Forskolin (Fsk) has been demonstrated to interact directly with the enzyme adenylyl cyclase (EC 4.6.1.1) in diverse tissues. However, the ability of Fsk to bind to and activate adenylyl cyclase varies depending on the tissue being studied. Different adenylyl cyclase subtypes have been cloned and expressed in a recombinant Sf9 expression system. This provides an opportunity to study the effects of chemically reactive derivatives of Fsk on individual adenylyl cyclase subtypes in the absence of Gs alpha. Reaction of type I adenylyl cyclase with an isothiocyanate derivative of Fsk (6-[[N-(2-isothiocyanatoethyl)amino]carbonyl]forskolin) causes irreversible inhibition of Fsk binding with an IC50 of 300 nM and irreversible inhibition of Fsk activation with an IC50 of 10 microM, suggesting that there are two sites of 6-[[N-(2-isothiocyanatoethyl)amino]carbonyl]forskolin interaction. These studies establish the usefulness of the isothiocyanate derivative of Fsk in localizing the site(s) of Fsk interaction with type I adenylyl cyclase.

An improved affinity support for the purification of adenylyl cyclase was prepared from 7-desacetyl-7-aminoethylaminocarbonyl forskolin. This analog allows convenient synthesis of an affinity matrix that is chemically stable, withstanding repeated use for up to two years, and efficient, yielding purifications of adenylyl cyclase from solubilized bovine brain membranes of 2,000-6,000 fold in a single step. Immunoblotting data suggest that the majority of the enzyme purified in this fashion differs from forms described previously. Since the specific activity of this preparation is substantially higher than that described in previous reports, it is possible that the purification described here selects, presumably on the basis of affinity for forskolin, for a form of adenylyl cyclase with higher specific activity than any described previously.

Three series of analogs were regioselectively prepared from a protected forskolin precursor to afford 7-carbamoyl-7-desacetylforskolins (series 1), 6-carbamoyl-7-desacetylforskolins (series 2), and 6-carbamoylforskolins (series 3). The analogs were pharmacologically evaluated for binding (IC50) to and activation (EC50) of type I adenylyl cyclase in membranes from stably transfected Sf9 cell lines expressing a single adenylate cyclase subtype. The following ranges were determined for the IC50's and EC50's of each individual series: series 1, IC50 = 43-1600 nM, EC50 = 0.5-9.6 microM; series 2, IC50 = 65-680 nM, EC50 = 0.63-6.5 microM; series 3, IC50 = 21-271 nM, EC50 = 0.5-8.1 microM (forskolin IC50 = 41 nM and EC50 = 0.5 microM). Activation paralleled binding; however, some analogs exhibited poor binding and good activation whereas others demonstrated good binding but poor activation. Steric bulk tended to diminish binding and activation when at the 6- or 7-position, although bulk was accommodated at the 6-position if the 7-site was reacetylated. Acylation of the 7-position by the carbamoyl linker or acetyl was important for obtaining good binding and activation; however, the effect was more pronounced with binding. For both binding and activation, small, linear, lipophilic substituents (propyl, allyl, isopropyl) are well tolerated at the 7-position but less so in the 6-position, even when the 7-site is reacetylated. Planar aromatic moieties (phenyl and 2-pyridinyl) demonstrated moderate to good potency for binding and activation when located at either the 6- or 7-positions. There is an overall trend toward increasing potency for both binding and activation with polar substituents.

To identify specific drug targets of the antimitotic drug estramustine, a photoaffinity analogue, 17-O-[[2-[3-(4-azido-3-[125I] iodophenyl)propionamido]ethyl]carbamyl]estradiol-3-N-bis(2- chloroethyl)carbamate, was synthesized and reacted in competition assays with cytoskeletal protein preparations. By attaching the photoaffinity ligand to the 17 beta-position of the steroid D-ring, the cytotoxic properties of the drug were maintained. In cytoskeletal protein preparations from human prostate carcinoma cells (DU 145) or a clonally selected, estramustine-resistant cell line (E4), the major microtubule-associated protein (MAP) present was MAP4. In both cytoskeletal fractions and reconstituted microtubules, 17-O-[[2-[3-(4-azido-3-[125I]iodophenyl)propionamido] ethyl]carbamyl]estradiol-3-N-bis(2-chloroethyl)carbamate bound to both MAP4 and tubulin. From competition assays, the apparent binding constant for MAP4 from DU 145 cells was 15 microM. Similar calculations for tubulin gave values of 13 microM (bovine brain), 19 microM (DU 145 wild-type cells), and 25 microM (E4 cells). The identification of these cytoskeletal proteins as specific drug targets provides a direct explanation for the antimicrotubule and antimitotic effects of estramustine.

