Neurosteroids stimulate G protein-coupled sigma receptors in mouse brain synaptic membrane.

Dehydroepiandrosterone, its sulfate (DHEAS) and pregnenolone sulfate, representative neurosteroids as well as (+)-pentazocine concentration-dependently stimulated the [35S]GTPgammaS binding in synaptic membranes of mouse prefrontal cortex. These stimulations were blocked by NE-100, a sigma-receptor antagonist, and by progesterone, another type of neurosteroid. The DHEAS-induced stimulation was blocked by the pertussis toxin (PTX)-treatment, and completely recovered by reconstitution of PTX-treated membranes with recombinant G(i1), but not with G(oA). DHEAS also stimulated the [35S]GTPgammaS binding in the coronal sections of mouse brain in NE-100- or progesterone-reversible manner. These findings suggest that some neurosteroids may act on metabotropic sigma receptors, and this study may be the first to show the coupling of neurosteroid binding site and G(i).

Metabotropic neurosteroid/sigma-receptor involved in stimulation of nociceptor endings of mice.

In peripheral nociceptive flexor test, SA4503, (+)-pentazocine, and (+)-3-(hydroxyphenyl)-N-(1-propyl)piperidine, representative sigma-receptor agonists, elicited dose-dependent flexor responses. These responses were blocked by sigma-receptor antagonists NE-100 or BD1063, but not by pretreatments with antisense oligodeoxynucleotide for sigma1 binding protein. The sigma-agonists' nociception is attributed to the substance P (SP) release from nociceptor endings through activations of Galpha(i1) and phospholipase C (PLC). On the other hand, attomolar doses of neurosteroids such as dehydroepiandrosterone sulfate (DHEAS) and pregnenolone sulfate caused similar nociception, and they were blocked by progesterone (PROG). However, DHEAS nociception was not affected by pertussis toxin, but was completely inhibited by a PLC inhibitor or thapsigargin. Although the nociception by lower doses of DHEAS was abolished by diphenhydramine (DPH), H1 antagonist, there were dose-dependent responses by high doses of DHEAS in the presence of DPH. The responses by DHEAS in the presence of DPH were blocked by NE-100, and those by (+)-pentazocine were blocked by PROG. All these findings suggest that two novel types of neurosteroid receptors exist, neuronal NS1/sigma-type, which mediates activation of Galpha(i1) by neurosteroids and sigma-agonists, followed by SP release from nociceptor endings; and NS2 type, which mediates histamine release from mast cells by very low doses of neurosteroids.

Binding of [35S]GTPgammaS stimulated by (+)-pentazocine sigma receptor agonist, is abundant in the guinea pig spleen.

Here we measured sigma receptor agonist, [3H](+)-pentazocine binding and (+)-pentazocine-stimulated [35S]GTPgammaS binding throughout brain regions and peripheral organs of mice and guinea pigs to investigate the distribution of G protein-coupled sigma receptors. There was no significant correlationship between both distributions, in which the [3H](+)-pentazocine binding is highest in the liver of each species, while the [35S]GTP-gammaS binding is highest in the guinea pig spleen. The agonist-stimulated [35S]GTPgammaS binding in the spleen was also confirmed by in situ autoradiography using sections. Thus it is suggested that there are at least two subtypes, metabotropic and nonmetabotropic sigma receptors, and the former ones are abundant in the guinea pig spleen.

Selective coupling of mouse brain metabotropic sigma receptor with recombinant Gi1.

Various sigma (sigma) ligands including (+)-pentazocine stimulated [35S]GTPgammaS binding in synaptic membranes from the mouse cerebellum. The (+)-pentazocine-stimulated [35S]GTPgammaS binding was blocked by the treatment of membranes with pertussis toxin (PTX), but completely recovered by the reconstitution of PTX-treated membranes with recombinant Gi1, but not with GoA. These findings suggest that metabotropic sigma receptors are selectively coupled to Gi1 protein.

Synthesis and quantitative structure-activity relationships of N-(3-oxo-3,4-dihydro-2H-benzo[1,4]oxazine-6-carbonyl)guanidines as Na/H exchange inhibitors.

