Carbachol and nitric oxide inhibition of Na,K-ATPase activity in bovine ciliary processes.

PURPOSE: Nitric oxide (NO) donors and cholinergic agents decrease intraocular pressure, in part because they induce a decrease in aqueous humor production. Because Na,K-adenosine triphosphatase (ATPase) is involved in aqueous humor formation, this study was conducted to investigate the hypothesis that NO and cholinomimetics regulate its activity in bovine ciliary processes. METHODS: Bovine tissue slices were incubated with agonists and antagonists in a physiological buffer in vitro. Na,K-ATPase activity was determined by assaying hydrolysis of adenosine triphosphate (ATP) in suspended permeabilized tissue slices. RESULTS: Carbachol-induced inhibition of Na,K-ATPase activity correlated with increases in cGMP. This inhibition was abolished by the muscarinic blocker atropine, the NO inhibitor N(w)-nitro-L-arginine (L-NAME) and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Sodium nitroprusside (SNP) mimicked the actions of carbachol. The SNP-induced decrease in Na,K-ATPase activity correlated with an increase in cGMP and was also abolished by ODQ. Both 8-bromo (Br)-cGMP and okadaic acid also inhibited Na,K-ATPase activity. CONCLUSIONS: Carbachol-induced inhibition of Na,K-ATPase activity involves muscarinic receptor activation. That SNP mimics and L-NAME reverses carbachol's effect on Na,K-ATPase activity suggests that the actions of carbachol are mediated by NO. Carbachol's and SNP's effects on Na,K-ATPase activity involved soluble guanylate cyclase and cGMP. Inhibition of Na,K-ATPase activity by 8-Br-cGMP and okadaic acid indicates that protein phosphorylation events may mediate SNP-induced inhibition of Na,K-ATPase activity.

Carbachol inhibits Na(+)-K(+)-ATPase activity in choroid plexus via stimulation of the NO/cGMP pathway.

Secretion of cerebrospinal fluid by the choroid plexus can be inhibited by its cholinergic innervation. We demonstrated that carbachol inhibits the Na(+)-K(+)-ATPase in bovine choroid tissue slices and investigated the mechanism. Many of the actions of cholinergic agents are mediated by nitric oxide (NO), which plays important roles in fluid homeostasis. The inhibition of Na(+)-K(+)-ATPase was blocked by the NO synthase inhibitor [N(omega)-nitro-L-arginine methyl ester] and was quantitatively mimicked by the NO agonists sodium nitroprusside (SNP) and diethylenetriamine NO. Inhibition by SNP correlated with an increase in tissue cGMP and was abolished by 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase. Inhibition was mimicked by the protein kinase G activator 8-bromo-cGMP and by okadaic acid, an inhibitor of protein phosphatases 1 and 2A. cGMP-dependent protein kinase inhibitors Rp-8-pCPT-cGMP (0.5-5 microM) and KT-5823 (2.0 microM) did not block the effects of SNP, but higher concentrations of the more selective inhibitor (Rp-8-pCPT-cGMP) had a pharmacological inhibitory effect on Na(+)-K(+)-ATPase. The data suggest that cholinergic regulation of the Na(+)-K(+)-ATPase is mediated by NO and involves activation of guanylate cyclase and elevation of cGMP.

Identification of an extensive system of nitric oxide-producing cells in the ciliary muscle and outflow pathway of the human eye.

PURPOSE: Nitric oxide (NO) is an important intracellular and intercellular regulator in nerve tissue as well as in vascular endothelium, smooth muscle, and certain other cell types. In the ocular anterior segment, studies in rat have revealed a comparatively restricted distribution of the NO synthetic enzyme, NO synthase (NOS-1). Recent physiological studies, however, have shown that NO-mimicking nitrovasodilators can alter intraocular pressure in monkeys through an action (at least in part) on outflow resistance. The current studies, which determine the sites of NO synthesis in the human outflow pathway, were performed to provide an anatomic and biochemical explanation for these observations. METHODS: The occurrence and distribution of sites of ocular NO production in postmortem human eyes were determined using the isozyme-independent NO-indicator marker, NADPH-diaphorase (NADPH-d), together with direct biochemical assay and immunocytochemical localization of specific NO synthase (NOS) isoforms. RESULTS: The ciliary muscle (CM) and outflow pathway of normal human eyes were found to be substantially enriched in NADPH-d, the majority of which, by immunological analysis, consisted not of NOS-1 (brain or bNOS) but rather of NOS type 3 (endothelial cell or ecNOS). Biochemical analysis confirmed the NADPH-dependent production of NO and, unlike the primarily soluble distribution of bNOS, activity was found in both particulate and soluble fractions. NO reactivity was enriched in major sites of outflow resistance (trabecular meshwork and Schlemm's canal) as well as in collecting channels and was particularly prominent in the CM, especially in the anatomically distinct longitudinal subgroup of CM fibers that insert near (and may normally play a role in regulating resistance in) the trabecular meshwork. CONCLUSION: The human outflow pathway and CM are enriched sites of NO synthesis. These sites are anatomically distributed in such a manner as to suggest that one possible role for NO in the anterior segment may be to modulate outflow resistance either directly at the level of the trabecular meshwork, Schlemm's canal and collecting channels, or indirectly through alteration in the tone of the longitudinal CM.

