The benefit of organ culture is to retain the original structural relationship between various cell species and their interactions and enable us to study the long-term effects of exogenous stimuli. Organ culture methods have been used especially in the studies of the proliferative vascular diseases, such as atherosclerosis and restenosis. We describe here that organ culture is a useful in vitro method to study the biology of vascular and other smooth muscle organs.
Erectile dysfunction (ED) is a common problem with a prevalence of approximately 50% in men aged 40 to 70. There are several etiologies for ED including vasculogenic, neurogenic, hormonal and /or psychogenic factors; one-fourth of ED cases can be drug-related. Penile erection involves a complex interaction between the CNS and local factors. It is a neurovascular event modulated by psychological and hormonal factors. Pharmacologically, neural modulation and endocrine status are very important to attaining penile erection. There have been several significant advances for the pharmacologic treatment of ED. Treatments include agents that are not only orally effective, but possess either local or central acting mechanisms of action. Apomorphine, a centrally-acting agent, is effective in the treatment of ED. Sildenafil, another orally effective agent, acts by inhibiting cyclic GMP-specific phosphodiesterase Type V. Testosterone can be effective transdermally. Non-orally active agents include alprostadil and papaverine. Phentolamine and trazodone are effective in selected cases. Some agents can interact with other medications. Several pharmacological agents, some with central-acting mechanisms and some with locally-acting vascular effects, are therapeutically useful in the treatment of ED.
Ipecac syrup, prepared from a galentical ipecac, contains the nauseant alkaloids cephaeline and emetine. The involvement of receptors and serotonin- and dopamine-metabolizing enzymes in the emesis induced by ipecac syrup and these components was investigated. 1) In ferrets, the selective 5-HT3-receptor antagonist ondansetron (0.5 mg/kg, p.o.) prevented each emesis induced by TJN-119 (0.5 mL/kg, p.o.), cephaeline (0.5 mg/kg, p.o.) and emetine (5.0 mg/kg, p.o.), but the intraperitoneal administration of the selective dopamine D2-receptor antagonist sulpiride failed to significantly suppress the TJN-119, cephaeline and emetine-induced emesis at a dose of 0.1 mg/kg that blocked apomorphine-induced emesis. 2) In the receptor binding assays, cephaeline and emetine had a distinct affinity to 5-HT4 receptor, but no or weak affinity to 5-HT1A, 5-HT3, nicotine, M3, β1, NK1 and D2 receptors. 3) Cephaeline and emetine did not affect activities of metabolic enzymes of 5-HT and dopamine (MAO-A, MAO-B, tryptophan 5-hydroxylase and tyrosine hydroxylase) in vitro. These results suggest that 5-HT3 receptor plays an important role in the emetic action of TJN-119, cephaeline and emetine, and the 5-HT4 receptor may be involved in their mechanisms.
The presence of nonischemic regional dysfunction at the adjacent region of the ischemic myocardium was demonstrated in clinical studies. Recent studies demonstrated an angiotensin II type 1 (AT1)-receptor antagonist reduced myocardial ischemia-reperfusion injury. We investigated the role of the adjacent region after reperfusion by studying the effects of AT1-receptor antagonist on myocardial function and infarct size. We investigated 12 open-chest anesthetized dogs undergoing 90 min of left anterior descending coronary artery occlusion followed by 4 h of reperfusion. Six dogs injected with an AT1-receptor antagonist (CV11974) immediately after reperfusion were compared with 6 control dogs. Percent systolic shortening (%SS) was measured by two sets of the pair sonomicrometer crystals implanted to adjacent and remote nonischemic myocardium. After 4 h of reperfusion, infarct size was measured. There were no significant differences of the %SS at baseline between two regions. In both groups, %SS at adjacent region after reperfusion was significantly decreased as compared with remote region. There were no significant differences between the two groups. Infarct size, as a percentage of the area at risk, was smaller in the AT1 group than in control group (25.49 ± 7.53% vs 68.58 ± 26.88% P<0.01). AT1-receptor antagonist reduces infarct size. This effect is not related to the change of regional myocardial function at adjacent region after reperfusion.
