Caffeine and related methylxanthines: possible naturally occurring pesticides.

Natural and synthetic methylxanthines inhibit insect feeding and are pesticidal at concentrations known to occur in plants. These effects are due primarily to inhibition of phosphodiesterase activity and to an increase in intracellular cyclic adenosine monophosphate. At lower concentrations, methylxanthines are potent synergists of other pesticides known to activate adenylate cyclase in insects. These data suggest that methylxanthines may function as natural insecticides and that phosphodiesterase inhibitors, alone or in combination with other compounds, may be useful in insect control.

Octopamine receptors, adenosine 3',5'-monophosphate, and neural control of firefly flashing.

An adenylate cyclase activated as much as 25-fold by low concentrations of octopamine has been identified in the firefly lantern. The relative potency of octopamine and various other amines in stimulating this enzyme, and effects of antagonists in blocking octopamine activation, correlate well with the known effects of these agents in affecting light production. In addition to suggesting a role for adenosine 3',5'-monophosphate (or pyrophosphate) in the neural control of firefly flashing, identification of this potent enzyme should facilitate the characterization of phenylethylamine receptors in excitable tissue.

Beta-adrenergic-sensitive adenylate cyclase in secretory cells of choroid plexus.

Biochemical evidence supporting the sympathetic control of cerebrospinal fluid production has been obtained through identification of a specific beta-adrenergic-sensitive adenylate cyclase in the choroid plexus. The enzyme, which is localized in the secretory epithelium, is activated by low concentrations of isoproterenol and norepinephrine and appears separate from beta-adrenergic-sensitive adenylate cyclase present in cerebral blood vessels.

Brain barrier tissues: end organs for atriopeptins.

Little is known about the pathophysiology of cerebral edema and other disturbances of water balance that involve the barrier tissues at the interface of blood and brain. The present experiments show that these barrier tissues contain receptors and second messenger systems for atriopeptins, recently identified cardiac peptides involved in peripheral water regulation. They also show that atriopeptins can alter the rate of cerebrospinal fluid production. Because the blood-brain and blood-cerebrospinal fluid barriers are involved in normal water movements in the central nervous system, these studies suggest that brain barrier tissues may be important end organs for the atriopeptins and that atriopeptins could have therapeutic application to disorders of water balance in the central nervous system. An isolated, purified population of atriopeptin receptor cells, obtained from choroid epithelium, was used in these experiments. This cell population may provide a valuable model system for investigating the intracellular biochemical mechanisms through which atriopeptins exert their actions.

Octopamine-sensitive adenylate cyclse: evidence for a biological role of octopamine in nervous tissue.

An adenylate cyclase that is activated specifically by very low concentrations of octopamine has been identified both in homogenates and in intact cells of the thoracic ganglia of an insect nervous system. This enzyme appears to be distinct from two other adenylate cyclases present in the same tissue, which are activated by dopamine and by 5-hydroxytryptamine, respectively. The data raise the possibility of a role of octopamine-sensitive adenylate cyclase in the physiology of synaptic transmission.

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).

Release of the phosphodiesterase activator by cyclic AMP-dependent ATP:protein phosphotransferase from subcellular fractions of rat brain.

The subcellular distribution of the endogenous phosphodiesterase activator and its release from membranes by a cyclic AMP-dependent ATP:protein phosphotransferase was studied in fractions and subfractions of rat brain homogenate. These fractions were obtained by differential centrifugation and sucrose density gradient; their identity was ascertained by electron microscopy and specific enzyme markers. In the subcellular particulate fractions, the concentration of activator is highest in the microsomal fraction, followed by the mitochondrial and nuclear fractions. Gradient centrifugation of the main mitochondrial subfraction revealed that activator was concentrated in those fractions containing mainly synaptic membranes. Activator was releasted from membranes by a cyclic AMP-dependent phosphorylation of membrane protein. The release of activator occurred mainly from the mitochondrial subfractions containing synaptic membranes and synaptic vesicles. The data support the view that a release of activator from membranes may be important in normalizing the elevated concentration of cyclic AMP following persistent transsynaptic activation of adenylate cyclase.

Isolation and N-terminal amino acid sequence of an octopamine ligand binding protein.

An octopamine receptor photoaffinity probe was used to label membranes from the light organs of Photinus pyralis, a tissue highly enriched in octopamine receptors. Labeling was concentrated in a glycoprotein of 75 +/- 2 kDa with lesser labeling of a 79 +/- 2 kDa component. Labeling could be displaced by prior incubation with octopamine, mianserin, cyproheptadine, phentolamine or propranolol, with a relative potency that correlated with the ability of these same agents to modulate light organ octopamine-sensitive adenylate cyclase. The 75 kDa binding protein was isolated and its N-terminal amino acid sequence was determined.

