For the teaching and/or learning about drug actions and for the discovery and development of new drugs, it is important to understand how drugs act on living bodies. So far, there has been no clear description on the general principle of drug action in pharmacology textbooks. We propose two principles to depict the action mechanism of drugs. The first is that most, if not all, drugs act on proteins at the molecular level, that is, enzymes, receptors, ion channels, and transporters. The second is that a drug may cause divergent or convergent responses, resulting in changes of a physiological or pathological function of the human body. The concept of divergence and convergence can be used to explain the complex individuality of drug actions.
The classical methods for hypophysectomy require a long training-period and at the end of each experiment, it is required to check any undue remnant of the hypophyseal tissue in the sella turcica, and there must be exclusion of such datum with the remnant from the experimental group. The present method for functional hypophysectomy by neck-strangulation in the rat is very simple to perform so that even an experimenter with little experience can become easily accustomed to the technique and obtain a number of functionally hypophysectomized preparations at any time he intends, with no need to check the remnant. The cerebral blood circulation of vertebrally dislocated rats was immediately intercepted by means of neck-strangulation and artificially respired. Fifteen minutes after intravenous injection of ACTH (0.1 – 3.2 mU/rat) or LH (312.5 – 2500 ng/rat), blood was sampled from the vena cava, and sera were examined for corticosterone or testosterone by radioimmunoassay. A linear dose-response relationship was obtained within a dose range of 0.2 – 1.6 mU/rat of ACTH and 312.5 – 1250 ng/rat of LH. In the ACTH assay, parapharyngeally hypophysectomized rats were compared. It was found that the sensitivity of functionally hypophysectomized preparations was 2.72 times higher than that of the parapharyngeally hypophysectomized rats.
Alternative splicing of the human telomerase reverse transcriptase subunit (hTERT) suppresses telomerase activity during the development of human fetal kidney cells into mature cells. Tumor cell differentiation is the process of turning abnormal tumor cells into ‘normal’ cells accompanied by down-regulation of telomerase activity. However, the precise mechanism of the regulation of telomerase activity in differentiated cells is not fully understood. In this study, we observed the role of alternative splicing of hTERT in the regulation of telomerase activity in all-trans-retinoic acid (ATRA)-induced, differentiated HL-60 cells. ATRA-induced down-regulation of telomerase activity in differentiated HL-60 cells was associated with a decrease in hTERT and an increase in human telomerase-associated protein-1 (hTP1) transcription. Expression of full length variant hTERT α+β+ mRNA decreased in a dose- and time-dependent manner. The drop of hTERT β− mRNA was time-dependent. hTERT α− and hTERT α−β− mRNA were reduced dramatically after ATRA treatment. In the dose-effect study, hTERT α+β+ and hTERT β− maintained a relatively stable ratio when telomerase activity decreased largely from treatment with 1 to 5 μM ATRA. Although the splicing pattern of hTERT mRNA was altered in time-effect research, the change was not related to the ATRA-treated decline of telomerase activity. The expression of alternative splicing variants of hTERT also decreased at the protein level. All these results suggested that alternative splicing of hTERT mRNA may not contribute to the suppression of telomerase activity during ATRA-induced HL-60 leukemia cell differentiation.
We investigated the actions of Gosha-jinki-gan, a traditional Japanese medicine containing processed Aconiti tubers, on urinary bladder function in anesthetized rats. In cystometrical investigations, Gosha-jinki-gan (1.0 g/kg, i.d.) increased bladder capacity as well as micturition threshold pressure. In addition, it decreased the frequency of distension-induced rhythmic bladder contractions. However, it did not influence the amplitude of bladder contractions induced by electrical stimulation of the pontine micturition center. The inhibitory effect of Gosha-jinki-gan on bladder motility was abolished by pretreatment with nor-binaltorphimine (10 mg/kg, s.c.), and was diminished by the concomitant use of anti-dynorphin A antiserum (10 μg, i.t.), yohimbine (10 μg, i.t.), or methysergide (20 μg, i.t.). Processed Aconiti tuber extract (27 mg/kg, i.d.) also suppressed bladder motility, and the effect was abolished by nor-binaltorphimine. These results suggest that Gosha-jinki-gan attenuates bladder sensation via the kappa-opioid receptor-stimulating action of processed Aconiti tuber. Gosha-jinki-gan may be a useful anti-pollakiuria agent that does not influence bladder contractility at micturition.
