Forskolin-induced Cyclic AMP Accumulation Research Articles

Selectivity and Specificity of Sphingosine-1-Phosphate Receptor Ligands: Caveats and Critical Thinking in Characterizing Receptor-Mediated Effects

Receptors for sphingosine-1-phosphate (S1P) have been identified only recently. Their medicinal chemistry is therefore still in its infancy, and few selective agonists or antagonists are available. Furthermore, the selectivity of S1P receptor agonists or antagonists is not well established. JTE-013 and BML-241 (also known as CAY10444), used extensively as specific S1P2 and S1P3 receptors antagonists respectively, are cases in point. When analyzing S1P-induced vasoconstriction in mouse basilar artery, we observed that JTE-013 inhibited not only the effect of S1P, but also the effect of U46619, endothelin-1 or high KCl; JTE-013 strongly inhibited responses to S1P in S1P2 receptor knockout mice. Similarly, BML-241 has been shown to inhibit increases in intracellular Ca2+ concentration via P2 receptor or α1A-adrenoceptor stimulation and α1A-adrenoceptor-mediated contraction of rat mesenteric artery, while it did not affect S1P3-mediated decrease of forskolin-induced cyclic AMP accumulation. Another putative S1P1/3 receptor antagonist, VPC23019, does not inhibit S1P3-mediated vasoconstriction. With these examples in mind, we discuss caveats about relying on available pharmacological tools to characterize receptor subtypes.

6-Amino-2-mercapto-3 H-pyrimidin-4-one derivatives as new candidates for the antagonism at the P2Y 12 receptors

P2Y 12 plays an important role in platelet aggregation, which makes it an interesting target for antithrombotic agents. Compounds that antagonize P2Y 12 include the active metabolites of thienopyridines and molecules that are structurally related to ATP, which is an antagonist of P2Y 12. During the last few years, our group has been working on the development of P2Y 12 receptors antagonists that are based on an extremely simple chemical structure, the 6-amino-2-mercapto-3 H-pyrimidin-4-one, variously substituted at the sulfur and oxygen functions. This nucleus represents the simplified combination of two known P2Y 12 antagonists: the active metabolite of the thienopyridines and ATP derivatives. The effects of the synthesized compounds were tested on ADP-induced human platelet aggregation, using light transmission aggregometry. None of the tested compounds induced platelet aggregation, while some of them, at concentration of 10 −4 M, partially inhibited platelet aggregation induced by ADP 10 −6 M. The most potent compound, 6b, antagonized the inhibitory effect of 2-methylthio-ADP on the forskolin-induced accumulation of cyclic-AMP in CHO FlpIN cells expressing recombinant human P2Y 12-receptors. In addition, none of the tested compounds, including 6b, interfered with ligand binding to P1 receptors. Our results suggest that some of the synthesized compounds are specific antagonists of P2 receptors, and in particular of P2Y 12 and suggest that further development of this structurally new series of compounds as P2Y 12 receptors antagonists is recommended.

A truncated form of CKbeta8-1 is a potent agonist for human formyl peptide-receptor-like 1 receptor.

1. Human formyl peptide-receptor-like-1 (FPRL-1) is a promiscuous G protein-coupled receptor (GPCR), and belongs to a chemoattractant receptor family protein. This receptor has been reported to interact with various host-derived peptides and lipids involved in inflammatory responses. We described here, a novel role for FPRL-1 as a high-affinity beta-chemokine receptor for an N-terminally truncated form of the CKbeta8 (CCL23/MPIF-1) splice variant CKbeta8-1 (22-137 aa). 2. RT-PCR analysis of mRNA derived from human tissues and cells revealed a predominant expression of FPRL-1 in inflammatory cells, particularly in neutrophils. 3. Intracellular calcium mobilisation assay, used as screening tool, in recombinant Chinese hamster ovary (CHO-K1) and human embryonic kidney (HEK293s) cells coexpressing FPRL-1 and Galpha(16), demonstrated FPRL-1 is a functional high-affinity receptor for CKbeta8-1 (46-137 aa, sCKbeta8-1), with pEC(50) values of 9.13 and 8.85, respectively. 4. The FPRL-1 activation in CHO-K1 cells is mediated by Galpha(i)/Galpha(o) proteins, as assessed by pertussis toxin sensitivity and inhibition of forskolin-induced cyclic AMP accumulation. 5. Binding experiments were performed with a radio-iodinated synthetic peptide, [(125-)I]-WKYMVm, a known potent FPRL-1 agonist. CHO-K1 cell membranes expressing FPRL-1 bound [(125-)I]-WKYMVm with a K(d) value of 9.34. Many known FPRL-1 agonists were tested and sCKbeta8-1 was the most effective nonsynthetic ligand in displacing the radiolabelled agonist, with a pIC(50) of 7.97. 6. The functional significance of sCKbeta8-1 interaction with FPRL-1 was further demonstrated by the activation of polymorphonuclear leukocytes (PMNs) calcium mobilisation and chemotaxis. These interactions were shown to be via FPRL-1 by specific blockade of PMNs activation in the presence of an FPRL-1 antibody.

