Adenylate Cyclase In Presence Research Articles

Shaping Signals

Although keeping up with them can present difficulty for some human brains, it is evident that there are not enough distinct signaling mechanisms to account for the elaborate range of regulatory events that are controlled in a living organism. Neves et al. show one way in which fine-tuning of the same signaling pathway can allow distinct patterns of control. They examined the way in which cell shape impacts a classical signaling pathway from the β-adrenergic receptor through the second messenger adenosine 3′,5′-monophosphate (cAMP) to activation of mitogen-activated protein kinases (MAPKs) in neurons. Neves et al. used a combination of mathematical modeling of the system with measurements of temporal and spatial dynamics of signaling in isolated mouse hippocampal neurons with fluorescence resonance energy transfer imaging techniques. Their results show that the presence of adenylate cyclase activated by β-adrenergic receptors at the cell surface, with phosphodiesterase activity (which degrades cAMP) in the cytosol, along with other properties of the system, led to activation of gradients of signaling through cAMP to MAPKs. Furthermore, in part because the surface-to-volume ratio is very different in the narrow dendrites of a neuron compared with that in the neuronal cell body, there was a distinctly stronger signal in the dendrites than in the cell body, even though receptors are evenly distributed on the surface of the cell. Modeling indicated that the negative regulators would be key factors in controlling the size of microdomains of activation and propagation of spatially distinct signals through the pathway, and experimental manipulations verified this, with pharmacological inhibition of phosphodiesterase 4 allowing MAP kinase activation in the cell body as well as the dendrites. Kholodenko and Kolch provide commentary. S. R. Neves, P. Tsokas, A. Sarkar, E. A. Grace, P. Rangamani, S. M. Taubenfeld, C. M. Alberini, J. C. Schaff, R. D. Blitzer, I. I. Moraru, R. Iyengar, Cell shape and negative links in regulatory motifs together control spatial information flow in signaling networks. Cell 133 , 666-680 (2008). [PubMed] B. N. Kholodenko, W. Kolch, Giving space to cell signaling. Cell 133 , 566-567 (2008). [PubMed]

Ultrastructural and cytochemical analysis for adenyl cyclase in frog choroid plexus

An ultrastructural and cytochemical analysis of the anterior choroid plexus in adult Rana esculenta was undertaken. The epithelial cells are implicated in the production of cephalorachidian liquid by transporting metabolites from the blood and by synthesizing and secreting activity. The epithelial cells are also capable of re-absorbing catabolites from the cephalorachidian liquid. The presence of adenylate cyclase, along the basal and lateral membranes in some epithelial cells and along the apical membranes of others, leads us to hypothesize that the epithelium of the plexus is made up of two cell types, one type with a secretory function and another type with an absorption function.

A cAMP independent inhibitory action of high doses of forskolin in rat Leydig cells

In addition to well known direct stimulatory and potentiatory actions of forskolin, we have previously reported that low doses of this diterpene (10 −9, 10 −12 M) markedly inhibit the production of cAMP and testosterone in rat Leydig cells through a pertussis toxin sensitive G-protein (A. Khanum and M. L. Dufau, J. Biol. Chem. 261, 1986). A different type of inhibitory effect of forskolin is described in this study. Forskolin (10 −5 M) markedly stimulates basal adenylate cyclase activity (about 200%) in rat Leydig cell membranes and potentiates the stimulatory effect of gonadotropin (10 −9, 10 −7 M) on adenylate cyclase in presence or in absence of GTP (10 −5 M). Similarly a time-dependent stimulation of forskolin (10 −5 M) alone is noted on all cAMP pools and testosterone production. Using a supramaximal steroidogenic dose of hCG (0.26 nM) or choleragen (0.1 μM), forskolin potentiates the gonadotrophin and toxin-induced responses of all cAMP pools significantly while inhibiting testosterone production. Moreover, forskolin also inhibits 8-Bromo-cAMP stimulated steroidogenesis. In contrast, pregnenolone synthesis was not altered by the diterpene. We have demonstrated in this study that the inhibitory effect of high doses of forskolin on steroidogenesis is distal to cAMP generation, and resulted from a steroidogenic block residing beyond pregnenolone synthesis.

