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The prostacyclin (IP) receptor agonists, treprostinil, iloprost and the selexipag metabolite, MRE-269 (ACT-333679) were evaluated in rat distal pulmonary blood vessels. Small pulmonary arteries and veins were pre-contracted with the thromboxane mimetic, U46619 (25 and 100nM, respectively), and relaxation determined with and without IP receptor antagonists, RO1138452 and RO3244794. In arteries, treprostinil was a more potent vasorelaxant than iloprost, while the efficacy of iloprost was greater. In pulmonary arteries, treprostinil-induced relaxation was essentially abolished by both IP antagonists (1μM), while responses to iloprost were partially inhibited. Both treprostinil and iloprost were equipotent, prominently relaxing pulmonary veins with responses being similarly and partially sensitive to IP antagonists. In contrast, RO1138452 failed to inhibit relaxations to MRE-269 in either pulmonary arteries or veins, suggesting no involvement of typical IP receptors. Thus, rat pulmonary tissues cannot be considered appropriate to assess classical IP receptors using the proposed highly selective non-prostanoid agonist MRE-269, contrasting with the IP receptor agonism profile of prostacyclin analogues, iloprost and treprostinil., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background and Purpose: Vascular ATP-sensitive potassium (K(ATP)) channels are activated by cyclic AMP elevating vasodilators through protein kinase A (PKA). Direct channel phosphorylation is a critical mechanism, though the phosphatase opposing these effects is unknown. Previously, we reported that calcineurin, a Ca(2+)-dependent phosphatase, inhibits K(ATP) channels, though neither the site nor the calcineurin isoform involved is established. Given that the type-2 regulatory (RII) subunit of PKA is a substrate for calcineurin we considered whether calcineurin regulates channel activity through interacting with PKA., Experimental Approach: Whole-cell recordings were made in HEK-293 cells stably expressing the vascular K(ATP) channel (K(IR)6.1/SUR2B). The effect of intracellular Ca(2+) and modulators of the calcineurin and PKA pathway on glibenclamide-sensitive currents were examined., Key Results: Constitutively active calcineurin A alpha but not A beta significantly attenuated K(ATP) currents activated by low intracellular Ca(2+), whereas calcineurin inhibitors had the opposite effect. PKA inhibitors reduced basal K(ATP) currents and responses to calcineurin inhibitors, consistent with the notion that some calcineurin action involves inhibition of PKA. However, raising intracellular Ca(2+) (equivalent to increasing calcineurin activity), almost completely inhibited K(ATP) channel activation induced by the catalytic subunit of PKA, whose enzymatic activity is independent of the RII subunit. In vitro phosphorylation experiments showed calcineurin could directly dephosphorylate a site in Kir6.1 that was previously phosphorylated by PKA., Conclusions and Implications: Calcineurin A alpha regulates K(IR)6.1/SUR2B by inhibiting PKA-dependent phosphorylation of the channel as well as PKA itself. Such a mechanism is likely to directly oppose the action of vasodilators on the K(ATP) channel.

Background: Reactive oxygen species (ROS) have been implicated in various forms of cellular injury. ROS may cause cell damage and are involved in the pathophysiology of several diseases, including atherosclerosis and chronic inflammation., Methods: Disturbances of intracellular ROS levels were investigated in 28 patients with end-stage renal failure. The intracellular ROS levels were measured in lymphocytes before and after hemodialysis using biocompatible membranes and were compared with those from 11 patients with end-stage renal failure, not yet on renal replacement therapy, and 27 healthy control subjects. ROS levels were measured spectrophotometrically using the intracellular dye dichlorofluorescin diacetate., Results: The spontaneous production of ROS was significantly higher in lymphocytes from patients with end-stage renal failure compared with healthy control subjects (P < 0.01). The addition of 100 nmol/L phorbol-myristate-acetate (PMA) produced a significant increase of ROS, both in lymphocytes from patients with end-stage renal failure and healthy control subjects. The PMA-induced ROS increase was significantly higher in lymphocytes from patients with end-stage renal failure compared with healthy control subjects (P < 0.01). In patients with end-stage renal failure, not yet on renal replacement therapy, the PMA-induced ROS was also significantly higher compared with healthy control subjects. The PMA-induced ROS increases were significantly inhibited by catalase, but not by superoxide dismutase or the superoxide dismutase mimetic, tempol. PMA-induced ROS was significantly reduced by tyrphostin A51 in lymphocytes from patients with end-stage renal failure and from healthy control subjects (each P < 0.01), indicating the involvement of a tyrosine kinase-dependent pathway. In patients with end-stage renal failure, the spontaneous and the PMA-induced production of ROS was not significantly different before and after hemodialysis., Conclusions: Regular hemodialysis sessions using biocompatible membranes have no effect on the elevated intracellular ROS in patients with end-stage renal failure.