A probe for octopamine receptors: synthesis of 2-[(4-azido-2,6-diethylphenyl)imino]imidazolidine and its tritiated derivative, a potent reversible-irreversible activator of octopamine-sensitive adenylate cyclase.

In order to develop an irreversible ligand for octopamine receptors, a highly potent azido-substituted 2-(phenyl-imino)imidazolidine (NC-5Z, 8) and its tritiated derivative (3H-NC-5Z, 11) have been designed and synthesized. Under reversible-binding conditions, NC-5Z is 50-100-fold more potent than octopamine in activating octopamine-sensitive adenylate cyclase in a variety of tissues. After photolysis, 3H-NC-5Z binds irreversibly to cell membranes, and this binding is reduced by preincubation with octopamine agonists and antagonists but not by norepinephrine, dopamine, serotonin, or histamine. NC-5Z should be useful both as a potent reversible octopamine agonist and as an affinity probe for characterizing and isolating octopamine-receptor proteins.

Atriopeptin-activated guanylate cyclase in the anterior segment. Identification, localization, and effects of atriopeptins on IOP.

Atriopeptins are a recently-discovered group of polypeptides secreted from cardiac myocytes in response to fluid overload. The present studies demonstrate that atriopeptin receptors, coupled to the activation of guanylate cyclase, are present in rabbit ciliary process. Rat atrial natriuretic peptide 1-28 (rANP) activated ciliary process guanylate cyclase activity with a Vmax of from 24-337% and with a Ka of from 0.4-4 nM, similar to that for atriopeptin receptors present in rabbit kidney. Activation was greater for the intact peptide than for rANP fragments 1-11 or 13-28, and stimulated activity was greater in isolated ciliary processes than in ciliary muscle or iris. Intravitreal injection of the complete peptide into living rabbits caused a marked decrease in IOP in the ipsilateral eye which persisted for more than 48 hr and occurred without evidence of an inflammatory response. There was a smaller decrease in IOP in the contralateral eye. Following intravitreal injection of rANP 1-28, atriopeptin levels in aqueous humor remained elevated for at least 44 hr. Injection of the biochemically less active rANP fragments 1-11 and 13-28 caused no decrease in IOP. These physiological data, together with the biochemical identification of atriopeptin receptors and second messenger systems in the ciliary process, suggest that certain tissues of the anterior segment may be atriopeptin end-organs and that agents acting at atriopeptin receptors may be able to regulate IOP.

Human ciliary process adrenergic receptor: pharmacological characterization.

To better understand the nature of interaction of various amines with adrenergic receptors in te human ciliary process, beta-adrenergic-stimulated adenylate cyclase activity was characterized in broken cell preparations of this tissue from donor eyes. Among various agonists, isoproterenol was the most potent activator of enzyme activity (Ka = 3.4 x 10(-7)M), followed in order by epinephrine (Ka = 2.7 x 10(-6)M), norepinephrine (Ka = 2.1 x 10(-5)M), and phenylephrine (Ka greater than 10(-4)M). Isoproterenol-stimulated enzyme activity was blocked by timolol (Ki = 3.4 x 10(-9)M), IPS 339 (Ki = 4.4 x 10(-9)M), H35/25 (Ki = 6.9 x 10(-7)M), and atenolol (Ki = 1.4 x 10(-5)M). These pharmacological characteristics indicate that the human ciliary processes contain a predominance of beta2-adrenergic receptors. The findings are relevant to physiological studies of aqueous humor secretion and to the potential development of adrenergic agents with greater specificity for the beta-adrenergic receptor.

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.

Epinephrine increases facility of outflow and cyclic AMP content in the human eye in vitro.

The physiologic mechanism that underlies the epinephrine-induced increase in facility of outflow (C) in glaucomatous human eyes and normal primate eyes is not completely understood. In this study, a recently developed in vitro human eye perfusion model was used to simultaneously monitor facility and cyclic adenosine monophosphate (AMP) changes in response to epinephrine (EPI). In this system, EPI (2.5 x 10(-5) mol/l) resulted in a maximal 44% increase in C, with an ED50 occurring at approximately 8 x 10(-6) mol/l. The C-increasing effect of 10(-5) mol/l EPI was unaffected by 10(-6) mol/l phentolamine. However, it was completely blocked in the presence of 10(-6) mol/l timolol or 2 x 10(-7) mol/l ICI118,551, suggesting the involvement of beta-2 adrenergic receptors. In biochemical studies, 10(-5) mol/l EPI induced a 12- to 14-fold increase in cyclic AMP in the perfusate of treated eyes. This increase was blocked by ICI118,551. In isolated intact human trabecular tissue, a 10 min incubation with 10(-5) EPI stimulated cyclic AMP by a factor of 2.7 over control levels. After 90 min, cyclic AMP levels were increased 4.2 fold over control levels. Collectively, these results show that the intraocular pressure lowering effect of EPI in the human eye is mediated, at least in part, by an increase in facility of outflow. Furthermore, the facility increase appears to be mediated by beta-2 adrenergic receptors and is correlated in time with increased cyclic AMP production.

