Diverse attention should be paid to evaluating NOx (NO2- and NO3-) in plasma as an index of endothelial nitric oxide (NO) formation in vivo. Nitric oxide, which subsequently appears as NOx, originates from different types of NO synthase and from nonenzymatic reactions. NOx also comes from exogenous sources such as food and gastrointestinal microorganisms. The fate of the NO incorporated into activation of guanylate cyclase, formation of nitrosyl hemoglobin (or nitrosohemoglobin), nitrosothiols, peroxynitrite and its derivatives and other possible compounds is not clear at present. However, some of these compounds would produce NOx as by-products or as final products through metabolism. Therefore, plasma NOx contains information about these pathways, although how extensively these factors contribute to plasma NOx has not been quantitatively defined. A theoretical simulation of NOx in the systemic circulation indicates that only small changes are expected by inhibition or stimulation of endothelial NO production. Measuring NOx production during coronary circulation has the advantage that some degree of NOx accumulation is expected from intact endothelial cells because an excretion system is absent in the heart.
The activity of voltage-dependent Ca2+ channels is highly regulated by neurotransmitter receptors coupled to heterotrimeric G-proteins. In the expression studies using cloned Ca2+ channel subunits, it has been clarified that the main mechanism of the inhibition of N-type channel current is mediated directly by G-protein βγ subunits in a membrane-delimited and voltage-sensitive manner. In addition, recent studies have also clarified that N-type channels are modulated by several G-protein α subunits in different ways. Among them, Gαo mediates a voltage-resistant inhibition of N-type current by neurotransmitters. This type of inhibition is more apparent in the case of P/Q-type channels in both native cells and expression systems. Moreover, other G-protein subunits, such as Gαq and Gαs, also seem to regulate N-type channels in a membrane-delimited manner. The fine tunings of Ca2+ channel activity by intracellular proteins have physiological and pathological meanings in the regulation of Ca2+ influx into excitable cells by neurotransmitters and pharmacological implications as novel drug targets for controlling Ca2+ influx.
The place preferences by some histamine H1 antagonists, such as tripelennamine, optical isomers of chlorpheniramine (dl-, d- and l-forms) and pyrilamine, in rats were evaluated with the conditioned place preference paradigm. In the present study, tripelennamine and all of the optical isomers of chlorpheniramine, but not pyrilamine, produced a significant place preference. The degree of the place preference induced by optical isomers of chlorpheniramine (6.0 mg/kg) did not correlate with the H1-antagonistic potency of these drugs, suggesting that H1-antagonist-induced place preferences are not mediated by H1-receptor blockade. The tripelennamine (3.0 mg/kg)- and dl-chlorpheniramine (6.0 mg/kg)-induced place preferences were completely abolished by pretreatment with the dopamine D1-receptor antagonist SCH23390 (0.05 mg/kg). Furthermore, the doses of H1 antagonists that induced a place preference significantly reduced the levels of DOPAC, which may be mediated by inhibition of dopamine uptake, in the limbic forebrain (including the nucleus accumbens and olfactory tubercle). These results suggest that some H1 antagonists induce rewarding effects, which may be mediated by the activation of dopamine D1 receptors, followed by the inhibition of dopamine uptake.
The effects of oral administration of two synthetic trypsin inhibitors (camostate and ONO-3403) and soybean trypsin inhibitor (SBTI) on cholecystokinin (CCK), secretin gene expression and pancreatic secretion were examined in CCK-A-receptor-deficient (OLETF) rats. The rats were fed chow containing 0.1% trypsin inhibitors for 7 days. To examine pancreatic secretion, the rats were prepared with cannulae to drain the bile and pancreatic juice separately, a duodenal cannula and an external jugular vein cannula. The animals were maintained in Bollman cages and the experiments were conducted 4 days after surgery. The levels of CCK mRNA were significantly increased by each treatment. The levels of secretin mRNA were significantly increased by camostate and SBTI, but not by ONO-3403. Bicarbonate secretion was significantly increased in rats treated with camostate and ONO-3403, but not SBTI, while protein secretion was not affected by any treatment. These observations suggest that increased bicarbonate secretion produced by synthetic trypsin inhibitors in CCK-A-receptor-deficient rats may not be due to secretin but due to ONO-3403 in the circulation.
The background for these investigations was the discovery that formation of angiotensin II by the renin angiotensin system can take place in extravascular tissues (e.g., cardiomyocytes and neurons) and within single cells. Consequently, the question arose about whether such tissue-based systems might be differentially influenced by angiotensin I-converting enzyme (ACE) inhibitors with distinct physicochemical properties. Therefore, the aim of this study was to investigate how the membrane penetration of various ACE inhibitors depends on their lipophilia. All diacid forms of ACE inhibitors are dissociated at a pH of 7.4 and scarcely extractable into octanol (extraction coefficient <10%). In contrast, the extraction coefficients of the parent substances showed marked differences in the following order of increasing lipophilia: enalapril=perindopril<captopril=ceranapril<ramipril<quinapril<HOE288=zofenopril<fosinopril<HOE065. For selected substances, the kinetics of diffusion through a monolayer of cultured bovine aortic endothelium were determined. The diffusion rates (expressed as half lives) of captopril (59.6 min), enalapril (53.4 min), enalaprilat (50.8 min), ramipril (56.9 min) and ramiprilat (51.1 min) are similar indicating: 1) that penetration is independent on lipophilia and 2) that endothelium constitutes no specific barrier for the passage of ACE inhibitors into the vessel wall.