Pharmacological profiles of a novel opioid receptor-like1 (ORL(1)) receptor antagonist, JTC-801.

Pharmacological effects of a novel opioid receptor-like1 (ORL(1)) receptor antagonist, [N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl) benzamide monohydrochloride] (JTC-801), were examined in in vitro and in vivo. JTC-801 inhibited the binding of [(3)H]-nociceptin to human ORL(1) receptors expressed in HeLa cells with a K(i) value of 44.5 nM. JTC-801 completely antagonized the suppression of nociceptin on forskolin-induced accumulation of cyclic AMP (IC(50) : 2.58 microM) using ORL(1) receptor expressing HeLa cells in vitro. In in vivo, when given intravenously at dosages of 0.01 mg kg(-1) and above, or orally at dosages 1 mg kg(-1) and above, JTC-801 antagonized the nociceptin-induced allodynia in mice. Effects of JTC-801 on various nociceptive models were examined. In mouse hot-plate test, JTC-801 prolonged escape response latency (ERL) to exposed heat stimulus with minimum effective doses (MED) of 0.01 mg kg(-1) by i.v. or 1 mg kg(-1) by p.o. In the rat formalin test, JTC-801 reduced both the first and second phases of the nociceptive response with MED of 0.01 mg kg(-1) by i.v. administration or 1 mg kg(-1) by p.o. administration. This anti-nociceptive action of JTC-801 was not inhibited by naloxone (10 mg kg(-1), s.c.). We have demonstrated that JTC-801 antagonizes the ORL(1) receptor response, and that JTC-801 has efficacious and potent anti-nociceptive effects in acute pain animal models not only by intravenous injection but also oral administration. These results suggest that JTC-801 may represent a new class of analgesics.

Postnatal Handling Induces Long-term Modifications in Central β-noradrenergic Signalling in Rats

Neonatal handling has been shown to induce a short-term reduction in the binding properties of g -adrenoceptors and in their primary biochemical responses in the young rat brain, which may account for the reduced responsiveness to stress observed in the handled rats. We have studied the persistence and duration of these changes in cerebral cortex, cerebellum and hippocampus at successive stages of life in neonatally handled rats. Binding properties of the g -adrenoceptors in handled animals were essentially similar to those of the control rats from 3 to 24 months of age. However, handling disturbed the efficacy of the g -adrenoceptor intracellular signalling since the isoprenaline-induced accumulation of cyclic AMP was reduced in cerebral cortex at 1, 12 and 24 months; in cerebellum at 12 and 18 months; and in hippocampus at 3 and 12 months. This effect might be partially dependent on a regulatory action of handling on the adenylyl cyclase enzyme itself since both basal and forskolin-induced accumulation of cyclic AMP were persistently reduced. Therefore, postnatal handling can be considered as an effective intervention that modifies sensitivity to various hormonal and neurochemical signals; these changes may be involved over a long period of time in protection against an excessive response to stress. These results suggest that the long-lasting adaptation of the adenylyl cyclase transduction system, which affects the primary biochemical response of the g -adrenoceptor, may participate in the effects of this manipulation.

Synthesis and structure-activity relationships of trisubstituted phenyl urea derivatives as neuropeptide Y5 receptor antagonists.