Postnatal development of striatal dopamine function. I. An examination of D 1 and D 2 receptors, adenylate cyclase regulation and presynaptic dopamine markers

We have characterized the postnatal development from 1 to 7 weeks after birth in rat striatal homogenates of D 1 and D 2 dopamine (DA) receptor sites, adenylate cyclase (AC) enzyme activity coupled to DA receptor function, guanine nucleotide binding sites and presynaptic markers of DA terminal function. D 1 receptor density, expressed per unit of membrane protein, does not increase over this developmental interval, while maximum DA-stimulated AC activity per mg membrane protein increases 50–100%. D 1 agonist affinity for D 1 receptor sites doubles by 7 weeks of age but is consistently reduced by guanine nucleotide during development. Guanine nucleotide stimulation of AC develops a biphasic dose-response curve after 3 weeks of age. Between 2 and 4 weeks postnatal age there is a rapid increase in AC catalytic component activity as manifested by the capacity of forskolin or manganese ion to stimulate AC in presence of guanine nucleotide and DA. Reversible [ 3H]GppNHp (guanyldiphosphonateimidophosphate) binding to striatal homogenates is dependent on Mg 2+, inhibited by Ca 2+ and GppNHp analogues, and occurs in about a 300-fold excess over D 1 sites. Presynaptic markers of dopaminergic function indicate a 7-fold increase in tissue DA levels, a 2-fold reduction in DA turnover and no apparent change in density of DA uptake sites, assayed by [ 3H]mazindol binding. Subcomponents of D 1 and D 2 DA receptors have distinct postnatal developmental profiles. Striatal D 1 sites do not change significantly during development, but D 2 receptors and GTP inhibition of AC increase and appear, respectively, at 3–4 weeks of age, at the same time as the massive matrix innervation of striatum by DA terminals.

Calcium transport through the placenta.

The calcium content of the foetus increases exponentially during gestation. The transfer of calcium from the mother to the foetus is believed to occur via an active mechanism, based on observations that the calcium concentration is higher in foetal than in maternal blood, and that perfusion in situ of the umbilical artery of the placenta results in a net increase in the calcium concentration of the perfusate even when the calcium content of the perfusate is threefold higher than in the maternal blood. The calcium concentration in the foetal blood is mainly regulated by foetal parathyroid hormone and the plasma concentration of 1.25-(OH)2 vitamin D3. Several experiments suggest that this vitamin D metabolite plays a key role in calcium transport through the syncytial cell. A vitamin D dependent calcium binding protein has been detected in the placenta, which might intervene in the hormonal control of this transport. The cellular mechanisms of calcium transport through the placenta are not clearly defined. An ATP-dependent calcium uptake by microsomal membrane vesicles from human placenta has been described by several investigators. This calcium transport is saturable, magnesium dependent, and displays Michaelis-Menten kinetics with a Km of approximately 70 nM ionized calcium. Two Ca2+ ATPases with low and high affinities for calcium are described. Since there are discrepancies concerning the exact localization of these enzymes in the syncytial cell, it has not been established whether they intervene in calcium transport through the placenta.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochemical localization of adenylate cyclase in bovine cumulus-oocyte complexes

The ultrastructural localization of adenylate cyclase was studied in bovine cumulus-oocyte complexes. Adenylate cyclase was observed on the plasma membrane of the oocyte and occasionally on the plasma membrane of cumulus cells. The cytochemical observations presented demonstrate that there is more adenylate cyclase in cumulus-oocyte complexes after in vitro stimulation with forskolin. The presence of adenylate cyclase upon the oocyte was more pronounced. In addition adenylate cyclase appeared to be localized on the cumulus cells, especially between junctional complexes of cumulus cells and on cumulus cell processes contacting the oocyte. The cumulus cells never showed the presence of adenylate cyclase in the absence of forskolin. No changes in the presence of adenylate cyclase were observed during in vitro meiotic maturation.

