the area postrema in the brain and the noradrenaline-producing cells in the interrenal gland of the kidney. Furthermore, we observed efferent projections of mlnr-expressing dopaminergic neurons in the lobus vagi (XL) and nucleus motorius nervi vagi (NXm) of the medulla oblongata by establishing a transgenic medaka expressing the enhanced green fluorescence protein driven by the mlnr promoter. The expression of dopamine receptor mRNAs in the XL and cholinergic neurons in NXm was confirmed by in situ hybridization. These results indicate novel sites of MLN activity other than the gastrointestinal tract. MLN may exert central and peripheral actions through the regulation of catecholamine release in medaka.

Epidemiology and Risk Factors for Giant Coronary Artery Aneurysms Identified After Acute Kawasaki Disease.

A giant coronary artery (CA) aneurysm is a potentially fatal cardiac complication resulting from Kawasaki disease (KD). We aimed to identify epidemiologic characteristics and potential risk factors associated with giant CA aneurysms identified after acute KD. We analyzed 90,252 patients diagnosed with KD from 2011 to 2018, using data obtained in nationwide KD surveys conducted in Japan. Multivariable logistic regression analyses were performed to evaluate potential risk factors associated with subsequent giant CA aneurysm complications (defined as lumen size ≥ 8 mm), adjusting for all potential factors. Giant CA aneurysms were identified in 144 patients (0.16%) after acute KD. The annual prevalence ranged from 0.07 to 0.20% during the study period. In the multivariate analyses, male sex (adjusted odds ratio 2.09 [95% confidence interval 1.41-3.11], recurrent KD (1.90 [1.09-3.33]), IVIG administration at 1-4 days of illness (1.49 [1.04-2.15]) and ≥ 8 days after KD onset (2.52 [1.38-4.60]; reference, 5-7 days), detection of CA dilatations and aneurysms at initial echocardiography (4.17 [1.85-5.41] and 46.5 [28.8-74.8], respectively), and resistance to IVIG treatment (6.09 [4.23-8.75]) were significantly associated with giant CA aneurysm complications identified after acute KD. The annual prevalence of giant CA aneurysms identified after acute KD did not increase during the study period. Patients with larger CA abnormalities detected at initial echocardiography were independently associated with progression to giant CA aneurysm complications after acute KD regardless of the number of days from onset at treatment initiation.

Critical role of V1a vasopressin receptor in murine parturition†.

The precise mechanisms of the reproductive physiological processes, such as labor initiation, are poorly understood. Oxytocin (OT) is one of the well-known uterotonics and is clinically adopted as a medication to facilitate childbirth. Vasopressin (VP), a posterior pituitary hormone similar to OT, has also been proposed to be involved in the reproductive physiology. In this study, we found that a total deficiency of V1a receptor subtype (V1aR) in mice resulted in a reduced number of pups, delayed labor initiation, and increased post-delivery hemorrhage compared with those in wild-type mice. Among the VP receptor subtypes, only V1aR was found to be expressed in the murine uterus, and its distribution pattern was different from that of the oxytocin receptor (OTR); V1aR expression was mainly distributed in the circular myometrium, whereas OTR was strongly expressed in both the circular and longitudinal myometrium. The maximum contractile force of the circular myometrium, induced by VP or OT, was attenuated in the pregnant uterus of Avpr1a-deficient mice. Contrarily, while OT expression was decreased in the Avpr1a-deficient uterus, OTR expression was significantly increased. These results suggest that V1aR deficiency not only reduces the uterine contractile force but also perturbs the expression of genes responsible for the reproductive physiology. Therefore, V1aR is necessary to exert the maximum contraction of the circular myometrium to deliver pups. This study revealed an important role of V1aR in physiological contraction and term parturition in mice.

Vasopressin Increases Urinary Acidification via V1a Receptors in Collecting Duct Intercalated Cells.