In order to increase the tissue level of tetrahydrobiopterin (BH4), supplementation with 6R-tetrahydrobiopterin (6RBH4) has been widely employed. In this work, the effectiveness of 6RBH4 was compared with 7,8-dihydrobiopterin (7,8BH2) and sepiapterin by administration to mice. Administration of 6RBH4 was the least effective in elevating tissue BH4 levels in mice while sepiapterin was the best. In all three cases, a dihydrobiopterin surge appeared in the blood. The appearance of the dihydrobiopterin surge after BH4 treatment suggested that systemic oxidation of the administered BH4 had occurred before accumulation of BH4 in the tissues. This idea was supported by the following evidences: 1) An increase in tissue BH4 was effectively inhibited by methotrexate, an inhibitor of dihydrofolate reductase which reduces 7,8BH2 to BH4. 2) When the unnatural diastereomer 6SBH4 was administered to mice, a large proportion of the recovered BH4 was in the form of the 6R-diastereomer, suggesting that this BH4 was the product of a dihydrofolate reductase process by which 7,8BH2 converts to 6RBH4. These results indicated that the exogenous BH4 was oxidized and the resultant 7,8BH2 circulated through the tissues, and then it was incorporated by various other tissues and organs through a pathway shared by the exogenous sepiapterin and 7,8BH2 in their uptake. It was demonstrated that maintaining endogenous tetrahydrobiopterin in tissues under ordinary conditions was also largely dependent on an methotrexate-sensitive process, suggesting that cellular tetrahydrobiopterin was maintained both by de novo synthesis and by salvage of extracellular dihydrobiopterin.
Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-dependent transcription factor involved in various processes including the inflammation and carcinogenesis. The aim of the present study was 1) to examine the mRNA and protein expression of PPARγ in gastric cancer (GC); 2) to evaluate the effect of PPARγ ligand (ciglitazone) on the proliferation and apoptosis of GC cell line; and 3) to assess the levels of gastric tissue proinflammatory cytokines, IL-1β and IL-8, and plasma gastrin in GC patients before and after Helicobacter pylori (H. pylori) eradication. The trial material included 30 H. pylori-negative controls and 30 sex- and age-matched GC patients without or with H. pylori before and after its eradication. Expression of tissue PPARγ, tissue levels of IL-1β and IL-8, and plasma concentration of gastrin were significantly higher in H. pylori-positive GC compared to controls, but H. pylori eradication significantly reduced these parameters. Kato III cells incubated with alive H. pylori upregulated PPARγ expression and ciglitazone inhibited cell proliferation and induced apoptosis. PPARγ, proinflammatory cytokines and plasma gastrin appear to be implicated in H. pylori-related gastric carcinogenesis and PPARγ agonists may have potential in cancer therapy.
We investigated the efficacy of a potent inhibitor of secretory phospholipase A2 (sPLA2), S-5920/LY315920Na, in an experimental model of acute pancreatitis in rats. Combined intraductal injection of sodium taurocholate (5 mg/rat) and porcine pancreatic sPLA2-IB (300 μg/rat) caused severe hemorrhagic necrotizing pancreatitis resulting in high mortality, along with rapid increases of catalytic PLA2 and lipase activities in plasma and ascites and with gradual increases of plasma amylase and aspartate aminotransferase levels over 9 h after the pancreatitis. Prophylactic intravenous treatment with S-5920/LY315920Na significantly reduced mortality at 7 days, and strongly abrogated PLA2 activities in both plasma and ascites along with significant reduction of lipase activity, amylase, aspartate aminotransferase, and hemorrhage at 6 h. It also significantly reduced histological damage such as edema and parenchymal and fat necroses of the pancreatic tissue. This sPLA2 inhibitor could become an effective agent for the treatment of severe acute pancreatitis.
We have reported that oridonin isolated from Rabdosia rubescens induces apoptosis of human melanoma A375-S2 cells within 12 h. In this study, TUNEL assay and flow cytometric analysis also indicate that one of the causes of A375-S2 cell death induced by oridonin was apoptosis. The cell death was preceded by the release of cytochrome c from the mitochondria. Twelve hours after treatment with oridonin, the ratio of Bax/Bcl-xL protein expression was increased and release of cytochrome c was decreased by an extracellular signal-regulated kinase (ERK) MAPK inhibitor (PD98059) and a phosphoinositide 3-kinases (PI3-K) inhibitor (wortmannin). A mitochondrial permeability transition (MPT) inhibitor, decylubiquinone, suppressed the release of cytochrome c without affecting Bax expression. The activation of p53 by oridonin was also blocked by wortmannin. In addtion, PD98059 and wortmannin significantly decreased oridonin-induced DNA fragmentation, but the p38 MAPK inhibitor (SB203580) did not after DNA fragmentation. Oridonin induced A375-S2 cell apoptosis by activating parallel p53 and ERK pathways, increasing the ratio of Bax/Bcl-xL protein expression, and promoting the release of cytochrome c into the cytosol, resulting in apoptotic cell death.
