Mabayoje A. Oriowo

Indications of the mechanisms involved in improved sperm parameters by zinc therapy.

Objective: To determine possible indications of the mechanisms involved in improved sperm parameters by zinc therapy in asthenozoospermic men. Subjects and Methods: Forty-five men with asthenozoospermia (≧40% immotile sperm) were randomized into four therapy groups: zinc only: n = 11; zinc + vitamin E: n = 12 and zinc + vitamins E + C: n = 14 for 3 months, and non-therapy control group: n = 8. Semen analysis was done according to WHO guidelines. Malone dialdehyde, tumour necrosis factor-α (TNF-α), total antioxidant capacity, superoxide dismutase (SOD) and glutathione peroxidase were determined in the semen and serum. Antisperm antibodies IgG, IgM and IgA were evaluated by immunobeads. Sperm chromatin integrity was determined by acid denaturation by acridine orange and sperm apoptosis by light and electron microscopy. The effect of zinc on in vitro induced sperm oxidative stress by NADH was evaluated. Results: Asthenozoospermia was significantly associated with oxidative stress with higher seminal malone dialdehyde (8.8 vs. 1.8 mmol/l, p < 0.001) and TNF-α (60 vs. 12 pg/l, p < 0.001), and low total antioxidant capacity (1.8 vs. 8.4, p < 0.01), SOD (0.8 vs. 3.1, p < 0.01) and glutathione peroxidase (1.6 vs. 4.2, p < 0.05), compared to normozoospermia. Zinc therapy alone, in combination with vitamin E or with vitamin E + C were associated with comparably improved sperm parameters with less oxidative stress, sperm apoptosis and sperm DNA fragmentation index (DFI). On the whole, there was no difference in the outcome measures between zinc only and zinc with vitamin E and combination of vitamins E + C. In the in vitro experiment zinc supplementation resulted in significantly lower DFI (14–29%, p < 0.05) compared to zinc deficiency. Conclusion: Zinc therapy reduces asthenozoospermia through several mechanisms such as prevention of oxidative stress, apoptosis and sperm DNA fragmentation.

Atypical β‐adrenoceptors in the rat isolated common carotid artery

1 The possible existence of atypical β‐adrenoceptors in vascular smooth muscle of the rat common carotid artery was examined in this study. 2 Isoprenaline produced concentration‐dependent relaxation of noradrenaline (10−7 m) precontracted ring segments of the carotid artery. The relaxation was not affected by endothelial denudation. 3 Propranolol (10−8 m‐3 × 10−7 m) shifted the isoprenaline curve to the right without suppressing the maximum response. However, the slope (0.74) of the Schild plot was significantly (P < 0.05) less than 1. 4 Salbutamol (fe), CGP 12177 and BRL 37344 (ft) also concentration‐dependently relaxed noradrenaline precontracted artery segments. These relaxations were not affected by propranolol (10−7 m). Pretreatment of the artery segments with BRL 37344 did not desensitize the tissue to the relaxant effect of isoprenaline, CGP 12177 and salbutamol. 5 It is concluded that atypical β‐adrenoceptors exist in vascular smooth muscle of the common carotid artery.

Heterogeneity of Postjunctional α1-Adrenoceptors in Mammalian Aortae: Subclassification Based on Chlorethylclonidine, WB 4101 and Nifedipine

The effects of chlorethylclonidine, WB 4101 and nifedipine on norepinephrine-induced contractions of rat, guinea-pig, rabbit and dog aortae were investigated in order to characterize the alpha 1-adrenoceptor subtype(s) present in the aortae of these different species. The putative alpha 1A-adrenoceptor antagonist, WB 4101, was significantly more potent in the rat aorta compared to the rabbit, guinea-pig and dog aortae which were not significantly different from each other. The calcium channel antagonist, nifedipine (1 microM), had little or no effect on norepinephrine-induced contractions in aortic segments from the rabbit, guinea pig and dog; whereas in the rat aorta, nifedipine significantly inhibited the response to norepinephrine. Based on the studies with WB 4101 and nifedipine, alpha 1-adrenoceptors in rat aorta would be tentatively classified as alpha 1A-adrenoceptors, whereas those in the guinea-pig, rabbit and dog aortae would be of the alpha 1B-adrenoceptor subtype. The putative irreversible alpha 1B-adrenoceptor antagonist, chlorethylclonidine, inhibited the response to norepinephrine in aortae from all species, but to dramatically different degrees. The response to norepinephrine was inhibited by 500-fold and 450-fold by chlorethylclonidine in the rat and dog aortae, respectively, whereas in the guinea-pig and rabbit aortae, the potency of norepinephrine was reduced by only 3- and 20-fold, respectively. Thus, based on studies with chlorethylclonidine, alpha 1-adrenoceptors in the rat and dog aortae would be classified as alpha 1B-adrenoceptors (i.e., chlorethylclonidine-sensitive), whereas alpha 1A-adrenoceptors (chlorethylclonidine-insensitive) would predominate in the guinea-pig aorta, and possibly both alpha 1A- and alpha 1B-adrenoceptors would coexist in the rabbit aorta.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclo-oxygenase-2, endothelium and aortic reactivity during deoxycorticosterone acetate salt-induced hypertension.

