Protective effect of magnesium acetyltaurate against endothelin-induced retinal and optic nerve injury.

Vascular dysregulation has long been recognized as an important pathophysiological factor underlying the development of glaucomatous neuropathy. Endothelin-1 (ET1) has been shown to be a key player due to its potent vasoconstrictive properties that result in retinal ischemia and oxidative stress leading to retinal ganglion cell (RGC) apoptosis and optic nerve (ON) damage. In this study we investigated the protective effects of magnesium acetyltaurate (MgAT) against retinal cell apoptosis and ON damage. MgAT was administered intravitreally prior to, along with or after administration of ET1. Seven days post-injection, animals were euthanized and retinae were subjected to morphometric analysis, TUNEL and caspase-3 staining. ON sections were stained with toluidine blue and were graded for neurodegenerative effects. Oxidative stress was also estimated in isolated retinae. Pre-treatment with MgAT significantly lowered ET1-induced retinal cell apoptosis as measured by retinal morphometry and TUNEL staining. This group of animals also showed significantly lesser caspase-3 activation and significantly reduced retinal oxidative stress compared to the animals that received intravitreal injection of only ET1. Additionally, the axonal degeneration in ON was markedly reduced in MgAT pretreated animals. The animals that received MgAT co- or post-treatment with ET1 also showed improvement in all parameters; however, the effects were not as significant as observed in MgAT pretreated animals. The current study showed that the intravitreal pre-treatment with MgAT reduces caspase-3 activation and prevents retinal cell apoptosis and axon loss in ON induced by ET1. This protective effect of ET1 was associated with reduced retinal oxidative stress.

Role of supraspinal vasopressin neurones in the effects of atrial natriuretic peptide on sympathetic nerve activity.

The aim of the present study was to determine if paraventricular-spinal vasopressin neurones participate in the sympatho-inhibitory effects of systemically administered atrial natriuretic peptide (ANP) on renal sympathetic nerve activity (RSNA). Experiments were carried out on male Sprague-Dawley rats anesthetized with 1.3 g/kg urethane. Changes in mean arterial pressure (mm Hg), heart rate (beats per minute) and RSNA (%) were measured following intravenous bolus administration of ANP (250 ng, 500 ng and 5 microg). Intrathecal application of selective V 1a receptor antagonist was performed to test for the involvement of supraspinal vasopressin pathways in mediating the effect on sympathetic outflow evoked by intravenous ANP administration. The results obtained demonstrated that both low and high doses of ANP caused renal sympathoinhibition (250 ng; - 7.5 +/- 1%, 500 ng; - 14.2 +/- 1%, 5 microg; - 16.4 +/- 2%), concomitant with vasodilation and bradycardia. After spinal vasopressin receptor blockade, the inhibitory effects of ANP were prevented and there was a small renal sympatho-excitation (250 ng; + 1.7 +/- 0.2%, 500 ng; + 6.1 +/- 0.03%, 5 microg; + 8.0 +/- 0.03%, P < 0.05). Therefore, the renal sympathetic nerve inhibition elicited by circulating ANP is dependent on the efficacy of a well established supraspinal vasopressin pathway. Since supraspinal vasopressin neurones without exception excite renal sympathetic neurones, it is suggested that ANP elicits this effect by activating cardiac vagal afferents that inhibit the spinally projecting vasopressin neurones at their origin in the paraventricular nucleus of the hypothalamus.

Tissue kallikrein increases duration of survival after prolonged coronary artery ligation in hypertensive rats.