Since increased cellular production of reactive oxygen species is a source of oxidative stress and thus may contribute to the development of diabetic complications, the baseline and stimulated concentrations of intracellular reactive oxygen species were measured in 16 patients with diabetes mellitus type 2 and 19 healthy control subjects. Reactive oxygen species and cytosolic calcium were monitored spectrophotometrically using dihydrorhodamine-123 and fura-2, respectively, in a suspension of mononuclear leukocytes. Measurements were made in the presence or absence of superoxide dismutase, sodium azide, genistein, or bisindolylmaleimide 1. Baseline reactive oxygen species concentrations were significantly higher in diabetic patients compared with control (p<0.001). Activation of mononuclear leukocytes by formyl-Met-Leu-Phenylalanine and phytohemagglutinin significantly increased reactive oxygen species in diabetic patients compared with control (p<0.05). The formyl-Met-Leu-Phenylalanine-induced increases were unchanged in the presence of superoxide dismutase, but dropped significantly in the presence of sodium azide by 80% and 73% in diabetic patients and control, respectively (each p<0.01). The formyl-Met-Leu-Phenylalanine-induced responses were significantly inhibited by genistein and bisindolylmaleimide 1 (p<0.01), suggesting the involvement of tyrosine kinase and protein kinase C. Resting calcium (p<0.05) and stimulated calcium were significantly greater in diabetic patients than in control. The results show that patients with diabetes mellitus type 2 generate increased reactive oxygen species under stimulated conditions, suggesting increased risk for oxidative stress and associated complications.

To evaluate whether increased levels of reactive oxygen species (ROS) are involved in the pathogenesis of essential hypertension (EH) and non-insulin-dependent diabetes mellitus (NIDDM), both resting and stimulated levels of intracellular ROS were measured in lymphocytes from patients with EH (n = 10), NIDDM (n = 16) and age-matched healthy individuals (control subjects, n = 19). ROS was monitored with the dye, dihydrorhodamine-123 (DHR; 1 micromol/L) in the presence or absence of superoxide dismutase (superoxide scavenger), sodium azide (singlet oxygen/hydrogen peroxide scavenger), genistein (tyrosine kinase inhibitor), or bisindolylmaleimide (protein kinase C inhibitor). Simultaneous monitoring of cytosolic [Ca2+]i was done with fura-2. Resting ROS levels were significantly higher in NIDDM (4.71+/-0.25 nmol/10(6) cells; mean +/- SEM, P<.05) compared with EH (4.03+/-0.22 nmol/10(6) cells) or controls (4.05+/-0.15 nmol/10(6) cells). The formyl-Met-Leu-Phenylalanine-(fMLP)-induced ROS generation was significantly higher in NIDDM (21.92+/-2.23 nmol/10(6) cells; P<.05) compared with EH (14.58+/-1.90 nmol/10(6) cells) or control (16.06+/-1.22 nmol/10(6) cells). The fMLP-induced ROS increase was significantly reduced in the presence of sodium azide in all groups (P<.01) but was largely unaffected in the presence of SOD. Genistein and bisindolylmaleimide significantly inhibited the fMLP-induced ROS in all groups. The fMLP-induced [Ca2+]i increase was significantly higher in NIDDM (71+/-12 nmol/L, P <.01) compared with EH (42+/-4 nmol/L) and control subjects (35+/-3 nmol/L). Phytohemagglutinin was more effective in increasing [Ca2+]i than ROS. It is concluded that ROS may play a role in the metabolic syndrome of NIDDM but not in EH.