Objective To test the hypothesis that the enhanced vascular responsiveness to norepinephrine that occurs during deoxycorticosterone acetate (DOCA)-salt induced hypertension is causally related to increased expression of cyclo-oxygenase (COX)-2 and oxidative stress, which diminishes the vasomodulatory influence of endothelium-derived nitric oxide. Methods Four groups of age-matched, male Sprague–Dawley rats were studied: Sham (normotensive); DOCA-salt (hypertensive); DOCA-salt treated with manganese(III) tetra(4-benzoic acid) porphyrin chloride [MnTBAP, an antioxidant; 15 mg/kg intraperitoneally (i.p.) for 21 days]; DOCA-salt treated with {N-[2-(cyclohexyloxy)-4-nitrophenyl]-methane sulfonamide} (NS-398, a COX-2 selective blocker; 5 mg/kg i.p. for 7 days). Contraction and relaxation were measured with FT03 force transducers coupled to a Grass polygraph in aortic rings bathed with physiologic salt solution (37°C) and bubbled with a 5%CO2/95%O2 gas mixture. Aortic sensitivities (pD2 values) to norepinephrine and serum isoprostanes (8-iso-prostaglandin F2α, a marker of oxidative stress) were measured for each experimental paradigm. Results NS-398 significantly reduced maximal contractions in response to norepinephrine in aortic rings from Sham (44 ± 3%) and DOCA-salt (96 ± 2%) group rats. Expression of COX-2 protein increased significantly in vessels from DOCA-salt rats compared with those from Sham group rats. Treatment of DOCA-salt rats with either MnTBAP or NS-398 alleviated hypertension, normalized aortic pD2 values for norepinephrine and restored serum 8-isoprostane concentrations towards those observed in Sham group rats. Conclusions COX-2 expression increases during DOCA-salt hypertension, and mediates production of factors that enhance rat aortic contractility in response to norepinephrine. Our data also suggest a role for increased oxidative stress, which is at least in part dependent on enhanced COX-2 expression, in the mechanism(s) of enhanced aortic contractility in response to norepinephrine during DOCA-salt hypertension.

Different atypical β-adrenoceptors mediate isoprenalineinduced relaxation in vascular and non-vascular smooth muscles

Atypical beta-adrenoceptors mediating smooth muscle relaxation were compared in several rat tissues including the distal colon, fundic strip, thoracic aorta and common carotid artery. Isoprenaline, CGP 12177 and BRL 37344 concentration-dependently relaxed longitudinal strips of the distal colon and fundus precontracted with carbachol (10(-6)M) as well as ring segments of the aorta and carotid artery precontracted with noradrenaline (10(-7)M). The rank order of potency was isoprenaline = BRL 37344 > CGP 12177 in the distal colon, isoprenaline = CGP 12177 > BRL 37344 in the aorta and carotid artery segments and isoprenaline > BRL 37344 > CGP 12177 in the fundic strip. Pretreatment with BRL 37344 induced a marked desensitization of the distal colon and fundic strips but not the aorta and carotid artery to isoprenaline. In the fundus and distal colon, pretreatment with CGP 12177 (10(-4)M abolished the effect of isoprenaline. Cyanopindolol (10(-6)M) shifted the isoprenaline curve to the right, without reducing the maximum response, in the distal colon fundic strip. -logKB values were 7.44 +/- 0.08 and 7.53 +/- 0.10 in the distal colon and fundic strip respectively. The same concentration of cyanopindolol did not inhibit the relaxant effect of isoprenaline in the aorta and carotid artery segments. It was therefore concluded that atypical beta-adrenoceptors in these preparations were not identical, indicating heterogeneity of atypical beta-adrenoceptors.