Alterations of ocular nitric oxide synthase in human glaucoma.

PURPOSE: The authors recently reported that sites of outflow resistance and regulation in the human eye are highly enriched in the endothelial isoform of nitric oxide (NO) synthase (ecNOS). In vasculature, ecNOS activation is associated with altered vascular resistance and, in the stomach, defects in NO are associated with pathologic gastric hypertension. Because human glaucoma sometimes is associated with an increase in intraocular pressure and resistance changes in the aqueous outflow pathway (OP), the authors have investigated the possibility that alterations in NO or defects in NO-synthesizing tissues might exist in glaucomatous eyes. METHODS: Occurrence, distribution, and extent of sites of ocular NO production in the anterior segments of 16 normal eyes (10 patients) and 17 eyes (12 patients) with a history of primary open-angle glaucoma (POAG) were determined using the NO-indicator marker, NADPH-diaphorase (NADPH-d), which is known to colocalize with ecNOS immunoreactivity. Analysis of NADPH-d reactivity in tissues was combined with examination of overall cell distribution and use of neuron-specific markers. RESULTS: The ciliary muscle (CM) and OP of glaucomatous eyes showed marked differences in the amount and distribution of NADPH-d and alterations in gross structure. NADPH-d reactivity was decreased in trabecular meshwork (TM) and Schlemm's canal, and there was a marked reduction of anterior longitudinal CM fibers that insert near (and may normally regulate resistance in) the TM. CONCLUSION: Abnormalities in NO or NO-containing cells occur in POAG. These abnormalities may be causally related to glaucoma or may be a manifestation of the disease or its treatment. In either case, such alterations, together with recent pharmacologic studies showing that NO-mimicking nitrovasodilators alter IOP, indicate that NO has relevance to the course, treatment, or both, or some forms of this disease.

Magnetic resonance imaging of brain death.

OBJECTIVE: To demonstrate the magnetic resonance imaging (MRI) appearance of the brain in patients with clinical brain death. PATIENTS AND METHODS: High-field (1.5-T) MRI was performed on five patients who were subsequently proven clinically brain dead. Conventional T1-weighted and T2-weighted imaging was performed. RESULTS: MRI exhibited similar features for all of the patients: loss of the subarachnoid spaces of the brain; slow flow in the intracavernous and cervical internal carotid arteries; and loss of flow void in the small and large intracranial arteries, as well as in the major intracranial venous sinuses. The differentiation between grey and white matter in the brain was preserved, although the brain had a "supernormal" appearance due to the absence of cerebrospinal fluid and arterial pulsations. These findings have not been observed in MRI of comatose patients who were not clinically brain dead. CONCLUSION: With the advent of MRI-compatible ventilators and noninvasive monitoring, which facilitate imaging of patients under intensive care, MRI may offer another method of confirming the clinical diagnosis of brain death.

The cellular Na+ pump as a site of action for carbon monoxide and glutamate: a mechanism for long-term modulation of cellular activity.

Carbon monoxide (CO) induces a long-lasting alteration in cerebellar alpha 3-Na,K-ATPase independent of [Na+] but linked to cGMP synthesis and localized to Purkinje neurons. The action of CO is absent in Purkinje neuron-deficient mice, mimicked by 8-Br-cGMP, and blocked by inhibition of PKG. Glutamate (Glu) and metabotropic agonists mimic the action of CO, an effect that requires PKC and is associated with CO synthesis. These data suggest that CO regulates Na,K-ATPase through cGMP and PKG, and that Glu regulates CO through mGluRs. This system is also modulated by NMDA agonists and nitric oxide, possibly via Glu release, as well as by free radicals. These findings offer a mechanism by which CO, Glu, and free radicals can exert specific effects on synaptic transmission (relevant to long-term changes in cell excitability), as well as more general actions on energy metabolism (relevant to the pathophysiology of excitotoxicity).