1-((1R,2R)-2-Hydroxy-1-methyl-2-phenylethyl)-1-methyl-3-(4-phenoxyphenyl)urea (1) was identified as a hit from the screening of the neuropeptide Y5 (NPY5) receptor. This lead was optimized for in vitro potency by changing the stereochemistry, the phenylethyl segment, the urea portion, and the 4-phenoxyphenyl group on the molecule. Over 40 analogues of 1 were prepared to study the structure-activity relationship for this series. The most potent compounds in this class have IC(50)s less than 0.1 nM at the NPY5 receptor (e.g., 40f, 44a, and 47). To determine the functional activity for this series of compounds, selected analogues were tested in a cellular assay measuring forskolin-induced cyclic AMP accumulation in 293 cells transfected with the human NPY5 receptor. All urea analogues tested in the functional assay acted as antagonists (e.g., 1, 32, 40a, and 44e).

Involvement of the cyclic AMP system in the switch from tolerance into supersensitivity to the antinociceptive effect of the opioid sufentanil.

1. We have previously demonstrated that chronic and simultaneous treatment of rats with the mu-opioid receptor agonist sufentanil and the Ca(2+) channel blocker nimodipine, not only prevented tolerance development, but the animals became supersensitive to the antinociceptive effect of the opioid. The focus of the present work was to determine the possible involvement of cross interactions between the adenylyl cyclase pathway and L-type voltage-sensitive Ca(2+)-channels, in modulating the switch from opioid tolerance into supersensitivity. 2. The modulatory effect of sufentanil on adenylyl cyclase activity was determined by measuring cyclic AMP production in slices from the cortex of rats rendered tolerant or supersensitive to the antinociceptive effect of the opioid. Tolerance was induced by chronic infusion of sufentanil, at a rate of 2 microg h(-1), for 7 days. Supersensitivity was induced by concurrent infusion of sufentanil (2 microg h(-1)) and nimodipine (1 microg h(-1)) for 7 days. Antinociception was evaluated by the tail-flick test. 3. Tolerance to the analgesic effect of sufentanil was associated with a significant reduction in the response of adenylyl cyclase to forskolin. Furthermore, the effect of the opioid on forskolin-induced cyclic AMP accumulation was abolished. On the other hand, supersensitivity to the analgesic effect of the opioid was associated with an increase in both, the adenylyl cyclase response to forskolin, and the opioid inhibition of cyclic AMP production. 4. We suggest that sustained L-type Ca(2+) channel blockade may result in changes in the adenylyl cyclase effector system triggered by mu-opioid receptor activation, leading to the switch from opioid tolerance into supersensitivity.

Supersensitization of the adenylyl cyclase system in Chinese hamster ovary cells co-expressing cloned opioid receptors and G z, a PTX-insensitive G protein

Chronic and/or sustained stimulation of opioid receptors has been shown to lead to an increase in activity of the adenylyl cyclase (AC) system via pertussis toxin (PTX)-sensitive G i/o, family G protein. In the present study, we examined whether supersensitization of the AC system is induced via a PTX-insensitive G protein, G z, activation of which leads to the inhibition of AC activity. In Chinese hamster ovary (CHO) cells expressing either μ- or κ-opioid receptors, acute treatment with morphine or U69,593, but not naloxone or norbinaltorphimine, respectively, suppressed forskolin-induced cyclic AMP (cAMP) accumulation, while sustained (4 h) treatment with the opioid agonists induced cAMP overshoot above the naive level (supersensitization of the AC system). Both effects were completely blocked by pretreatment with PTX. In CHO cells co-expressing μ- or κ-opioid receptors and α z, inhibitory effects of cAMP accumulation by acute treatment with the opioid agonists and supersensitization of the AC system by sustained treatment with them were induced despite pretreatment with PTX. These data suggest that supersensitization of the AC system is induced by sustained opioid agonist treatment not only via G i/o but also via G z.

