Hiroyoshi Tsuchiya

Prostaglandin E2 Attenuates Preoptic Expression of GABAA Receptors via EP3 Receptors

Prostaglandin E2 (PGE2) has been shown to produce fever by acting on EP3 receptors within the preoptic area of the brain. However, there is little information about the molecular events downstream of EP3 activation in preoptic neurons. As a first step toward this issue, we examined PGE2-induced gene expression changes at single-cell resolution in preoptic neurons expressing EP3. Brain sections of the preoptic area from PGE2- or saline-injected rats were stained with an anti-EP3 antibody, and the cell bodies of EP3-positive neurons were dissected and subjected to RNA amplification procedures. Microarray analysis of the amplified products demonstrated the possibility that gene expression of γ-aminobutyric acid type A (GABAA) receptor subunits is decreased upon PGE2 injection. Indeed, we found that most EP3-positive neurons in the mouse preoptic area are positive for the α2 or γ2 GABAA receptor subunit. Moreover, PGE2 decreased the preoptic gene expression of these GABAA subunits via an EP3-dependent and pertussis toxin-sensitive pathway. PGE2 also attenuated the preoptic protein expression of the α2 subunit in wild-type but not in EP3-deficient mice. These results indicate that PGE2-EP3 signaling elicits Gi/o activation in preoptic thermocenter neurons, and we propose the possibility that a rapid decrease in preoptic GABAA expression may be involved in PGE2-induced fever.

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.

Subcellular localization and internalization of the vasopressin V1B receptor

Only limited information is available on agonist-dependent changes in the subcellular localization of vasopressin V1B receptors. Our radioligand binding study of membrane preparations and intact cells revealed that a large fraction of the V1B receptor is located in the cytoplasm in unstimulated CHO cells, which is in contrast to the plasma membrane localization of the V1A and V2 receptors. Moreover, when the affinity of radiolabeled arginine-vasopressin ([3H]AVP) was compared between membrane preparations and intact cells, the affinity of [3H]AVP to the cell surface V1B receptors, but not the V1A receptors, was significantly reduced. Although the number and affinity of cell surface V1B receptors decreased, they became extensively internalized upon binding with [3H]AVP. Approximately 87% of cell surface-bound [3H]AVP was internalized and became resistant to acid wash during incubation with 1 nM [3H]AVP. By contrast, less ligand (35%) was internalized in the cells expressing the V1A receptor. Extensive internalization of the V1B receptors was partially attenuated by inhibitors of cytoskeletal proteins, siRNA against β-arrestin 2, or the removal of sodium chloride from the extracellular buffer, indicating that this internalization involves clathrin-coated pits. Together, these results indicate that the mechanism that regulates the number and affinity of V1B receptors in the plasma membrane is markedly distinct from the corresponding mechanisms for the V1A and V2 receptors and plays a critical role under stress conditions, when vasopressin release is augmented.

Chronic ritodrine treatment induces refractoriness of glucose-lowering β2 adrenoceptor signal in female mice.

Adverse events in tocolytic therapy with β2-adrenergic agents compromise cardiovascular and non-cardiovascular functions, including blood glucose regulation and liver function. Here, we have examined the effects of the β2 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 β2 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 β2-adrenoceptor signaling in response to β2 agonists during the course of treatment.

Proximal tubules and podocytes are toxicity targets of bucillamine in a mouse model of drug-induced kidney injury

Effective detection of potential nephrotoxicity is crucial for pre-clinical drug development. We evaluated a sensitive animal model for drug-induced kidney injury, which includes hemi-nephrectomy of mice. Although bucillamine and d-penicillamine are used for the treatment of rheumatoid arthritis in Japan, drug-related adverse effects on the kidney can limit their therapeutic utilities. When bucillamine (1000 or 2000 mg/kg/day) or d-penicillamine (2000 mg/kg/day) were orally administered to hemi-nephrectomised BALB/c mice, the urinary protein levels of bucillamine-treated mice, but not of those treated with d-penicillamine, the vehicle, or in bucillamine treated unnephrectomized mice, were significantly increased and remained high during the 4-week drug-loading period. Membranous glomerulonephropathy occasionally seen in bucillamine/d-penicillamine-treated arthritis patients was not reproduced in mice. Instead, our mouse model showed proximal tubular injury and podocyte foot process effacement in the bucillamine-treated kidneys. These two cell types are also the primary targets of the experimental Heymann membranous glomerulonephropathy. Gene expression profiling of the bucillamine-treated mice identified lipocalin 2 as a significantly up-regulated transcript together with cytochrome P450 CYP4a14, a group-specific component, and proprotein convertase subtilisin/kexin type 9. Moreover, large amounts of lipocalin 2 were detected in the urine of the bucillamine-treated mice, but not in the hemi-nephrectomised control mice. These results indicate that hemi-nephrectomy effectively promotes acute kidney injury by bucillamine, which is accompanied by up-regulation of the urinary biomarker lipocalin 2. Our mouse model with initial stage of kidney injury should be useful to analyse the pathogenesis of drug-induced glomerular and tubular injuries.

Pharmacological lineage analysis revealed the binding affinity of broad-spectrum substance P antagonists to receptors for gonadotropin-releasing peptide.

A group of synthetic substance P (SP) antagonists, such as [Arg(6),D-Trp(7,9),N(Me)Phe(8)]-substance P(6-11) and [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]-substance P, bind to a range of distinct G-protein-coupled receptor (GPCR) family members, including V1a vasopressin receptors, and they competitively inhibit agonist binding. This extended accessibility enabled us to identify a GPCR subset with a partially conserved binding site structure. By combining pharmacological data and amino acid sequence homology matrices, a pharmacological lineage of GPCRs that are sensitive to these two SP antagonists was constructed. We found that sensitivity to the SP antagonists was not limited to the Gq-protein-coupled V1a and V1b receptors; Gs-coupled V2 receptors and oxytocin receptors, which couple with both Gq and Gi, also demonstrated sensitivity. Unexpectedly, a dendrogram based on the amino acid sequences of 222 known GPCRs showed that a group of receptors sensitive to the SP antagonists are located in close proximity to vasopressin/oxytocin receptors. Gonadotropin-releasing peptide receptors, located near the vasopressin receptors in the dendrogram, were also sensitive to the SP analogs, whereas α1B adrenergic receptors, located more distantly from the vasopressin receptors, were not sensitive. Our finding suggests that pharmacological lineage analysis is useful in selecting subsets of candidate receptors that contain a conserved binding site for a ligand with broad-spectrum binding abilities. The knowledge that the binding site of the two broad-spectrum SP analogs partially overlaps with that of distinct peptide agonists is valuable for understanding the specificity/broadness of peptide ligands.