This study examined the role of calcium in the generation of reactive oxygen species (ROS) in human lymphocytes activated by the chemoattractant formyl-Met-Leu-Phe (fMLP) and the T-cell mitogen phytohaemagglutinin (PHA). The concentrations of cytosolic calcium ([Ca2+]i) and ROS were monitored simultaneously with a fluorescence spectrophotometer after the cells had been incubated in fura-2 (calcium-sensitive dye) and 2',7'-dichlorofluorescein diacetate (DCF-DA, ROS-sensitive dye). The lymphocytes were stimulated with fMLP (200 nmol l-1) or PHA (10 micromol l-1) in the absence and presence of extracellular calcium. A dose-response test was also conducted for extracellular calcium. fMLP and PHA significantly increased both [Ca2+]i (P < 0.001) and ROS concentrations (P < 0. 001) above the control levels in the presence of extracellular calcium. However, such increases were abolished in the absence of extracellular calcium, suggesting total dependence of the responses to both fMLP and PHA on transplasma-membrane calcium influx. There were also graded increases in ROS with increasing concentrations of extracellular calcium. The results show that transplasma-membrane calcium influx is essential for fMLP- and PHA-induced generation of reactive oxygen species in human lymphocytes.

This study examined the antihypertensive efficacy of nifedipine (a calcium channel blocker) in salt-loaded alloxan-diabetic rats. Significant increases in basal mean arterial blood pressures were observed after six weeks of high salt (8% NaCl) feeding in both diabetic (p < 0.05) and nondiabetic (p < 0.05) rats. The values were 129.95+/-3.14 mmHg for control (C); 149.22+/-8.83 mmHg for nondiabetic salt-fed (N-SF) and 150.60+/-8.01 mmHg for diabetic salt-fed (D-SF) groups. The non-salt-fed diabetic group (D) had a pressure of 136.75+/-6.66 mmHg. The maximum mean arterial blood pressures in response to noradrenaline (10(-9)-10(-5) M) infusion were significantly (p < 0.05) higher in the N-SF and D-SF groups than in the control. Nifedipine (100 microg/Kg) reduced significantly the pressures (both before and following noradrenaline infusion) in the salt-fed groups (p < 0.001). The inhibitory effect of nifedipine was more marked (p < 0.01) in the diabetic salt-fed than in the nondiabetic salt-fed. It is therefore suggested that nifedipine is effective in lowering salt-induced high blood pressure in diabetic rats.

This study examined the signaling mechanism involved in the generation of reactive oxygen species (ROS) in human lymphocytes activated by formyl-Met-Leu-Phenylalanine (fMLP; 200 nmol/L) or phorbol-myristate-acetate (PMA; 100 nmol/L). ROS were monitored spectrophotometrically using dichlorofluorescin diacetate. fMLP and PMA significantly increased ROS above the control levels (p<0.05 and 0.001, respectively). These increases were significantly inhibited by catalase, sodium azide, and dimethylsulfoxide but not by superoxide dismutase, suggesting that the ROS apparently included hydrogen peroxide, singlet oxygen and hydroxyl ion but not superoxide anion. PMA-induced responses were reduced by tyrphostin (p<0.01), ST-638 (p<0.05), KN-62 (p<0.001), bisindolylmaleimide (p<0.001), RO-31-8220 (p<0.001), and by LY-83583 (p<0.001), suggesting significant involvement of tyrosine kinase, calcium/calmodulin kinase II, protein kinase C and guanylyl cyclase. fMLP-induced responses were significantly reduced by only tyrphostin (p<0.001), ST-638 (p<0.05), and KN-62 (p<0.01). The results show that tyrosine kinase and calcium/calmodulin kinase II are common signalling components in the production of reactive oxygen species in activated lymphocytes.