There is evidence that the kallikrein-kinin system (KKS) is an important mediator in the regulation of blood pressure, and cardiac and renal hemodynamics. The present study was designed to examine the effect of tissue kallikrein and Trasylol, an inhibitor of tissue kallikrein, on survival time after continuous (prolonged) coronary artery ligation in spontaneously hypertensive rats (SHR). Tissue kallikrein (8 and 16 microg/kg, i.v.) treatment caused significant (p < 0.05) increases in the survival time of SHR as compared with the saline-treated control SHR. Trasylol pretreatment abolished (p < 0.05) the beneficial effect of tissue kallikrein on survival time. The tissue kallikrein treatment resulted in a significant (p < 0.05) reduction in systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR) of SHR as compared to the saline-treated control SHR. Trasylol (6 microg/kg) treatment antagonized the effects of tissue kallikrein associated with survival time, SBP, DBP and HR. Ligation of the coronary artery caused a significant (p < 0.001) reduction in the SBP, DBP and HR of SHR, when the mean values were compared between before coronary artery ligation and after coronary artery ligation. However, there was no significant difference (p > 0.05) in SBP, DBP and HR between saline and kallikrein-treated SHR after coronary artery ligation. These findings may suggest that tissue kallikrein is able to act as a cardioprotective agent as demonstrated by an increase in survival time of SHR with prolonged coronary artery ligation.

ANN modeling of the penetration across a polydimethylsiloxane membrane from theoretically derived molecular descriptors.

A quantitative structure-permeability relationship was developed using Artificial Neural Network (ANN) modeling to study penetration across a polydimethylsiloxane membrane. A set of 254 compounds and their experimentally derived maximum steady state flux values used in this study was gathered from the literature. A total of 42 molecular descriptors were calculated for each compound. A genetic algorithm was used to select important molecular descriptors and supervised ANN was used to correlate selected descriptors with the experimentally derived maximum steady-state flux through the polydimethylsiloxane membrane (log J). Calculated molecular descriptors were used as the ANN's inputs and log J as the output. Developed model indicates that molecular shape and size, inter-molecular interactions, hydrogen-bonding capacity of drugs, and conformational stability could be used to predict drug absorption through skin. A 12-descriptor nonlinear computational neural network model has been developed for the estimation of log J values for a data set of 254 drugs. Described model does not require experimental parameters and could potentially provide useful prediction of membrane penetration of new drugs and reduce the need for actual compound synthesis and flux measurements.

Theoretically-derived molecular descriptors important in human intestinal absorption.

A quantitative structure-human intestinal absorption relationship was developed using artificial neural network (ANN) modeling. A set of 86 drug compounds and their experimentally-derived intestinal absorption values used in this study was gathered from the literature and a total of 57 global molecular descriptors, including constitutional, topological, chemical, geometrical and quantum chemical descriptors, calculated for each compound. A supervised network with radial basis transfer function was used to correlate calculated molecular descriptors with experimentally-derived measures of human intestinal absorption. A genetic algorithm was then used to select important molecular descriptors. Intestinal absorption values (IA%) were used as the ANN's output and calculated molecular descriptors as the inputs. The best genetic neural network (GNN) model with 15 input descriptors was chosen, and the significance of the selected descriptors for intestinal absorption examined. Results obtained with the model that was developed indicate that lipophilicity, conformational stability and inter-molecular interactions (polarity, and hydrogen bonding) have the largest impact on intestinal absorption.

Effects of Eurycoma longifolia Jack (Tongkat Ali) on the initiation of sexual performance of inexperienced castrated male rats.

We studied the effects of Eurycoma longifolia Jack, commonly known as Tongkat Ali in Malaysia, on the initiation of sexual performance and the weights of sexual accessories in inexperienced castrated male rats. The doses of 200, 400 and 800 mg/kg body weight, which were extracted from E. longifolia Jack, were orally administered to the rats twice daily for 10 days prior to the tests and continued throughout the test period. Testosterone was used as a positive control after injecting 15 mg/kg daily subcutaneously for 32 days. Results showed that E. longifolia Jack produced a dose-dependent increase in sexual performance of the treated animals, but the E. longifolia Jack groups showed lower sexual performance in mounting, intromission and ejaculation than the testosterone group. Further results also showed that E. longifolia Jack promoted the growth of both ventral prostate and seminal vesicles as compared with the control, but the growth of sexual accessories at 800 mg/kg of butanol, methanol, water and chloroform fractions of E. longifolia Jack was less than that of testosterone treated group. The present study therefore gives further evidence of the folkuse of E. longifolia as an aphrodisiac.

