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Recent studies indicate a risk of learning and memory impairments when patients with senile dementia are treated with antimuscarinic drugs. In this study, we compared the effectiveness of propiverine hydrochloride (propiverine) and oxybutynin chloride (oxybutynin) on the increased urinary frequency and cognitive impairment induced by nucleus basalis magnocellularis (nBM) lesioning in conscious and nonrestrained rats. For examination of bladder function, nBM-lesioned rats were given total parenteral nutrition regimens for 8 days. Propiverine administered orally at 0.3, 3 and 30 mg/kg on the postoperative day 7 significantly lessened the increase in the frequency of voiding caused by the nBM lesion, whereas oxybutynin administration did not show any improvement at 0.1 or 1 mg/kg but did so at 10 mg/kg. To examine the memory impairment, we trained nBM-lesioned rats in an 8-arm radial maze task for 20 days and then evaluated the effectiveness of oral drug administration on 19th and 20th radial maze performance. The higher rate of errors caused by nBM lesioning was significantly aggravated by oxybutynin at 30 and 100 mg/kg. Propiverine showed slight aggravation of errors, but with no statistical significance at any dose, 30, 100 or 300 mg/kg. These results suggest that propiverine has comparatively less effect on the cognitive impairment than oxybutynin.

The effect of phosphorothioated antisense oligodeoxynucleotide (AS ODN) against the mu-opioid receptor (MOR) on MOR mRNA level in the periaqueductal gray (PAG) of rat brain was investigated. The MOR mRNA levels at 3, 6, 12, 24, 48 and 72 h after MOR AS ODN microinjection into the PAG were determined by reverse transcriptase-polymerase chain reaction. The MOR mRNA level was significantly decreased only at 12 h after the injection of 10 microg MOR AS ODN. When 10 microg MOR AS ODN was given three times at the interval of 48 h, MOR mRNA levels were significantly decreased at 6, 12 and 24 h after the last injection of the AS ODN. However, MOR mRNA levels were not significantly changed by three injections at 48-h interval of MOR sense ODN or AS ODNs against delta- and kappa-opioid receptors, although the two latter AS ODNs significantly reduced the respective targeted mRNA levels. In conclusion, the present results show that the selective decrease in MOR mRNA is at least one reason why the reported diminished effects of MOR agonists are produced in animals pretreated with MOR AS ODN, although they could be produced through several mechanisms in which MOR mRNA level does not change.

The immunological barrier remains the major obstacle to the widespread use of transplantation as a replacement therapy for terminal organ failure. Since the first successful renal transplant, performed by Hume et al. (1952), there has been an elusive search for agents that can render the immune mechanism unresponsive to the specific alloantigen stimulus of the engrafted organ, while sparing non-specific host resistance. Immunosuppressive therapies in organ transplantation can be divided into the following four main classes: chemical (pharmaceutical), biological (immunological), physical (radiological) and surgical. Of these, chemical agents (drugs) have continued to play a principal role. The discovery of new immunosuppressive drugs such as corticosteroids, ciclosporin, azathioprine and FK506 have been epoch-making discoveries at each stage in the history of clinical organ transplantation. The recent immunosuppressants were designed to focus their action selectively on T and/or B cells by inhibiting cytokine synthesis (ciclosporin, FK506), cytokine action (rapamycin), or cell differentiation (15-deoxyspergualin) pathways, rather than to act on immune systems in a non-selective fashion. At the present time, however, there is no single panacea. To achieve the maximum preventive and therapeutic effects with the minimum toxicity, two or more immunosuppressive drugs are used appropriately in combination, taking the mechanisms of action of each into consideration.

