Role of hydrogen sulfide production in inhibitory action of L-cysteine on isolated porcine irides.

PURPOSE: To investigate the direct pharmacological actions of L-cysteine, a substrate for the production of H(2)S, on isolated porcine irides in the presence of tone induced by muscarinic receptor stimulation. Furthermore, we examined the underlying mechanism of action of L-cysteine in this smooth muscle. METHODS: Isolated porcine iris muscle strips were set up in organ baths containing oxygenated Krebs buffer solution at 37 degrees C. Longitudinal isometric tension was recorded via a grass FT03 Force-Displacement Transducer and analyzed using the PolyView computer software. The relaxant action of L-cysteine on carbachol-induced tone was studied in the absence and presence of inhibitors of enzymes of the biosynthetic pathways for H(2)S, and prostanoids. In addition, we also examined the effect of ATP-sensitive K(+) (K(ATP)) channel antagonist, glibenclamide on relaxations induced by L-cysteine. RESULTS: L-cysteine (30 nM-1 mM) evoked concentration-dependent relaxations of carbachol-induced tone in isolated porcine irides, reaching a maximum inhibition of 43% at 1 mM. This response was enhanced significantly (P < 0.001) in the presence of the COX inhibitor, flurbiprofen (3 microM). Additionally,in the presence of flurbiprofen, the H(2)S donors, NaHS and Na(2)S, mimicked the relaxations produced by L-cysteine, yielding IC(50) values of 5.8 microM and 180 microM, respectively. Both the inhibitor of cystathionine beta-synthase, AOA (30 microM) and the K(ATP) channel antagonist, glibenclamide (100 microM) caused significant (P < 0.001) rightward shifts in the concentration-response curves to L-cysteine and attenuated the maximum inhibitory response. Conversely, the inhibitor of cystathionine gamma-lyase, PAG (1 mM) blocked only relaxations caused by high concentrations of L-cysteine (> 100 microM). CONCLUSIONS: The inhibitory action of L-cysteine in isolated porcine irides is dependent on the endogenous production of H(2)S by cystathionine gamma-lyase and cystathionine beta-synthase. Furthermore, prostanoids and K(ATP) channels are involved in the inhibitory action of L-cysteine in this tissue.

Effect of hydrogen sulfide on cyclic AMP production in isolated bovine and porcine neural retinae.

Hydrogen sulfide (H(2)S) has been reported to exert pharmacological effects on neural and non-neural tissues from several mammalian species. In the present study, we examined the role of the intracellular messenger, cyclic AMP in retinal response to H(2)S donors, sodium hydrosulfide (NaHS) and sodium sulfide (Na(2)S) in cows and pigs. Isolated bovine and porcine neural retinae were incubated in oxygenated Krebs buffer solution prior to exposure to varying concentrations of NaHS, Na(2)S or the diterpene activator of adenylate cyclase, forskolin. After incubation at different time intervals, tissue homogenates were prepared for cyclic AMP assay using a well established methodology. In isolated bovine and porcine retinae, the combination of both phosphodiesterase inhibitor, IBMX (2 mM) and forskolin (10 microM) produced a synergistic increase (P < 0.001) in cyclic AMP concentrations over basal levels. NaHS (10 nM-100 microM) produced a time-dependent increase in cyclic AMP concentrations over basal levels which reached a maximum at 20 min in both bovine and porcine retinae. At this time point, both NaHS and Na(2)S (10 nM-100 microM) caused a significant (P < 0.05) dose-dependent increase in cyclic AMP levels in bovine and porcine retinae. For instance, NaHS (100 nM) elicited a four-fold and three-fold increase in cyclic AMP concentrations in bovine and porcine retinae respectively whilst higher concentrations of Na(2)S (100 microM) produced a much lesser effect in both species. In bovine and porcine retinae, the effects caused by forskolin (10 microM) on cyclic AMP production were not potentiated by addition of low or high concentrations of both NaHS and Na(2)S. We conclude that H(2)S donors can increase cyclic AMP production in isolated neural retinae from cows and pigs. Bovine retina appears to be more sensitive to the stimulatory effect of H(2)S donors on cyclic nucleotide production than its porcine counterpart indicating that species differences exist in the magnitude of this response. Furthermore, effects produced by forskolin on cyclic AMP formation were not additive with those elicited by H(2)S donors suggesting that these agents may share a common mechanism in their action on the adenylyl cyclase pathway.