Acute renal failure in 2K2C Goldblatt hypertensive rats during antihypertensive therapy: comparison of an angiotensin AT1 receptor antagonist and clonidine analogues.

1. This study compared the effect of a non-peptide angiotensin II receptor antagonist and a series of clonidine analogues on blood pressure and renal function in a two-kidney two-clip Goldblatt rat model of hypertension subjected to 2 weeks of dietary sodium deprivation. 2. Animals received either vehicle, the angiotensin II antagonist, ZD7155 or structural analogues derived from clonidine (AL-11, AL-12 and CN-10) at 10 mg kg-1 day-1 for 4 days. 3. All groups of rats had systolic blood pressure in the hypertensive range (160-180 mmHg). ZD7155 caused a 33-mmHg fall in blood pressure (P < 0.05) and raised plasma urea and creatinine four- to six-fold. 4. AL-12 decreased blood pressure by 30 mmHg (P < 0.05), but had no effect on water intake, urine flow or plasma urea and creatinine. AL-11 and CN-10 had minimal effects on blood pressure and water intake and while CN-10 decreased urine flow on the third treatment day, AL-11 markedly reduced urine flow by some 70%. 5. These data show that in this sodium deficient renovascular model of hypertension, blockade of angiotensin II receptors normalizes blood pressure but causes renal failure, whereas the vasodepressor action of the clonidine analogue AL-12 occurs without detriment to renal function. These findings imply that angiotensin II receptor antagonists could lead to renal failure if used as antihypertensive agents in renovascular hypertension whereas this would be avoided with the use of clonidine-like analogues.

The effect of bradykinin and its antagonist on survival time after coronary artery occlusion in hypertensive rats.

It is known that BK does play a role in the cardioprotective effect of angiotensin converting enzyme (ACE) inhibitors. The present study therefore was conducted to examine the effects of bradykinin (BK) and its antagonist on survival time in spontaneously hypertensive rats (SHR) with coronary artery ligation for 15 min and continuously. We also evaluated the heart rate and blood pressure (BP) in the presence and absence of BK and BK2 receptor antagonist, D-Arg-[Hyp-D-Phe7]BK. Coronary artery was ligated in anaesthetized rats and they were artificially ventilated with room air (stroke volume, 4 ml; 48 strokes/min) as described by the previous investigators. Lead II elecrocardiogram (ECG) was recorded from subcutaneous steel needle electrodes. Results of this investigation indicated that BK treatment 4 microg/kg (i.v.) and 8 microg/kg (i.v.) caused significant (P < 0.05) increase in survival time in SHR with coronary artery ligation for 15 min and continuously as compare to their respective saline-treated controls. However, BK antagonist treatment 4 microg/kg (i.v.) abolished the increase in survival time caused by BK treatment. The mean values of survival time between the saline-treated and BK antagonist plus BK-treated rats did not differ significantly (P > 0.05). The heart rate and BP responses were greatly reduced (P < 0.001) in the presence of coronary artery ligation. These findings suggest that BK might have cardioprotective effect to increase the survival time in rats by activating BK2 receptors after coronary artery ligation.

Altered cardiac tissue and plasma kininogen levels in hypertensive and diabetic rats.

The present investigation was aimed at evaluating the cardiac and total plasma kininogen levels, as well as LVWT in hypertensive and diabetic rats. STZ-induced diabetes produced a significant (P < 0.001) rise in mean arterial blood pressure (BP). The LVWT increased (P < 0.001) in SHR with and without diabetes) and diabetic WKYR. The cardiac tissue, as well as total plasma kininogen levels fell significantly (P < 0.001) in diabetic WKYR and SHR with and without diabetes compared to the control WKYR. These findings suggest that reduced kininogen levels may indicate a deficiency in kinin generation in the heart and in the peripheral circulation in diabetic and hypertensive rats. This effect may contribute to the development of LVH.

