Forskolin, adenylyl cyclase

Forskolin Adenylyl cyclase activator FRTL5 rat cell line X X X... 

FIGURE 9.14 Effects of adenosine receptor agonist 2-chloro-adenosine on vascular perfusion pressure of isolated perfused rat kidneys. Minor effects seen in untreated kidneys (filled circles) and pronounced vasoconstriction while vasodilatation in kidneys coperfused with subthreshold concentrations of a-adrenoceptor vasoconstrictor methoxamine and vasodilatatory activation of adenylyl cyclase with forskolin (open circles). Redrawn from [49]. 

Adenylyl Cyclases. Figure 4 Regulation of adenylyl cyclases by G-proteins. Abbreviations Hs, Hj, Rs, and Rj denote hormones and receptors that lead to stimulation or inhibition, respectively, of adenylyl cyclases, Ca and Ci are active and inactive configurations of adenylyl cyclase, Fo forskolin binding site, Gs and Gj are GTP-dependent regulatory proteins comprising their respective as, and (3y subunits. 

Adenylyl Cyclases. Table 5 Prodrug inhibition of [3H] cAMP formation in intact cells. Cells were prelabeled for 2 h with [3H]adenine before 50 pM forskolin and pronucleotides were added. After a 15 min incubation the newly formed [3H]cAMP was extracted and quantified as in (7)... 

Forskolin is a diteipene derivative from the plant Coleus forskohlii. It activates all mammalian isozymes of adenylyl cyclase except AC9 and AC 10. 

Active derivatives of forskolin include 7-deacetyl-forskolin (EC5o 20(llM), 6-acetyl-7-deacetyl-forskolin (EC5o 40(llIVI), 7-deacetyl-7-0-hemisuccinyl-forskolin (EC50 50(llIVI). The last of these has been used as an immobilized affinity chromatography ligand for the purification of adenylyl cyclases from tissues. 

P-site ligands inhibit adenylyl cyclases by a noncompetitive, dead-end- (post-transition-state) mechanism (cf. Fig. 6). Typically this is observed when reactions are conducted with Mn2+ or Mg2+ on forskolin- or hormone-activated adenylyl cyclases. However, under- some circumstances, uncompetitive inhibition has been noted. This is typically observed with enzyme that has been stably activated with GTPyS, with Mg2+ as cation. That this is the mechanism of P-site inhibition was most clearly demonstrated with expressed chimeric adenylyl cyclase studied by the reverse reaction. Under these conditions, inhibition by 2 -d-3 -AMP was competitive with cAMP. That is, the P-site is not a site per se, but rather an enzyme configuration and these ligands bind to the post-transition-state configuration from which product has left, but before the enzyme cycles to accept new substrate. Consequently, as post-transition-state inhibitors, P-site ligands are remarkably potent and specific inhibitors of adenylyl cyclases and have been used in many studies of tissue and cell function to suppress cAMP formation. 

Not all opiates induce an increase in forskolin stimulation of adenylyl cyclase. Buprenorphine, which is used to treat morphine dependence, does not cause a compensatory rise in cyclase activity [80]. Co-treatment of morphine with buprenorphine prevented morphine from increasing cyclase activity suggesting that bu-... 

Biochemical and molecular cloning studies indicate the existence of nine separate and unique forms of adenylyl cyclase (AC), which comprise a distinct enzyme family, referred to as AC1-AC9 [1, 2]. These members of the adenylyl cyclase superfamily are all membrane-bound. There is also an additional soluble form, sAC, that has catalytic activity similar to the others but is genetically the most divergent member of the family. All the membrane-bound forms of adenylyl cyclase are activated by the stimulatory G protein Gas (see Ch. 19), and all with the exception of AC9 are stimulated by forskolin. The soluble form sAC is not stimulated by either Gas or forskolin but is sensitive to bicarbonate levels. All known forms of... 

