Inhibition of adenylate cyclase of catfish and rat hepatocyte membranes by 9-(tetrahydro-2-furyl)adenine (SQ 22536).

The adenosine analogue 9-(Tetrahydro-2-furyl)adenine, SQ 22536, inhibited adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity of crude membrane preparations from catfish (Ictalurus melas) and rat isolated hepatocytes in a non-competitive manner. The IC50s were reduced in the presence of NaF. SQ 22536 reduced the activity of adenylate cyclase also in the presence of increasing concentrations of GTP, as well as Mg++ and Mn++. In the presence of catecholamines (epinephrine, norepinephrine, isoproterenol, phenylephrine) SQ 22536 reduced their activating effect on adenylate cyclase in both catfish and rat membranes. SQ 22536 also inhibited the effect of glucagon (0.1 microM) on rat membrane cyclase activity.     

The effects of inhibitors of adenylate cyclase and phosphodiesterase on D. discoideum aggregation.

The action of two adenine analogues on the aggregation of D. discoideum amebae was examined. SQ22536 and SQ20009 are inhibitors of adenylate cyclase and phosphodiesterase, respectively, in higher eukaryotes. Both compounds are shown here to inhibit the differentiation of cells to aggregation-competence. SQ22536-treated cells exhibited normal accumulation of their adenylate cyclase activity as measured in cell lysates but the amebae did not synthesize cAMP. The ability of this drug to compete for cAMP surface-binding sites, and the observation that the effects of the drug could be reversed by imposed pulses of cAMP, suggest that SQ22536 functions as a cAMP antagonist. The effects of SQ20009 on cell differentiation did not appear to be mediated by an inhibition of phosphodiesterase activity or cAMP binding to the cell surface. Amebae were arrested at a very early stage in development and remained unresponsive to external cAMP.     

Alterations in lipid peroxidation and antioxidant status in pregnancy with preeclampsia

Soy consumption is associated with a lower risk of atherosclerotic disease in the oriental population. Genistein is a soy isoflavone bearing estrogenic properties. Previous experiments in our laboratory demonstrated the potentiation of endothelium-independent relaxation of coronary artery by both estrogen and genistein. The potentiating effects of both estrogen and genistein were mediated through the cAMP-signaling pathway. We hypothesize that genistein could enhance protein kinase A (PKA) activity in porcine coronary artery smooth muscle, thereby offering a mechanism for the potentiation of vascular relaxation by genistein. In our study, a high concentration of genistein (10^−4.5 M) significantly increased PKA activity in porcine coronary artery rings. While genistein at 10^−5.5 M and forskolin at 10⁻⁷ M had no effect on PKA activity, the combination of the two compounds at the prescribed concentrations caused a significant increase in PKA activity. The increase in PKA activity by genistein was abolished by SQ 22536 (adenylate cyclase blocker), but not by NF 449 (Gs protein blocker) or ICI 182780 (estrogen receptor antagonist). Our results suggest that the action of genistein is mediated via adenylate cyclase, but does not appear to involve Gs protein or ICI 182780-sensitive estrogen receptor.     

Regulation of ethanol-sensitive EAAT2 expression through adenosine A1 receptor in astrocytes

Adenosine-regulated glutamate signaling in astrocytes is implicated in many neurological and neuropsychiatric disorders. In this study, we examined whether adenosine A1 receptor regulates EAAT2 expression in astrocytes using pharmacological agents and siRNAs. We found that adenosine A1 receptor-specific antagonist DPCPX or PSB36 decreased EAAT2 expression in a dose-dependent manner. Consistently, knockdown of A1 receptor in astrocytes decreased EAAT2 mRNA expression while overexpression of A1 receptor upregulated EAAT2 expression and function. Since A1 receptor activation is mainly coupled to inhibitory G-proteins and inhibits the activity of adenylate cyclase, we investigated the effect of forskolin, which activates adenylate cyclase activity, on EAAT2 mRNA levels. Interestingly, we found that forskolin reduced EAAT2 expression in dose- and time-dependent manners. In contrast, adenylate cyclase inhibitor SQ22536 increased EAAT2 expression in dose- and time-dependent manners. In addition, forskolin blocked ethanol-induced EAAT2 upregulation. Taken together, these results suggest that A1 receptor-mediated signaling regulates EAAT2 expression in astrocytes.     