BACKGROUND: Antagonists of the V1a vasopressin receptor (V1aR) are emerging as a strategy for slowing progression of CKD. Physiologically, V1aR signaling has been linked with acid-base homeostasis, but more detailed information is needed about renal V1aR distribution and function. METHODS: We used a new anti-V1aR antibody and high-resolution microscopy to investigate Va1R distribution in rodent and human kidneys. To investigate whether V1aR activation promotes urinary H+ secretion, we used a V1aR agonist or antagonist to evaluate V1aR function in vasopressin-deficient Brattleboro rats, bladder-catheterized mice, isolated collecting ducts, and cultured inner medullary collecting duct (IMCD) cells. RESULTS: Localization of V1aR in rodent and human kidneys produced a basolateral signal in type A intercalated cells (A-ICs) and a perinuclear to subapical signal in type B intercalated cells of connecting tubules and collecting ducts. Treating vasopressin-deficient Brattleboro rats with a V1aR agonist decreased urinary pH and tripled net acid excretion; we observed a similar response in C57BL/6J mice. In contrast, V1aR antagonist did not affect urinary pH in normal or acid-loaded mice. In ex vivo settings, basolateral treatment of isolated perfused medullary collecting ducts with the V1aR agonist or vasopressin increased intracellular calcium levels in ICs and decreased luminal pH, suggesting V1aR-dependent calcium release and stimulation of proton-secreting proteins. Basolateral treatment of IMCD cells with the V1aR agonist increased apical abundance of vacuolar H+-ATPase in A-ICs. CONCLUSIONS: Our results show that activation of V1aR contributes to urinary acidification via H+ secretion by A-ICs, which may have clinical implications for pharmacologic targeting of V1aR.

Vasopressin V1a and V1b receptors: from molecules to physiological systems.

The neurohypophysial hormone arginine vasopressin (AVP) is essential for a wide range of physiological functions, including water reabsorption, cardiovascular homeostasis, hormone secretion, and social behavior. These and other actions of AVP are mediated by at least three distinct receptor subtypes: V1a, V1b, and V2. Although the antidiuretic action of AVP and V2 receptor in renal distal tubules and collecting ducts is relatively well understood, recent years have seen an increasing understanding of the physiological roles of V1a and V1b receptors. The V1a receptor is originally found in the vascular smooth muscle and the V1b receptor in the anterior pituitary. Deletion of V1a or V1b receptor genes in mice revealed that the contributions of these receptors extend far beyond cardiovascular or hormone-secreting functions. Together with extensively developed pharmacological tools, genetically altered rodent models have advanced the understanding of a variety of AVP systems. Our report reviews the findings in this important field by covering a wide range of research, from the molecular physiology of V1a and V1b receptors to studies on whole animals, including gene knockout/knockdown studies.

Dysfunction of lipid sensor GPR120 leads to obesity in both mouse and human.

Free fatty acids provide an important energy source as nutrients, and act as signalling molecules in various cellular processes. Several G-protein-coupled receptors have been identified as free-fatty-acid receptors important in physiology as well as in several diseases. GPR120 (also known as O3FAR1) functions as a receptor for unsaturated long-chain free fatty acids and has a critical role in various physiological homeostasis mechanisms such as adipogenesis, regulation of appetite and food preference. Here we show that GPR120-deficient mice fed a high-fat diet develop obesity, glucose intolerance and fatty liver with decreased adipocyte differentiation and lipogenesis and enhanced hepatic lipogenesis. Insulin resistance in such mice is associated with reduced insulin signalling and enhanced inflammation in adipose tissue. In human, we show that GPR120 expression in adipose tissue is significantly higher in obese individuals than in lean controls. GPR120 exon sequencing in obese subjects reveals a deleterious non-synonymous mutation (p.R270H) that inhibits GPR120 signalling activity. Furthermore, the p.R270H variant increases the risk of obesity in European populations. Overall, this study demonstrates that the lipid sensor GPR120 has a key role in sensing dietary fat and, therefore, in the control of energy balance in both humans and rodents.

Prostaglandin D2 elicits the reversible neurite retraction in hypothalamic cell line.

Prostaglandins (PGs) play important roles in diverse physiological processes in the central nervous system. PGD2 is the most abundant PG in the brain and acts through specific receptors, DP1 and CRTH2. We investigated the effects of PGD2 on the morphology of the hypothalamic cell line mHypoE-N37 (N37). In N37 cells, serum starvation induced neurite outgrowth and PGD2 elicited neurite retraction, although we failed to detect transcripts for DP1 and CRTH2. Such an effect of PGD2 was efficiently mimicked by its metabolite, 15-deoxy-Δ(12,14)-prostaglandin J2. N-acetyl cysteine completely abolished the effect of PGD2, and reactive oxygen species (ROS) were considered to be important. Notably, neurite outgrowth was restored by PGD2 removal. These results suggest that PGD2 induces reversible neurite retraction in a ROS-mediated mechanism that does not involve any known receptor.

Identification of protein arginine N-methyltransferase 5 (PRMT5) as a novel interacting protein with the tumor suppressor protein RASSF1A.