Two types of serotonin 2C subtype receptor mRNA, receptor-type and short variant, has been reported. The expression of the receptor-type mRNA could be detected as well as the short variant in NG108-15 cells by using a high temperature stable reversetranscriptase and the expression of the receptor-type mRNA was enhanced in drug-induced neuronal differentiated cells. The deleted sequence of the short variant include the RNA editing site by adenosine deaminase. Analysis of the sequence at the editing site revealed that the mRNA of undifferentiated cells was highly edited at sites A and B and that cytosine deaminase activity may also be involved in neuronal differentiation.
Urocortin has a high affinity for the corticotropin-releasing factor receptor type 2β (CRF-R2β). This study was conducted to reveal the role of CRF-R2β in blood vessels. CRF-R2β expressions were detected both in smooth muscle and endothelium from Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) aortas, and there was no significant difference between them. Urocortin reduced phenylephrine-induced contraction of aorta with endothelium dose-dependently in both rats. However, deendothelialization significantly but not completely (about 50%) reduced the vasodilation. The reduction of vasodilatory action of urocortin by deendothelialization was age-dependent in SHR. An adenylyl cyclase inhibitor, SQ22536, significantly inhibited urocortin-induced relaxation in denuded WKY and SHR aortas, while in preparations with endothelium, neither SQ22536 nor L-NMMA reduced the relaxation. However, simultaneous addition of both drugs significantly reduced the relaxation. In contrast to young rats (7-week-old), in aged rats (19-week-old), L-NMMA successfully reduced urocortin-induced relaxation of aorta with endothelium. These results suggest that urocortin relaxes aorta at least partly via two signal pathways, that is, an increase in intracellular cAMP by binding to CRF-R2β expressed in smooth muscle cells and NO production from endothelium evoked by binding to the receptors expressed in endothelium and that aging increases the role of the latter system.
In this study, we investigated the acute hemodynamic effects of an infusion of the endothelin-1 (ET-1)-A-selective receptor antagonists BQ-610 and BQ-123 in heatstroke rats with circulatory shock and cerebral ischemia. Heatstroke was induced by putting the anesthetized adult Sprague-Dawley rats into an ambient temperature of 42°C. The moment in which the mean arterial pressure dropped irreversibly from the peak for an extent of 25 mmHg was taken as the onset of heatstroke. The interval between initiation of heat exposure and heatstroke onset was found to be about 80 min for rats treated with vehicle solution. When the animals were exposed to 42°C for 80 min, hyperthermia, arterial hypotension, decrement of cardiac output (due to decreased stroke volume and decreased total peripheral resistance), increment of plasma ET-1 and tumor necrosis factor-α, and increment of cerebral ischemia and injury markers were manifested. Prior antagonism of ET-1 A receptors with BQ-610 (0.5 mg/kg, i.v.) or BQ-123 (1 mg/kg, i.v.), but not ET-1B receptors with BQ-788 (0.5 mg/kg, i.v.), 60 min before the initiation of heat exposure, appreciably alleviated hyperthermia, arterial hypotension, decreased cardiac output, increment of tumor necrosis factor-α, and increment of cerebral ischemia (e.g., glutamate and lactate/pyruvate ratio) and injury (e.g., glycerol) markers exhibited during heatstroke. The data indicates that ET-1A receptor antagonism may maintain appropriate levels of mean arterial pressure and cerebral circulation during heatstroke by reducing production of tumor necrosis factor-α.
Evidence suggests that S-nitrosylation is a biological process involved in cerebral ischemia. The aim of the present study was to elucidate the effects of S-nitrosylated (SNO) polyethylene glycol-conjugated (PEG) hemoglobin (Hb) developed as an artificial oxygen carrier, which can absorb free NO and translocate NO to a sulfhydryl (SH) moiety, on ischemic cerebral dysfunction. Long-term potentiation (LTP) in the perforant path-dentate gyrus synapses of the rat hippocampus was evaluated as functional outcome 4 days after transient incomplete cerebral ischemia (2-vessel occlusion: 2VO, 10 min). SNO-PEG-Hb (250 mg/kg, i.v.) administered on Day 0, 1, 2, or 4 (immediately, 24 h, 48 h, or 96 h after reperfusion, respectively) alleviated 2VO-induced LTP impairment with a therapeutic time window. The effect was significant when SNO-PEG-Hb was administered on Day 1 or 2. SNO-PEG-Hb altered NOS features observed in the vehicle-treated 2VO rat, upregulation of eNOS, nNOS, and iNOS expressions at mRNA and protein levels; SNO-PEG-Hb further upregulated eNOS and nNOS and downregulated iNOS expressions. These findings suggest that SNO-PEG-Hb might have protective effects on the rat hippocampus from ischemia/reperfusion-induced functional damages, thereby increasing the therapeutic potential as an artificial oxygen carrier for use in the area of oxygen therapy.
