The Na⁺ -Ca²⁺ exchanger in the plasma membrane is a bidirectional electrogenic ion transporter that couples the translocation of Na⁺ in one direction with that of Ca²⁺ in the opposite direction. This system is involved in regulation of intracellular Ca²⁺ concentration via the forward mode (Ca²⁺ extrusion) or the reverse mode (Ca²⁺ influx). There are two types of the plasma membrane Na⁺ -Ca²⁺ exchanger in an animal, and they are called the cardiac type and rod outer segment type. In addition, there is an electroneutral Na⁺ -Ca²⁺ exchanger present in mitochondria. Recent studies by the molecular biology technique show that there are at least 8 isoforms of the cardiac type (NCX1), and there are two other exchangers in the brain (NCX2 and NCX3). Due to new techniques of molecular biology and electrophysiology, much evidence is accumulating with respect to the structure, mechanism, regulation, and physiological and pathological roles of the Na⁺ -Ca²⁺ exchanger. This review summarizes recent progress in this research field that is of nharmacolorical interest.

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It has been reported that amiodarone induces disorders of alveolar macrophages and pulmonary fibrosis, but the mechanism is not well-understood. This study was performed to elucidate the toxic mechanism from the standpoint of cellular function. Using alveolar macrophages obtained from a male Slc:ICR mouse, several injuries caused by amiodarone were compared to those caused by amantadine and mianserin as cationic amphiphilic drugs (CADs). As parameters for the drug effects, H⁺-ATPase and acid sphingomylinase activities, cellular pH, cytokine and prostaglandin releases, phagocytosis and neutral red uptake were measured. Amiodarone decreased H⁺-ATPase activity initially and subsequently increased cellular pH and decreased acid sphingomyelinase activity. These changes, which were also observed with amantadine and mianserin, were considered to be CAD-related. Amiodarone increased cytokine and prostaglandin releases and suppressed neutral red uptake and phagocytosis. These changes, being not induced by amantadine and mianserin, were considered to be specific for amiodarone. The above data suggest that amiodarone has two types of toxic effects on alveolar macrophages.

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The effects of nicardipine and nifedipine on the urinary excretion of urate were examined in rats and compared with those of diltiazem. Test drugs were administered to urethane-anesthetized oxonate-loaded rats by continuous i.v. infusion. Diltiazem (10 μg/rat/min), nicardipine (0.3 μg/rat/min) and nifedipine (1.0 μg/rat/min) caused similar reductions of systemic blood pressure and increased total renal blood flow. Diltiazem did not increase urine volume significantly. However, this drug produced obvious uricosuria, with a significant increase in the ratio of urate clearance to inulin clearance (Cua/Cin), which resulted from an increase in Cua, but not from changes in the glomerular filtration rate (GFR). Nicardipine had clear diuretic and uricosuric effects, with similar increases in Cua and GFR and, thus, no change in Cua/Cin. On the other hand, nifedipine did not have any significant effect on the renal handling of urate. These results suggest that nicardipine produces uricosuria in rats via alterations in renal hemodynamics, while the uricosuric effect of diltiazem involves the tubules, as well as alterations in renal hemodynamics.

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SM-10902 ((+)-methyl [2-[(2R, 3aS, 4R, 5R, 6aS)-octahydro-5-hydroxy-4-[(E)-(3S, 5S)-3-hydroxy-5-methyl-1-nonenyl]-2-pentalenyl]ethoxy]acetate)and its free acid, SM-10906 are new stable 3-oxamethano prostaglandin (PG)I₁ analogs. Their affinities for [³H]iloprost and [³H]PGE₂ binding sites in human platelets and human umbilical vascular endothelial cells were compared with those of the PGI₂ analog iloprost, PGE₁ and PGE₂ by the radioligand binding assay method. The cyclic AMP (cAMP)synthesis activity of these drugs were also determined in human umbilical vascular endothelial cells. We found that SM-10906 apparently displaced [³H]iloprost binding to the membrane fractions in those cells since the pK_i values were 6.30 in platelets, 7.52 in vein endothelial cells and 6.31 in the arterial endothelial cells. The pK_i values of SM-10906 for [³H]PGE₂ binding sites were significantly lower than those obtained for [³H]iloprost binding. SM-10902, which is a prodrug of SM-10906, showed low affinity for [³H]iloprost binding sites in those cells. SM-10906 also dose-dependently enhanced the cAMP level in the vascular endothelial cells. Thus, these findings indicate that SM-10906 binds to [³H]iloprost binding sites and exhibits pharmacological functions such as an anti-platelet action and a cytoprotective action in endothelial cells through the elevation of intracellular cAMP contents.