The candidate tumor suppressor gene RASSF1A (Ras-association domain family 1, isoform A) is inactivated in many types of adult and childhood cancers. However, the mechanisms by which RASSF1A exerts tumor suppressive functions have yet to be elucidated. In this report, we sought to identify candidate proteins that interact with RASSF1A using proteomic screening. Using peptide mass fingerprinting, we identified protein arginine N-methyltransferase 5 (PRMT5), a type II protein arginine N-methyltransferase that monomethylates and symmetrically dimethylates arginine residues, as a novel protein that interacts with RASSF1A. The association between the two proteins was confirmed by co-immunoprecipitation and immunofluorescence staining. Co-expressing RASSF1A and PRMT5 led to a redistribution of PRMT5 from the cytosol to stabilized microtubules, where RASSF1A and PRMT5 became co-localized. Our results demonstrate that PRMT5 translocates to bundled microtubules on stabilization by RASSF1A expression. Our results show that the tumor suppressor RASSF1A interacts with PRMT5 in vivo and in vitro. Notably, this is the first demonstration of RASSF1A-dependent microtubule recruitment of PRMT5, suggesting a novel role for RASSF1A in the anchoring of cytosolic PRMT5 to microtubules.

Computer vision analysis of mother-infant interaction identified efficient pup retrieval in V1b receptor knockout mice.

Close contact between lactating rodent mothers and their infants is essential for effective nursing. Whether the mother's effort to retrieve the infants to their nest requires the vasopressin-signaling via V1b receptor has not been fully defined. To address this question, V1b receptor knockout (V1bKO) and control mice were analyzed in pup retrieval test. Because an exploring mother in a new test cage randomly accessed to multiple infants in changing backgrounds over time, a computer vision-based deep learning analysis was applied to continuously calculate the distances between the mother and the infants as a parameter of their relationship. In an open-field, a virgin female V1bKO mice entered fewer times into the center area and moved shorter distances than wild-type (WT). While this behavioral pattern persisted in V1bKO mother, the pup retrieval test demonstrated that total distances between a V1bKO mother and infants came closer in a shorter time than with a WT mother. Moreover, in the medial preoptic area, parts of the V1b receptor transcripts were detected in galanin- and c-fos-positive neurons following maternal stimulation by infants. This research highlights the effectiveness of deep learning analysis in evaluating the mother-infant relationship and the critical role of V1b receptor in pup retrieval during the early lactation phase.

Metastasis-associated protein, S100A4 mediates cardiac fibrosis potentially through the modulation of p53 in cardiac fibroblasts.

Metastasis-associated protein, S100A4 is suggested as a marker for fibrosis in several organs. It also modulates DNA binding of p53 and affects its function. However, the functional role of S100A4 in the myocardium has remained unclear. Therefore, we investigated the role of S100A4 and its relationship with p53 in cardiac fibrosis. In Dahl-rat hypertensive heart disease model, S100A4 was upregulated in the hypertrophic myocardium and further activated during transition to heart failure (HF). It was expressed in various cells including fibroblasts. In in vitro cardiac fibroblasts, the knockdown of S100A4 significantly suppressed both cell proliferation and collagen expressions. S100A4 co-localized and interacted with p53 in the nucleus. S100A4 knockdown increased the expression of p53-downstream genes, p21 and mdm2, and concomitant knockdown of p53 recovered cell proliferation and collagen expression. Transverse aortic constriction (TAC) was performed in S100A4 knockout (KO) mice, which showed a similar baseline-phenotype to wild type (WT) mice. Although there was no difference in hypertrophic response, KO mice showed reduced interstitial fibrosis, decreased myofibroblasts, and suppressed expressions of collagens and profibrotic cytokines in the left ventricle. Also, DNA microarray analysis showed that S100A4 knockout in vivo had a significant impact on expressions of p53-associated genes. These findings suggest that S100A4 modulates p53 function in fibroblasts and thereby mediates myocardial interstitial fibrosis through two distinct mechanisms; cell proliferation and collagen expression. Blockade of S100A4 may have therapeutic potential in cardiac hypertrophy and HF by attenuating cardiac fibrosis.

Voluntary locomotion linked with cerebral activation is mediated by vasopressin V1a receptors in free-moving mice.