An iodinated derivative of forskolin, 6-O-[[2-[3-(4-azido-3-[125I] iodophenyl)propionamido]ethyl]carbamyl]forskolin ([125I]6-AIPP-Fsk), photolabels the multidrug efflux pump P-glycoprotein in membranes prepared from the multidrug-resistant cell lines KB-V1 and KB-C1. The labeling site for [125I]6-AIPP-Fsk was localized by immunoprecipitation of tryptic fragments of P-glycoprotein labeled in KB-C1 membranes. A 6-kDa, photolabeled, tryptic fragment was immunoprecipitated by antiserum raised against residues 348-419 of P-glycoprotein, PEPG9, but not by antisera raised against flanking regions PEPG7 and PEPG11. A peptide that corresponds to residues 343-359 of P-glycoprotein inhibited immunoprecipitation of the 6-kDa fragment by antiserum against PEPG9 but had no effect on the immunoprecipitation of photolabeled fragments by antiserum against PEPG7. A second peptide, corresponding to residues 360-376, had no effect on the immunoprecipitation by antiserum against PEPG9. [125I]6-AIPP-Fsk labels the carboxyl-terminal half of P-glycoprotein, because low molecular mass tryptic fragments were immunoprecipitated by three carboxyl-terminal antisera. Therefore, [125I]6-AIPP-Fsk labels both halves of P-glycoprotein, and labeling in the amino-terminal half can be localized to residues 291-359, which span proposed transmembrane regions 5 and 6. KB-V1 membranes photolabeled with [125I]6-AIPP-Fsk and [125I]iodoarylazidoprazosin were digested with either Staphylococcus aureus V8 protease or chymotrypsin and had similar digestion patterns, suggesting that the two drugs label the same sites on P-glycoprotein.

Several forms of adenylyl cyclase (types I, II, V, and VI) have been expressed using the recombinant baculovirus expression system in Sf9 cells. The activation of type I adenylyl cyclase by forskolin and G(s alpha) was not greater than additive. In contrast, there was synergistic activation of type II, V, and VI adenylyl cyclases by G(s alpha) and forskolin. G(s alpha) potentiated the effect of forskolin on type II adenylyl cyclase to the greatest extent. Type I and II adenylyl cyclases were photolabeled specifically by an iodinated photoaffinity derivative of forskolin ([I-125]-6-AIPP-Fsk). Type I adenylyl cylcase was photolabeled efficiently in the absence of G(s alpha), and the addition of G(s alpha) only slightly increased the labeling efficiency. In contrast, type II adenylyl cyclase was not photolabeled efficiently in the absence of G(s alpha), and the addition of G(s alpha) greatly enhanced the labeling efficiency. Potolabeling of type V and VI adenylyl cyclases was detected only in the presence of G(s alpha). Neither carcium/calmodulin nor G protein beta gamma subunits modulated the photolabeling of type I or II adenylyl cyclases. Another iodinated derivative of forskolin, [I-125]-6-IHPP-Fsk, bound to Sf9 cell membranes expressing type I adenylyl cyclase with high affinity in a filtration binding assay, and the specific binding was not enhanced by the addition of G(s alpha). In contrast, specific binding of [I-125] -6-IHPP-Fsk to membranes expressing type II adenylyl cyclase was detected only in the presence of G(s alpha). [I-125]-6- IHPP-Fsk bound to membranes expressing type I adenylyl cyclase with a K-d of 8 nM and a B-max of 6.4 pmol/mg protein. The K-d for binding of [(125)]-6-IHPP-Fsk to membranes expressing type II adenylyl cyclase in the presence of G(s alpha) was 134 nM, and the B-max was 11.2 pmol/mg protein. Deoxy and desacetyl derivatives of forskolin displaced the binding of [I-125]-6-IHPP-Fsk to membranes expressing type I and II adenylyl cyclases with a similar rank order of potency. However, the affinity of these derivatives for the type I enzyme was between 6- and 20-fold higher than that for the type II enzyme.