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We have investigated opioid mechanisms concerning regulation of urine production in the hypothalamic supraoptic nucleus (SON). In this study, the effect of [D-Ala², N-Me-Phe⁴, G1y⁵-ol]enkephalin (DAMGO), a potent selective μ-opioid agonist, microinjected into the SON of anesthetized hydrated rats, on the urine outflow rate was examined. DAMGO caused a dose-dependent decrease in the urine outflow rate with no significant changes in blood pressure nor heart rate. The ED₅₀ value for the antidiuresis was calculated to be 0.055 nmol from the dose-response curve. The antidiuresis elicited by DAMGO (0.1 nmol)was partially inhibited by intra-SON pre-injection of naloxone (3 nmol), a relatively μ-selective opioid antagonist, and timolol (100 nmol), a β-adrenoceptor antagonist, but not by intra-SON pre-injection of phenoxybenzamine (20 nmol), an α-adrenoceptor antagonist, nor atropine (300 nmol), a muscarinic antagonist. Intravenous injection of d(CH₂)₅-D-Tyr(Et)VAVP (16.7 μg), a vasopressin receptor antagonist, did not influence the DAMGO-induced antidiuresis. These findings suggest that antidiuresis mediated through μ-opioid receptors in the SON involves β-adrenoceptors in the nuclei, but does not involve an increase in vasopressin release.

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We have investigated the effects of two novel antiparkinsonian drugs, talipexole (Domin®)and pramipexole, on MPTP-induced dopamine (DA)reduction in the striatum of C57BL/6N mice in comparison with those of bromocriptine. Fifteen days after MPTP treatment (25 mg/kg, i.p., given daily for 5 days), the DA content in the striatum was decreased to 40-60010 of the control value. Among the three dopamine receptor agonists, talipexole and pramipexole (1 mg/kg, i.p., once a day for 20 days)more significantly suppressed the MPTP-induced DA reduction in the striatum than bromocriptine (10 mg/kg, i.p., once a day for 20 days). Talipexole did not influence [³H]MPP⁺ uptake into striatal synaptosomes. These results suggest that talipexole and pramipexole have a protective effect against MPTP-induced DA reduction in the striatum of C57BL/6N mice.

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We studied the pharmacological effects of dalteparin (low-molecular-weight heparin)and heparin on bone metabolism in rats. After their 28 days of consecutive intravenous injections, significant loss of bone weight and mineral contents was observed in the heparin-treated rats, whereas dalteparin slightly reduced bone mass. By the end of the experiment, the femora of 7 out of 8 rats fractured in the heparin (10, 000 U/kg/day)-treated group, but none had broken in the control and dalteparin-treated groups. Serum osteocalcin levels were significantly decreased in the former group. The growth plate width of the tibia was increased in a dose-dependent manner, especially in the heparin-treated group. Histomorphometric assessment of tibia showed that the osteoid surface and mineral apposition rates were significantly reduced in the heparin-treated group, whereas the eroded surface was significantly increased in the heparin-treated group. The above results suggest that heparin not only augments bone resorption but also suppressed bone formation and that dalteparin has a weaker suppressive effect on bone formation compared with heparin.

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The present study was carried out to clarify whether orally administered 5-{3-[((2S)-1, 4-benzodioxan-2-ylmethyl)amino]propoxy}-1, 3-benzodioxole (MKC-242), a serotonin_1A (5-HT_1A)-receptor agonist having potent anxiolytic-like and antidepressant-like effects in animal models, binds to 5-HT1A receptors in rat brain. Quantitative autoradiography showed that orally administered MKC-242 (0.1-0.5 mg/kg)caused a significant decrease in [³H]8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT)binding in the hippocampus and dorsal raphe nucleus sections. The decrease in the binding by MKC-242 was observed up to 4 hr after administration, and the effective doses were similar to those in its anxiolytic-like effect in the animal models. Repeated treatment of MKC-242 (0.5 mg/kg/day, p.o.)or buspirone (30 mg/kg/day, p.o.)for 2 weeks did not affect [³H]8-OH-DPAT binding in both sections. These results suggest that orally administered MKC-242 at the low doses that do not show 5-HT1A-receptor-mediated in vivo responses such as the hypothermic effect, adrenocortical effect and the decrease in 5-HT turnover passes the blood-brain barrier and subsequently binds to 5-HT_1A receptors in rat brain. In addition, they indicate that repeated stimulation of the receptors by the agonists does not affect the number of the binding sites.