We previously reported that cerebral activation suppressed baroreflex control of heart rate (HR) at the onset of voluntary locomotion. In the present study, we examined whether vasopressin V1a receptors in the brain were involved in these responses by using free-moving V1a receptor knockout (KO, n = 8), wild-type mice locally infused with a V1a receptor antagonist into the nucleus tractus solitarii (BLK, n = 8) and control mice (CNT, n = 8). Baroreflex sensitivity (HR/MAP) was determined from HR response (HR) to a spontaneous change in mean arterial pressure (MAP) every 4 s during the total resting period, which was ∼8.7 h, of the 12 h measuring period in the three groups. HR/MAP was determined during the periods when the cross-correlation function (R(t)) between HR and MAP was significant (P < 0.05). Cerebral activity was determined from the power density ratio of to δ wave band (/δ) on the electroencephalogram every 4 s. Spontaneous changes in /δ were significantly correlated with R(t) during 62 ± 3% of the total resting period in CNT (P < 0.05), but only 38 ± 4% in KO and 47 ± 2% in BLK (vs. CNT, both P < 0.001). When R(t) and HR/MAP were divided into six bins according to the level of /δ, both were positively correlated with /δ in CNT (both P < 0.001), while neither was correlated in KO or BLK (all P > 0.05). Moreover, the probability that mice started to move after an increase in /δ was 24 ± 4% in KO and 24 ± 6% in BLK, markedly lower than 61 ± 5% in CNT (both P < 0.001), with no suppression of the baroreflex control of HR. Thus, central V1a receptors might play an important role in suppressing baroreflex control of HR during cerebral activation at the onset of voluntary locomotion.

Vasopressin V1a receptor and oxytocin receptor regulate murine sperm motility differently.

Specific receptors for the neurohypophyseal hormones, arginine vasopressin (AVP) and oxytocin, are present in the male reproductive organs. However, their exact roles remain unknown. To elucidate the physiological functions of pituitary hormones in male reproduction, this study first focused on the distribution and function of one of the AVP receptors, V1a. In situ hybridization analysis revealed high expression of the Avpr1a in Leydig cells of the testes and narrow/clear cells in the epididymis, with the expression pattern differing from that of the oxytocin receptor (OTR). Notably, persistent motility and highly proportional hyperactivation were observed in spermatozoa from V1a receptor-deficient mice. In contrast, OTR blocking by antagonist atosiban decreased hyperactivation rate. Furthermore, AVP stimulation could alter the extracellular pH mediated by the V1a receptor. The results highlight the crucial role of neurohypophyseal hormones in male reproductive physiology, with potential contradicting roles of V1a and OTR in sperm maturation. Our findings suggest that V1a receptor antagonists are potential therapeutic drugs for male infertility.

The roles of V1a vasopressin receptors in blood pressure homeostasis: a review of studies on V1a receptor knockout mice.

A prompt rise in blood pressure occurs when arginine-vasopressin is administered in quantities adequate to activate vascular V1a subtype vasopressin receptors. However, it has been controversial whether the endogenous vasopressin-V1a system contributes to the maintenance of basal blood pressure during normal development and aging. Mutant mice lacking the V1a receptor gene (V1a(-/-)) show significantly lower blood pressure compared to control mice, without a notable change in heart rate. In V1a(-/-) mice, arterial baroreceptor reflexes were attenuated due to malfunctioning baroreflex center, and the mice's circulating blood volume was significantly reduced. In line with this reduction in circulating blood volume, adrenocortical hormone release was attenuated; plasma aldosterone levels were reduced and adrenocorticotropic hormone-stimulated corticosteroid release was attenuated. In addition, V1a receptor expression was detected in macula densa cells of the kidneys, which may have facilitated renin production from the juxtaglomerular cells. Deletion of the V1a receptor appears to impact the renin-angiotensin-aldosterone system. Studies on V1a(-/-) mice revealed that non-vascular V1a receptors in the central nervous system and peripheral tissues play critical roles in the maintenance of blood pressure homeostasis.

Chronic ritodrine treatment induces refractoriness of glucose-lowering ß2 adrenoceptor signal in female mice.