E4177, 3-[(2'-carboxybiphenyl-4-yl)methyl]-2-cyclopropyl-7-methyl-3H- imidazo[4,5-b]pyridine, was characterized by in vitro autoradiography and by examining functional antagonism upon angiotensin II (Ang II)-induced contraction of isolated vessels. In rat adrenal cortex and liver, E4177 competitively inhibited the specific binding of 125I-[Sar1,Ile8]Ang II, with IC50 being (5.2 +/- 1.0) x 10(-8) M for the adrenal cortex and (1.2 +/- 0.3) x 10(-7) M for the liver. These IC50 values were similar to those for losartan, which showed an IC50 of (6.0 +/- 0.9) x 10(-8) M for the adrenal cortex and (1.3 +/- 0.5) x 10(-7) M for the liver. In contrast, E4177 and losartan had little effect on the binding to rat adrenal medulla where AT2-receptors predominate. These results indicate that E4177 is AT1-specific as is losartan. E4177 and losartan competitively antagonized the Ang II-induced contraction of human and rabbit arterial strips without any agonistic action. The obtained IC50 values indicated that E4177 was twice as potent as losartan in human arteries and three times more so in rabbit aortic strips. Responses to norepinephrine, serotonin, histamine or KCl were not affected by E4177. In addition, E4177 (10(-5) M) had no effect on angiotensin-converting enzyme activity. These data indicate that E4177 is a potent AT1 Ang II-receptor antagonist that may be clinically useful for the treatment of cardiovascular diseases such as hypertension.

Using isolated arteries, we demonstrated a marked difference in the angiotensin II-forming systems between human and rodent vessels. In human arteries, only 30-40% of the conversion of angiotensin I to angiotensin II depended on the angiotensin-converting enzyme (ACE), and the rest of the angiotensin II formation was ascribed to chymostatin-sensitive angiotensin II-generating enzyme (CAGE). On the contrary, angiotensin II formation in rodent arteries totally depended upon ACE, without any sign of CAGE involvement. Such a marked species-difference can be relevant to the reported difference between humans and rodents in the ACE inhibitor effects on the myointimal hyperplasia after intimal balloon injury.

The effect of Ba2+ on the catecholamine biosynthetic activity was studied by measuring the formation of [14C]catecholamines from L-[14C]tyrosine in cultured adrenal chromaffin cells. In the absence of Ca2+, [14C]catecholamine formation was markedly stimulated by Ba2+, and this stimulation was observed in a manner dependent on its concentration. The stimulation of [14C]catecholamine formation by relatively low concentrations of Ba2+ was suppressed by polymyxin B, a typical inhibitor of Ca2+/phospholipid-dependent protein kinase (protein kinase C); and this inhibitory action of polymyxin B was attenuated by increasing the Ba2+ concentration. On the other hand, a tendency toward the enhancement of Ba2+-stimulated [14C]catecholamine formation was observed by a protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (TPA). In contrast to the acute effect of TPA, [14C]catecholamine formation stimulated by Ba2+ was reduced by long-term exposure of chromaffin cells to a high concentration of TPA, which has already been reported to cause the reduction of protein kinase C activity as a result of the down-regulation of this enzyme. These findings suggest that Ba2+ stimulates catecholamine biosynthesis, probably through its direct action on protein kinase C in adrenal chromaffin cells.

The intra-third-ventricular (i.t.v.) administration of [Met5]-enkephalin (enk) to rats pretreated i.t.v. with three peptidase inhibitors (PIs), amastatin, captopril and phosphoramidon, inhibited the tail-flick response. The enk-induced inhibition was augmented by increasing the doses of the three PIs, with the maximum inhibition being attained at the doses of 10 nmol each. The enk-induced inhibition in rats pretreated with any combination of two PIs, however, were markedly smaller than that in rats pretreated with all three PIs, indicating that three kinds of enzymes all played important roles in the inactivation of enk. The inhibitory effect of enk on the tail-flick response in rats pretreated with the three PIs at doses of 10 nmol each was approximately tenfold higher than that of morphine. The relative anti-nociceptive potencies of enk and morphine were similar to the relative inhibitory potencies obtained previously with the isolated guinea pig ileum pretreated with the three PIs, indicating that the hydrolysis of the i.t.v. administered enk was largely prevented by the three PIs. However, the magnitude of the enk-induced inhibition in rats pretreated s.c. with the three PIs indicated that the hydrolysis of enk injected i.t.v. was not largely prevented by the s.c. administration of three PIs at doses up to 10 micromol each/kg.

