Platelets play a central role in the hemostatic process and consequently are similarly involved in the pathological counterpart, thrombosis. They adhere to various subendothelial proteins, exposed either by injury or disease, and subsequently become activated by the thrombogenic surface or locally produced agonists. These activated platelets aggregate to form a platelet plug, release agonists which recruit more platelets to the growing thrombus, and provide a catalytic surface for thrombin generation and fibrin formation. These platelet-rich thrombi are responsible for the acute occlusion of stenotic vessels and ischemic injury to heart and brain. A range of anti-platelet drugs are currently used, both prophylactically and therapeutically, in regimens to manage thrombo-embolic disorders. These include inhibitors of the generation, or effects, of locally produced agonists; several large clinical trials have supported roles for cyclooxygenase inhibitors, which prevent thromboxane generation, and thienopyridine derivatives, which antagonize ADP receptors. Similarly intravenous αIIbβ3 antagonists have been shown to be effective anti-thrombotics, albeit in highly selective situations; in contrast, to date studies with their oral counterparts have been disappointing. Recent advances in understanding of platelet physiology have suggested several novel, if yet untested, targets for anti-platelet therapy. These include the thrombin receptor, the serotonin handling system, and the leptin receptor.
The vascular endothelium is involved in the release of various vasodilators, including nitric oxide (NO), prostaglandins, and endothelium-derived hyperpolarizing factor as well as vasoconstrictors. NO plays an important role in the regulation of vascular tone, the inhibition of platelet aggregation, and the suppression of smooth muscle cell proliferation. Several diseases are associated with changes in endothelial function mediated through reduced NO bioavailability. In addition, endothelial dysfunction is an early feature of atherosclerosis and vascular diseases in humans. Therefore, it is clinically important to estimate the degree of endothelial dysfunction. Several methods have been used to assess endothelial function in humans. Recently, we have evaluated the effects of intra-arterial infusion of infusion of NO agonists, such as acetylcholine, methacholine, and bradykinin, and NO antagonists on forearm blood flow using mercury-filled Silastic strain-gauge plethysmography. The response to the intra-arterial infusion of vasoactive agents should be considered the gold standard in assessing endothelial function, because the use of agonists to stimulate NO release allow us to draw more specific conclusions concerning the role of basal and stimulated NO release. However, the invasive method is time-consuming and is a burden for patients. A noninvasive method of measuring forearm blood flow response to reactive hyperemia also is useful in assessing endothelial function. In this review we would like to explain in detail the methods of assessing endothelial function in humans using strain-gauge plethysmography.
Evidence has accumulated that impairment of vascular endothelial function is the initial step in the development of atherosclerosis. One important finding is impairment of the release of endothelium-dependent relaxing factor, which is now thought to be nitric oxide or its related substances, from endothelial cells. Flow-mediated dilatation has been known to be endothelium-dependent, and this can be detected during reactive hyperemia by high-resolution ultrasound in superficial arteries. Several coronary risk factors have been reported to be significantly related with decreased flow-mediated dilatation. We studied the association between the accumulation of coronary risk factors (hyperlipidemia, diabetes mellitus, hypertension, and current smoking habitus) and vascular endothelial function. The lower incidence of atherosclerosis in women before the menopause than that in men is an established epidemiological observation. Short-term estrogen therapy improves endothelial function in postmenopausal women. However, there are few reports on its long-term effects on endothelial function. Furthermore, we determined whether a reduced dosage of estrogen may maintain its beneficial effects. A similar improvement was also observed while women were on hormone replacement therapy even at the reduced dosage. Our results indicate that even at half the dose of estrogen, hormone replacement therapy may improve endothelial function in postmenopausal women.
Although plasma NOx (NO2− and NO3−) has been used as an index of nitric oxide (NO) formation in vivo, many unreasonable results appeared even after active elimination of NOx contamination from laboratory ware. For example, plasma NOx concentrations did not increase during vasodilation mediated by the NO/cGMP pathway or after organ perfusion. A possible shift of NOx from plasma to erythrocytes (RBCs) as a cause of these phenomena has been excluded, leaving the destination of NOx (after leaving plasma) unknown. Kinetic analyses have revealed that steady state NOx concentrations in plasma and whole blood did not correlate with the NOx formation rate, but rather with the NOx elimination rate. Therefore, the supposition that the NO status is directly reflected by plasma NOx concentrations appears untenable. As nitrosothiols (R-SNOs), possible carriers of NO bioactivity, have been flagged as alternative indices of NO status in vivo, efforts have been made to detect these substances. When interference by ultrafiltration was eliminated, low molecular weight R-SNOs such as nitrosocystein and nitrosogluthathione were undetectable. However, a high-molecular weight R-SNO, nitrosoalbumin, was detected in human blood. Further research is required into the significance and practical use of nitrosoalbumin as a marker of NO in vivo.