Effect of endothelin on outflow facility and accommodation in the monkey eye in vivo.

The effect of the vasoactive peptide, endothelin, on facility of outflow, accommodation, and pupil diameter was measured in the monkey eye in vivo. Endothelin increased the outflow facility 22-71% at approximate anterior chamber concentrations ranging from 10(-10)-10(-7) M. Endothelin-induced accommodation was modest but consistent and statistically significant ranging from 1.61 to a maximum of 2.43 diopters at 10(-9) M and 10(-7) M endothelin, respectively. No change in pupil diameter was noted with any of the administered doses. These data demonstrate an action of endothelin on outflow facility and, together with prior evidence indicating the presence of endothelin receptors on the ciliary muscle, suggest that, like cholinergic agonists, the observed effects of endothelin on outflow facility may be mediated, at least in part, through an action on the ciliary muscle.

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.

Effects of a potent and specific beta 2-adrenoceptor antagonist on intraocular pressure.

1 Physiological studies support the possibility of adrenergic regulation of aqueous humor secretion from the ciliary process, a tissue which has recently been shown to contain a predominance of beta 2-adrenoceptors. 2 The present experiments examine the effects of a potent and highly specific beta 2-antagonist (IPS 339) on intraocular pressure in the normal rabbit eye. 3 The observed decrease in pressure caused by IPS 339 suggests that highly specific beta 2-antagonists may be useful in decreasing elevated intraocular pressure.

ICI 118,551: an effective ocular hypotensive agent with selectivity for the ciliary process beta 2-adrenoceptor and with minimal cardiac side effects.

Prior biochemical studies have shown that the ciliary process epithelium, which is involved in the secretion of aqueous humour, is rich in beta-adrenoceptors with pharmacological characteristics similar to those of the beta 2 subclass. The present experiments demonstrate that the beta-adrenoceptor antagonist, ICI 118,551, is a potent inhibitor of isoprenaline-stimulated adenylate cyclase activity measured in broken cell preparations of rabbit ciliary process. In rabbit cardiac muscle, however, ICI 118,551 is a relatively weak antagonist of isoprenaline-stimulated adenylate cyclase, being approximately 100 fold less potent than the non-selective beta-adrenoceptor antagonist, timolol. ICI 118,551 is also less potent than timolol in inhibiting isoprenaline-sensitive adenylate cyclase of rabbit lung. ICI 118,551 applied topically to eyes of unanaesthetized rabbits causes a dose-dependent decrease in intraocular pressure. Furthermore, in a blind crossover study in rabbits, topically applied ICI 118,551 decreased intraocular pressure for more than 6 h and was more effective than an identical dose of the clinically effective anti-glaucoma agent, timolol. Systemic absorption from topically-applied timolol, but not ICI 118,551, is sufficient to alter cardiac response to subcutaneous administration of isoprenaline. Furthermore, dose-response studies, using direct systemic administration of the two beta-adrenoceptor antagonists, revealed that ICI 118,551 is about 60 times less potent than timolol in blocking isoprenaline-induced cardio-acceleration. ICI 118,551, applied to one eye, causes a decrease in intraocular pressure in the contralateral eye, and systemic administration of ICI 118,551 results in decreased intraocular pressure in both eyes, data indicating that at least part of the ocular hypotensive effect of topical ICI 118,551 is mediated through systemic absorption. 8 These findings provide biochemical and physiological evidence that selective beta 2-adrenoceptor blockers such as ICI 118,551, used topically or systemically, may be useful as ocular hypotensive agents with decreased cardiac side-effects.

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.

Galanin stimulates rat pituitary growth hormone secretion in vitro.

The effect of galanin on growth hormone (GH) secretion was investigated in monolayer cultures of rat anterior pituitary cells. Galanin caused a gradual increase in GH concentrations into the culture medium that was maximal at 90 minutes and sustained after 180 minutes. The ED50 for galanin-stimulated GH secretion was approximately 200 nM compared to an ED50 for rat GH-releasing factor (rGRF)-stimulated GH secretion of 10pM. Galanin and rGRF were additive in increasing GH release into the incubation medium. These data indicate that porcine-derived galanin has a direct effect on pituitary GH secretion in vitro.