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We examined whether opioids, especially morphine, would centrally elicit scratching in mice and determined some characteristics of the scratch-inducing action of opioids. When intracisternally (i.c.) injected, morphine (0.1 3 nmol/mouse) produced a dose-dependent increase in scratching of the face, but not of the ears, head and body trunk. When injected intradermally into the rostral part of the back, morphine (at most potent i.c. dose of 3 nmol/mouse or higher) did not increase the scratching of the injected site. Facial scratching of the mouse induced by i.c. injection of morphine (0.3 nmol/mouse) was almost abolished by distraction and by naloxone (1 mg/kg, s.c.). [D-Ala², N-Me-Phe⁴, GIy⁵-ol]Enkephalin (DAMGO)(0.03-2 nmol), but not [D-Pen^{2, 5}]enkephalin (DPDPE)and U-50, 488, dose-dependently elicited facial scratching by i.c. injection. These results suggest that morphine and DAMGO increased facial scratching, probably mediated by central opioid μ-receptors in mice, and such scratching was due to a sensation, probably itching. The present animal model may be useful for analyzing opioid-mediated central itching.

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We investigated the interaction between nitric oxide and the renin angiotensin system in regulating isolated aortic tension and mean arterial pressure in renal hypertensive rats (RHR). Acetylcholine (ACh) relaxed aorta precontracted with norepinephrine from RHR significantly less than that from normotensive rats (NR)(E_max: 34.3% and 86.0%, respectively, P<0.01). The ACh-induced relaxation was significantly enhanced by losartan (P <0.05) and completely abolished by removal of endothelium or N^G-nitro-L-arginine methyl ester (L-NAME). ACh lowered the mean arterial pressure slightly less effectively in RHR than in NR (6.8 and 13.0 mmHg, respectively, at 0.1 μg/kg), whereas the depressor effect was reduced by L-NAME (-15.5 and 10.3 mmHg, respectively, at 0.1 μg/kg), but rather enhanced by further treatment with losartan (9.9 (P < 0.05) and 17.3 mmHg, respectively, at 0.1 μg/kg). Angiotensin II induced similar contractile and pressor responses in both RHR and NR, and these effects were significantly enhanced by L-NAME, except for the pressor effect in RHR. L-NAME induced a similar pressor response in RHR and NR (15.9 and 15.2 mmHg, respectively, at 0.1 mg/kg), the effect being decreased by pretreatment with losartan. Losartan induced a depressor response that was smaller in RHR than in NR (34.0 and 48.8 mmHg, respectively, at 0.3 mg/kg), and the response was significantly reduced by L-NAME. These results suggest that nitric oxide interacts with the renin angiotensin system to control the vascular tension and systemic arterial circulation in RHR.

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We examined the effect of KW-5092, a gastroprokinetic agent with acetylcholinesterase inhibitory and acetylcholine release facilitatory activities, on the peristalsis of isolated guinea pig ileum. KW-5092 (10^-9-3 × 10^-6 M)increased the frequency of the peristaltic wave without changing its amplitude. Neostigmine increased the frequency at 10^-7 M, but domperidone (10^-8-3 × 10^-6 M)had no effect on the peristalsis. The present results suggest that KW-5092 enhances the peristalsis via the inhibition of acetylcholinesterase, resulting in the intestinal propulsion.

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Microinjections of α-adrenoceptor agonists into the paraventricular nucleus (PVN)of Wistar Kyoto rats (WKY)decreased the urine outflow rate in dose and time-dependent manners. The order of the antidiuretic potency is norepinephrine (an α-agonist)> phenylephrine (an α₁-agonist)>> clonidine (an α₂-agonist). The phenylephrine-induced effect was inhibited by WB4101 (an α₁-antagonist), but not by yohimbine (an α₂-antagonist). d(CH₂)₅-D-Tyr(Et)VAVP (a vasopressin antagonist)blocked the antidiuretic effect of phenylephrine. In spontaneously hypertensive rats (SHR), norepinephrine and phenylephrine produced weaker antidiuretic effects than in WKY. These findings suggest that the α₁-subtype of the PVN decreases urine production mediated through vasopressin release. This mechanism is more feeble in SHR than WKY.