This minireview describes the practical use of assay systems to detect nitric oxide (NO) by electron paramagnetic resonance (EPR) spectroscopy for evaluation of endothelial functions. The iron(II)-dithiocarbamate complexes, such as iron(II)-(N-methyl-D-glucamine dithiocarbamate), are commonly used in EPR detection of NO both in vivo and in vitro. However, due to their redox activity, these complexes have some drawbacks that limit their usefulness for the detection of NO. On the other hand, the measurement of hemoglobin-NO adduct (HbNO) in whole blood by the EPR method seems relevant for the assessment of systemic NO levels. However, ceruloplasmin and an unknown radical species overlapping the same magnetic field as that of HbNO, which makes it physically impossible to measure small amounts of HbNO. Thus, to reveal the EPR spectrum of HbNO, we developed the EPR signal subtraction method, which is based on the computer-assisted subtraction of the digitized EPR spectrum of HbNO-depleted blood from that of the sample blood using software. Using this technique, we succeeded in measuring the steady blood HbNO level as an index of NO by the EPR HbNO signal subtraction method. We also demonstrated that temocapril reduces abnormalities of NO dynamics in the L-NAME (Nω-nitro-L-arginine-methylester)-induced endothelial dysfunction of rats using the EPR HbNO signal subtraction method.
The G-protein activation induced by μ-opioid receptor agonists was determined in spinal cord membranes from two types of μ-opioid receptor knockout mice: mice with a disruption of exon 1 (MOR (Exon 1)-KO) or exons 2 and 3 (MOR (Exons 2 and 3)-KO) of the μ-opioid receptor gene. The G-protein activation induced by the opioid agonists was measured by monitoring the increases of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding. The μ-opioid receptor agonists (D-Ala2,N-MePhe 4,Gly-ol5]enkephalin, endomorphin-1, endomorphin-2, morphine, morphine-6β-glucuronide, and fentanyl produced concentration-dependent increases of [35S]GTPγS binding to spinal cord membranes in wild-type mice, but not in MOR (Exon 1)-KO mice or MOR (Exons 2 and 3)-KO mice. On the other hand, the δ-opioid receptor agonist [D-Pen 2,5]enkephalin, the κ-opioid receptor agonist (−)U50,488H, or the ORL1-receptor agonist nociception increased [35S]GTPγS binding in the spinal cord membranes from both MOR (Exon 1)-KO mice and MOR (Exons 2 and 3)-KO mice to the same extent as in the corresponding wild-type mice. The results provide further information about the important roles of the sequences encoded within exon 1 and exons 2 and 3 of μ-opioid receptor gene for the activation of G-proteins by μ-opioid receptor agonists in the mouse spinal cord.
The purpose of the present study was to elucidate the expression of human organic anion transporter 1 (hOAT1) and hOAT3 in the choroid plexus of the human brain and their interactions with neurotransmitter metabolites using stable cell lines. Immunohistochemical analysis revealed that hOAT1 and hOAT3 are expressed in the cytoplasmic membrane and cytoplasm of human choroid plexus. Neurotransmitter metabolites, namely, 5-methoxyindole-3-acetic acid (5-MI-3-AA), homovanillic acid (HVA), vanilmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindole-3-acetic acid (5-HI-3-AA), N-acetyl-5-hydroxytryptamine (NA-5-HTT), melatonin, 5-methoxytryptamine (5-MTT), 3,4-dihidroxymandelic acid (DHMA), 5-hydroxytryptophol, and 5-methoxytryptophol (5-MTP), but not methanephrine (MN), normethanephrine (NMN), and 3-methyltyramine (3-MT), at 2 mM, inhibited para-aminohippuric acid uptake mediated by hOAT1. On the other hand, melatonin, 5-MI-3-AA, NA-5-HTT, 5-MTT, 5-MTP, HVA, 5-HI-3-AA, VMA, DOPAC, 5-hydroxytryptophol, and MN, but not 3-MT, DHMA, and NMN, at 2 mM, inhibited estrone sulfate uptake mediated by hOAT3. Differences in the IC50 values between hOAT1 and hOAT3 were observed for DHMA, DOPAC, HVA, 5-HI-3-AA, melatonin, 5-MI-3-AA, 5-MTP, 5-MTT, and VMA. HOAT1 and hOAT3 mediated the transport of VMA but not HVA and melatonin. These results suggest that hOAT1 and hOAT3 are involved in the efflux of various neurotransmitter metabolites from the cerebrospinal fluid to the blood across the choroid plexus.