N-(3-Oxo-3,4-dihydro-2H-benzo[1,4]oxazine-6-carbonyl)guanidines 4 were prepared and tested for Na/H exchange inhibitory activities in order to clarify the structure-activity relationship (SAR). Quantitative SAR (QSAR) analysis of 6-carbonylguanidines 4 indicated that the length of the 4-substituent was parabolically related to activity and that the calculated optimum 4-substituents were propyl, ethyl and isopropyl groups. This SAR was similar to the SAR of the 2- and 4-substituents of 7-carbonylguanidine derivatives 3, although the position relative to the essential guanidinocarbonyl group was different. Larger 2-substituents, such as a phenyl group were unfavorable. The most potent derivative in this series was N-(4-isopropyl-2,2-dimethyl-3-oxo-3,4-dihydro- 2H-benzo[1,4]oxazine-6-carbonyl)guanidine 4 g, with an IC50 value of 0.12 microM. The methanesulfonate salt (KB-R9032) of 4g had excellent water-solubility and showed anti-arrhythmia activity against a rat acute myocardial infarction model. KB-R9032 was selected for further investigation as a therapy for ischemia-reperfusion induced injury.

Design, synthesis and quantitative structure-activity relationship study of N-(3-oxo-3,4-dihydro-2H-benzo[1,4]oxazine-7-carbonyl)guanidine derivatives as potent Na/H exchange inhibitors.

Inhibition of the Na/H exchanger is a promising approach for treating ischemia-reperfusion injury, but no clinical agent is yet available. Recently, we established the structural requirements for potent inhibitors of the Na/H exchanger. In the present work, we designed N-(3-oxo-3,4-dihydro-2H-benzo[1,4]oxazine-7-carbonyl)guanidine 3a as a new lead compound for potent inhibitors with good water-solubility, based on the previous information. During the structural optimization, care was taken to keep the hydrophobicity (clogP) in the range of about 1.5-2.0, which is considered optimum for good bioavailability. Various derivatives of 3a were synthesized and the quantitative structure-activity relationship (QSAR) was studied. The QSAR result indicated that the lengths of the substituents at the 2- and the 4-positions of the 2H-benzo[1,4]oxazine ring are parabolically related to activity. The most potent compounds were (R) and/or (S)-N-(2-ethyl-4-isopropyl(or ethyl)-3-oxo-3,4-dihydro-2H-benzo[1,4]oxazine-7-carbonyl)guanidines 3q-t with IC50 values of 0.036-0.073 microM. The water-solubility of the hydrochlorides and methanesulfonates is 3-5 mg/ml, which is sufficient for therapeutic use.

Expression and characterization of lacrimal gland water channels in Xenopus oocytes.

Lacrimal glands transport fluid for secretion as tears. To examine whether the expression of the aquaporin family of water channels is essential for water transport in lacrimal glands, we determined the water permeability in Xenopus oocytes injected with rat lacrimal gland poly(A)+ RNA. In oocytes injected with poly(A)+ RNA, osmotic water permeability was 4-fold higher than that observed in vehicle-injected controls. The enhanced water permeability was inhibited by 65% by coinjection of poly(A)+ RNA with antisense aquaporin-5 but not antisense aquaporin-1 oligonucleotide. To detect aquaporin mRNA in rat lacrimal glands, we performed reverse transcription-polymerase chain reaction analysis. PCR products were detected using specific aquaporin-5 primers. Our results strongly suggest that rat lacrimal glands express aquaporin-5 water channels for lacrimation.

Non-selective effects of amiloride and its analogues on ion transport systems and their cytotoxicities in cardiac myocytes.