Anti-inflammatory activity of Crinum asiaticum plant and its effect on bradykinin-induced contractions on isolated uterus.

Crinum asiaticum Linn plant is used in Malaysia as a rheumatic remedy and to relieve local pain. In the present study, we examined the anti-inflammatory effects of this plant extract on carrageenan-induced hind paw oedema in mice. C. asiaticum was serially extracted with petroleum ether, followed by chloroform and lastly, methanol. The chloroform and methanol extracts of the plant given orally (50 mg kg-1) caused significant (p < 0.05; n = 7) reduction in paw oedema but the petroleum ether extract did not induce significant effect (p > 0.05) on paw oedema. The methanol extract was then dissolved in water and extracted consecutively with chloroform, ethyl acetate and butanol. The chloroform fraction of methanol extract (CFME) treatment (50 mg kg(-1)) significantly reduced (p < 0.05; n = 7) the acute paw oedema. This may indicate that active anti-inflammatory compounds are present in the CFME. In an attempt to study the mechanism of action of its anti-inflammatory activity, the effects of CFME on BK- and histamine-induced contractions were investigated in isolated rat uterus and guinea-pig ileum preparations, respectively. It was found that CFME caused dose-dependent reduction (p < 0.05; n = 6) of the contractile response induced by BK and shifted the log dose-response curve of histamine to the right. The present findings suggest that C. asiaticum possessed an anti-inflammatory activity as suggested by its use in traditional medicine. The anti-inflammatory activity of this plant could not have been due to its anti-bradykinin activities as CFME non-specifically inhibited BK-induced contraction. It also suggest that CFME may contain compound(s) with anti-histaminic properties. The significance of these findings is discussed.

Left ventricular hypertrophy and its relation to the cardiac kinin-forming system in hypertensive and diabetic rats.

We investigated the cardiac tissue kallikrein and kininogen levels, left ventricular wall thickness and mean arterial blood pressure of Wistar Kyoto and spontaneously hypertensive rats with and without streptozotocin-induced diabetes. The mean arterial blood pressure was highly elevated (P<0.001) in Wistar Kyoto diabetic and spontaneously hypertensive diabetic rats as compared with their respective controls. The cardiac tissue kallikrein and kininogen levels were reduced significantly (P<0.001) in diabetic Wistar Kyoto, spontaneously hypertensive and diabetic spontaneously hypertensive compared with Wistar Kyoto control rats. In addition, the left ventricular thickness was found to be increased (P<0.001) in diabetic Wistar Kyoto and spontaneously hypertensive rats in the presence and in the absence of diabetes. Our results indicate that reduced activity of the kinin-forming system may be responsible for inducing left ventricular hypertrophy in the presence of raised mean arterial blood pressure in diabetic and hypertensive rats. Thus, the kinin-forming components might have a protective role against the development of left ventricular hypertrophy. The possible significance of these findings is discussed.

Potentiation of bradykinin-induced responses by indomethacin in the intact and denuded epithelium of guinea pig tracheal preparations.

We studied the effect of indomethacin, a cyclooxygenase inhibitor, on bradykinin-induced responses in the intact and denuded epithelium of the isolated tracheal smooth muscle in guinea pigs. Epithelium removal alone did not alter the responsiveness to bradykinin. Indomethacin (2.8 microM) enhanced the sensitivity to bradykinin of both intact and denuded preparations. This finding suggests that the tracheal epithelial may have no protective effect on the contractile responses induced by bradykinin. This may be due to the presence of high amounts of bradykinin-inactivating enzymes in the tracheal smooth muscle. Indomethacin-medicated potentiation caused by bradykinin in epithelium intact and denuded preparations may be an indication of removal of the bronchodilator prostaglandin biosynthesis. The significance of these findings is discussed.