In general, the 10 different forms of adenylyl cyclase can be divided into three major and two minor groups based on their functional attributes as well as on their sequence homology. The three major families are (1) the Ca2+/calmodulin-stimulated isoforms, which include AC1, AC3, and AC8 certain of these enzymes are inhibited by GPt (2) the G[ -stimulated isoforms, which include AC2, AC4, and AC7 and (3) the Ca2+ and Gai inhibited isoforms, which are AC5 and AC6. The other two categories each have a single member (4) AC9, which is the most divergent of the membrane-bound isoforms, is stimulated by G(IS, inhibited by calcineurin (a protein phosphatase), and is insensitive to forskolin (5) sAC, the only soluble isoform, is the most divergent and is most similar to the adenylyl cyclase found in Cyanobacteria. 

There are also many neurotransmitter and hormone receptors that contribute to the fine control of cAMP formation by inhibition of adenylyl cyclase. The action of inhibitory receptors is mediated by several different forms of the Gai family, specifically the Gail, Gai2, Gai3, Gao and Goa subtypes. The Ga subunits of these isoforms can inhibit the catalytic activity of adenylyl cyclase when the enzyme is activated by either Gas or forskolin. The inhibition of catalytic activity does not occur via competition with Gas but appears to occur by an interaction at a symmetric site on the AC molecule. Gai-mediated inhibition of adenylyl cyclase is most dramatic for AC5 and AC6. A few other forms of adenylyl cyclase, most notably AC1, can be inhibited by Gao but this effect is not as potent as the inhibition of AC5 and AC6 by Gai isoforms. The GTPase activity of Gai family members can be accelerated by a large family of RGS proteins (see Chapter 19). 

Fig. 12.4 Functional coupling of the Gly389 and Arg389 receptors to adenylyl cyclase. (Reproduced from ref. 26 with permission of the American Society for Biochemistry and Molecular Biology.) Shown are the results from studies with clonal lines expressing each receptor at matched levels and the data presented as absolute activities (A) and normaUzed to the stimulation by forskolin (B). The results of similar studies with two other clonal lines are shown in C and D. The Arg389 demonstrated small increases in basal activities and marked increases in agonist-stimulated activities compared with the Gly389 receptor. Shown are the mean results from four independent experiments carried out with each line. Absent error bars denote that standard errors were smaller than the plotting symbol...

Another excellent example of synergism is the activation of a soluble adenylyl cyclase by forskolin and the a-... 

One of the best-characterized effectors and second messenger systems is the cAMP cascade that can be either activated or inhibited by neurotransmit-ter/neuropeptide receptors, including those implicated in anxiety/stress such as CRE Receptors that activate cAMP synthesis couple with the stimulatory G protein, Gsa, and those that inhibit this second messenger couple with the inhibitory G protein, Gia, and these either stimulate or inhibit adenylyl cyclase, the effector enzyme responsible for synthesis of cAMP (Duman and Nestler 1999). There are at least nine different forms of adenylyl cyclase that have been identified by molecular cloning, each with a unique distribution in the brain. The different types of adenylyl cyclase are activated by Gsa as well as the diterpene forskolin, but are differentially regulated by Gia, the Py subunits, Ca, and by phosphorylation. This provides for fine control of adenylyl cyclase enzyme activity and regulation by other effector pathways. 

A common feature of the different adenylyl cyclases is the stimulation of their enzyme activity by the GTP-bound form of the a-subimit of the Gs-protein. Furthermore, all subtypes are stimulated by the diterpene forskolin. In addition to the central regulation by the activated a-subunit, there are other stimulatory or inhibitory influences on the different subtypes of adenylyl cyclase, in a manner characteristic for the particular subtype. The various subtypes differ in these regulatory influences, whereby none of the different subtypes have an identical pattern of regulation. 