The effects of amiloride and its analogues (3',4'-dichlorobenzamil (DCB), 2',4'-dimethylbenzamil (DMB), 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and 5-(N-methyl-N-isobutyl)amiloride (MIBA)) on cardiac ion transporters (Na+/Ca2+ exchanger, Na+/H+ exchanger, Na+ pump and Ca2+ pump) and their cytotoxicities were tested in cardiac myocytes. All the tested compounds showed concentration-dependent inhibitory effects on the ion transporters studied in canine cardiac sarcolemmal vesicles. The concentrations (microM) of amiloride, DCB, DMB, EIPA and MIBA required to produce 50% inhibition were > 1000, 19, 10, 83 and 84, respectively, for the Na+/Ca2+ exchanger; 130, 73, 63, 16 and 14 for the Na+/H+ exchanger; > 1000, 72, > 300, > 300 and > 300 for the Na+ pump; and > 1000, 37, 93, 90 and 70 for the Ca2+ pump, respectively. Furthermore, these agents induced cell death in isolated rat cardiac myocytes and the 50% lethal concentrations (microM) were > 1000, 9.2, 30, 16 and 17, respectively. These findings demonstrate that amiloride and its analogues have non-selective inhibitory effects on cardiac ion transporters and cytotoxicity in cardiomyocytes. When these drugs are employed as experimental tools to investigate the involvement of ion transporters in cell functions, the results must be interpreted with caution.

Regulation of Na+/glucose cotransport in LLC-PK1 cells by glucose in culture medium: involvement of Gi protein.

The reduction of the glucose concentrations in the culture medium results in an increase in Na+/glucose cotransport activity in the epithelial cells. To study this regulation of Na+/glucose cotransport in LLC-PK1 cells, we have attempted to examine the involvement of Gi protein. The immunoblotting with an antibody for the carboxy-terminal of alpha subunit of Gi protein (Gi alpha) revealed no significant change in the level of Gi alpha in cells cultured with low (5 mM) or high (25 mM) glucose concentrations. In contrast, the cultured with low glucose concentrations induced the increases of both islet activating protein (IAP)-catalyzed ADP-ribosylation of Gi alpha and Na+/glucose cotransport activity. Furthermore, this increase in the transport activity was blocked by the presence of IAP in a dose dependent manner. These results suggest that there is a close relationship between the concentration of the heterotrimeric form of Gi protein (Gi alpha beta gamma) and the activity of Na+/glucose cotransport in LLC-PK1 cells.

Preparations, solution conformation and molecular structures of N,N'-ethylene-bridged dipeptides and their derivatives.

The solution conformations of novel dipeptides, methyl (2S, 3'S)-3-methyl-2-(2'-oxo-3'-isopropyl-1'-piperazinyl)-butanoate (EVV-OCH3), methyl (2S, 3'S)-3-phenyl-2-(2'-oxo-3'-benzyl-1'-piperazinyl) propionate (EFF-OCH3), and their derivatives (Boc-Gly-EVV-OH, Boc-Gly-EVV-Gly-OH, and Boc-Gly-EFF-OH), were studied by 1H NMR measurements and molecular mechanics calculations (1). The molecular structures of Boc-Gly-EVV-OH, Boc-Gly-EFF-OH, and the hydrochloride of EVV-OCH3 were determined by X-ray analyses. The conformations of the piperazinone rings and the side chains of these oligopeptides were clarified.

Insulin regulates Na+/glucose cotransporter activity in rat small intestine.

In order to examine the involvement of insulin in the activity of Na+/glucose cotransporter in rat small intestine, we compared Na(+)-dependent uptake of D-glucose by brush-border membrane vesicles prepared from control, streptozotocin-induced diabetic, insulin-treated diabetic and starved diabetic rats. In four groups, the uptake of D-glucose showed a transient overshoot in the presence of Na+ gradient between medium and vesicles (medium greater than vesicles). The overshoot magnitude was increased (1.8-fold of controls) in diabetic brush border membrane vesicles and recovered to the control level by the treatment of diabetic rats with insulin. In contrast, increased uptake of D-glucose in diabetic rats was not recovered by the starvation of diabetic rats although the blood glucose level was the same as that of controls. Furthermore, we attempted to examine phlorizin binding activities among four groups. Scatchard analysis indicated that phlorizin binding to diabetic brush border membrane vesicles was increased (1.6-fold of controls) without a change of the affinity for phlorizin as compared with controls. Increased binding of phlorizin to diabetic brush border membrane vesicles was also recovered to the control level by the treatment of diabetic rats with insulin, but not by starvation. These results suggested that the increased activity of Na+/glucose cotransporter in diabetic rats was due to the increase of the number of cotransporter and that intestinal cotransporter was physiologically controlled by insulin, but not by blood glucose levels.