Whether a state of nasal hyperresponsiveness influences antigen-induced biphasic nasal blockage and sneezing were examined using a guinea pig model of allergic rhinitis. Sensitized animals were challenged with an antigen, Japanese cedar pollen, once every week. Before the 13th challenge, the animals were randomly divided into 2 groups, and then the 13th challenge was performed (Groups A-0 and B-0). The 14th challenge was done on day 2 (Group A-2) and on day 7 (Group B-7) after the 13th challenge, on which nasal hyperresponsiveness was present and absent, respectively. Biphasic nasal blockage and sneezing after the challenge in Group A-2 were more severe than those in Group A-0, while those of Group B-7 were almost the same as those of Group B-0. An anti-histaminic, mepyramine, inhibited sneezing but not the biphasic nasal blockage in Group B-7. A cysteinyl leukotriene (CysLT) antagonist, pranlukast, suppressed the late nasal blockage but not the early blockage and sneezing in Group B-7. In contrast, in Group A-2, mepyramine significantly attenuated not only sneezing but also the early nasal blockage. Pranlukast significantly inhibited both nasal blockage and sneezing in Group A-2. In conclusion, nasal hyperresponsiveness aggravated the antigen-induced nasal responses, to which histamine and CysLTs considerably contributed.
Effects of the serotonergic anxiolytic buspirone on plasma glucose and glucose-induced hyperglycemia were studied in mice. Buspirone did not affect plasma glucose levels of non-fasted mice, while it increased serum insulin levels. In fasted mice, buspirone significantly reduced glucose-induced hyperglycemia and enhanced insulin release elicited by glucose. This suggests that buspirone enhances insulin release, resulting in inhibition of glucose-induced hyperglycemia. The major metabolite of buspirone, 1-(2-pyrimidinyl)piperazine (1-PP) increased serum insulin levels and induced a slight hypoglycemia in non-fasted mice. 1-PP decreases glucose-induced hyperglycemia and amplifies insulin release elicited by glucose in fasted mice. Since buspirone is mainly metabolized to 1-PP and formation of 1-PP occurs quickly, the inhibitory effect of buspirone on glucose-induced hyperglycemia is likely mediated by 1-PP.
Repetitive transcranial magnetic stimulation (rTMS) changes the function of the cortex. This study clarified the effects of acute rTMS treatment on extracellular serotonin (5-HT) concentrations in the rat prefrontal cortex (PFC) by using in vivo microdialysis methods. Each rat received acute rTMS treatment of the frontal brain at 500 stimuli from twenty trains applied at 25 Hz for 1 s at 1-min intervals between trains. Sham-treated rats received the same handling procedure and sound of the stimulator. Sham treatment increased the extracellular 5-HT levels compared with the non-treated group. However, rTMS treatment using the stimulation intensity of 110% motor threshold eliminated the increase in 5-HT levels induced by the sham treatment. Acute rTMS treatment of the frontal brain is related to the serotonergic neuronal system in the rat PFC, and it may have therapeutic implications for emotional disorders.
Stress is a global menace fortified by the advancement of industrialization. Failure of stress management is due to lack of proper evaluation of anti-stress products. We explored the anti-stress potential of the Ginkgo biloba (G. biloba, 30 mg/kg, p.o.) and compared it with that of Panax ginseng (P. ginseng, 100 mg/kg, p.o.) against acute stress (AS) and chronic stress (CS) models in rats. Immediately after AS and CS, the rats were sacrificed, and adrenal glands and stomach were dissected out for weight determination and scoring of the ulcer index (UI), respectively, as well as changes in biochemical parameters like plasma glucose (GL), triglycerides (TG), cholesterol (CL), creatine kinase (CK), and serum corticosterone (CORT) were also estimated. AS significantly increased UI, adrenal gland weight (AGW), GL, CK activity, and CORT, whereas G. biloba significantly reduced them. P. ginseng significantly reverted GL and CK activity. In CS, a significant increase was found in the UI, AGW, CK activity, and CORT with a decrease in the level of CL and TG. G. biloba did not produce any significant effect on CS-induced alterations. P. ginseng reduced the UI, AGW, plasma GL, TG, CK activity, and CORT level significantly. From the above study, G. biloba is more effective in AS, whereas for CS, P. ginseng will be a better option. Hence these extracts possess significant anti-stress properties and can be used for the treatment of stress-induced disorders.