Atrial natriuretic peptide modulates sodium and potassium-activated adenosine triphosphatase through a mechanism involving cyclic GMP and cyclic GMP-dependent protein kinase.

Prior studies indicate that the natriuretic effects of atrial natriuretic peptide (ANP) are due, in part, to an inhibition of the passive movement of sodium ions from tubular lumen through apical cation channels into renal tubular epithelium. The present work demonstrates that ANP also exerts a potent inhibitory effect on the active pumping of sodium ions by renal tubular sodium and potassium-activated adenosine triphosphatase (Na, K-ATPase). This action of ANP is relatively long lasting, is due to a change in enzyme Vmax and is specific for ouabain-sensitive activity. Enzyme modulation occurs with an EC50 for ANP of 0.1 nM, is independent of intracellular [Na+] and is associated with an increase in tissue cyclic GMP (cGMP), but not cyclic AMP (cAMP). Modulation of Na, K-ATPase by ANP is mimicked by 8-bromo-cGMP and okadaic acid (OA) and is blocked by KT 5823, a selective inhibitor of cGMP-dependent protein kinase (PKG), but not by KT 5720, a selective inhibitor of cyclic AMP-dependent protein kinase (PKA), which suggests that the action of ANP on the sodium pump involves cGMP-mediated changes in protein phosphorylation. Regulation of renal Na, K-ATPase activity also occurs with nitric oxide-generating compounds, such as nitroglycerin and sodium nitroprusside (SNP). However, the ability of ANP to modulate Na, K-ATPase does not appear to involve this latter pathway because the effects of ANP on the sodium pump cannot be blocked by either N omega-nitro-L-arginine, an inhibitor of NO synthase, or hemoglobin, which blocks NO through binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Topically applied hydralazine: effects on systemic cardiovascular parameters, blood-aqueous barrier, and aqueous humor dynamics in normotensive humans.

Local application of hydralazine has been found to alter intraocular pressure in animal eyes. This study was undertaken to determine, in normotensive humans, the effects of topically-applied hydralazine on systemic cardiovascular parameters, blood-aqueous barrier, and aqueous humor flow. Two different concentrations of hydralazine were used: 0.03%, and 0.1%. Twenty healthy normotensive subjects were studied. Blood pressure, pulse rate, and intraocular pressure were measured every hour for six hours after hydralazine or placebo was instilled into the conjunctival sac in a double-masked, randomized fashion. With either hydralazine dose, there was no significant change in systemic blood pressure or pulse. In addition, spectrophotometrically-measured polarization of fluorescence and flare failed to show any significant breakdown of the blood-aqueous barrier. In most individuals, application of hydralazine was followed by a brief, mild to moderate, conjunctival hyperemic response. Compared to placebo at the same time of day, a small increase in intraocular pressure was observed with 0.03% hydralazine (p < .05). At 0.1%, this increase tended to be less. There was no statistically significant difference in aqueous humor flow between hydralazine-treated and placebo-treated eyes at the lower concentration (3.20 +/- 0.63 vs. 3.05 +/- 0.61 microL/min (mean +/- S.D.) or at the higher concentration (3.37 +/- 0.53 vs. 3.28 +/- 0.60 microL/min) of hydralazine. These results suggest that acute topical application of 0.1% hydralazine to the eyes of normal humans does not cause clinically significant cardiovascular effects or significant ocular toxicity.

Nitric oxide, cGMP, and hormone regulation of active sodium transport.

The inter- and intracellular regulator nitric oxide (NO) has been suggested to play a role in the modulation of cellular excitability, but the mechanism(s) by which this occurs remain unclear. Using the kidney as a model system, we report here evidence that NO, produced in response to various hormones and cytokines, can effect long-term alterations in the activity of the membrane sodium pump. This regulation of Na, K-ATPase, which occurs in a system of NO-containing renal tubules, involves cGMP and cGMP-dependent protein kinase. Na, K-ATPase can also be regulated by alterations of cGMP initiated through NO-independent factors, such as atriopeptin, and in nonrenal tissues, such as cerebellum. Regulation of the membrane sodium pump by NO and cGMP, therefore, represents a mechanism for hormonal modulation of ion gradients, not only in kidney but also in other organs, including brain, where NO and cGMP play a prominent role in cellular function.

Photoaffinity labeling of a neuronal octopamine receptor.