The antinephritic effects of ONO-1301 ([7,8-dihydro-5-[(E)-[[a-(3-pyridyl)benzylidene]-aminooxy]ethyl]-1 -naphtyloxy]acetic acid) on crescentic-type anti-glomerular basement membrane (GBM) nephritis in rats were investigated. ONO-1301 was orally given to crescentic-type anti-GBM nephritic rats for 40 days after the induction of nephritis. ONO-1301 (30 mg/kg) suppressed the elevation of protein excretion into urine. In the ONO-1301-treated rats, cholesterol and urea nitrogen content in the plasma was lower than that of the nephritic control rats. Histological observation demonstrated that ONO-1301 suppressed the incidence of crescent formation and adhesion of capillary wall to Bowman's capsule. However, ONO-1301 failed to inhibit the antibody production against rabbit IgG and the rat-IgG deposition on the GBM. The increase in very late antigen-4 (CD49b, VLA-4)-positive cells in nephritic glomeruli was significantly reduced by ONO-1301 treatment on day 5. cAMP-elevating agents inhibited the up-regulation of vascular cell adhesion molecule-1 (VCAM-1) expression on the surface of human umbilical vein endothelial cells (HUVECs) mediated by tumor necrosis factor (TNF)-alpha. These findings suggest that the antinephritic action of ONO-1301 is due to, at least in part, inhibition of intraglomerular accumulation of leukocytes through the prevention of the up-regulation of VCAM-1.

In cultured bovine adrenal chromaffin cells, pituitary adenylate cylase-activating polypeptide (PACAP) stimulated [ 14 C]catecholamine synthesis from [ 14 C]tyrosine (but not from [ 14 C]DOPA) in a concentration-dependent manner, causing maximal stimulation at 10 -7 M. The stimulatory action of PACAP was not affected by staurosporine (an inhibitor of protein kinase C) or in the cells in which protein kinase C was down-regulated by prolonged exposure to TPA (an activator of protein kinase C), whereas it was partially attenuated in Ca 2+ -free medium. PACAP (10 -7 M) increased the formation of [ 3 H]inositol phosphates, [Ca 2+ ] i and 45 Ca 2+ uptake as well as cAMP. The peptide also stimulated the phosphorylation of tyrosine hydroxylase, the enzyme catalyzing the rate-limiting step in catecholamine synthesis. Catecholamine synthesis and tyrosine hydroxylase phosphorylation stimulated by the maximal effective concentration of dibutyryl cAMP or high K + , which activates Ca 2+ uptake, were further enhanced by PACAP, suggesting that both cAMP- and Ca 2+ -dependent protein kinases may be involved in the stimulation of tyrosine hydroxylase phosphorylation and catecholamine synthesis caused by PACAP

The stimulatory actions of veratridine (VTD) on catecholamine secretion and Na+ influx in cultured bovine adrenal chromaffin cells were studied in the presence of high concentrations of Ca2+ in the incubation mixture. Catecholamine secretion evoked by VTD was reduced by elevating the external Ca2+ concentration to higher than 2 mM. Under the same conditions, VTD-stimulated 22Na+ uptake into the cells was also reduced by elevating the extracellular Ca2+ concentration. In contrast, the secretory action of VTD was not significantly suppressed by preloading Ca2+ to the cells. Furthermore, the effects of extracellular Ca2+ on the sensitivities of the cells to VTD and tetrodotoxin (TTX) were examined, and neither their sensitivities to VTD nor those to TTX were shown to be significantly altered by elevating the Ca2+ concentration in the incubation mixture. These results seem to indicate that the elevation of extracellular Ca2+ concentration may cause the inhibition of VTD-induced catecholamine secretion as a consequence of the inactivation of voltage-dependent Na+ channels, and suggest that Ca2+ may directly act on the cell surface, and the site of Ca2+ action is presumably distinct from the sites of both VTD and TTX actions in the plasma membranes of adrenal chromaffin cells.