7-Bromoacetyl-7-desacetylforskolin (BrAcFsk), an alkylating derivative of forskolin, activated adenylyl cyclase and irreversibly blocked high affinity forskolin binding sites in human platelet membranes and rat brain membranes (Laurenza et al., 1990). Photoincorporation of an iodinated arylazido derivative of forskolin, 125I-6-AIPP-Fsk, into adenylyl cyclase in bovine brain membranes was irreversibly inhibited by BrAcFsk but not by 1,9-dideoxy-BrAcFsk, suggesting that BrAcFsk was reacting specifically with a nucleophilic group(s) at the forskolin binding site of adenylyl cyclase. Immunoblotting with antiforskolin antiserum demonstrated that partially purified bovine brain adenylyl cyclase had incorporated BrAcFsk. The interaction of BrAcFsk with the glucose transporter in human erythrocyte membranes was examined in a similar manner. Photoincorporation of 125I-7-AIPP-Fsk, an iodinated arylazido derivative of forskolin which is specific for the glucose transporter, into the glucose transporter was not irreversibly inhibited by BrAcFsk, suggesting that, in contrast to adenylyl cyclase, there is no reactive nucleophilic group at the forskolin binding site on the human erythrocyte glucose transporter. The immunoblotting procedure with antiforskolin antiserum confirmed that BrAcFsk was not covalently attached to human erythrocyte glucose transporter.

7-(2-Aminoethyl)aminocarbonyl-7-desacetylforskolin (7-AEC-Fsk) and 6-(2-aminoethyl)aminocarbonylforskolin (6-AEC-Fsk) were synthesized and tested for their ability to activate adenylyl cyclase and inhibit the high affinity binding of [3H]forskolin to bovine brain membranes. Forskolin and 7-AEC-Fsk were equipotent in activating adenylyl cyclase, with EC50 values of about 4 microM, whereas 6-AEC-Fsk had an EC50 of about 2 microM. 6-AEC-Fsk and 7-AEC-Fsk stimulated adenylyl cyclase about 7-fold over basal levels at 100 microM, whereas forskolin produced a 5-fold stimulation. Forskolin and 6-AEC-Fsk inhibited the binding of [3H]forskolin to bovine brain membranes with Kd values of 41 nM and 28 nM, respectively, whereas 7-AEC-Fsk had a Kd of 83 nM. The 3-(3-iodo-4-hydroxyphenyl)propionamide derivative of 6-AEC-Fsk (6-I-HPP-Fsk) was more potent than forskolin in inhibiting [3H]forskolin binding to bovine brain membranes, with a Kd of 14 nM. 6-AEC-Fsk was reacted with 125I-labeled Bolton-Hunter reagent to produce 6-125I-HPP-Fsk with a specific activity of 2175 Ci/mmol. 6-125I-HPP-Fsk bound to bovine brain membranes with a Kd of 13 nM and a Bmax of 3.8 pmol/mg of protein. Forskolin inhibited the binding of 6-125I-HPP-Fsk to bovine brain membranes with a Kd of 31 nM, whereas 1,9-dideoxyforskolin only slightly inhibited the binding at 10 microM. The binding of 6-125I-HPP-Fsk was not inhibited by agents that inhibit forskolin binding to the glucose transporter, such as D-glucose or cytochalasin B. There was no displaceable binding of 6-125I-HPP-Fsk to red blood cell membranes, which contain a large concentration of the glucose transporter. Pretreatment of bovine brain membranes with an alkylating derivative of forskolin, 7-bromoacetyl-7-desacetylforskolin (BrAcFsk), led to an irreversible decrease in the binding of [3H]forskolin and 6-125I-HPP-Fsk. The time dependence and concentration dependence for the BrAcFsk-induced decrease in [3H]forskolin binding sites were identical to those observed for the decrease in 6-125I-HPP-Fsk binding sites. 6-125I-HPP-Fsk binding was determined in human platelet membranes in the presence of Mg2+ alone and in combination with guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or AIF4-. The presence of GTP gamma S or AIF4- increased the binding of 6-125I-HPP-Fsk by 4.5-fold and 4-fold, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