Adverse events in tocolytic therapy with ß2-adrenergic agents compromise cardiovascular and non-cardiovascular functions, including blood glucose regulation and liver function. Here, we have examined the effects of the ß2 agonist ritodrine on glucose metabolism and liver injury in mice. Under fasting conditions, ritodrine significantly increased serum insulin levels and decreased glucose concentrations. This contrasts with the ß2 agonist-induced hyperglycemia observed in previous studies on humans and other animals. After 14 days of ritodrine treatment, the mice showed a decrease in the total mass of epididymal fat pads, whereas their body weights increased significantly. Chronic ritodrine treatment attenuated the glucose-lowering effect observed during acute administration. Ritodrine also significantly increased serum levels of liver enzymes, which returned to control levels after 14 days of treatment. Thus, ritodrine responsiveness changes between acute and chronic treatment, indicating that close monitoring of blood glucose and serum liver enzymes is necessary in patients with reduced glucose tolerance. The findings reported here of glucose homeostasis in mice provide a unique opportunity to understand refractoriness of ß2-adrenoceptor signaling in response to ß2 agonists during the course of treatment.

Serum alanine aminotransferase level and intravenous immunoglobulin resistance in patients with kawasaki disease.

INTRODUCTION/OBJECTIVES: Serum alanine aminotransferase (ALT) elevation is considered a risk factor for resistance to initial intravenous immunoglobulin (IVIG) treatment in patients with Kawasaki disease (KD). However, serum ALT levels change dramatically during acute KD illness. We tested the hypothesis that risk assessment for initial IVIG resistance based on serum ALT elevation may differ by examination day after KD onset. METHODS: We analyzed 18,492 population-based patients who developed KD throughout Japan. First, we epidemiologically evaluated the serum ALT variation at 1‒10 days after disease onset. Second, we conducted multivariable logistic regression to determine the association between serum ALT level and initial IVIG resistance according to timing of initial hospital visit by stratifying the patients into an early group (1‒5 days after onset) and a late group (6‒10 days after onset). RESULTS: Serum ALT rapidly increased after KD onset, peaked at day 4 of illness, and then declined regardless of IVIG responsiveness. The adjusted odds ratio (OR) increased with increasing serum ALT in the early group (adjusted OR [95% CI]: 1.44 [1.25-1.66], 1.94 [1.65-2.28], and 2.22 [1.99-2.48] for serum ALT 50-99, 100-199, and ≥ 200 IU/L, respectively; reference ALT level: 1-49 IU/L). No significant association was observed in the late group. CONCLUSIONS: The findings indicate that risk assessment for initial IVIG resistance based on serum ALT level may only be reliable for patients with KD who visit hospitals during early illness, specifically 1-5 days after disease onset. Key Points Serum alanine aminotransferase level differed markedly according to examination days after Kawasaki disease onset. Serum alanine aminotransferase level declined toward normal range after day 5 of illness regardless of intravenous immunoglobulin responsiveness. Elevated serum alanine aminotransferase level was no longer a significant risk factor for initial intravenous immunoglobulin resistance when measured on delayed hospital visits. Risk assessment for initial intravenous immunoglobulin resistance based on serum alanine aminotransferase level may only be reliable for patients who visit hospitals during early illness, specifically 1-5 days after disease onset.

Serum sodium level associated with coronary artery lesions in patients with Kawasaki disease.

INTRODUCTION/OBJECTIVES: Hyponatremia is a potential risk factor for the development of coronary artery lesions (CALs) identified after acute Kawasaki disease (KD). However, the serum sodium distribution corresponding to the reference intervals differs between infants (< 1 year of age) and older children. We hypothesized the association of serum sodium level with CAL complications differs between infants and older patients with KD. METHODS: We analyzed 21,610 population-based patients who developed KD throughout Japan during 2013-2014. We performed multivariable logistic regression analyses to evaluate the association between serum sodium and CAL complications. Additionally, we stratified the serum sodium distribution associated with CAL complications by infants and older patients. RESULTS: CALs were identified in 158 (3.6%) infants and 302 (1.8%) older patients. Infants were more likely to develop CALs when within the normal sodium range compared with older patients (75% vs. 29%), whereas most older patients developed CALs with a sodium level lower than the reference interval. Serum sodium ≤ 130 mEq/L indicated significantly higher risk for development of CALs in both groups (adjusted odds ratio [95% confidence intervals] = 3.21 [1.65-6.25] in infants and 1.74 [1.18-2.57] in older patients). CONCLUSIONS: Serum sodium distribution associated with CAL complications differed greatly between infants and older patients. Older patients developed CALs with sodium levels lower than the reference interval; however, among infants, hyponatremia was not necessarily a risk factor for developing coronary artery lesion. When considering risk assessments for CALs using serum sodium levels, infants with KD should be distinguished from older patients. Key Points • Hyponatremia is a potential risk factor for the development of coronary artery lesions (CALs) among patients with Kawasaki disease. • However, the serum sodium distribution corresponding to the reference intervals differs between infants (< 1 year of age) and older children. • Most infants developed CALs within the normal sodium range, whereas older patients developed at a range lower than the reference interval. • These findings highlight that when considering risk assessments for CALs using serum sodium levels, infants should be distinguished from older patients.