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Lecithinized-superoxide dismutase (PC-SOD), which is synthesized with a lecithin derivative bound covalently to recombinant human Cu, Zn-SOD, has a longer half-life in blood and higher cell affinity than unmodified SOD. The effects of PC-SOD were evaluated using the rat ulcerative colitis model induced by 3% dextran sulfate sodium. Intravenous injection of rats with 0.5 or 1 mg/kg of PC-SOD suppressed the progression of bloody stools, the formation of erosion, and the infiltration of the colon with inflammatory cells. Furthermore, it also reduced the increase of leukocytes in blood. Thus, PC-SOD may have therapeutic potential in the treatment of ulcerative colitis.

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We examined the implication of a nitric oxide (NO)-guanosine 3'', 5''-cyclic monophosphate (cGMP)cascade in the suppression of gastric vagal afferents due to ecabapide in anesthetized rats using a standard extracellular method of multi-unit recording. Sodium nitroprusside (SNP, 0.5 mg/kg), an NO donor, depressed the afferent discharge rate of the vagus nerve, like ecabapide (60 μg/kg). On the other hand, N^G-nitro-L-arginine (L-NNA, 5 mg/kg), an NO biosynthesis inhibitor, significantly elevated its discharge rate. Pretreatment with L-NNA completely blocked the action of ecabapide. Atropine (0.05 mg/kg), a competitive antagonist of muscarinic cholinoceptors, showed no effect on the afferent firing. These results suggest that ecabapide may suppress the activation of vagal afferents in gastric inhibitory vago-vagal reflex pathways through the NO-cGMP cascade.

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We examined whether FR149175 (ethyl-[(S)-8-[(R)-2-(3-chlorophenyl)-2-hydroxyethylamino]6, 7, 8, 9-tetrahydro-5H-benzocyclohepten-2-yloxy]acetate monohydrochloride monohydrate), a selective agonist for the β₃-adrenoceptor, is a possible therapeutic agent for non-insulin-dependent diabetes mellitus (NIDDM). FR149175 had hypoglycemic effects with an increase in the level of plasma insulin in normal rats. In Zucker fatty rats, an animal model of NIDDM, repeated administration of the drug improved hyperinsulinemia and showed a tendency to decrease the area under the curve (AUC)for plasma glucose levels in the glucose tolerance test. Moreover, FR149175 decreased plasma triglyceride, free fatty acid and total cholesterol levels in the rats. Body weight gain in the rat was suppressed by FR149175 as well. These results suggest that FR149175 has antiobesity and antidiabetic effects and that this drug may be useful for treating NIDDM.

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Acetylcholine (ACh)-induced relaxation in the aortae precontracted with norepinephrine was significantly enhanced in the aortae from estrus (E)rats, compared with that in those from metestrus (D-1), diestrus (D-2)and proestrus (PE)rats. N^G-Nitro-L-arginine methyl ester (L-NAME)inhibited the endothelium-dependent relaxation in E rats. These results suggest that there is a difference in ACh-induced relaxation of the thoracic aorta during the sexual cycle of rats, and the relaxation is greatest in E of the sexual cycle; this may be due to a difference in nitric oxide synthesis in the endothelium in the sexual cycle.

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Manganese-hyaluronate conjugate (Mn-HA) was synthesized from a diethylenetriaminepentaacetic acid derivative of hyaluronic acid and manganese ion. The conjugate markedly scavenged superoxide anion in vitro and exhibited much higher anti-inflammatory activity than superoxide dismutase in suppressing paw edema in mice when intravenously injected 30 min before the initiation of ischemia.

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We have developed a simple and semi-quantitative method for mRNA determination in single cells using the reverse transcription and polymerase chain reaction (RT-PCR). The distinct features of this method are the highly efficient RNA harvest from whole dissociated cells and the ability to perform all RT procedures in one tube that allowed semi-quantitative determination of mRNA in dissociated cells. This method revealed that histamine H₁-receptor mRNA was highly expressed in 5/28 small and 1/26 large dorsal root ganglion neurons of the mouse.

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