Particulate adenylate cyclase plays a key role in human sperm olfactory receptor-mediated chemotaxis

Human sperm chemotaxis is a critical component of the fertilization process, but the molecular basis for this behavior remains unclear. Recent evidence shows that chemotactic responses depend on activation of the sperm olfactory receptor, hOR17-4. Certain floral scents, including bourgeonal, activate hOR17-4, trigger pronounced Ca(2+) fluxes, and evoke chemotaxis. Here, we provide evidence that hOR17-4 activation is coupled to a cAMP-mediated signaling cascade. Multidimensional protein identification technology was used to identify potential components of a G-protein-coupled cAMP transduction pathway in human sperm. These products included various membrane-associated adenylate cyclase (mAC) isoforms and the G(olf)-subunit. Using immunocytochemistry, specific mAC isoforms were localized to particular cell regions. Whereas mAC III occurred in the sperm head and midpiece, mAC VIII was distributed predominantly in the flagellum. In contrast, G(olf) was found mostly in the flagellum and midpiece. The observed spatial distribution patterns largely correspond to the spatiotemporal character of hOR17-4-induced Ca(2+) changes. Behavioral and Ca(2+) signaling responses of human sperm to bourgeonal were bioassayed in the presence, or absence, of the adenylate cyclase antagonist SQ22536. This specific agent inhibits particulate AC, but not soluble AC, activation. Upon incubation with SQ22536, cells ceased to exhibit Ca(2+) signaling, chemotaxis, and hyperactivation (faster swim speed and flagellar beat rate) in response to bourgeonal. Particulate AC is therefore required for induction of hOR17-4-mediated human sperm behavior and represents a promising target for future design of contraceptive drugs.     

Muscarinic Receptors Modulate Dopamine-Activated Adenylate Cyclase of Rat Striatum

We investigated the effect of acetylcholine (ACh) on the activation of adenylate cyclase by dopamine (DA) in a lysed synaptosomal preparation from rat striatum. ACh reduced both basal and the DA-activated adenylate cyclase with an apparent IC50 of approximately 1 microM. From a kinetic analysis it appeared that ACh reduced the Vmax for activation by DA but not the activation constant for DA. For most preparations the Vmax was reduced by 30-40%. The presence of atropine did not affect the activation of the enzyme by DA but it blocked the inhibition by ACh. Following 6-hydroxydopamine lesion of the nigrostriatal pathway, the enzyme became supersensitive to activation by DA and also more sensitive to inhibition by ACh. Inhibition of adenylate cyclase by ACh appeared to be rather specific for activation by DA, as ACh had no effect on activation of adenylate cyclase by the adenosine analogue N6-(L-2-phenylisopropyl)adenosine. These results indicate that some striatal muscarinic and dopaminergic receptors are probably coupled to the same adenylate cyclase domain. Moreover, they suggest a biochemical model for the dynamic balance of cholinergic and dopaminergic neurons that innervate the striatum.     

Effects of sodium fluoride on the mechanical activity in mouse gastric preparations

The aim of the present study was to investigate the responses induced by sodium fluoride (NaF) on gastric mechanical activity, using mouse whole-stomach preparations. The mechanical activity was recorded in vitro as changes of intraluminal pressure. In most of the preparations, NaF induced a tetrodotoxin-insensitive biphasic effect characterized by early relaxation followed by slowly developing contractile response. The contraction was dependent on the concentration of NaF, whereas the relaxation was observed at only 10-30 mmol/L NaF. The contractile effect was significantly reduced by nifedipine (an L-type Ca(2+) channel blocker), ryanodine or ruthenium red (inhibitors of Ca(2+) release from sarcoplasmic reticulum), and GF109203X (a protein kinase C inhibitor). Moreover, it was abolished by neomycin (an inhibitor of phospholipase C) and potentiated by SQ22536 (an inhibitor of adenylyl cyclase). All the drugs significantly increased the relaxation, except SQ22536, which abolished it. The present results suggest that NaF causes a complex mechanical response in the whole-stomach, which might explain gastric discomfort after fluoride ingestion. The relaxation appears owing to production of cAMP, while the contractile effects imply activation of phospholipase C, protein kinase C, influx of Ca(2+), and release of Ca(2+) from ryanodine-sensitive intracellular store.     