The effects of centrally injected nitric oxide (NO) donors on gastric acid secretion were investigated in continuously perfused stomach of anesthetized rats. The lateral cerebroventricular (LV) injection of NOC5 (30 – 100 μg) and NOC12 (10 – 100 μg) dose-dependently stimulated gastric acid secretion. The LV injection of NOC18 (30 μg) also stimulated gastric acid secretion. The other type of NO donor, sodium nitroprusside (3 – 30 μg, LV), also dose-dependently stimulated gastric acid secretion. The effect of NOC5 at 100 μg was blocked by carboxy-PTIO, an NO scavenger, and by cervical vagotomy. Furthermore, NOC12 (30, 100 μg) dose-dependently stimulated gastric acid secretion in pylorus-ligated conscious rats. These results suggest that centrally injected NO donors stimulate gastric acid secretion in both conscious and anesthetized rats through vagus activation.
We investigated the laxative and anti-diarrheal activity of polycarbophil, an insoluble hydrophilic polymer, in comparison with other agents used for treating functional bowel disorder (FBD). In naive rats, polycarbophil (500 mg/kg) increased fecal weight and water contents without producing diarrhea. Carboxymethylcellulose (CMC) did not produce evident changes in bowel movement. Picosulfate markedly produced diarrhea. Loperamide, trimebutine and granisetron decreased stool output dose-dependently. Constipation, indicated by decrease in fecal weight, was produced by loperamide and clonidine in rats. Polycarbophil (500 mg/kg) and CMC increased fecal weight without diarrhea. Conversely trimebutine further decreased fecal weight in constipated rats. Polycarbophil (500 mg/kg) suppressed diarrhea induced by castor oil, and at 250 – 500 mg/kg, it produced shaped stools in animals with stools loosened by prostaglandin E 2, serotonin or carbachol in mice. Polycarbophil (500 mg/kg) also reduced stools in rats with stool output increased by wrap restraint stress (WRS). CMC had no effect in the diarrhea models, except for carbachol-induced diarrhea, and WRS-induced evacuation. Loperamide, trimebutine and granisetron inhibited diarrhea production and WRS-induced evacuation, except for carbachol-induced diarrhea. The results show that polycarbophil prevents constipation and diarrhea without inducing diarrhea or constipation, which is different from the other agents. Hydrophilic polymers such as polycarbophil will be promising agents for the treatment of FBD.
The antioxidative and hypolipidemic effects of barley leaf essence (BL) were investigated in a rabbit model of atherosclerosis. Twenty-four New Zealand White male rabbits were assigned randomly into four dietary groups. The normal group was fed regular rabbit chow and the control group was fed a chow containing 0.5% cholesterol and 10% corn oil. The BL group and the probucol group were fed the same diet as the control group plus 1% (w/w) BL or 1% (w/w) probucol, respectively. The plasma levels of total cholesterol, triacylglycerol, lucigenin-chemiluminescence (CL) and luminol-CL were increased in the control group compared to the normal group; and they were decreased in the BL group and the probucol group compared to the control group. The value of T50 of red blood cell hemolysis and the lag phase of low-density lipoprotein oxidation increased in the BL group and in the probucol group compared to the controls. Ninety percent of the intimal surface of the thoracic aorta was covered with atherosclerotic lesions in the control group, but only 60% of the surface was covered in the BL group. This 30% inhibition of hyperlipidemic atherosclerosis by BL was associated with a decrease in plasma lipids and an increase in antioxidative abilities (as measured by T50, lag phase and CL). These results suggest that the antioxidant and hypolipidemic effects of BL could be useful in the prevention of cardiovascular disease in which atherosclerosis is important.