Homologous regulation of the α2C‐adrenoceptor subtype in human hepatocarcinoma, HepG2

1. Previous studies of the regulation of the alpha2C-adrenoceptor in OK and in transfected cells have led to discrepant conclusions. In the present work, we examined the homologous regulation of the human alpha2C-adrenoceptor in the hepatocarcinoma cell-line, HepG2; a model which expresses this subtype spontaneously. 2. Short-period treatment of the cells with UK14304 provoked neither a diminution of the potency of the alpha2-agonist to inhibit forskolin-induced cyclic AMP-accumulation nor a change in the degree of receptor coupling to G-proteins. 3. Long-period exposure to UK14304 resulted in a large reduction of [3H]MK912 binding sites (55% decrease). The action of UK14304 was dose-dependent (EC50 = 190 +/- 45 nM), rapid (t1/2 = 4.2 h) and reversible. Receptor down-regulation was also observed with clonidine or (-)adrenaline (38 and 36% decrease, respectively) and was blocked by the addition of alpha2-antagonists. 4. Conversely to that observed with alpha2-agonists, treatment of the cells with RX821002 or yohimbine alone, but not with phentolamine, promoted a significant increase of the receptor expression. 5. The observed alterations of receptor density are not the reflection of changes at the alpha2C4 mRNA level. Estimation of the receptor protein turnover and measurement of its half-life demonstrated that down-regulation by alpha2-agonists and up-regulation by alpha2-antagonists, with inverse-agonist efficacy, are respectively the consequence of increased and decreased rate of receptor degradation. 6. In conclusion, our data show that alpha2C-adrenoceptor does not undergo desensitization but is down-regulated in HepG2. The lack of desensitization agrees with previous results obtained in cells transfected with the alpha2C4 gene, but not with observations made in OK cells. Inversely, down-regulation fits with results obtained in OK but not in transfected cells. The reasons for these discrepancies are discussed. Our results also demonstrated that certain alpha2-antagonists behave as inverse agonist on the HepG2 model and thus provide for the first time evidence of inverse efficacy of antagonists on a cellular model expressing physiological level of a wild-type alpha2-adrenoceptor.

Adenylate cyclases in keratinocytes: FRSK cells express types I, II, III, IV, VI and VIII, and 1,25(OH)2D3, retinoic acid and TPA augment forskolin-induced cyclic AMP accumulation in the absence of altered isozyme expression.

Molecular cloning analysis has detected at least nine adenylate cyclase isozymes in mammalian tissues. Using fetal rat skin keratinocytes (FRSK), we investigated adenylate cyclase expression and its modulation by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), a retinoid (Ro10-1670), and 12-O-tetradecanoylphorbol-13-acetate (TPA). Reverse transcription polymerase chain reaction (RT-PCR) indicated that FRSK contain adenylate cyclases I, II, III, IV, VI and VIII. Treatment with 1,25(OH)2D3 (1 x 10(-7) M), Ro10-1670 (1 x 10(-6) M), and TPA (100 ng/ml) resulted in increased forskolin-induced cyclic AMP accumulation by FRSK cells and normal human keratinocytes (NHK). Quantitative RT-PCR and Western blot analysis, however, detected no alteration in mRNA and protein levels of each adenylate cyclase isozyme for at least 48 h. These results indicate that FRSK contain at least six (I, II, III, IV, VI and VIII) adenylate cyclase isozyme mRNAs, suggesting a complex regulatory mechanism of cyclic AMP generation in keratinocytes. Although 1,25(OH)2D3, Ro10-1670, and TPA augmented forskolin-induced cyclic AMP accumulation, they do not seem to affect the expression of specific adenylate cyclase isozymes by FRSK cells.

Functional alteration of guanine nucleotide binding proteins (Gs and Gi) in psoriatic epidermis

Psoriatic involved epidermis has been characterized by a defective β-adrenergic adenylate cyclase response. It is also characterized by increased cholera toxin- and forskolin-induced cyclic AMP accumulations. Due to the fact that receptor signals are transduced to adenylate cyclase through guanine nucleotides binding proteins (G-proteins), that affect cholera toxin- and forskolin-induced cyclic AMP accumulations, possible alterations of G-proteins of psoriatic involved and perilesional uninvolved epidermis by using toxin-catalyzed ADP-ribosylation and immunoblot analyses was investigated. Cholera toxin catalyzes ADP-ribosylation of stimulatory guanine nucleotides binding protein (Gs) in either trimeric (inactive) or monomeric (active) form, while islet activating protein (IAP) catalyzes ADP ribosylation of inhibitory guanine nucleotide binding protein (Gi) in only trimeric (inactive) form. Results indicate that although the psoriatic involved epidermis shows increased cholera toxin- and IAP-catalyzed ADP-ribosylations, the amounts of immunoreactive Gs α or Gi α are not significantly altered. The increase in IAP-catalyzed ADP-ribosylation indicates increased inactive Gi, explaining the increased forskolin-induced cyclic AMP accumulation. The increase in cholera toxin-catalyzed ADP-ribosylation of Gs explains the increased cholera toxin-induced cyclic AMP accumulation in the psoriatic involved epidermis.