Differential Modulation of D1 and D2 Dopamine-Sensitive Adenylate Cyclases by 17β-Estradiol in Cultured Striatal Neurons and Anterior Pituitary Cells

Primary cultures of anterior pituitary cells from female rats and of mouse embryonic striatal neurons were used to study the effects of 17 beta-estradiol on D1- and D2-dopamine (DA)-sensitive adenylate cyclase. 17 beta-Estradiol pretreatment (10(-9) M, 72 h) suppressed the D2-DA-induced inhibition of adenylate cyclase activity in anterior pituitary cells. The steroid (10(-9) M, 24 h) also blocked the D2-DA-evoked response in striatal neurons whereas it enhanced by twofold the D1-DA-induced stimulation of the enzyme activity in these neurons. All these effects of the steroid were dose dependent and specific, as neither 17 alpha-estradiol, dexamethasone, nor progesterone used at the same concentration (10(-9) M) was effective. Furthermore, the modulation of DA-sensitive adenylate cyclases by the steroid required long-term exposure of living cells to 17 beta-estradiol since neither 17 beta-estradiol pretreatment for 4 h nor its addition to broken cells directly into the adenylate cyclase assay induced any alteration in the DA-sensitive adenylate cyclase activity. These results are in agreement with a genomic effect of the steroid. Using both anterior pituitary cells and striatal neurons in culture, 17 beta-estradiol affected neither the total number of DA (D1 and D2) receptors nor the estimated number of adenylate cyclase catalytic units. Therefore, it is suggested that the steroid modifies the coupling process by a mechanism that still has to be elucidated. These results demonstrate an effect of 17 beta-estradiol on DA target cells in both systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of adenylate cyclase by adenosine analogues in preparations of broken and intact human platelets. Evidence for the unidirectional control of platelet function by cyclic AMP.

Whereas adenosine itself exerted independent stimulatory and inhibitory effects on the adenylate cyclase activity of a platelet particulate fraction at low and high concentrations respectively, 2-substituted and N6-monosubstituted adenosines had stimulatory but greatly decreased inhibitory effects. Deoxyadenosines, on the other hand, had enhanced inhibitory but no stimulatory effects. The most potent inhibitors found were, in order of increasing activity, 9-(tetrahydro-2-furyl)adenine (SQ 22536), 2',5'-dideoxyadenosine and 2'-deoxyadenosine 3'-monophosphate. Kinetic studies on prostaglandin E1-activated adenylate cyclase showed that the inhibition caused by either 2',5'-dideoxyadenosine or compound SQ 22536 was non-competitive with MgATP and that the former compound, at least, showed negative co-operativity; 50% inhibition was observed with 4 micron-2',5'-dideoxyadenosine or 13 micron-SQ 22536. These two compounds also inhibited both the basal and prostaglandin E1-activated adenylate cyclase activities of intact platelets, when these were measured as the increases in cyclic [3H]AMP in platelets that had been labelled with [3H]adenine and were then incubated briefly with papaverine or papaverine and prostaglandin E1. Both compounds, but particularly 2',5'-dideoxyadenosine, markedly decreased the inhibition by prostaglandin E1 of platelet aggregation induced by ADP or [arginine]vasopressin as well as the associated increases in platelet cyclic AMP, so providing further evidence that the effects of prostaglandin E1 on platelet aggregation are mediated by cyclic AMP. 2'-Deoxyadenosine 3'-monophosphate did not affect the inhibition of aggregation by prostaglandin E1, suggesting that the site of action of deoxyadenosine derivatives on adenylate cyclase is intracellular. Neither 2',5'-dideoxyadenosine nor compound SQ 22536 alone induced platelet aggregation. Moreover, neither compound potentiated platelet aggregation or the platelet release reaction when suboptimal concentrations of ADP, [arginine]vasopressin, collagen or arachidonate were added to heparinized or citrated platelet-rich plasma in the absence of prostaglandin E1. These results show that cyclic AMP plays no significant role in the responses of platelets to aggregating agents in the absence of compounds that increase the platelet cyclic AMP concentration above the resting value.     