A frog used for "hunting magic" by several groups of Panoan-speaking Indians in the borderline between Brazil and Peru is identified as Phyllomedusa bicolor. This frog's skin secretion, which the Indians introduce into the body through fresh burns, is rich in peptides. These include vasoactive peptides, opioid peptides, and a peptide that we have named adenoregulin, with the sequence GLWSKIKEVGKEAAKAAAKAAGKAALGAVSEAV as determined from mass spectrometry and Edman degradation. The natural peptide may contain a D amino acid residue, since it is not identical in chromatographic properties to the synthetic peptide. Adenoregulin enhances binding of agonists to A1 adenosine receptors; it is accompanied in the skin secretion by peptides that inhibit binding. The vasoactive peptide sauvagine, the opioid peptides, and adenoregulin and related peptides affect behavior in mice and presumably contribute to the behavioral sequelae observed in humans.

It has been inferred that residue changes that affect T cell recognition of synthetic peptides will have a similar effect in the intact protein. However, since small peptides do not require antigen processing it is possible that residue changes in synthetic peptides will not have an equivalent effect in the intact protein. Mutant proteins of staphylococcal nuclease (Nase) and 15mer synthetic peptides with corresponding substitutions were compared to determine if residue changes within an immunodominant epitope have an effect on the generation of naturally processed peptides. Five different substitutions in the synthetic peptide resulted in loss of reactivity of individual Nase-specific clones. When the same single amino acid changes were made in the intact protein, the naturally-processed peptides were also unable to stimulate the Nase-specific clones. However, two other substitutions in the synthetic peptide were stimulatory for a T cell clone even though the same changes in the intact protein were non-stimulatory. These results suggest that certain residue changes affect recognition of the naturally processed peptide but not the synthetic peptide with the same amino acid change. In addition, these results demonstrate that the effects of amino acid substitutions in synthetic peptides on T cell recognition may not always reflect the effects of these substitutions in the intact protein. Substitutions located outside Nase-specific T cell epitopes were also examined. Thirty different mutant proteins were all stimulatory. Moreover, a number of these mutants proteins were 50- to 100-fold more efficient in their stimulatory capacity than the native Nase protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoalkylcarbamate derivatives of forskolin have been synthesized at the 6- and 7-hydroxyl positions which have different selectivity for adenylyl cyclase and a glucose transporter, respectively. They were radioiodinated using the Bolton-Hunter reagent to yield [125I]-2-[3-(4-hydroxy-3-iodophenyl)propanamido]-N-ethyl-6- (aminocarbonyl)forskolin ([125I]6-IHPP-Fsk) and [125I]-2-[3-(4-hydroxy-3-iodophenyl)(propanamidol]-N-ethyl-7- (aminocarbonyl)-7-desacetylforskolin ([125I]7-IHPP-Fsk) and tested as autoradiographic probes for adenylyl cyclase and a glucose transporter. In slide-mounted rat brain sections [125I]6-IHPP-Fsk binding was potently inhibited by 1 microM 6-HPP-Fsk (95%) but unaffected by 500 mM D-glucose. In contrast, [125I]7-IHPP-Fsk was only partially inhibited by 1 microM 6-HPP-Fsk (37%), but residual [125I]7-IHPP-Fsk binding was further inhibited 56% by 500 mM D-glucose. These data suggest that while [125I]6-IHPP-Fsk binds exclusively to adenylyl cyclase, a significant fraction of [125I]7-IHPP-Fsk is binding to a glucose transporter in brain. Autoradiographic patterns of [125I]6-IHPP-Fsk and glucose-sensitive [125I]7-IHPP-Fsk binding were different. [125I]6-IHPP-Fsk binding was heterogeneously distributed and resembled [3H] forskolin binding. Highest densities of binding sites were noted in olfactory tubercle, caudate putamen, nucleus accumbens, pyramidal and granule cell layers of hippocampus, molecular layer of cerebellum and substantia nigra. In contrast, of glucose-sensitive [125I]7-IHPP-Fsk, binding appeared more homogeneous and similar to [3H]cytochalasin B, a compound which inhibits glucose transport. Highest densities of binding were noted in caudate putamen, nucleus accumbens, cerebral cortex and molecular layer of cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)