Characterization of LY344864 as a pharmacological tool to study 5-HT 1F receptors: Binding affinities, brain penetration and activity in the neurogenic dural inflammation model of migraine

LY344864 is a selective receptor agonist with an affinity of 6 nM (K i) at the recently cloned 5-HT 1F receptor. It possesses little affinity for the 56 other serotonergic and non-serotonergic neuronal binding sites examined. When examined for its ability to inhibit forskolininduced cyclic AMP accumulation in cells stably transfected with human 5-HT 1F receptors, LY344864 was shown to be a full agonist producing an effect similar in magnitude to serotonin itself. After an intravenous dose of 1 mg/kg, rat plasma LY344864 levels declined with time whereas brain cortex levels remained relatively constant for the first 6 hours after injection. Oral and intravenous LY344864 administration potently inhibited dural protein extravasation caused by electrical stimulation of the trigeminal ganglion in rats. Taken together, these data demonstrate that LY344864 is a selective 5-HT 1F receptor agonist that can be used to explore both the in vitro and in vivo functions of this receptor.

Recognition and activation of the opioid receptor-like ORL1 receptor by nociceptin, nociceptin analogs and opioids

Nociceptin, also known as orphanin FQ, was recently identified as the naturally occurring agonist of orphan opioid receptor-like ORL1 receptor (Meunier et al., 1995, Nature 377, 532; Reinscheid et al., 1995, Science 270, 792). Nociceptin is a heptadecapeptide which, although it resembles dynorphin A, the endogenous agonist of the κ-opioid receptor, displays very low potency in competing with binding of [ 3H]diprenorphine to or inhibiting adenylate cyclase via μ-, δ- and κ-opioid receptors. Tritium-labeled nociceptin ([ 3H]nociceptin) was used here to establish a pharmacological profile in vitro of the ORL1 receptor. In membranes from recombinant Chinese hamster ovary (CHO) cells expressing the ORL1 receptor, equilibrium binding of [ 3H]nociceptin is highly specific, saturable ( B max in the range 1.3–1.8 pmol/mg protein) and of high affinity ( K d ≈ 0.1 nM). It is selectively decreased in the presence of Na + ions and/or of the GTP analog 5′-guanylylimido-diphosphate, an allosteric regulation that is analogous to that of opiate binding to opioid receptors. A few opiates, namely lofentanil, a 4-anilinopiperidine derivative and etorphine, a 6,14- endo-ethenotetrahydrothebaine derivative, were found to be quite potent not only in competing with binding of [ 3H]nociceptin at the ORL1 receptor but also in inhibiting forskolin-induced accumulation of cyclic AMP in intact recombinant CHO cells. In a preliminary attempt to delineate active parts of the neuropeptide, nociceptin analogs were also tested, including N- and C-terminal truncation products. Our results suggest that the highly basic, internal core of nociceptin might be essential in conferring on the peptide both affinity for and activity at the ORL1 receptor. In this respect, the message and address division of dynorphin A, nociceptin's closest structural analog, do not seem to apply to nociceptin.