Effects of forskolin, prostaglandin F2 alpha, and Ba2+ on the short-circuit current of the isolated rabbit iris-ciliary body.

To gain information on the role of cyclic AMP (cAMP), ion transport and cell membrane permeability on aqueous humor formation, agents with well-known effects on transport properties in other epithelia were tested on the isolated rabbit iris-ciliary body. Forskolin stimulated the short-circuit current (SCC) by 37.5% when added to the aqueous-side solution. Forskolin was ineffective when added to the blood-side solution or when HCO3- was absent from the bathing solutions. The effect of forskolin confirms the presence of adenylate cyclase in the ciliary epithelium and the involvement of cAMP in ion transport. In HCO3- -rich media, 5 X 10(-5) M prostaglandin F2 alpha (PGF2 alpha), produced a prompt 25% increase in the SCC when added to the aqueous-side, and a small stimulatory SCC response when added to the blood-side. No change in SCC occurred when PGF2 alpha was added to either side of a HCO3- -free bathing solution. It is implied that cAMP acts on a HCO3- dependent transport system. These results are consistent with the previously observed stimulation of the SCC by 8Br-cAMP. BaCl2, 2.5 mM, on the aqueous-side increased the SCC by 240.5%, but reduced the SCC by 26% when added to the blood-side solution. The Ba2+ effects indicate the presence of high conductance K+ channels in the basolateral membranes of both the pigmented and non-pigmented cell layers.

Cytochemical localization of adenylate cyclase in the dense tubule system of human blood platelets stimulated by forskolin, prostacyclin and prostaglandin D 2

Platelets were briefly fixed in paraformaldehyde/glutaraldehyde and then incubated with 5′-adenylyl imidodiphosphate under conditions suitable for the cytochemical detection of adenylate cyclase activity. The adenylate cyclase activity of these platelets retains the ability to respond to prostaglandins E 1, D 2, I 2 (prostacyclin), forskolin and fluoride. Sites of stimulated adenylate cyclase activity were localized cytochemically by the reaction of lead with the reaction product imidodiphosphate to form deposits of lead imidodiphosphate that are visible in the electron microscope. Reaction product deposition was seen only in the dense tubule system of human platelets when the incubation medium contained forskolin, prostacyclin, or prostaglandin D 2 at concentrations known to stimulate the enzyme in intact platelets. Epinephrine, an antagonist of adenylate cyclase inhibited the cytochemical reaction stimulated by prostacyclin. The fact that the cytochemical reaction was induced by agonists that stimulate the enzyme through two different types of prostaglandin receptors and by forskolin, which acts distal to the receptors, confirms that the method specifically detects adenylate cyclase. The presence of adenylate cyclase in the dense tubules may be significant for the regulation of intracellular Ca 2+ and arachidonic acid metabolism by this membrane system.

Adenylate cyclase in the microvessels of the rat brain. A histochemical study with light and electron microscopy.

The presence of adenylate cyclase (AC) in the microvessels of the rat brain was studied by a new histochemical method for light and electron microscopy. The method is based on the precipitation of strontium and the subsequent conversion of the formed strontium salt into lead phosphate. Isoproterenol and 5-guanylylimidodiphosphate were used as enzyme activators. In the light microscope, the final reaction product was detected in the choroid plexus as well as in the walls of the microvessels in the brain parenchyma. In the electron microscope, both the luminal and abluminal endothelial membrane as well as the basal lamina of the parenchymal microvessels displayed reaction product. The observations demonstrate that isoproterenol-stimulated AC is located in the endothelium of the rat brain microvessels and suggest that the enzyme may play a role in the receptor-mediated regulation of endothelial functions.

Cyclic AMP and anaerobic gene expression in E. coli

The expression of the fumarate reductase system of Escherichia coli is completely dependent on the presence of adenylate cyclase or cyclic AMP, but the cyclic AMP receptor protein (CRP) is not required. This suggests that cyclic AMP may function as an effector for a second gene activator protein, possibly Fnr, and thus form part of a redox-sensitive regulatory mechanism controlling the expression of anaerobic respiratory functions.