The invertebrate aminergic neurotransmitter and neuromodulator octopamine (OA) acts at both neuronal and nonneuronal receptors that appear to have distinct pharmacological characteristics. The current work uses a potent and specific OA photoaffinity ligand, tritiated 2(2,6-diethyl-4-azidophenylimino)imidazolidine ([3H]NC-5Z), to identify and characterize a putative neuronal OA receptor protein in membranes from nerve tissue of the desert locust, Schistocerca gregaria. Under nonphotolyzing conditions, [3H]NC-5Z demonstrated high-affinity binding (KD = 2.5 +/- 0.3 nM; Bmax = 702 fmol/mg of protein) to a single class of noninteracting sites. The absolute and rank order potency of binding of both agonists and antagonists was highly correlated (r = 0.99) with their known ability to displace [3H]OA binding to locust neuronal membranes and was consistent with the labeling of a class 3 OA receptor. Under photolyzing conditions, [3H]NC-5Z demonstrated irreversible binding that was resistant to trichloroacetic acid and methanol, displaceable by OA and other octopaminergic agonists and antagonists, soluble in sodium dodecyl sulfate, and only sparingly soluble in nonionic detergents. Membrane-bound [3H]NC-5Z, solubilized with Nonidet P-40, bound specifically only to immobilized concanavalin A or lentil lectin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of photolyzed proteins under reducing conditions revealed a single peak of radioactivity with a molecular mass of 53 +/- 5 kDa. Taken together, these biochemical and pharmacological results support the identity of this protein peak as that of the neuronal OA3 receptor.

Monoamine uptake in insect synaptosomal preparations.

Biochemical studies of mammalian synaptosomal nerve fractions indicate the existence of multiple transporter proteins important for the termination of synaptic transmission by each of several monoamines. In insects, however, data on monoamine uptake has been limited to the study of whole tissue preparations, making it unclear whether neuronal (as opposed to glial) uptake is a significant mechanism in the insect. The present experiments elucidate the difficulties that have limited the use of insect synaptosomal preparations for characterizing amine reuptake. Key procedural improvements, including the utilization of carrier protein for tracer separation and the use of receptor antagonists to decrease non-specific membrane binding are described. With these and other modifications, reproducible sodium-dependent and cocaine-inhibitable dopamine and octopamine uptake are described in synaptosomal-containing preparations from insect brain and ganglia. These studies therefore support the existence of specific Na(+)-dependent uptake mechanisms in insect neurons.

Adrenergic regulation of aqueous outflow.

Epinephrine increased outflow facility and cyclic AMP in the in vitro perfused human anterior segment with a maximal facility increase of 44% occurring at approximately 2 x 10(-5) M. Cyclic AMP measured in the perfusate from anterior segments increased by 12-14 fold after administration of 10(-5) M epinephrine. Both the facility increase and cyclic AMP rise were blocked by the beta-2 selective antagonist, ICI118,551. While there was a correlation between the facility increase and elevation in cyclic AMP levels, the rise in cyclic AMP preceded the facility increase by about 1 hour, suggesting that the ultimate effect of epinephrine involved a rather slow event such as synthesis and release of prostaglandins or protein synthesis. Subsequent perfusion studies showed that very large concentrations of indomethacin were necessary to block the outflow facility effect of epinephrine, suggesting that prostaglandin synthesis did not underlie the facility effect in this system. However, 5 x 10(-5) M cyclohexamide blocked the effect on outflow facility of both epinephrine and forskolin, but did not block the rise in cyclic AMP. These studies suggest that protein synthesis may play a role in the epinephrine-induced facility increase at some point beyond the second messenger level.

Nitrovasodilator effects on intraocular pressure and outflow facility in monkeys.

The nitrovasodilators nitroglycerin (NTG) and hydralazine (HZN) were investigated for their effects on intraocular pressure (IOP) and outflow facility in the cynomolgus monkey eye in vivo. Fifty microliters of 0.1% HZN were administered topically in masked, randomized, crossover, placebo controlled trials, and by intracameral perfusion. Both NTG and HZN decreased IOP (mean IOP decrease with NTG 4.40 +/- 2.11 (S.E.M.) (+/- 4.7, S.D.) mmHg, P < 0.01, n = 5, with HZN 3.15 +/- 0.85 (S.E.M.) (+/- 2.3, S.D.) mmHg, P = 0.01, n = 7). Outflow facility increased by 92% (P < 0.05) after intracameral bolus injection of 10(-3) mol l-1 NTG, but not at lower doses. Intracameral HZN caused a significant increase in facility of outflow of 28% (P < 0.05) when perfused at a constant concentration of 10(-5) mol l-1, but not at certain other concentrations. These results demonstrate the capacity for topically applied NTG and HZN to reduce IOP in the living primate eye. They further suggest that, at certain drug doses, but not others, the IOP reduction may be mediated, in part, by an action on the outflow apparatus.

Cocaine as a naturally occurring insecticide.