Identification and characterization of an endogenous ligand for opioid receptor homologue ROR-C: its involvement in allodynic response to innocuous stimulus

We reported here purification and characterization of a novel heptadecapeptide in bovine brain as an endogenous ligand for ROR-C, an opioid receptor homologue cloned from rat cerebrum. The amino acid sequence of the peptide that we purified is identical to those recently identified as nociceptin in rat brain and orphanin FQ in porcine brain. The peptide inhibited the forskolin-induced cyclic AMP accumulation in ROR-C expressing Chinese hamster ovary cells. Studies on inhibitory activity of cyclic AMP accumulation and Northern blot analysis showed that the peptide and its precursor mRNA are present in a number of brain regions, less abundant in the spinal cord, and negligible in the cerebellum. In situ hybridization analysis revealed that hybridization-positive neurons were distributed in the superficial layer (lamina I) of the dorsal horn and were also interspersed between the tract of Lissauer in the spinal cord. Intrathecal administration of the peptide into conscious mice induced allodynia, a pain response to innocuous tactile stimuli, in a bell-shaped manner. These results demonstrate that the peptide exists in the brain and spinal cord and plays an important role in pain transmission.

KW‐3902, a selective high affinity antagonist for adenosine A1 receptors

1. We demonstrate that 8-(noradamantan-3-yl)-1,3-dipropylxanthine (KW-3902) is a very potent and selective adenosine A1 receptor antagonist, assessed by radioligand binding and cyclic AMP response in cells. 2. In rat forebrain adenosine A1 receptors labelled with [3H]-cyclohexyladenosine (CHA), KW-3902 had a Ki value of 0.19 nM, whereas it showed a Ki value of 170 nM in rat striatal A2A receptors labelled with [3H]-2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoad enosine (CGS21680), indicating 890 fold A1 receptor selectivity versus the A2A receptor. KW-3902 at 10 microM showed no effect on recombinant rat A3 receptors expressed on CHO cells. 3. Saturation studies with [3H]-KW-3902 revealed that it bound with high affinity (Kd = 77 pM) and limited capacity (Bmax = 470 fmol mg-1 of protein) to a single class of recognition sites. A high positive correlation was observed between the pharmacological profile of adenosine ligands inhibiting the binding of [3H]-KW-3902 and that of [3H]-CHA. 4. KW-3902 showed potent A1 antagonism against the inhibition of forskolin-induced cyclic AMP accumulation in DDT1 MF-2 cells by the A1-selective agonist, cyclopentyladenosine with a dissociation constant (KB value) of 0.34 nM. KW-3902 antagonized 5'-N-ethylcarboxamidoadenosine-elicited cyclic AMP accumulation via A2B receptors with a KB value of 52 nM. 5. KW-3902 exhibited marked species-dependent differences in the binding affinities. The highest affinity was for the rat A1 receptor (ki = 0.19 nM) and these values for guinea-pig and dog A1 receptors were 1.3 and 10 nM, respectively.

1,25-Dihydroxyvitamin D 3 increased β-adrenergic adenylate cyclase response of fetal rat keratinizing epidermal cells (FRSK cells)

Using fetal rat keratinizing epidermal cells (FRSK), the effects of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) on adenylate cyclase system were investigated. The β-adrenergic adenylate cyclase response was significantly increased by the stimulation of 1 × 10 −7 M 1,25(OH) 2D 3. The effect was observed by 6 h and continued for at least 48 h. The 1,25(OH) 2D 3-induced β-adrenergic augmentation effect was dose-dependent and the maximal response was observed at a concentration of 1 × 10 −7 M 1,25(OH) 2D 3. Other adenylate cyclase systems (adenosine, prostaglandin E 2 and histamine) were not affected by treatment with 1,25(OH) 2 D 3. The thymidine incorporation in FRSK cells was not significantly affected by 1,25(OH) 2D 3 treatment. Forskolin-induced cyclic AMP accumulation was significantly increased by 1,25(OH) 2D 3 treatment. Cholera toxin-induced cyclic AMP accumulation was moderately increased, but this was statistically not significant. Northern blot hybridization showed that none of the mRNAs (the β 2-adrenergic receptor, the α subunits of the stimulatory or inhibitory guanine nucleotide binding proteins, Gsα, Gi2α, Gi3α) were significantly altered by 1,25(OH) 2D 3 treatment. We have already reported that the β-adrenergic response was increased by dexamethasone and inhibited by retinoids in FRSK cells. The addition of both 1,25(OH) 2D 3 and dexamethasone to the incubation medium resulted in an additive augmentation. On the other hand, the β-adrenergic augmentation by the 1,25(OH) 2D 3 treatment was suppressed by the addition of all trans-retinoic acids. Our results indicate that 1,25(OH) 2D 3 induces β-adrenergic augmentation without an alternation of thymidine incorporation of FRSK cells.