A novel murine model of aging (kl/kl mice) has been developed by in vivo mutagenesis. We analyzed endothelial function in this strain. Ring preparations of the thoracic aorta were obtained from 6- to 9-week old wild-type (+/+) and heterozygous (kl/+) klotho mice. The aortas of kl/+ mice showed an exaggerated contractile response to norepinephrine and attenuated vasodilator responses to acetylcholine and lecithinized superoxide dismutase (SOD) compared to +/+ mice. The response to sodium nitroprusside was unaltered in kl/+ mice. The contraction in response to norepinephrine was augmented by treatment with NG-nitro-L-arginine methyl ester (L-NAME, 10−5 M) to a greater extent in +/+ mice than in kl/+ mice. Treatment with L-NAME abolished the vasodilator responses to both acetylcholine and lecithinized SOD. NO metabolites (NO2− and NO3−) and cGMP concentrations in the urine were significantly reduced in kl/+ mice compared to +/+ mice. However, the urinary excretion of 6-keto-prostaglandin F1α was unaltered. There was little immunostaining for NO synthase and vascular endothelial growth factor (VEGF) in the aorta of kl/+ mice. No immunostaining for NO synthase was noted in the aorta of kl/kl mice. The expression of the klotho gene product may have a role in the regulation of VEGF expression and is tightly linked to endothelial release of NO.
Overproduction of nitric oxide (NO) from inducible nitric oxide synthase (iNOS) is importantly involved in the pathogenesis of endotoxemia and atherosclerosis. Calcium antagonists are commonly used as cardiovascular drugs and have a beneficial effect on prolonging survival in various models of endotoxin shock. The present study was to investigate the effect of a calcium antagonist amlodipine on nitrite, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) formation and iNOS induction both in lipopolysaccharide (LPS) and interferon-γ (IFN-γ)-treated rat aortic smooth muscle cells (RASMC) and in a rat model of endotoxemia. Incubation with amlodipine (0.1 – 10 μM) for 24 h resulted in a significant and dose-dependent attenuation in medium nitrite, TNF-α and IL-1β formation as well as iNOS protein expression in LPS/IFN-γ-treated RASMC. In addition, amlodipine inhibited leucigenin-induced superoxide formation in RASMC. In the rat endotoxic model, the serum nitrite/nitrate, TNF-α and IL-1β levels as well as iNOS protein expression of lungs were also suppressed by administration of amlodipine (50 μg/kg, i.v.). These results suggest that amlodipine may exert vascular beneficial effects by suppressing pro-inflammatory cytokines and free radical generation as well as iNOS induction in smooth muscle cells during activation of inflammatory mechanism.
In the present study, the effects on drug oxidations in rat liver microsomes in vitro using 126 Kampo extracts were investigated. Although the effects of inhibition on drug oxidations were dependent on the Kampo extracts and probe reactions studied, most of the Kampo extracts showed inhibitory effects on both N-demethylations of aminopyrine and erythromycin in rat liver microsomes. Among the Kampo extracts studied herein, Daio-kanzo-to exhibited the most remarkable inhibitory effect on both reactions. The Rhei Rhizoma extract inhibited not only aminopyrine and erythromycin N-demethylations, but also phenacetin O-deethylation, 7-ethoxycoumarin O-deethylation, ethanol oxidation and tolbutamide 4-hydroxylation in rat liver microsomes. The Glycyrrhizae Radix extract also showed a remarkable inhibitory effect on phenacetin O-deethylation as well as aminopyrine and erythromycin N-demethylations. In contrast, the Glycyrrhizae Radix extract virtually showed no effect on ethanol oxidation.
We investigated whether inhaled pinacidil and moguisteine inhibit capsaicin-induced coughs in guinea pigs. Inhaled pinacidil (15 – 60 μg/ml), an ATP-sensitive K+ channel opener, and moguisteine (15 – 60 μg/ml) each dose-dependently inhibited the number of capsaicin-induced coughs. The antitussive effects of pinacidil and moguisteine were significantly antagonized by pretreatment with glibenclamide (10 mg/kg, i.p.), an ATP-sensitive K+ channel blocker. However, pretreatment with naloxone methiodide (10 mg/kg, s.c.) had no significant effect on the antitussive effects of either pinacidil or moguisteine. On the other hand, inhaled dihydrocodeine (15 – 60 μg/ml) also dose-dependently suppressed the number of capsaicin-induced coughs. The antitussive effect of inhaled dihydrocodeine was significantly antagonized by pretreatment with naloxone methiodide (10 mg/kg, s.c.), but not by glibenclamide (10 mg/kg, i.p.). These results indicate that inhaled pinacidil and moguisteine both attenuate capsaicin-induced coughs. Pinacidil and moguisteine may exert their antitussive effects through the activation of ATP-sensitive K+ channels in the tracheobronchial tract. Furthermore, it is possible that ATP-sensitive K+ channels may be involved in the antitussive effects of peripherally acting non-narcotic antitussive drugs.