We developed a new coughing model that evoked coughs by microinjection of citric acid into the larynx in unanesthetized unrestrained guinea pigs; additionally, we recorded synchronous sounds and waveforms of coughing utilizing built-in microphones and a whole body plethysmograph. The coughing model was able to distinguish a coughing response from other expiratory responses, such as an expiratory reflex or a sigh, by examining the waveform of the expiratory response and the existence of sound. It was not necessary to distinguish a cough from a sneeze, since the administration site was restricted to the larynx. Microinjection of 0.4 M citric acid, total of 20 μl (10 times, 2 μl at 30-s intervals), induced coughs (27.03 ± 4.03 coughs in 10-min observation) that were stable and independent of the inhalation volume. In the inhalation studies, animals were exposed to citric acid only once, because the number of coughs remarkably decreased with repeated administration at intervals of 24 h (tachyphylaxis). However our coughing model was able to repeatedly challenge the microinjection of citric acid at an interval of 24 h. These results indicated that this coughing model was highly sensitive and correctly assessed the cough response.
Interleukin (IL)-6 has been shown to protect neuronal cells from cell death induced by various stimulants. Although neuronal cells including PC12 cells were shown to produce IL-6, little is known about the effects of dopaminergic neurotoxins, 1,2,3,4-tetrahydroisoquinoline (TIQ) and 1-methyl-4-phenylpyridinium ion (MPP+), on IL-6 expression in PC12 cells. In the present study, we investigated the role of IL-6 in the TIQ- and MPP+-induced cell death in PC12 cells. Treatment with 3.2 mM TIQ for 24 h caused a delayed cell death (lactate dehydrogenase (LDH) leakage and nuclear DNA fragmentation) markedly 72 h after the addition. Addition of 0.4 mM MPP+ caused LDH leakage and nuclear DNA fragmentation 24 h after the addition. The cell death induced by MPP+ was inhibited by an inhibitor of caspases, z-Val-Ala-Asp(OMe)-fluoromethylketone. The cell death induced by TIQ or MPP+ was inhibited by nerve growth factor and 10% serum and significantly enhanced by the treatment with anti-IL-6 antibody. Both neurotoxins decreased the IL-6 mRNA level in PC12 cells without changing the other tested mRNA levels (IL-1α, β-actin, etc.). These findings suggest that dopaminergic neurotoxins cause cell death in PC12 cells at least partially by changing IL-6 expression.
Cardiovascular diseases are the most frequent and costly complication of diabetes. Many previous studies showed that ATP-sensitive potassium channels (KATP) and inward rectifier potassium channels (Kir) play important regulatory roles in functions of cardiovascular tissues. It’s still not very clear how these potassium channels are involved in cardiovascular complications of diabetes. We used the streptozotocin (STZ)-induced diabetic rats model to study the expressions of KATP and Kir channel subtypes in diabetic cardiovascular tissues. The mRNA expression levels of Kir2.1, Kir3.1, Kir6.1, Kir6.2, and sulfonylurea receptor (SUR) 2A and 2B subunits in heart and aortal smooth muscles were determined by the reverse-transcription polymerase chain reaction. The results showed that in comparison with the control rats, mRNA expression of SUR 2A was reduced significantly in the diabetic heart (SUR 2A/GAPDH, 1.04 ± 0.16 vs 0.38 ± 0.09, P<0.01, n = 3); SUR 2B was reduced markedly in the aortal smooth muscle of diabetic rats (SUR 2B/GAPDH, 1.13 ± 0.14 vs 0.35 ± 0.07, P<0.01, n = 3). However, there are no significant expression changes of Kir2.1, Kir3.1, Kir6.1, and Kir6.2 in diabetic rats. These results suggested that expression of specific KATP channel subunits were altered in the heart and aorta of diabetic rats.