Regulation of norepinephrine release from isolated bovine irides by histamine.

In the present study, we investigated the effect of histamine on sympathetic neurotransmission from isolated, superfused bovine irides. We also studied the pharmacology of prejunctional histamine receptors that regulate the release of norepinephrine (NE) from this tissue. The effect of exogenous histamine and various histamine receptor agonists was examined on the release of [(3)H]-norepinephrine ([(3)H]NE) triggered by electrical field stimulation using the Superfusion Method. Histamine receptor agonists caused a concentration-dependent inhibition of field-stimulated [(3)H]NE overflow with the following rank order of potency: imetit > histamine > R-alpha-methylhistamine. In all cases, the inhibitory action of histamine receptor agonists was attenuated at high concentrations of these compounds. The histamine receptor antagonists, clobenpropit (H(3)-antagonist/H(4)-agonist) and thioperamide (H(3)-antagonist) blocked the inhibitory response elicited by R-alpha-methylhistamine and imetit, respectively. Inhibitory effects of R-alpha-methylhistamine and clonidine were not additive suggesting that prejunctional H(3)- and alpha(2)-adrenoceptors coexist at neurotransmitter release sites. We conclude that histamine produces an inhibitory action on sympathetic neurotransmission in the bovine iris, an effect mimicked by selective H(3)-receptor agonists and blocked by H(3)-antagonists.

Pharmacological consequences of oxidative stress in ocular tissues.

The eye is a unique organ because of its constant exposure to radiation, atmospheric oxygen, environmental chemicals and physical abrasion. That oxidative stress mechanisms in ocular tissues have been hypothesized to play a role in diseases such as glaucoma, cataract, uveitis, retrolental fibroplasias, age-related macular degeneration and various forms of retinopathy provides an opportunity for new approaches to their prevention and treatment, In the anterior uvea, both H2O2 and synthetic peroxides exert pharmacological/toxicological actions tissues of the anterior uvea especially on the sympathetic nerves and smooth muscles of the iris-ciliary bodies of several mammalian species. Effects produced by peroxides require the presence of trace amounts of extracellular calcium and the functional integrity of mitochondrial calcium stores. Arachidonic acid metabolites appear to be involved in both the excitatory action of peroxides on sympathetic neurotransmission and their inhibitory effect on contractility of the iris smooth muscle to muscarinic receptor activation. In addition to the peroxides, isoprostanes (products of free radical catalyzed peroxidation of arachidonic acid independent of the cyclo-oxygenase enzyme) can also alter sympathetic neurotransmission in anterior uveal tissues. In the retina, both H2O2 and synthetic peroxides produced an inhibitory action on potassium depolarization induced release of [3H] D-aspartate, in vitro and on the endogenous glutamate and glycine concentrations in vivo. Effects caused by peroxides in the retina are mediated, at least in part, by second messengers such as nitric oxide, prostaglandins and isoprostanes. The ability of H2O2 to alter the integrity of neurotransmitter pools from sympathetic nerves in the anterior uvea and glutaminergic nerves in the retina could underlie its role in the etiology of glaucoma.

Arachidonic acid metabolites and peroxide-induced inhibition of [3H]D-aspartate release from bovine isolated retinae.