Endothelin-1 (ET-1), angiotensin II (Ang II), and phenylephrine, an α1-adrenoceptor agonist, share the common signaling process, resulting in activation of Gq protein-coupled receptor (GqPCR) to activate the hydrolysis of phosphoinositide (PI). They do not elicit any inotropic effect in isolated dog ventricular muscle. In the presence of forskolin or IBMX (3-isobutyl-1-methylxanthine), ET-1 produced a dual effect, that is, a positive inotropic effect (PIE) and/or a negative inotropic effect (NIE) depending on concentrations of forskolin or IBMX present simultaneously with ET-1. Phenylephrine produced a definite PIE and Ang II induced a small and transient PIE in the presence of forskolin or IBMX, but they did not elicit a NIE. Facilitation of Ca2+ influx via L-type Ca2+ channel may play a crucial role in the crosstalk because GqPCR agonists produced, likewise a PIE in the presence of Bay k 8644. GqPCR agonists failed to induce a PIE in the presence of dihydroouabain or elevated [Ca2+]o. These findings indicate that the accumulation of cAMP or activation of L-type Ca2+ channels markedly modulates the inotropic response to GqPCR agonists in a manner that leads to a PIE in dog ventricular myocardium. In addition, ET-1, but not Ang II or phenylephrine, activates the signal transduction process that results in a NIE.
Neural stem cells (NSCs) were isolated from the mouse cortex on embryonic day 12.5 and cultured by neurosphere formation in serum-free medium in the presence of basic fibroblast growth factor (bFGF). When NSCs were inoculated in collagen gels with 10% fetal bovine serum (FBS) and bFGF and incubated for 10 days, vessel-like tube structures consisting of PECAM-1- or VE-cadherin-immunoreactive cells were formed in the gels. Moreover, the formation of vascular tube-like structures with a massive investment of α-smooth muscle actin-immunoreactive or GFAP-immunoreactive cells was occasionally observed, indicating angiogenesis identical to cerebral vascular development in vivo. To examine whether NSCs are capable of producing endothelial cells, differentiation was induced by the addition of 10% FBS after bFGF withdrawal. Most of the cells displayed a cobblestone-like morphology. Immunological analyses and RT-PCR indicated that NSCs expressed endothelial cell-specific marker proteins such as PECAM-1, VE-cadherin, and Flk-1; and these expressions were maintained or up-regulated during differentiation. Similar tube structures were also observed when the differentiated cells were inoculated in collagen gels and incubated for 5 days. These results suggested that NSCs give rise to two types of vascular cells, endothelial cells and mural cells in vitro, which have the ability to form vascular tubes.
Cardiovascular effects of cilnidipine, a dual L/N-type Ca2+ channel blocker, were evaluated in the chronic atrioventricular block dogs, of which systemic blood pressure and plasma catecholamine levels significantly increased in the pre-drug control. Administration of antihypertensive doses of cilnidipine (1 and 3 μg/kg, i.v.) significantly decreased the total peripheral vascular resistance, mean blood pressure, and atrial rate and increased the cardiac output. These results suggest that cilnidipine not only decreases the blood pressure, but also decreases the sinus automaticity in the in vivo hypertensive condition with increased adrenergic tones.
Modulation of the spontaneous activity of rat medial vestibular nuclear neurons by nitric oxide was investigated using the whole-cell patch-clamp technique. The spike frequency was increased by sodium nitroprusside (SNP), a nitric oxide liberating agent, and it was also increased by another nitric oxide liberating agent, sodium-nitroso-N-acetylpenicillamine. L-Arginine, the substrate of nitric oxide synthase, increased the firing of the neurons. The increased SNP-induced firing was inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinozalin-1-one (ODQ), a specific inhibitor of guanylate cyclase. These results suggest that nitric oxide increases the neuronal excitability of the neurons by a cGMP-dependent mechanism.