We assessed the effects of OPC-51803 ((5R)-2-[1-(2-chloro-4-(1-pyrrolidinyl)benzoyl)-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]-N-isopropylacetamide), a nonpeptide vasopessin V2-receptor agonist, on micturition frequency in female homozygous Brattleboro rats (strain carries hereditary diabetes insipidus) and aged male Sprague-Dawley rats with polyuria. Female homozygous Brattleboro rats exhibited more diuresis and a larger micturition frequency over a 24-h period than did the heterozygous controls. In Brattleboro rats, an oral administration of OPC-51803 at 0.03 and 0.3 mg/kg significantly decreased urinary frequency and was accompanied by decreased urine volume. However, little effect was seen in the mean and maximal micturition volume. Aged male Sprague-Dawley rats (25-month-old) showed a significant increase in urine volume throughout a 0- to 24-h period compared with mature (6-month-old) rats. Orally administered OPC-51803 at 0.3 mg/kg decreased not only urine volume but also urinary frequency in aged rats. Furthermore, OPC-51803 prolonged the time prior to the first micturition. Therefore, OPC-51803 decreased micturition frequency in both rat species by reducing urine outflow. This suggests that the compound will be useful for treating micturition disorders that result in frequent micturition, such as that from polyuria, nocturnal polyuria, and some kinds of urinary incontinence.
In this study, we investigated whether carbofuran, a commonly used carbamate pesticide, and N-nitrosocarbofuran (NOCF), the N-nitroso metabolite of carbofuran, have cytotoxicity in mouse brain microvascular endothelial cells (bEnd.3). Results from the MTT assay in bEnd.3 cells showed that NOCF but not carbofuran caused a remarkable decrease in cell viability. The cell death induced by NOCF appeared to involve apoptosis, based on our results from annexin V staining and electron microscopy. To investigate the mechanism of the NOCF-induced cell death, we examined the effects of selective inhibitors for MAP kinase pathways, PD98059 (for MEK/ERK), SB202190 (for p38 MAP kinase), and SP600125 (for JNK), on the NOCF-induced cell death. The NOCF-induced cell death was significantly reduced by PD98059, but not by SB202190 or SP600125. NOCF increased ERK phosphorylation as early as 15 min after the treatment and this increase was maintained for 2 h. In summary, our results suggest that NOCF can induce apoptotic cell death, at least in part, through the ERK pathway in brain microvascular endothelial cells.
Fourteen chemical agents used in dental practice were assessed for their cell-transforming activity using the Syrian hamster embryo (SHE) cell transformation assay system. The cell-transforming activity was quantitatively assessed by the frequency of morphological transformation (MT) in SHE cells induced by these agents. MT was induced by m-cresol, guaiacol, formaldehyde, sodium hypochlorite, hydrogen peroxide, sodium arsenite, acid fuchsin, and basic fuchsin, but not by p-chlorophenol, p-phenolsulfonic acid, glutaraldehyde, and erythrosine B. Iodine and chlorhexidine exhibited positive and pseudopositive responses, respectively. The chemical agents exhibiting a negative or pseudopositive response neither induced nor enhanced MT even in the presence of exogenous metabolic activation.
In the present study, the hepatoprotective effect of metformin (Met), a dimethylbiguanide anti-hyperglycemic, was examined in a mouse model of liver damage induced by chronic repeated administration of carbon tetrachloride (CCl4) (5 μl/kg, twice a week for 12 weeks). Met, when given orally in drinking water at an estimated daily dose of 25 or 50 mg/kg for 10 weeks starting 2 weeks after CCl4 challenge, protected against CCl4 hepatotoxicity. The results indicate that the hepatoprotection afforded by Met treatment at a dose of 25 mg/kg against CCl4 toxicity may at least in part be mediated by the enhancement of mitochondrial glutathione redox status.
We examined the effects of chlorpromazine on NO3− transport between erythrocytes (RBCs) and extracellular fluid. Chlorpromazine (10 μg/ml) did not influence NO3− movement in both whole blood and RBC suspension. Though an anion exchanger (AE1) inhibitor DIDS (4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid, 100 μM) did not alter NO3− movement in whole blood, it inhibited the movement in a concentration-dependent manner in the RBC suspension. The inhibition was abrogated by plasma and albumin concentration-dependently. Our results indicated that chlorpromazine had no effect on NO3− transport through AE1 and that the inertness of DIDS on AE1 in whole blood is due to interference by albumin in plasma.