Objective: To compare the normal respiratory function values in young Kenyans with those reported for regional African neighbours and Caucasians., Design: Forced expiratory volume in one second (FEV1), forced vital capacity (FVC) and peak expiratory flow rate (PEFR) were measured in a non-randomised sample of young indigenous Kenyans and the values were compared with those of age-matched regional African neighbours (Ethiopians and South Africans) and Caucasians (Australians)., Setting: Faculty of Health Sciences, Moi University, Eldoret., Subjects: Eighty eight apparently healthy young Kenyan university students (64 males, 20-25 years and 24 females, 19-23 years) were examined., Results: The mean values (males versus females) were FEV1, 3.95 +/- 0.07 versus 2.97 +/- 0.08, FVC, 4.31 +/- 0.08 versus 3.19 +/- 0.09, and PEFR, 586.30 +/- 8.54 versus 438.30 +/- 8.55. The values for females were 25-26% less than those for males. The expiratory ratios (FEV1/FVC x 100) were 92.22 +/- 0.60% and 93.44 +/- 0.61% for males and females respectively, well within normal range. The FEV1, FVC and PEFR for both males and females correlated positively with heights., Conclusion: These results were comparable to values reported for age-matched regional neighbours (Ethiopians and South Africans) but were lower than those reported for Australian caucasians. This highlights the need for the Kenyan population to have its own local reference values.

The effectiveness of insulin replacement therapy in the prevention of salt-induced hypertension in diabetes mellitus was examined using Alloxan diabetic rats. Early daily (eight units/day) treatment with insulin prevented the development of high blood pressure after six weeks of high-salt feeding. The mean arterial pressure (MAP) for the early insulin-treated and salt-fed group (DET-SF) was 123.37 +/- 6.37 mmHg which was close to the value for normal (control) rats 128.17 +/- 4.84 mmHg, but significantly (p < 0.001) less than that of the untreated diabetic salt-fed group (DSF) which was 164.58 +/- 8.33 mmHg. The nondiabetic salt-fed (NDSF) group had MAP of 150.27 +/- 4.24 mmHg. Late commencement of insulin therapy did not significantly affect the sensitivity of the diabetic rats to high-salt diet. The results suggest that early commencement of insulin therapy could prevent the development of high blood pressure in diabetic rats.

Responses of the aorta and portal veins isolated from rats to aqueous extract of plantain (Musa paradisiaca) were studied. The extract produced concentration-dependent relaxation in both noradrenaline- and KCl-contracted aortic rings. The maximum relaxation in noradrenaline-contracted rings was 52.49 +/- 6.63% and in KCl-contracted rings was 77.51 +/- 2.52% of the initial tensions developed in response to the contractile agents. The extract also produced significant (P < 0.01) inhibition of the maximum aortic contractile response to noradrenaline and completely abolished the spontaneous contractions of the portal veins. Serotonin (5-hydroxytryptamine), unlike the extract, produced contraction rather than relaxation of the aortic rings. The results show a non-specific relaxing and inhibiting effect of plantain extract on aortic and portal smooth muscles, an effect that is not attributable to its abundant serotonin content.

The responsiveness of the rat aorta after chronic consumption of 15% (wt/wt) fresh and thermally oxidized palm oil diets was studied under standard organ bath procedures. Aortic rings from the oxidized oil-fed group showed significantly (P < 0.05) enhanced vascular responses to noradrenaline and potassium chloride when compared with the control and fresh palm oil-fed groups. The maximum tensions were 285.10 +/- 30 mg/mg tissue weight for the oxidized oil-fed group and 148.98 +/- 36 mg/mg for the control in response to noradrenaline. The fresh oil-fed group produced maximum tension of 133.9 +/- 20 mg/mg which was not significantly different from the control. The trend was similar with potassium chloride. The maximum tensions were 206.31 +/- 25 mg/mg for the oxidized oil-fed group and 93.33 +/- 13 mg/mg for the control group. The fresh oil-fed group produced maximum tension of 109.31 +/- 7.8 mg/mg which was not significantly different from the control. Relaxation to acetylcholine was significantly (P < 0.01) attenuated in the aortic rings obtained from the oxidized palm oil-fed group when compared with the control and fresh palm oil-fed groups. The percentage maximum relaxations to acetylcholine were 28.1 +/- 6.7% in the oxidized oil-fed group, 71.4 +/- 6.0% in control and 78.2 +/- 6.0% in the fresh oil-fed groups. The relaxation in the fresh oil-fed group was not significantly different from control. These results suggest that functional changes occur in rat blood vessels after chronic consumption of thermally oxidized palm oil.