The kinin antagonist hoe 140 reduces acute paw oedema in rats caused by carrageenan, bradykinin and kaolin.

The present study aimed to evaluate the effect of Hoe 140, a BK receptor B(2) antagonist, on acute oedema induced by carrageenan, BK and kaolin in male Wistar Kyoto rats. Hoe 140 (0.2 mg/kg and 20 mg/kg) given ip caused significant (p<0.05 and p<0.01) inhibition of carrageenan and BK-induced paw oedema. This suggests that BK is the prime inflammatory mediator of carrageenan oedema, and that it is also a specific blocker of oedema caused by BK. Furthermore, Hoe 140 was found to be less effective in reducing kaolin-induced oedema in rats. This might reflect that BK is not a prime inflammatory mediator of kaolin-induced oedema. The possible significance of these findings is discussed.

Pro-inflammatory properties of the kallikrein-kinin system: potential for new drug therapy.

Components of the kallikrein-kinin system are activated in response to noxious stimuli (chemical, physical or bacterial), which may lead to excessive release of kinins in the synovial joints that may produce inflammatory joint disease. The inflammatory changes observed in synovial tissue may be due to activation of B(2) receptors. Kinins also stimulate the synthesis of other pro-inflammatory agents (PGs, LTs, histamine, EDRF, PGI(2) and PAF) in the inflamed joint. B(2)-receptor antagonists may provide valuable agents as new analgesic drugs. Furthermore, it is suggested that substances to reduce activation of the kallikrein-kinin system (KKS) may provide a pharmacological basis for the synthesis of novel antirheumatic or anti-inflammatory drugs.

Patterns of activity in sympathetic postganglionic nerves to skeletal muscle, skin and kidney during stimulation of the medullary raphe area of the rat.

In chloralose/urethane anaesthetised rats multi and single fibre activity was recorded in renal sympathetic nerves and in sympathetic nerves to skeletal muscle and to the skin of the hind limb. The activity was pulse-modulated and inhibited by stimulating baroreceptors with an increase in blood pressure following i.v. phenylephrine. The study investigated the effect of stimulating medial regions of the lower brainstem on activity in these sympathetic nerves. Comparing renal nerve and skin sympathetic nerve activity two main patterns of response were seen, either an inhibition of both or excitation of both. In contrast when comparing renal nerve activity with muscle sympathetic nerve activity a third pattern was evoked from a few sites located in nucleus raphe obscurus, an inhibition of renal nerve activity and excitation of muscle sympathetic activity. The latter is the same pattern as that evoked in the cat. Unlike the cat, however, the decerebrate rat was not observed to generate this differential pattern of activity either spontaneously or following the administration of physostigmine. It is argued that the activation of cell bodies located in raphe obscurus is responsible for inducing a pattern of sympathetic activity similar to that occurring during paradoxical sleep in other animals.

A comparison of the effects of intrathecally administered 5-hydroxytryptamine and thyrotropin-releasing hormone on renal and muscle sympathetic nerve activity.

Thyrotropin-releasing hormone (TRH) has been shown to coexist with 5-hydroxytryptamine (5-HT) in spinally projecting raphe neurones, some of which terminate in the sympathetic nuclei of the spinal cord. In an attempt to mimic the actions of these neurones, the effects of intrathecal administration of 5-HT was compared to that of TRH on activity in two sympathetic postganglionic nerves, the renal nerve and sympathetic fibres to skeletal muscle of the hind limb. In chloralose-urethane anaesthetised rats intrathecal infusion of TRH at the T9 level in doses of 5-30 micrograms increased activity in renal nerve as did low doses (20-100 micrograms) of 5-HT. In contrast, intrathecal infusion of TRH in doses of 5-30 micrograms and 5-HT in doses of 20-800 micrograms at L4 level decreased activity in sympathetic fibres to muscle. The results show that TRH, unlike 5-HT, has an excitatory but not an inhibitory action on renal sympathetic activity whereas both substances have only an inhibitory action on muscle sympathetic activity.