We have previously shown that hydrogen peroxide (H2O2) can inhibit K+-depolarization-evoked [3H]D-aspartate release from bovine isolated retinae. In the present study, we investigated the role of arachidonic acid metabolites in the inhibitory response elicited by H2O2 in the bovine retinae. Furthermore, we examined the direct effect of H2O2 on the production of prostaglandins and isoprostanes in this tissue. Isolated bovine retinae were prepared for studies of [3H]D-aspartate release using the Superfusion Method. Release of [3H]D-aspartate was elicited by Krebs solution containing an iso-osmotic concentration of KCl (50 mM). A direct action of H2O2 on prostaglandin E2 (PGE2) and 8-isoprostane F2alpha (8-iso-PGF2alpha) was also measured by enzyme-linked immunosorbant assay (ELISA). The cyclooxygenase inhibitor, flurbiprofen (3 microM), or the thromboxane-receptor antagonist, SQ 29548 (10 microM) had no significant (p > 0.05) effect on K+-evoked [3H]D-aspartate release. On the other hand, both flurbiprofen (3 microM) and SQ 29548 (10 microM) blocked the inhibition of K+-evoked [3H]D-aspartate induced by H2O2 (30 microM). In concentrations up to 100 microM, H2O2 caused an increase in PGE2 and 8-iso-PGF2alpha over basal levels. For instance, H2O2 (100 microM) increased PGE2 and 8-iso-PGF2alpha over basal levels by 348 +/- 41% and 185 +/- 26 (n = 4), respectively. We conclude that the peroxide-mediated inhibition of [3H]D-aspartate may involve the production of prostaglandins and isoprostanes in the bovine isolated retinae.

Effect of peroxides on [3H]D-aspartate release from bovine isolated retinae.

In the present study, we investigated the effect of naturally occurring and synthetic peroxides on K+-depolarization-evoked release of [3H]D-aspartate from bovine isolated retinae. Furthermore, effect of peroxides on endogenous glutamate concentrations were measured by HPLC in bovine neural retinae and vitreous humor of eyes treated with hydrogen peroxide (H2O2) ex vivo. Both naturally occurring H2O2 (1-100 microM) and synthetic (cumene hydroperoxide, cuOOH; 1-100 microM) peroxides caused a concentration-dependent inhibition of K+-evoked [3H]D-aspartate release without affecting basal tritium efflux. The antioxidant, trolox (2 mM) prevented the inhibition of evoked [3H]D-aspartate overflow elicited by both H2O2 (30 microM) and cuOOH (10 microM). Inhibition of catalase by 3-amino-triazole (3- AT 100 mM) enhanced an inhibitory effect of a low concentration of H2O2 (1 microM) but antagonized the effect of H2O2 (30 microM) on K+-induced [3H]D-aspartate release. In ex vivo experiments, exogenously applied H2O2 (1-100 microM) also caused a concentration-related decrease in glutamate levels in the bovine retina. We conclude that peroxides can inhibit K+-evoked release of [3H]D-aspartate and also decrease endogenous glutamate concentrations in the bovine retina.

Glucose-deprivation-induced [3H]D-aspartate release from isolated bovine and human retinae.

The glucose deprivation-induced release of [3H]D-aspartate was studied in bovine and human retinas in a superfusion apparatus. [3H]D-aspartate release was significantly increased upon omitting glucose in the superfusion buffer. This effect was dependent on external Ca2+ because L- and N-type Ca2+-channel blockers, such as diltiazem (1 microM), nitrendipine (1 microM), and omega-conotoxin (100 nM), significantly reduced the effect of glucose-deprivation induced release of [3H]D-aspartate. Furthermore, while glutamate receptor agonists (L-glutamate, N-methyl-D-aspartate, but not kainate) potentiated the effects of glucose deprivation, antagonists (MK-801, MCPG, ifenprodil, and L-AP3) at these receptors blocked the glucose deprivation-induced release process. Taken together, these studies have demonstrated that under conditions of glucose deprivation, as may happen during ischemic events in vivo, the retinal glutamatergic nerve endings and/or glial cells promote the efflux of [3H]D-aspartate into the extracellular environment. This process appears to be receptor-mediated and dependent on extracellular Ca2+ and is similar to previous reports pertaining to brain tissues.

Safety and mechanism of appetite suppression by a novel hydroxycitric acid extract (HCA-SX).