Chronic and/or sustained opioid treatment has been shown to result in development of sensitization of the adenylyl cyclase (AC) system or cAMP overshoot. In this study, we investigated the molecular mechanism responsible for sensitization of the AC system using CHO cells co-expressing cloned κ-opioid receptor and some chimeric G protein αi2/αq subunits. In CHO cells co-expressing the κ-opioid receptor and pertussis toxin-insensitive chimeric αi2/αq subunits with αi2 residues Met244 - Asn331, despite pretreatment with pertussis toxin, acute treatment with the κ-opioid-receptor - selective agonist U69, 593 suppressed forskolin-stimulated cAMP accumulation, while sustained treatment with U69, 593 (4 h) induced cAMP overshoot over the naive level by the κ-opioid-receptor - selective antagonist norbinaltorphimine (sensitization of the AC system). On the other hand, in CHO cells co-expressing the κ-opioid receptor and pertussis toxin-insensitive chimeric αi2/αq subunits without αi2 residues Met244 - Asn331, pretreatment with pertussis toxin completely blocked these acute and sustained effects of U69, 593 on cAMP accumulation. These results suggested that the presence of the specific region of αi2 (Met244 - Asn331), which was reported to be responsible for the inhibition of AC, and continuous inhibition of AC by αi2 is necessary for the development of sensitization.
The present study was designed to examine the role of basally released nitric oxide in relaxations to an ATP-sensitive K+ channel opener. Whether relaxations to levcromakalim are modulated by endothelial removal or the inhibitors of vasodilator effects of endothelium-derived nitric oxide, were investigated in the rat aorta. During contractions to phenylephrine (3×10-7 to 10-6 M), levcromakalim (10-8 to 10-5 M) or a nitric oxide donor, 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC-7, 10-9 to 10-5 M), was added in a cumulative fashion. Relaxations to levcromakalim (10-8 to 10-5 M) were significantly reduced by the endothelium-removal. In aortas with endothelium, relaxations in response to levcromakalim were decreased by selective inhibitors of nitric oxide synthase (NG-nitro-L-arginine methyl ester, 10-4 M) and soluble guanylate cyclase (1H-[1, 2, 4]oxadiazolo[4, 3-a]quinoxaline-1-one; ODQ, 10-5 M) and a scavenger of nitric oxide (carboxy-PTIO, 10-3 M). Relaxations to levcromakalim in aortas treated with these inhibitors are comparable to those seen in aortas without endothelium. KCl (30 mM) and an ATP-sensitive K+ channel inhibitor, glibenclamide (10-5 M), abolished relaxations to levcromakalim in aortas with or without endothelium, whereas glibenclamide did not alter relaxations to NOC-7 (10-9 to 10-5 M) in aortas without endothelium. These results suggest that in rat aortas, inhibition of vasodilator effects of basally released nitric oxide can reduce relaxations via ATP-sensitive K+ channels, although these channels do not mediate relaxations to exogenously applied nitric oxide.
We investigated the antinociceptive effect of systemic injection of calcitonin and its mechanisms of action in rats. Subcutaneous injection of [Asu1, 7]eel calcitonin (ECT, 4 U·kg-1·day-1) daily for 7 days suppressed nociceptive hypersensitivity induced by formalin (and by carrageenan); the effect was gradually increased by the repeated injections and significant effects were observed after administration for more than 4 days. The antinociceptive action of ECT (4 U·kg-1·day-1 for 7 days) was inhibited by intracerebroventricular injection of the serotonergic neurotoxin 5, 7-dihydroxytryptamine and serotoninreceptor antagonists methiothepin, cyproheptadine and ketanserin; methysergide showed an inhibitory tendency. Intrathecal injections of 5, 7-dihydroxytryptamine, methiothepin, cyproheptadine and ketanserin were without effects on the ECT action. The results suggest the involvement of serotonin in the brain, but not in the spinal cord, in the ECT antinociception. Intracerebroventricular or intrathecal injection of the catecholaminergic neurotoxin 6-hydroxydopamine and intracerebroventricular injection of the α-adrenoceptor antagonist phentolamine were also without effects on the ECT action. A subcutaneous infusion of the opioid receptor antagonist naloxone inhibited the antinociceptive action of morphine, but not that of ECT. Thus, adrenergic and opioidergic systems may not play important roles in the ECT antinociception. The present results suggest that repeated systemic injection of ECT produces analgesia and that the brain serotonergic terminals are involved in this action.