Protective effect of α-lipoic acid against arsenic trioxide-induced acute cardiac toxicity in rats.

The clinical use of arsenic trioxide (ATO) is often limited because of its adverse effects. We examined whether α-lipoic acid (LA) protects against the ATO-induced cardiac toxicity. In the chronic study, two of four rats suddenly died by the repeated dosing of ATO, whereas no deaths were observed in combination with LA. In the acute study, continuous ECG recording revealed that intravenous injection of ATO caused transient ST-T change, whereas pretreatment with LA abolished the ATO-induced ECG abnormality in all animals. These results suggest that LA protects against the ATO-induced acute cardiac toxicity and subsequent sudden death in rats.

Protective Effect of α-Lipoic Acid Against Arsenic Trioxide-Induced Acute Cardiac Toxicity in Rats.

The clinical use of arsenic trioxide (ATO) is often limited because of its adverse effects. We examined whether α-lipoic acid (LA) protects against the ATO-induced cardiac toxicity. In the chronic study, two of four rats suddenly died by the repeated dosing of ATO, whereas no deaths were observed in combination with LA. In the acute study, continuous ECG recording revealed that intravenous injection of ATO caused transient ST-T change, whereas pretreatment with LA abolished the ATO-induced ECG abnormality in all animals. These results suggest that LA protects against the ATO-induced acute cardiac toxicity and subsequent sudden death in rats.

Agonist dependency of the second phase access of ß-arrestin 2 to the heteromeric µ-V1b receptor.

During the development of analgesic tolerance to morphine, the V1b vasopressin receptor has been proposed to bind to ß-arrestin 2 and the µ-opioid receptor to enable their interaction. However, direct evidence of such a high-order complex is lacking. Using bioluminescent resonance energy transfer between a split Nanoluciferase and the Venus fluorescent protein, the NanoBit-NanoBRET system, we found that ß-arrestin 2 closely located near the heteromer µ-V1b receptor in the absence of an agonist and moved closer to the receptor carboxyl-termini upon agonist stimulation. An additive effect of the two agonists for opioid and vasopressin receptors was detected on the NanoBRET between the µ-V1b heteromer and ß-arrestin 2. To increase the agonist response of NanoBRET, the ratio of the donor luminophore to the acceptor fluorophore was decreased to the detection limit of luminescence. In the first phase of access, ß-arrestin 2 was likely to bind to the unstimulated V1b receptor in both its phosphorylated and unphosphorylated forms. In contrast, the second-phase access of ß-arrestin 2 was agonist dependent, indicating a possible pharmacological intervention strategy. Therefore, our efficient method should be useful for evaluating chemicals that directly target the vasopressin binding site in the µ-V1b heteromer to reduce the second-phase access of ß-arrestin 2 and thereby to alleviate tolerance to morphine analgesia.

Inhibition of heat shock protein 90 attenuates adenylate cyclase sensitization after chronic morphine treatment.

Cellular adaptations to chronic opioid treatment result in enhanced responsiveness of adenylate cyclase and an increase in forskolin- or agonist-stimulated cAMP production. It is, however, not known whether chaperone molecules such as heat shock proteins contribute to this adenylate cyclase sensitization. Here, we report that treatment of cells with geldanamycin, an inhibitor of heat shock protein 90 (Hsp90), led to effective attenuation of morphine-induced adenylate cyclase sensitization. In SK-N-SH human neuroblastoma cells, morphine significantly increased RNA transcript and protein levels of type I adenylate cyclase, leading to sensitization. Whole-genome tiling array analysis revealed that cAMP response element-binding protein, an important mediator for cellular adaptation to morphine, associated with the proximal promoter of Hsp90AB1 not only in SK-N-SH cells but also in rat PC12 and human embryonic kidney cells. Hsp90AB1 transcript and protein levels increased significantly during morphine treatment, and co-application of geldanamycin (0.1-10 nM) effectively suppressed the increase in forskolin-activated adenylate cyclase activation by 56%. Type I adenylate cyclase, but not Hsp90AB1, underwent significant degradation during geldanamycin treatment. These results indicate that Hsp90 is a new pharmacological target for the suppression of adenylate cyclase sensitization induced by chronic morphine treatment.