Salt-induced high blood pressure in diabetic rats was compared with that in non-diabetic (control) rats. Before the rats had eaten the high-salt diet, the diabetic group exhibited significantly greater pressor response to noradrenaline (100 ng/100 g body weight) than the controls (P < 0.05). When the rats were given a high-salt diet, a significant increase in blood pressure was recorded 4 weeks earlier in the diabetic than in the control group (P < 0.05); this increase occurred after 2 weeks in the diabetic group, but after 6 weeks of high-salt diet in the controls. The enhanced state of vascular reactivity in the diabetic rats was probably responsible for their enhanced sensitivity to salt-induced high blood pressure.

Sodium-potassium ATPase activity and transmembrane calcium influx in the aortic smooth muscle from control and diabetic rats were assessed indirectly through the measurement of KCl relaxation and contractile responses to CaCl2 in attempts to explain the contractile responses to KCl following streptozotocin-induced diabetes mellitus. There were no significant changes in the maximum contractile responses of the aortas from 4 and 12 week diabetic rats to KCl even when significant increases in calcium influx were demonstratable. On the other hand, the diabetic aortas were significantly (P < 0.05) more sensitive to KCl-induced relaxations than the controls. This provides an indirect evidence for increased activity of the sodium-postassium ATPase enzyme in the aortas from streptozotocin diabetic rats. This may, atleast in part, explain the inability of KCl to produce greater than normal contractions of the aortas from diabetic rats.

1. The relaxations of aortic preparations from 1, 4 and 12 week diabetic rats to acetylcholine and histamine were studied. 2. The relaxation to acetylcholine but not to histamine was significantly attenuated in the 4th and 12th weeks of the disease. In addition, the tissues became less sensitive to acetylcholine but not to histamine with increasing duration of the disease. 3. The results show that there is differential alteration in the responsiveness of the aorta from diabetic rats to both agents.

This work investigated the bronchodilator effect of Garcinia Kola (Bitter Kola) in normal Nigerians. Nineteen undergraduate male students (17-25 years) were used for the study which involved the consumption of Garcinia Kola (15 grammes per subject) and the measurement of peak expiratory flow rate (PEFR), forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) at intervals of 30 minutes up to a maximum of 90 minutes. Each subject served as his own control. There was time-dependent increases in the measured parameters. However, only the PEFR showed significant (p < 0.05) increase and this was observed at the 60th minute. This probably shows a mild bronchodilator effect of this Kola.

1. The responsiveness of aortic rings from 4 and 12 week streptozotocin-induced diabetic rats to D600 (Gallopamil) and nifedipine was studied. 2. The sensitivity and responsiveness to D600 were significantly enhanced (P < 0.05; 5-test, ANOVA; 9 d.f.) only in the 4 week diabetic preparations precontracted with noradrenaline. 3. Nifedipine-induced relaxations were significantly enhanced (P < 0.05-0.01; t-test, ANOVA; 8-12 d.f.) in all the diabetic (4 and 12 weeks) aortic preparations precontracted with both noradrenaline (10(-7) mol/L) and KCl (40 mmol/L) when compared with controls. 4. D600, unlike nifedipine, did not produce significant relaxation of diabetic aortic preparations precontracted with KCl (40 mmol/L) at both week 4 and 12 of the disease when compared with controls. 5. These results suggest that there is differential responsiveness of streptozotocin diabetic rat aorta to D600 and nifedipine.

1. The responsiveness of isolated aortic rings from 1, 4 and 12 week streptozotocin-induced diabetic rats to noradrenaline (NA) and 5-hydroxytryptamine (5-HT) were compared with those of non-diabetic controls under standard organ bath procedure. 2. There were significant increases in the maximum contractile responses to both agents after 1 and 4 weeks but not after 12 weeks of diabetes mellitus. 3. The variable responses show that duration-dependent functional changes occur in the course of streptozotocin diabetes in rats.

Nutritional and environmental factors are now known to affect the blood pressure and pulse rate of man. The present study investigated the effect of nicotiana tabacum on these parameters. Thirty-three male Nigerians (20-68 years) living in Calabar, Nigeria were used for this study. The subjects were treated at three different periods with snuff containing varied concentrations of Natron (0.5g and 10g/100g of snuff) and their systolic and diastolic pressures as well as pulse rate were measured at intervals of 30 minutes up to a maximum of 90 minutes. Each subject served as his own control. There were significant and time-dependent increases in the parameters measured. In addition, the increases were more marked in the absence of Natron than in its presence.