Excitatory and inhibitory actions of intrathecally administered 5-hydroxytryptamine on sympathetic nerve activity in the rat.

In chloralose/urethane anaesthetised rats the effect of intrathecal (i.t.) administration of 5-hydroxytryptamine (5-HT) on activity in sympathetic nerves to the kidney was studied. Intrathecal 5-HT (20-100 micrograms, 10 microliter) increased activity in renal nerves (mean change for 50 micrograms dose 157% +/- 39). At higher doses of 5-HT the initial excitatory response was followed by inhibition 64% +/- 15. Intrathecally, alpha methyl 5-HT (10-400 micrograms) mimicked only the inhibitory action of 5-HT in a dose-dependent manner. These inhibitions were completely antagonised by i.t. ketanserin (25-100 micrograms). Intrathecal 2 methyl 5-HT (10-200 micrograms) had little effect on renal nerve activity. Intrathecal 5 carboxyamidotryptamine (10-200 micrograms) had a more powerful excitatory action on renal nerve activity than did 5-HT. Intrathecally, 8 OH-DPAT (10-200 micrograms) was without effect. Neither the inhibitory action of 5-HT or the excitatory action was affected by i.t. ICS 205 930 (1-10 micrograms) or MDL 72222 (200 micrograms). It was concluded that the actions of 5-HT were within the spinal cord and that the inhibitory effect on sympathetic activity is mediated via a 5-HT2-like receptor whilst the receptors involved in the excitatory action of 5-HT are not M- or 5-HT3-like but appear to be more like the 5-HT1 subtype.

The action of a substance P antagonist on sympathetic nerve activity in the rat.

In anaesthetised rats recordings were made of sympathetic activity in renal nerves whilst studying the effects of intrathecal injection of the substance P antagonist (D-Arg1, D-Pro2, D-Trp7,9, Leu11)-substance P on the responses to, stimulation of the ventrolateral medulla, to intrathecal injection of substance P, serotonin and glutamate. All responses were abolished by the antagonist.

Autonomic neuropathy in the alloxan-diabetic rat.

This study was designed to correlate functional and ultrastructural examination of the innervation of the atria and vas deferens of rats with long-term alloxan-induced diabetes mellitus. After 7-8 months of diabetes the responses of preparations in vitro to nerve stimulation and to exogenous autonomic transmitter were compared with those from age-matched controls. Right atria from controls and diabetics were equally responsive to noradrenergic nerve stimulation and to exogenous noradrenaline. Left atria from diabetic rats were supersensitive to both noradrenaline and acetylcholine. The left atria gave normal inotropic responses to noradrenergic nerve stimulation but responses to cholinergic nerve stimulation were absent. The vasa deferentia from both groups gave similar responses to noradrenergic nerve stimulation and to noradrenaline. The electron microscope revealed many abnormal, degenerate noradrenergic nerve profiles in both right atria and vas deferens. No cholinergic terminals were found in the right atria. These findings indicate that this form of experimental diabetes causes parasympathetic denervation of the heart with some indications of degeneration of sympathetic nerves.

A method for the measurement of growth of noradrenergic axons in tissue culture and the effects of colchicine thereon.

Explants of superior cervical ganglia from 2-day-old mice have been cultured on collagen-coated glass coverslips in modified L15 culture medium (with foetal calf serum) for 5-day periods. Noradrenergic axons were visualized by uptake of alpha-methyl noradrenaline and subsequent treatment with buffered 2% glyoxylic acid for fluorescence microscopy. An index of axon growth was obtained by a point-counting method using a coherent multi-purpose test system graticule. The growth indices thus obtained for a sample of control cultures were normally distributed. Examination of a single batch of cultures showed that good agreement was obtained between successive counts by one observer and between counts by two observers. The method gave sufficient resolution to detect with statistical significance a small inhibition of growth by colchicine at 2 ng/ml and to obtain a concentration-effect plot for this drug. The acceptability of the method for routine use is discussed.