An inhibitory effect of 8-OH-DPAT on forskolin-stimulated adenylyl cyclase activity is observed in hippocampal membranes of the guinea pig and rat, and has been used as an index of the functional activities of 5-HTIA receptors [178]. Cold stress induces a reduction of the inhibitory effect of 8-OH-DPAT in the hippocampus isolated from 18-month-old rats, although it has no influence on either the affinity or number of [3H]8-OH-DPAT binding sites. The administration of EGb (50 mg/kg p.o. for 14 days) prevents the cold stress-induced reduction in the inhibitory effect of 8-OH-DPAT on forskolin-stimulated adenylyl cyclase activity in old rats. These results indicate that EGb prevents the stress-induced desensitization of hippocampal 5-HTu receptors thus, its effects might explain anti-stress and antidepressant properties of EGb [177]. 

FIGURE 12-13 Interaction of Gs with adenylyl cyclase. (PDB ID 1AZS) The soluble catalytic core of the adenylyl cyclase (AC, blue), severed from its membrane anchor, was cocrystallized with G,, (green) to give this crystal structure. The plant terpene forskolin (yellow) is a drug that strongly stimulates the enzyme, and GTP (red) bound to Gsa triggers interaction of Gsa with adenylyl cyclase. 

Despite this A3 AR desensitisation, the adenylyl cyclase activity is not reduced, but almost a twofold increase is detected in the presence or in the absence of forskolin. This effect seems not to be mediated by e new receptor synthesis, but is probably due to an increased coupling efficiency between Gs and adenylyl cyclase. The sensitization of adenylyl cyclase activity is evidence that acute and chronic treatment of adenosine receptors might lead to opposite effects (Palmer et al. 1997). 

Fig. 5. The open (inactive) and closed (active) conformations of the adenylyl cyclase catalytic domains bound to GocGTPyS. Helices are shown as cylinders and [j strands as ribbons. The C2 domains of the two mAC molecules are superimposed. The open state of mAC, which was crystallized in the absence of an ATP analogue, is colored gray (PDB ID, 1AZS) the closed state, colored blue, was crystallized with ATPaS bound to the AC catalytic site, shown as a stick model (1CJT). Forskolin is present in both complexes. Switch II of Gas is colored red. Note that binding of ATP is accompanied by segmental movement of residues in both domains toward the catalytic site.

Dessauer, C. W., Scully, T. T., and Gilman, A. G. (1997). Interactions of forskolin and ATP with the cytosolic domains of mammalian adenylyl cyclase. / Biol. Chem. 272, 22272-22277. 

Efforts may now have been successful Whereas normal tobacco cells require auxin for division, sequence tagged (TDNA) lines encoding an adenylyl cyclase were obtained which were auxin-independent but cAMP-dependent. From one line (axi 141), a complementary DNA encoding adenylyl cyclase has been isolated with characteristic leucine repeats and similarity to yeast adenylyl cyclase (Ichikawa et al., 1997). The result seems not to be the expression of an alternative division pathway from the normal auxin-driven division since it is blocked by auxin inhibitors and is activated by cAMP and the cyclase activator forskolin. Perhaps a link to G-protein at the membrane will now bring plant growth regulation even closer to that of animals. 

Laurenza A, Sutkowski EM and Seamon KB (1989) Forskolin a specific stimulator of adenylyl cyclase or a diterpene with multiple sites of action Trends Pharmacol Sci 10, 442-447. 

In general, the MC2 receptor is Gs protein-coupled (Table 1), and two studies in the rabbit pulmonary artery indicate that this is also true for the presynaptic receptor. The evidence is, indirect, however, in that it suggests activation of adenylyl cyclase, the typical transduction step downstream from Gs. In the study by Gothert and Hentrich (1984), the facilitatory effect of ACTH was increased by simultaneous administration of forskolin, an activator of the catalytic subunit of adenylyl cyclase, and AH 21-132, a phosphodiesterase inhibitor. In the study by Costa and Majewski (1988), the facilitatory effect of ACTH was occluded when the vessel was super-fused with a lipid-soluble cAMP analogue. 

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