Amelioration of experimental colitis by Na-H exchanger-1 inhibitor amiloride is associated with reversal of IL-1β and ERK mitogen-activated protein kinase

Objective Na-H exchanger-1 (NHE-1) is induced in experimental colitis. It has not yet been established whether its inhibition ameliorates colitis. The effects of amiloride, an inhibitor of NHE-1, on colitis were examined in this study. Levels of mitogen-activated protein (MAP) kinases ERK, p38 and interleukin 1β which participate in intestinal inflammation were also examined in the colonic smooth muscle of rats with colitis. Material and methods Colitis was induced in Sprague-Dawley male rats by intrarectal administration of trinitrobenzenesulphonic acid (TNBS) and treated daily with amiloride (3, 5, and 10 mg/kg b.w. (body-weight), orally) starting 1 h before induction of colitis. The animals were sacrificed on day 5 post-TNBS. Controls received phosphate buffered saline in a similar manner. Results The highest dose of amiloride (10 mg/kg) was lethal. The lowest dose (3 mg/kg) was tolerated and was used in this study. Amiloride significantly reversed the colitis-reduced contractility and induction of MPO activity, NHE-1, IL-1β and ERK, but not of p38 in inflamed colonic smooth muscle. Splenomegaly, increased colonic mass and decreased sodium pump activity were significantly reversed by amiloride treatment. There was no recovery of b.w. loss in the treated colitic animals. Urine output was increased, whereas food and water intake remained unchanged following amiloride treatment. Conclusions These findings suggest that the beneficial effects of NHE-1 inhibition in experimental colitis are mediated through IL-1β and ERK MAP kinase.

Receptor Protection Studies with Phenoxybenzamine Indicate that a Single α1-Adrenoceptor May Be Coupled to Two Signal Transduction Processes in Vascular Smooth Muscle

Full alpha 1-adrenoceptor agonists, such as (-)-norepinephrine, produce vasoconstriction in the rat aorta primarily through the mobilization of intracellular stores of calcium, whereas partial alpha 1-adrenoceptor agonists, such as (-)-dobutamine, produce vasoconstriction primarily through the translocation of extracellular calcium. The different pools of calcium utilized by full and partial alpha 1-adrenoceptor agonists have been proposed to result from the activation of different alpha 1-adrenoceptor subtypes. The irreversible alpha 1-adrenoceptor antagonist, phenoxybenzamine, selectively eliminates only that component of an alpha 1-adrenoceptor-mediated response in the rat aorta that is dependent upon the mobilization of intracellular stores of calcium. In order to determine whether in the rat aorta there exist two distinct alpha 1-adrenoceptor subtypes linked separately to the mobilization of intracellular and extracellular calcium, we utilized the full and partial alpha 1-adrenoceptor agonists, (-)-norepinephrine and (-)-dobutamine, respectively, and the irreversible antagonist, phenoxy-benzamine, as pharmacologic tools in a classical receptor-protection study to probe these alpha 1-adrenoceptor-mediated vasoconstrictor process(es). Our logic was that if the intracellular and extracellular pools of calcium were coupled to different alpha 1-adrenoceptor subtypes, then only (-)-norepinephrine, and not (-)-dobutamine, would protect against alpha 1-adrenoceptor alkylation by phenoxybenzamine, since phenoxybenzamine only eliminates the process that depends on intracellular calcium. Alternatively, if both (-)-norepinephrine and (-)-dobutamine produce a similar degree of alpha 1-adrenoceptor protection against phenoxybenzamine, our results would suggest that a single alpha 1-adrenoceptor subtype exists which activates both the translocation of extracellular calcium and the mobilization of intracellular calcium. Phenoxybenzamine (30 nM) abolished contractions of the rat aorta produced by (-)-norepinephrine, as expected. Pretreatment of the tissues with either (-)-norepinephrine or (-)-dobutamine, at concentrations that produced equivalent degrees of alpha 1-adrenoceptor occupancy, resulted in equal protection against alkylation of alpha 1-adrenoceptors by phenoxybenzamine, arguing against the existence of two distinct alpha 1-adrenoceptor subtypes in the rat aorta. These results are consistent with our previous hypothesis that two different signal-transduction processes may be activated in the rat aorta by a single alpha 1-adrenoceptor population, with the intrinsic efficacy of the agonist determining which signal-transduction process is activated.

Late administration of Mn porphyrin-based SOD mimic enhances diabetic complications

Mn(III) N-alkylpyridylporphyrins (MnPs) have demonstrated protection in various conditions where increased production of reactive oxygen/reactive nitrogen species (ROS/RNS), is a key pathological factors. MnPs can produce both pro-oxidative and antioxidative effects depending upon the cellular redox environment that they encounter. Previously we reported (Free Radic. Res. 39: 81–8, 2005) that when the treatment started at the onset of diabetes, Mn(III) meso-tetrakis(N-methylpyridinium-2-yl)porphyrin, MnTM-2-PyP5+ suppressed diabetes-induced oxidative stress. Diabetes, however, is rarely diagnosed at its onset. The aim of this study was to investigate if MnTM-2-PyP5+ can suppress oxidative damage and prevent diabetic complications when administered more than a week after the onset of diabetes. Diabetes was induced by streptozotocin. The MnP-based treatment started 8 days after the onset of diabetes and continued for 2 months. The effect of the treatment on activities of glutathione peroxidase, superoxide dismutase, catalase, glutathione reductase, glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and glyoxalases I and II as well as malondialdehyde and GSH/GSSG ratio were determined in kidneys. Kidney function was assessed by measuring lysozyme and total protein in urine and blood urea nitrogen. Vascular damage was evaluated by assessing vascular reactivity. Our data showed that delayed administration of MnTM-2-PyP5+ did not protect against oxidative damage and did not prevent diabetic complications. Moreover, MnTM-2-PyP5+ contributed to the kidney damage, which seems to be a consequence of its pro-oxidative action. Such outcome can be explained by advanced oxidative damage which already existed at the moment the therapy with MnP started. The data support the concept that the overall biological effect of a redox-active MnP is determined by (i) the relative concentrations of oxidants and reductants, i.e. the cellular redox environment and (ii) MnP biodistribution.