Although cocaine has a fascinating and complex medicinal history in man, its natural function in plants is unknown. The present studies demonstrate that cocaine exerts insecticidal effects at concentrations which occur naturally in coca leaves. Unlike its known action on dopamine reuptake in mammals, cocaine's pesticidal effects are shown to result from a potentiation of insect octopaminergic neurotransmission. Amine-reuptake blockers of other structural classes also exert pesticidal activity with a rank order of potency distinct from that known to affect vertebrate amine transporters. These findings suggest that cocaine functions in plants as a natural insecticide and that octopamine transporters may be useful sites for targeting pesticides with selectivity toward invertebrates.

Characterization of insect neuronal octopamine receptors (OA3 receptors).

Octopamine receptors in the nervous tissue of insects were investigated using a ligand-receptor assay with [3H]NC-5Z or [3H]octopamine as the radioligands. Both ligands recognized a homogeneous class of binding sites with the properties of an octopamine receptor. This receptor has been characterized pharmacologically. Both high-affinity agonists (e.g. NC 7, K1 = 0.3 nM) and antagonists (e.g. maroxepine, K1 = 1.02 nM) were investigated. The neuronal octopamine receptor belongs to a receptor class that can easily be distinguished from peripheral octopamine receptors. Initial investigations of the localization of octopamine receptors within the insect nervous tissue show the greatest receptor density in the optic lobes.

Identification of octopaminergic agonists with selectivity for octopamine receptor subtypes.

Previous studies utilizing relatively nonselective octopamine (OA) agonists have suggested that differences may exist between the pharmacological responses of Gs-linked OA receptors found in various insect tissues and species. The present experiments identify and characterize two structurally related phenyl(imino)imidazolidine (PII) derivatives that demonstrate a significant degree of OA receptor subtype selectivity and, when used in concert, are able to distinguish tissue- and/or species-related differences in OA receptor response. In membrane preparations, 2,4,6-triethyl-PII (TEP) showed > 60-fold selectivity for activating adenylate cyclase in the firefly light organ vs. that in Manduca sexta nerve cord, whereas 2,4,6-trimethyl-PII (TMP) showed > 2-fold selectivity for activating adenylate cyclase in Manduca nerve cord vs. that in the light organ. Within the same tissue, the potency ratio for TEP/TMP was 29 in the light organ and 0.2 in the nerve cord, a degree of selectivity much greater than that of other PII analogs examined. Corresponding physiological measurements of octopaminergic response correlated well with biochemical measurements. TEP was 24-fold more potent than TMP for OA-mediated light emission in intact firefly light organs, whereas TMP was more than 3-fold more potent than TEP as an antifeeding agent in Manduca. These results indicate that the PII analogs, TEP and TMP, should prove quite useful for characterizing and differentiating OA receptor subtypes in various tissues and species. In addition, these findings provide further evidence that light emission in fireflies and disruption of feeding in Manduca involve Gs-linked OA receptors, subtypes of which are distributed differently in the end organs of these two species.

Influence of in vivo reproductive endocrine state on growth hormone-releasing factor stimulated adenylate cyclase activity in anterior pituitary fragments.

The growth hormone releasing factor (GRF) stimulated adenylate cyclase activity was evaluated in membrane fractions of anterior pituitary glands from male and female rats and in gonadectomized rats following in vivo gonadal steroid treatments. The baseline adenylate cyclase activity was lower in random estrous cycle female rats as compared with males. When estrous cycle phases were evaluated, diestrus 1 females had a lower basal activity as compared with males, while proestrus females were similar to males. The maximal stimulation of adenylate cyclase activity by GRF (i.e. Vmax) was lower in random estrous cycle female rats than in males. This lower Vmax, relative to males, was more pronounced in diestrus 1 than in proestrus females. There was little difference in the ED50 for GRF-stimulated adenylate cyclase activity among these groups. The adenylate cyclase activity was altered 1 week after gonadectomy or 1 week after gonadectomy plus simultaneous in vivo gonadal steroid treatment. The expression of data as a function of whole tissue (content) or as a function of protein (concentration) influenced magnitude and direction of these treatment effects. This may reflect the proliferation of nonsomatotroph cell populations and altered protein synthetic activity following reproductive endocrine manipulations. When expressed as a whole tissue content, the baseline adenylate cyclase activity was unchanged after gonadectomy when compared to same-sex, gonadal-intact cohorts. However, an increase in the Vmax for GRF-stimulated adenylate cyclase activity was found in gonadectomized rats relative to sham-operated, gonadal-intact cohorts.(ABSTRACT TRUNCATED AT 250 WORDS)