A growing body of evidence demonstrates the efficacy of Garcinia cambogia-derived natural (-)-hydroxycitric acid (HCA) in weight management by curbing appetite and inhibiting body fat biosynthesis. However, the exact mechanism of action of this novel phytopharmaceutical has yet to be fully understood. In a previous study, we showed that in the rat brain cortex a novel HCA extract (HCA-SX, Super CitriMax) increases the release/availability of radiolabeled 5-hydroxytryptamine or serotonin ([3H]-5-HT), a neurotransmitter implicated in the regulation of eating behavior and appetite control. The aim of the present study was 2-fold: (a) to determine the effect of HCA-SX on 5-HT uptake in rat brain cortex in vitro; and (b) to evaluate the safety of HCA-SX in vivo. Isolated rat brain cortex slices were incubated in oxygenated Krebs solution for 20 min and transferred to buffer solutions containing [3H]-5-HT for different time intervals. In some experiments, tissues were exposed to HCA-SX (10 microM - 1 mM) and the serotonin receptor reuptake inhibitors (SRRI) fluoxetine (100 microM) plus clomipramine (10 microM). Uptake of [3H]-5-HT was expressed as d.p.m./mg wet weight. A time-dependent uptake of [3H]-5-HT occurred in cortical slices reaching a maximum at 60 min. HCA-SX, and fluoxetine plus clomipramine inhibited the time-dependent uptake of [3H]-5-HT. At 90 min, HCA-SX (300 microM) caused a 20% decrease, whereas fluoxetine plus clomipramine inhibited [3H]-5-HT uptake by 30%. In safety studies, acute oral toxicity, acute dermal toxicity, primary dermal irritation and primary eye irritation, were conducted in animals using various doses of HCA-SX. Results indicate that the LD50 of HCA-SX is greater than 5,000 mg/kg when administered once orally via gastric intubation to fasted male and female Albino rats. No gross toxicological findings were observed under the experimental conditions. Taken together, these in vivo toxicological studies demonstrate that HCA-SX is a safe, natural supplement under the conditions it was tested. Furthermore, HCA-SX can inhibit [3H]-5-HT uptake (and also increase 5-HT availability) in isolated rat brain cortical slices in a manner similar to that of SRRIs, and thus may prove beneficial in controlling appetite, as well as treatment of depression, insomnia, migraine headaches and other serotonin-deficient conditions.

Pharmacology of serotonin receptors modulating electrically-induced [3h]-norepinephrine release from isolated mammalian iris-ciliary bodies.

The pharmacology of prejunctional serotonin (5-HT) heteroreceptors that regulate the release of norepinephrine (NE) was studied in isolated bovine and human iris-ciliary bodies. The effect of exogenous 5-HT and various 5-HT receptor agonists was examined on the release of [3H]-norepinephrine ([3H]NE). Both 5-HT and m-chlorophenyl-biguanide (m-CPBG) caused enhancement in the field-stimulated release of [3H]NE from bovine tissues whereas 5-carboxamidotryptamine (5-CT) had no such effect. On the other hand, 8hydroxy-dipropylaminotetralin (8-OH-DPAT), caused a significant dose-related inhibition of evoked [3H]NE release. In human iris-ciliary bodies, 5-HT caused an inhibitory response on electrically-evoked [3H]NE release at low concentrations but produced an excitatory action at concentrations greater than 3 microM. To further confirm the nature of the prejunctional 5-HT heteroreceptors regulating [3H]NE release, effects of 5-HT3, 5-HT6 and 5-HT7 receptor antagonists were examined on a standard response to 5-HT. All antagonists examined caused a concentration-dependent inhibition of the response elicited by the standard 5-HT-induced response with the following rank order of potency (as measured by IC30 values): MDL-72222 >> SB-258719 > RO-04-690. We conclude that the excitatory prejunctional 5-HT heteroreceptors present in bovine iris-ciliary bodies belong to the 5-HT3 receptor subtype.