The lipopolysaccharide (LPS) released by Porphyromonas gingivalis, a Gram-negative bacterium found in the periodontal pockets of patients with periodontitis, induces bone resorbing activity in vivo. We previously showed that a receptor for LPS on human gingival fibroblasts and gingival epithelial cells is CD14. In this study, we established a mouse model of experimental periodontitis by applying a P. gingivalis LPS solution to the buccal region of mice. P. gingivalis LPS-induced bone resorption and interleukin-6 production in the gingival tissues were significantly inhibited by pretreatment with anti-CD14 antibody for 5 weeks prior to LPS treatment. This result suggests that anti-CD14 antibody may be usable as a prototype for the development of drugs for the treatment of periodontal disease.
Protease-activated receptor-2 (PAR-2), expressed in sensory neurons, triggers thermal hyperalgesia, nociceptive behavior and spinal Fos expression in rats. In the present study, we examined if the nociceptive processing by PAR-2 is mediated by trans-activation of capsaicin receptors. The thermal hyperalgesia following an intraplantar (i.pl.) administration of the PAR-2-activating peptide SLIGRL-NH2 was completely abolished by the capsaicin receptor antagonist capsazepine. In contrast, neither the nociceptive behavior nor spinal Fos expression in response to i.pl. SLIGRL-NH2 were attenuated by capsazepine. Our data imply that trans-activation of capsaicin receptors by PAR-2 might be involved in the PAR-2-triggered thermal hyperalgesia, but not nociception.
Isolated dog splenic arteries were perfused with Krebs-Henseleit solution at 37°C, using the cannula inserting method. Periarterial nerve electrical stimulation (10-V amplitude; 1-ms duration; 30-s trains of pulses; 1, 4 and 10 Hz) readily caused double peaked vasoconstrictions, i.e., 1st peaked response was mostly inhibited by α,β-methylene ATP and the 2nd one, by prazosin. These responses were consistently inhibited by ω-conotoxin GVIA (ω-CTX), whereas they were facilitated by BIIE 0246, a neuropeptide Y (NPY) Y2-receptor antagonist. The ω-CTX-induced blocking effects of transmitter release were significantly antagonized by BIIE 0246. It is possible that the NPY Y2 receptor activity may partially be linked to presynaptic Ca2+ channels.
L-158,809 is a new angiotensin II type 1 receptor antagonist. We simultaneously assessed its antagonistic potency and cardiovascular effects with the halothane-anesthetized in vivo canine model (n = 5). L-158,809 was intravenously infused over 10 min at escalating doses of 0.03, 0.3 and 3 mg/kg. Angiotensin II (0.1 μg/kg, i.v.)-induced vasopressor and negative inotropic responses were significantly suppressed from the low dose L-158,809. Meanwhile, L-158,809 did not affect any of the cardiovascular parameters except that QTc was slightly shortened after the high dose administration. These results support the previous in vitro knowledge that L-158,809 is a highly selective angiotensin II receptor antagonist.
In dog pulmonary arterial and venous strips without endothelium under treatment with prazosin, nicotine induced relaxation that was abolished by NG-nitro-L-arginine, hexamethonium and methylene blue. L-Arginine antagonized the NG-nitro-L-arginine action. Neurogenic relaxations tended to be more evident in the vein. Nitric oxide (NO)-induced relaxations were greater in the veins than in the arteries. Concentrations of NO to induce the same magnitude of relaxation as that to nicotine were higher in the arteries. In conclusion, dog pulmonary arteries and veins are innervated by nitroxidergic (nitrergic) nerves, and NO is released by nerve stimulation with nicotine in a larger amount in the artery than the vein.