Two radioiodinated derivatives of forskolin, [125I]6-IHPP-Fsk and [125I]7-IHPP-Fsk, were synthesized as specific ligands for adenylyl cyclase and glucose transporter, respectively. [125I]6-IHPP-Fsk bound to bovine brain homogenates with a Kd of 9 nM and binding was inhibited by forskolin but not 1,9-dideoxyforskolin, cytochalasin B, ord-glucose. [125I]7-IHPP-Fsk bound to bovine brain homogenates at two classes of binding sites with Kd's of 56 nM and 4.7 μM; cytochalasin B andd-glucose inhibited 75% of the high affinity binding while having no effect on the low affinity binding. [125I]6-IHPP-Fsk and [125I]7-IHPP-Fsk were used to localize adenylyl cyclase and glucose transporter in rat brain by receptor autoradiography. The pattern of binding obtained with [125I]6-IHPP-Fsk was similar to that observed using [3H]forskolin to detect adenylyl cyclase. In contrast, the pattern of binding obtained with [125I]7-IHPP-Fsk was similar to that observed by others using [3H]cytochalasin B to detect glucose transporter. These iodinated ligands are selective for adenylyl cyclase and glucose transporter and require significantly shorter exposure times to yield autoradiographs than tritiated ligands.

Neutrophils (PMN) treated with cAMP elevating agents were evaluated for their chemotactic responsiveness to FMLP and leukotriene B4 (LTB4). PGE1 and isoproterenol, increased PMN cyclic AMP production and inhibited chemotaxis to both FMLP and LTB4. In contrast, forskolin, which activates adenylate cyclase directly, inhibited chemotaxis to FMLP but not to LTB4. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), was required for inhibition of PMN chemotaxis to FMLP by forskolin, PGE1, and isoproterenol. Isoproterenol and PGE1 inhibited PMN chemotaxis to LTB4 in the absence of IBMX and chemotaxis was further inhibited in the presence of IBMX. PMN cAMP levels were stimulated 2- to 3-fold with isoproterenol, 6- to 10-fold with PGE1, and 5- to 7-fold with forskolin over basal levels in the presence of IBMX. These observations demonstrate that total cellular cAMP concentration is not correlated with inhibition of PMN chemotaxis to all stimuli; forskolin, which increased cyclic AMP 5- to 7-fold over basal levels, did not inhibit chemotaxis to LTB4, whereas isoproterenol, which increased cyclic AMP only 2- to 3-fold over basal levels, inhibited chemotaxis to LTB4. PMN cAMP extrusion was determined under basal conditions and in the presence of PGE1, isoproterenol, or forskolin. PMN extruded cAMP under all conditions examined.