Tacrine-induced tachyphylaxis in gastric smooth muscles

Tacrine is a medication applied in cases of mild to moderate dementia in Alzheimer??s disease. By blocking acetylcholinesterase activity the drug increases the concentration of acetylcholine, whose effects influence the functions of different organs and systems of the body. The effect of tacrine on smooth muscle preparations isolated from rat stomach was studied by isometric registration of muscle contractility. Our investigations found a specific significant systematic decrease in the strength of consecutive tacrine-induced contractions of smooth muscle preparations, a phenomenon known as tachyphylaxis. The tacrineinduced tachyphylaxis was significantly inhibited by SQ22536 (inhibitor of adenylate cyclase activity), by blockers of nitric oxide synthase and KT5823 (inhibitor of protein kinase G). The process was not influenced by cyclopiazonic acid (specific blocker of sarco/endoplasmic reticulum Ca²⁺-ATPase,) and atropine (blocker of M-cholinergic receptors). We hypothesize that the overlapping and different time-development of the two opposing processes: smooth muscle contraction caused by acetylcholinesterase inhibition and tacrine-induced relaxation influenced by synthesis of nitric oxide, results in tachyphylaxis.     

Membrane transduction pathway in the neuronal control of protein secretion by the albumen gland in Helisoma (Mollusca)

The albumen gland in Helisoma secretes a perivitelline fluid which surrounds each egg and is made up of several 66 kDa protein subunits and polysaccharide complexes. Forskolin, an adenylate cyclase activator, stimulated the secretion and release of the perivitelline fluid. An acidic extract of the central nervous system increased the intracellular concentration of cAMP in the albumen gland and this results in the release of the 66 kDa molecule and other proteins. Digestion of the brain extract with proteases abolished this activity, suggesting that the factor is a peptide. Cyclic AMP analogues and [BMX also stimulated the protein secretion in dose-dependent manner. Forskolin when added with the brain factor had an additive response. SQ22536, a non-competitive inhibitor of adenylate cyclase, inhibited brain extract dependent adenylate cyclase activity whereas aluminum fluoride, a G protein activator, was found to stimulate adenylate cyclase. Dopamine also stimulates protein secretion by the albumen gland and through the application of various agonists and antagonists of dopamine, it was established that the neurotransmitter acts via D1-like receptors by stimulating adenylate cyclase.     

Long-term estradiol treatment improves VIP-mediated vasodilation in atherosclerotic proximal coronary arteries

The aim of the present study was to evaluate the impact of long-term estrogen replacement therapy (ERT) on the vasodilatory effect of the two peptides vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) in atherosclerotic coronary and cerebral arteries.Female ovariectomized homozygous Watanabe heritable hyperlipidemic rabbits were randomized to 16 weeks treatment with 17beta-estradiol or placebo. The diet was semisynthetic, thereby avoiding the influence of phytoestrogens. Artery ring segments were mounted for isometric tension recordings in myographs. Following precontraction, the dose-response relationships for VIP and PACAP were evaluated.Treatment with 17beta-estradiol significantly improved the maximum VIP-mediated vasodilation (E(max), percentage of precontraction) in proximal coronary arteries (45.8+/-9.6% vs. 24.1+/-3.7%, p<0.05). In the same artery segment, 17beta-estradiol induced a significant decrease in the relative ratio between the repeated contractile response to potassium 30 and 120 mM (100+/-7% vs. 132+/-11%, p<0.05). For distal coronary arteries, there was a tendency to similar changes, but no statistical differences for the potassium or VIP responses in cerebral or distal coronary arteries were found between the two groups. 17beta-estradiol induced no changes in the PACAP-mediated vasodilation.These results suggest that long-term treatment with 17beta-estradiol improves the VIP-mediated but not the PACAP-mediated vasodilation in atherosclerotic proximal coronary arteries.     

Comparative vasodilation of peroxynitrite and 3-morpholinosydnonimine.