Protein kinase C-dependent modulation of stimulatory guanine nucleotide binding protein of fetal rat skin keratinocytes.

Although the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA) has been known to induce heterologous desensitization of the epidermal adenylate cyclase, the precise mechanism of PMA action remains unknown. Effects of PMA on the receptor-G-protein-adenylate cyclase system of fetal rat skin keratinocytes (FRSK) were investigated. Choleratoxin catalysed the ADP ribosylation of 45 kDa and 52 kDa membrane proteins and islet activating protein (IAP) catalysed the ADP ribosylation of a 40 kDa membrane protein. Incubation of FRSK with PMA decreased the cholera toxin-catalysed ADP ribosylation of the membrane protein, but not the IAP-catalysed ADP ribosylation. The effect of PMA on the cholera toxin-catalysed ADP ribosylation was inhibited by the PKC inhibitor, H-7 (1-(5-isoquinolinesulfonyl)-2-methyl piperazine dihydrochloride). 1-Oleoyl-2-acetylglycerol (OAG), a membrane-permeable diacylglycerol analogue, also decreased the cholera toxin-catalysed ADP ribosylation, but 4-0-methyl PMA, a very weak PKC activator, had no effect. Keratinocytes are known to express the guanine nucleotide binding proteins, Gsalpha, Gi2alpha, and Gi3alpha. Immunoblot analysis of the PMA-treated FRSK showed no detectable difference in the amount of Gsalpha, Gi2alpha, Gi3alpha, or the beta subunit of the G-protein. PMA significantly decreased the beta-adrenergic adenylate cyclase response and cholera toxin-induced cyclic AMP accumulation, while it markedly increased forskolin-induced cyclic AMP accumulation. These results indicate that phorbol esters affect the stimulatory guanine nucleotide binding protein (Gs) of FRSK via a PKC-dependent pathway.

Long‐term carbachol treatment‐induced down‐regulation of muscarinic M2‐receptors but not m2 receptor mRNA in a human lung cell line

1. The molecular mechanisms involved in the regulation of muscarinic receptor gene expression are poorly understood. We have investigated the effect of homologous stimulation on the regulation of M2 muscarinic receptor protein and gene in human embryonic lung fibroblasts (HEL 299 cells). 2. Saturation studies performed with the non-selective hydrophilic ([3H]-N-methyl-scopolamine, [3H]-NMS) and lipophilic (3H]-quinuclidinyl benzilate, [3H]-QNB) muscarinic antagonists revealed a single class of high affinity binding sites. 3. Carbachol (1 mM) induced a rapid down-regulation of [3H]-NMS binding sites. Within 12 h, the process had approached steady state with 40 to 60% loss of receptors at 12 and 24 h. 4. The loss of [3h]-QNB binding sites (40% reduction at 24 h) occurred at a slower rate than did loss of [3H]-NMS binding sites as a result of receptor sequestration. 5. Carbachol treatment was accompanied by a functional desensitization of the receptor after 24 h of agonist treatment. In untreated cells, forskolin induced a large increase in cyclic AMP accumulation which was inhibited significantly by carbachol. The inhibitory effect of carbachol on forskolin-induced cyclic AMP accumulation was lost following 24 h carbachol stimulation. 6. The steady state level of muscarinic m2 mRNA measured by Northern blot analysis was not affected by carbachol had no effect on the stability of m2 mRNA. 7. The rate of transcription of m2 muscarinic receptor gene as measured by nuclear RNA run-on assay was unaltered by carbachol stimulation. 8. These results suggest that homologous sequestration, desensitization, and down-regulation of M2 modifications of m2 muscarinic receptor mRNAs.

Protein kinase A and nicotinic activation of bovine adrenal tyrosine hydroxylase.