Electrical field stimulation (EFS) caused contraction of isolated tail arteries of rats. The EFS-induced contraction showed frequency-dependence and was entirely abolished by the sodium channel blocker tetrodotoxin (1×10-7 M). The EFS-induced (at 20 Hz) contraction was reduced by about 60% in the presence of phentolamine (1×10-6 M). Therefore, later experiments were carried out in the presence of phentolamine. Pyridoxal-phosphate-6-azophenyl-2′, 4′-disulphonic acid (PPADS) (1×10-8 - 1×10-6 M) and basilen blue E-3G (3×10-5 - 5×10-5 M), P2-receptor antagonists, significantly inhibited the contraction evoked by EFS. In addition, PPADS significantly inhibited the contractions induced by ATP (1×10-4 M) and a selective P2x-receptor agonist, α, β-methylene ATP (1×10-6 M). In contrast, basilen blue E-3G did not inhibit α, β-methylene ATP-induced contraction. The ecto-ATPase activator apyrase (5 and 10 U/ml) significantly reduced the EFS-induced contractions. These findings suggest that endogenous ATP released by EFS causes contractions of rat tail artery via both the P2x-receptors and P2y-receptors.
Using distention of the small intestine as a visceral pain model, we investigated the effect of zaldaride maleate (ZAL), a selective inhibitor of calmodulin, on the depressor response. In pentobarbital-anesthetized rats, small intestine distention was induced by rapid application of intraluminal pressures of 40 cmH2O causing a reflex fall in arterial blood pressure. The depressor response to intestinal distention was abolished by intraperitoneal administration of capsaicin (5 mg/rat), which depletes neuropeptides such as substance P from the sensory neurons, on the mesenteric stalk and by neonatal pretreatment with capsaicin (50 mg/kg, s.c.). Morphine (20 mg/kg, s.c.) reduced the depressor response following intestinal distention. At doses of 3 mg/kg (i.v.) and higher, ZAL significantly reduced depressor response. The effect of morphine was reversed by naloxone (5 mg/kg, i.v.); the effect of ZAL was not affected. These results suggest that ZAL helps reduce the visceral pain induced by noxious stimulus and that the antinociceptive effect of ZAL is not mediated by opioid receptors.
The structural organization and 5′-flanking region of the mouse V1a and V1b vasopressin receptor genes were determined. The mouse V1a receptor gene was located within an 8-kb XbaI fragment, and the mouse V1b receptor gene was located within a 14-kb EcoRV fragment. Both genes were comprised of two coding exons that were separated by a 2.3-kb and a 8.0-kb intron, respectively, located before the respective seventh transmembrane domain of the receptor sequence. The availability of these genes would allow us to study the functional role of V1a and V1b receptors by disrupting the gene in mice.
The differential display method was used to identify the intrinsic factor that changes its mRNA expression level in rat brain after a 14-day oral administration of 20 mg/kg imipramine. The expression of a 180-bp band was markedly enhanced by imipramine. The results of sequencing and a data base search revealed that the isolated clone was glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with a one-base difference. Enhancement of the expression by imipramine was observed in the amygdala. Quantitative PCR showed that imipramine treatment significantly elevated the GAPDH/β-actin ratio in the cortex. These findings suggest that long-term treatment with imipramine stimulates GAPDH mRNA expression.
The antidiarrheal action of zaldaride maleate (ZAL) after oral, intravenous and subcutaneous administration was examined to determine whether ZAL acts systemically or locally in the intestine of rats. Oral administration of ZAL inhibited castor oil- and 16, 16-dimethyl prostaglandin E2-induced diarrhea; however, intravenous or subcutaneous administration of ZAL was ineffective. When ZAL was orally administered, the area under the plasma concentration time curve of the compound was lower than that of ZAL following intravenous or subcutaneous administration at the maximum doses studied. The antidiarrheal effect of ZAL was not dependent on its plasma concentration level. These results suggest that ZAL acts locally in the intestinal tract in rats.
We examined the effect of benidipine, a 1, 4-dihydropyridine calcium channel blocker, on depolarizing stimulation-induced increases of intracellular calcium concentration ([Ca2+]i) in cultured mouse hippocampal neurons in comparison with those of nicardipine and nilvadipine. Benidipine (0.1 - 10 μM) inhibited the [Ca2+]i increase compared with the no drug control response. This effect was stronger than those of nicardipine and nilvadipine. The inhibitory effect of benidipine lasted even after washing out the drug for 125 min, while those of nicardipine and nilvadipine disappeared more rapidly. This is the first report that demonstrates that benidipine inhibits the [Ca2+]i increase in the neuron itself.
Chymase is a chymotrypsin-like protease localized in mast cells in complexes with heparin. In the present study, we demonstrated that suramin, a hexasulfonated naphthylurea used as an anti-cancer drug, inhibits the activity of purified human chymase in vitro. The inhibition was ionic-strength-dependent. It was observed that suramin competed with heparin-Sepharose gel for binding to chymase and the inhibition of chymase activity by suramin was partially impaired by heparin. Our results show that suramin may become a prototype of a new type of chymase inhibitor because of its unique character.