Molecular basis for the effects of zinc deficiency on spermatogenesis: An experimental study in the Sprague-dawley rat model

Introduction: The objective of this study is to investigate the molecular mechanisms underlying the effects of zinc deficiency on spermatogenesis in the Sprague-Dawley (SD) rat. Materials and Methods: Three groups of eight adult male SD rats were maintained for 4 weeks on a normal diet as control, zinc deficient diet and zinc deficient diet with zinc supplementation of 28 mg zinc/kg body weight respectively. Using standard techniques, the following parameters were compared between the three groups of experimental animals at the end of 4 weeks: (a) Serum zinc, magnesium (Mg), copper (Cu), selenium (Se) and cadmium (Cd), (b) serum sex hormones, malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPX), (c) interleukin-4 (IL-4), tumor necrosis factor-alpha (TNF-α), Bcl-2, Bax and caspase-3 expression in the testes, (d) assessment of apoptosis of testicular cells using electron microscopy and (e) testicular volume and histology using the orchidometer and Johnsen score, respectively. Results: The zinc deficient group showed a reduction of testicular volume, serum concentrations of Zn, Cu, Se, Mg, SOD, GPX, IL-4, Bcl-2 and testosterone (P < 0.05), as well as increased levels of serum Cd, MDA and tissue TNF-α, Bax, caspase-3 and apoptosis of the germ cells (P < 0.05) compared with control and zinc supplementation groups. Conclusion: Zinc deficiency is associated with impaired spermatogenesis because of reduced testosterone production, increased oxidative stress and apoptosis. These findings suggest that zinc has a role in male reproduction.

Histamine-induced vasodilatation in the perfused mesenteric arterial bed of diabetic rats

In this study, we have examined the contribution of endothelium-derived nitric oxide (EDNO) and endothelium-derived hyperpolarizing factor (EDHF) to histamine-induced endothelium-dependent relaxation in the perfused mesenteric arterial bed of rats treated with streptozotocin (STZ) to induce diabetes. Histamine (10(-10) to 5 x 10(-6) mol) produced dose-dependent vasodilator response in the perfused mesenteric arterial bed of both control and diabetic animals. In order to isolate the EDHF component of histamine-induced vasodilator response, NG-nitro-L-arginine-methyl ester hydrochloride (L-NAME) (10(-4) M) and indomethacin (10(-6) M) were added to the Krebs solution throughout the experiment. Histamine induced vasodilatation in the perfused mesenteric bed in preparations from both control and diabetic rats. The vasodilator response to histamine was slightly potentiated in the diabetic rat preparations. Sodium nitroprusside (SNP)-induced relaxation was similar in diabetic and control rats. The role of EDNO in histamine-induced vasodilatation was also examined. Vascular preparations were perfused with 20 mM K(+)-Krebs solution to inhibit the EDHF contribution to histamine-induced vasodilatation. Under this condition, histamine induced a vasodilator response in preparations from both control and diabetic rats. However, relative to nondiabetic control animals, histamine-induced maximal response was significantly reduced in preparations from diabetic animals. Pretreatment with L-NAME (10(-4) M) attenuated histamine-induced vasodilatation in both preparations, indicating an NO-mediated vasodilator response. There was a significant attenuation in histamine-induced vasodilatation in the vascular preparations from diabetic rats. The vasodilator effect of calcium ionophore A23187 was investigated in preparations from control and diabetic rats to investigate receptor dysfunction associated with diabetes. A23187 (10(-11) to 10(-7) mol)-induced vasodilator response was not significantly different in the preparations from control and diabetic animals. In conclusion, our results indicated that histamine-induced vasodilation in the perfused mesenteric arterial bed of the STZ-induced diabetic rats is mediated by two vasodilator components, namely EDHF and EDNO. Under diabetic conditions, the EDHF component was potentiated, while histamine-induced vasodilation mediated by the EDNO component was attenuated.