E4177, 3-[(2'-carboxybiphenyl-4-yl)methyl]-2-cyclopropyl-7-methyl-3H- imidazo[4,5-b]pyridine, was characterized by in vitro autoradiography and by examining functional antagonism upon angiotensin II (Ang II)-induced contraction of isolated vessels. In rat adrenal cortex and liver, E4177 competitively inhibited the specific binding of 125I-[Sar1,Ile8]Ang II, with IC50 being (5.2 +/- 1.0) x 10(-8) M for the adrenal cortex and (1.2 +/- 0.3) x 10(-7) M for the liver. These IC50 values were similar to those for losartan, which showed an IC50 of (6.0 +/- 0.9) x 10(-8) M for the adrenal cortex and (1.3 +/- 0.5) x 10(-7) M for the liver. In contrast, E4177 and losartan had little effect on the binding to rat adrenal medulla where AT2-receptors predominate. These results indicate that E4177 is AT1-specific as is losartan. E4177 and losartan competitively antagonized the Ang II-induced contraction of human and rabbit arterial strips without any agonistic action. The obtained IC50 values indicated that E4177 was twice as potent as losartan in human arteries and three times more so in rabbit aortic strips. Responses to norepinephrine, serotonin, histamine or KCl were not affected by E4177. In addition, E4177 (10(-5) M) had no effect on angiotensin-converting enzyme activity. These data indicate that E4177 is a potent AT1 Ang II-receptor antagonist that may be clinically useful for the treatment of cardiovascular diseases such as hypertension.

Using isolated arteries, we demonstrated a marked difference in the angiotensin II-forming systems between human and rodent vessels. In human arteries, only 30-40% of the conversion of angiotensin I to angiotensin II depended on the angiotensin-converting enzyme (ACE), and the rest of the angiotensin II formation was ascribed to chymostatin-sensitive angiotensin II-generating enzyme (CAGE). On the contrary, angiotensin II formation in rodent arteries totally depended upon ACE, without any sign of CAGE involvement. Such a marked species-difference can be relevant to the reported difference between humans and rodents in the ACE inhibitor effects on the myointimal hyperplasia after intimal balloon injury.

The effect of idebenone, an agent improving cerebral metabolism, on catecholamine secretion was examined using primary cultures of bovine adrenal chromaffin cells. Catecholamine secretion evoked by acetylcholine was markedly inhibited by idebenone, and this effect was concentration-dependent. In contrast, other cerebral metabolism-improving agents, such as hopantenate and propentofylline, failed to cause any significant effect on the secretion in the same concentration range. Furthermore, idebenone inhibited the secretion evoked by high K+, veratridine, and Ba2+, but failed to inhibit the secretion evoked by the Ca(2+)-ionophore A23187. Idebenone also inhibited the radioactive Ca2+ uptake stimulated by acetylcholine or high K+ under the conditions in which its inhibitory action on the secretion was observed. Nifedipine, a typical voltage-dependent Ca2+ channel blocker, inhibited the secretion evoked by high K+, and this inhibitory action on the secretion was markedly reduced by the presence of idebenone. The present results suggest that idebenone may inhibit the influx of extracellular Ca2+ into the cells presumably through its blocking action on the voltage-dependent Ca2+ channels, resulting in the inhibition of catecholamine secretion in the adrenal chromaffin cell.

The stimulatory action of Ba2+ on catecholamine secretion from cultured bovine adrenal chromaffin cells was studied to elucidate a possible relationship between Ba2+ action and the Ca2(+)-mediated secretory mechanism. Catecholamine secretion was dramatically stimulated by Ba2+ in the absence of external Ca2+, and this stimulatory action was observed in a concentration-dependent manner. Ba2+ evoked the concomitant release of dopamine beta-hydroxylase in a similar manner to the Ca2(+)-dependent secretion. The stimulation of catecholamine secretion by low concentrations of Ba2+ was markedly inhibited by protein kinase inhibitors, polymyxin B and trifluoperazine (TFP). The inhibitory action of polymyxin B, but not that of TFP, on the Ba2+ action was attenuated by elevating the concentration of Ba2+ in the incubation mixture. The stimulatory action of Ba2+ was enhanced by a protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (TPA). In contrast to the acute effect of TPA, chronic exposure of chromaffin cells to high concentration of TPA reduced catecholamine secretion stimulated by Ba2+ as well as high K+ and carbamylcholine. These findings suggest the possibility that Ba2+ may activate Ca2(+)-mediated secretory processes presumably through its action on protein kinase C, thus resulting in the stimulation of catecholamine secretion from bovine adrenal chromaffin cells.