1. Stimulation of nicotinic cholinoceptors on bovine chromaffin cells increases phosphorylation of three serine residues in tyrosine hydroxylase (TOH) and activates TOH. One of the serines is a target for protein kinase A phosphorylation, and phosphorylation of this serine is adequate alone to cause TOH activation. The role of protein kinase A in nicotinic activation of TOH was therefore investigated. 2. TOH activity was studied in situ in intact, cultured, bovine adrenal chromaffin cells, by measuring 14CO2 evolved following the hydroxylation and rapid decarboxylation of [14C]-tyrosine offered to the cells. 3. Nicotine (5 microM), forskolin (1 microM) and 8-bromo-cyclic AMP (8-Br-cyclic AMP, 1 mM) each increased TOH activity by up to 200% over 10 min. The effect of nicotine was completely abolished by removal of extracellular Ca2+. 4. TOH activation by all three drugs was blocked by H89 (3-20 microM), which inhibits protein kinase A by competing for the ATP binding site on the kinase. Adenosine 3':5'-cyclic monophosphorothioate Rp-diastereomer (Rp-cAMPS) (1 mM), an inhibitor of protein kinase A that competes with cyclic AMP for the regulatory subunit of the kinase, abolished the activation of TOH by nicotine, and reduced that by forskolin and 8-Br-cyclic AMP. Both H89 and Rp-cAMPS inhibited basal TOH activity by 50-80%. 5. A structural analogue of H89, H85 (3-20 microM), which lacks activity as a protein kinase A inhibitor, did not inhibit either the activation of TOH by nicotine (5 microM) or basal TOH activity. Neither sodium nitroprusside (0.3-1O microM) nor 8-Br-cyclic GMP (1 mM) increased TOH activity.6. In digitonin-permeabilized chromaffin cells, forskolin (3 microM), cyclic AMP (10 microM) and Ca2+ (approx.2 micro M free Ca2+) each increased TOH activity. The response to all three drugs was blocked by H89(10 microM), which also reduced basal TOH activity in the permeabilized cells.7. Maximal activation of TOH by forskolin was achieved with 10 micro M forskolin. This concentration was less than the EC50 for forskolin-induced cyclic AMP accumulation in these cells. The activations of TOH by forskolin (1O microM) and nicotine (5 microM) were additive.8. The results indicate that both basal TOH activity and nicotinic activation of TOH in bovine chromaffin cells require protein kinase A activity. However, it is unlikely that nicotinic activation of TOH is directly mediated by an activation of protein kinase A in response to elevated cyclic AMP levels.It is possible that protein kinase A plays a permissive role in allowing nicotinic cholinoceptors to activate TOH by another signalling pathway.

Protein kinase C-dependent modulation of stimulatory guanine nucleotide binding protein of fetal rat skin keratinocytes

Although the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA) has been known to induce heterologous desensitization of the epidermal adenylate cyclase, the precise mechanism of PMA action remains unknown. Effects of PMA on the receptor-G-protein-adenylate cyclase system of fetal rat skin keratinocytes (FRSK) were investigated. Choleratoxin catalysed the ADP ribosylation of 45 kDa and 52 kDa membrane proteins and islet activating protein (IAP) catalysed the ADP ribosylation of a 40 kDa membrane protein. Incubation of FRSK with PMA decreased the cholera toxin-catalysed ADP ribosylation of the membrane protein, but not the IAP-catalysed ADP ribosylation. The effect of PMA on the cholera toxincatalysed ADP ribosylation was inhibited by the PKC inhibitor, H-7 (1-(5-isoquinolinesulfonyl)-2-methyl piperazine dihydrochloride). 1-Oleoyl-2-acetylglycerol (OAG), a membrane-permeable diacylglycerol analogue, also decreased the cholera toxin-catalysed ADP ribosylation, but 4-O-methyl PMA, a very weak PKC activator, had no effect. Keratinocytes are known to express the guanine nucleotide binding proteins, Gsα, Gi2α and Gi3α. Immunoblot analysis of the PMA-treated FRSK showed no detectable difference in the amount of Gsα, Gi2α, Gi3α or the β subunit of the G-protein. PMA significantly decreased the β-adrenergic adenylate cyclase response and cholera toxin-induced cyclic AMP accumulation, while it markedly increased forskolin-induced cyclic AMP accumulation. These results indicate that phorbol esters affect the stimulatory guanine nucleotide binding protein (Gs) of FRSK via a PKC-dependent pathway.