Brefeldin A (BFA) causes rapid redistribution of Golgi proteins into the ER, leaving no definable Golgi apparatus, and blocks transport of proteins into post-Golgi compartments in the cell. In this study we follow the disassembly of the Golgi apparatus in BFA-treated, living cells labeled with NBD-ceramide and demonstrate that forskolin can both inhibit and reverse this process. Long, tubular processes labeled with NBD-ceramide were observed emerging from Golgi elements and extending out to the cell periphery in cells treated with BFA for 5 min. With longer incubations in BFA, the NBD label was dispersed in a fine reticular pattern characteristic of the ER. Treatment with forskolin inhibited these effects of BFA as well as BFA's earliest morphologic effect on the Golgi apparatus: the redistribution to the cytosol of a 110-kD Golgi peripheral membrane protein. In addition, forskolin could reverse BFA's block in protein secretion. Forskolin inhibition of BFA's effects was dose dependent and reversible. High concentrations of BFA could overcome forskolin's inhibitory effect, suggesting forskolin and BFA interact in a competitive fashion. Remarkably, in cells already exposed to BFA, forskolin could reverse BFA's effects causing the 110-kD Golgi peripheral membrane protein to reassociate with Golgi membrane and juxtanuclear Golgi complexes to reassemble. Neither membrane permeant cAMP analogues nor cAMP phosphodiesterase inhibitors could replicate or enhance forskolin's inhibition of BFA. 1,9-Dideoxyforskolin, which does not activate adenylyl cyclase, was equally as effective as forskolin in antagonizing BFA. A derivative of forskolin, 7-HPP-forskolin, that is less potent than forskolin at binding to adenylyl cyclase, was also equally effective as forskolin in antagonizing BFA. In contrast a similar derivative, 6-HPP-forskolin, that is equipotent with forskolin at binding to adenylyl cyclase, did not inhibit BFA's effects. These results suggest that forskolin acts as a competitive antagonist to BFA, using a cAMP-independent mechanism to prevent and reverse the morphologic effects induced by BFA.

(Aminoalkyl)carbamates of forskolin were synthesized at the 6- and 7-hydroxyl positions of forskolin with the length of the alkyl chain varying from ethyl to heptyl. Two of these derivatives, 7-[[(2-aminoethyl)amino]carbonyl]-7-desacetylforskolin (2) and 6-[[(2-aminoethyl)amino]carbonyl]forskolin (3), were used to synthesize iodinated derivatives of forskolin that bind with high affinity to adenylyl cyclase in bovine brain membranes and the glucose transporter in human erythrocyte membranes, respectively. Hydroxyphenyl derivatives of forskolin were prepared from the (aminoalkyl)carbamates and tested for their ability to bind to adenylyl cyclase in bovine brain membranes and the glucose transporter in human erythrocyte membranes. The 6-derivative (18) of forskolin had a Kd of 9 nM at adenylyl cyclase and was more potent than either the 7-derivatives or the 6-derivatives of 7-desacetylforskolin. The 7-derivatives were more potent at binding to the glucose transporter than forskolin. In contrast, the 6-derivatives had Kd's greater than 100 microM at the glucose transporter. Isothiocyanates and N-bromoacetyl derivatives were synthesized from 2 and 3 as potential alkylating agents for forskolin binding sites. The alkylating agents produced an irreversible loss of forskolin binding to adenylyl cyclase. In contrast, the alkylating agents bound reversibly to the glucose transporter.

Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6-AIPP-Fsk) and N-(3-(4-azido-3-125I-phenyl)propionamide)-7-aminoethylcarbamyl-7- desacetylforskolin (125I-7-AIPP-Fsk) were synthesized with specific activities of 2200 Ci/mmol and used to label adenylyl cyclase and the glucose transporter. The affinities of the photolabels for adenylyl cyclase were determined by their inhibition of [3H]forskolin binding to bovine brain membranes. 6-AIPP-Fsk and 7-AIPP-Fsk inhibited [3H]forskolin binding with IC50 values of 15 nM and 200 nM, respectively. 125I-6-AIPP-Fsk labeled a 115-kDa protein in control and GTP gamma S-preactivated bovine brain membranes. This labeling was inhibited by forskolin but not by 1,9-dideoxyforskolin or cytochalasin B. 125I-6-AIPP-Fsk labeling of partially purified adenylyl cyclase was inhibited by forskolin but not by 1,9-dideoxyforskolin. 125I-7-AIPP-Fsk specifically labeled a 45-kDa protein and not a 115-kDa protein in control and GTP gamma S-preactivated brain membranes. This labeling was inhibited by forskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose but not cytochalasin E or L-glucose. Human erythrocyte membranes were photolyzed with 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk. 125I-7-AIPP-Fsk, but not 125I-6-AIPP-Fsk, strongly labeled a broad 45-70-kDa band. Forskolin, 7-bromoacetyl-7-desacetylforskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose, but not cytochalasin E or L-glucose, inhibited 125I-7-AIPP-Fsk labeling of the 45-70-kDa band. 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk are high affinity photolabels with specificity for adenylyl cyclase and the glucose transporter, respectively.