Properties of adenylate cyclase and cyclic nucleotide phosphodiesterase in hamster isolated capillary preparations

Capillaries isolated by collagenase digestion of hamster epididymal fat pads were used to examine the properties of endothelial adenylate cyclase and cyclic nucleotide phosphodiesterase. Adenylate cyclase activity in capillary homogenates was increased by 10 μM GTP or 100 μM isoproterenol. Lower concentrations of the catecholamine and 5.7 μM prostagladin E 1 did not stimulate endothelial adenylate cyclase activity unless GTP was included in the assay system. The effects of isoproterenol on capillary adenylate cyclase activity were blocked by propranolol, but were not affected by phentolamine. Phosphodiesterase activity in endothelial homogenates showed anomalous kinetic behavior with either cyclic AMP or cyclic GMP as the enzyme substrate. At substrate concentrations below 1 μM, capillary phosphodiesterase activity hydrolyzed cyclic GMP 2–6 times faster than cyclic AMP. However, at high substrate levels, e.g., 100 μM, cyclic AMP and cyclic GMP were degraded at similar rates. Hydrolysis of 1 μM cyclic AMP by capillary homogenates was stimulated by 0.1 and 1 μM cyclic GMP. Caffeine, 1-methyl-3-isobutylxanthine, papaverine and dipyridamole SQ 20009 were effective inhibitors of capillary phosphodiesterase activity. In contrast, imidazole enhanced the activity of the enzyme. The presence of adenylate cyclase and phosphodiesterase activities in enzyme. The presence of adenylate cyclase and phosphodiesterase activities in hamster isolated capillaries is consistent with a role for cyclic AMP in the regulation of endothelial function. Moreover, the experiments described here indicate that hamster isolated capillaries are useful model systems for studying the metabolism of vascular endothelium.

Studies on the cytochemical localization of adenylate-cyclase activity in Dugesia lugubris s.I.

The localization of adenylate-cyclase activity in Dugesia lugubris s.1. has been investigated cytochemically using 5'-adenylyl-imidodiphosphate as substrate. The enzyme was localized in mucous gland cells, in rhabdite cells, in intercellular spaces and also in nerve endings of this planarian. The presence of adenylate-cyclase on the membrane suggests that it might mediate different stimulus-secretion coupling by increasing cyclic AMP synthesis in specialized areas of the planarian.

Demonstration of adenylate cyclase activity in human red blood cell ghosts

The presence of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) activity was demonstrated in human erythrocyte ghosts and was found to be around 3 pmol adenosine ′,5′-monophosphatase (cyclic AMP) · 2 h −1 · mg −1 protein. This enzymatic activity is strongly stimulated by NaF and 5′-guanylimidodiphosphate, is slightly stimulated by epinephrine, norephrine, soproterenol, and prostaglandin E, and is inhibited by calcium. The hormone stimulation is not potentiated by 5′-guanylylimidodiphosphate.

Adenyl Cyclase in Normal and Psoriatic Skin

After the incubation of slices of human skin with adenine-2,8- 3 H in the presence of theophylline, 3 H was found in cAMP, indicating the presence of adenyl cyclase in human skin. This activity was stimulated by epinephrine and NaF. Specimens of psoriatic skin were significantly less effective in incorporating adenine-2,8- 3 H into camp, less responsive to the stimulation of epinephrine and unresponsive to NaF. The unaffected skin of the psoriatic patients had normal activity and normal responses to epinephrine and NaF. The 650 × g pellets from homogenates of unaffected skin and affected skin from apsoriatic patient were prepared and incubated with 2 mM ATP-α- 32 P. The amounts of cAMP- 32 P formed were compared. The preparation from the unaffected skin produced 7.5 pmoles of cAMP per min per mg protein, while the affected skin had approximately half the capability. These results suggest deficiency and defects of adenyl cyclase in psoriatic skin.