Vasorelaxation mediated by peroxynitrite (ONOO-) and 3-morpholinosydnonimine (SIN-1) were investigated in isolated bovine intramammary arteries. Both ONOO- and SIN-1 relaxed U 46619-precontracted rings in a dose-dependent, endothelium-independent manner. Pretreatment with an adenylyl cyclase inhibitor, SQ 22536 [(9-tetrahydro-2-furyl)adenine], resulted in an enhanced ONOO--mediated relaxation, but did not modulate the response to SIN-1. ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), a potent and selective inhibitor of soluble guanylyl cyclase (sGC), did not significantly affect relaxant actions of ONOO-, but ODQ markedly attenuated SIN-1-elicited relaxation with a rightward shift in the dose-response curve and an unaltered maximal response. In the presence of carboxy-PTIO (2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide), a putative nitric oxide scavenger and ONOO- inactivator, the relaxant response to ONOO- was abolished, while relaxant actions of SIN-1 appeared to be unaffected. The results reveal a difference between ONOO- and SIN-1-mediated relaxation with regards to the role of the sGC and suggest that ONOO--evoked relaxation may not be associated with sGC activity, but rather depends on an sGC-independent mechanism triggered by ONOO- and/or NO itself. It also re-emphasizes that SIN-1 induces a vasorelaxant response, in part, via stimulation of sGC.     

Role of cyclic nucleotide phosphodiesterase in the effect of estradiol on uterine tissue

It is shown that 17 beta-estradiol (10(-7)--10(-5) M) inhibited phosphodiesterase activity of the preparations (supernatant 100000 epsilon, 1 h) obtained from uterine tissue of sexually mature rats and did not affect adenylate cyclase activity of crude membrane fraction of this tissue. The hormone did not change phosphodiesterase activity of the preparations obtained from the brain, heart and outer segments of the retinal rods. Cytosol preparations from uterine tissue were demonstrated to be able to specific hormone binding. The antiestrogen clomifen completely blocked the binding. In the presence of clomifen estradiol had no effect on phosphodiesterase activity. It is suggested that estrogen receptors are necessary for the effect of 17 beta-estradiol on phosphodiesterase to be realized in uterine tissue.     

Pituitary adenylate cyclase activating peptide (PACAP) in guinea-pig lung: distribution and dilatory effects.

The lower airways of guinea-pigs were analyzed for pituitary adenylate cyclase activating peptide (PACAP) using immunocytochemistry. In the trachea a moderate supply of PACAP-immunoreactive nerve fibers occurred around smooth muscle bundles, glands and small blood vessels. In the lung, PACAP-immunoreactive nerve fibers were distributed around small glands and bronchi. A rich supply of PACAP immunoreactive nerve fibers was found around blood vessels in the lungs. PACAP-suppressed smooth muscle responses were analysed using isolated circular segments of trachea, pulmonary arteries and aorta of guinea-pigs. In both airways and arteries PACAP caused a concentration-dependent relaxation of precontracted segments. The maximal relaxation effects were more pronounced in the airways than in the arteries while the order of potency was aorta greater than pulmonary artery greater than trachea. The effect of PACAP was compared to those of acetylcholine (ACh) and vasoactive intestinal peptide (VIP). In the pulmonary artery the vasomotor responses expressed as maximal dilatation had the order: ACh greater than VIP = PACAP while the order of potency was PACAP = VIP greater than ACh. In the trachea, PACAP was slightly more potent than VIP. The relaxatory responses to PACAP in the trachea and the intrapulmonary arteries were unaffected by pretreatment with atropine, prazosin, yohimbine, propranolol, mepyramine, cimetidine and Spantide. Removal of the endothelium abolished PACAP-induced vascular relaxation. Conceivably, PACAP-containing nerve fibers play a role in the regulation of airway resistance and local blood flow.     

Estrogen can regulate the cell cycle in the early G1 phase of yeast by increasing the amount of adenylate cyclase mRNA

The effects of beta-estradiol (estrogen; a minor component of yeast cells) on S. cerevisiae cells in the G0 and G1 phases were examined. Results showed that estrogen stimulated the recovery of growth from G0 arrest induced by nutrient limitation or ts mutation of cdc35 (adenylate cyclase) in the early G1 phase, and inhibited entry into the resting G0 phase by increasing the intracellular cAMP level. However, estrogen had no effect on late G1 arrest induced by the alpha factor or ts mutation of cdc36. Estrogen was found to lead to higher steady-state levels of adenylate cyclase mRNA but not to affect the expression of the RAS1 and RAS2 genes, although these can also alter the intracellular cAMP level. These results suggest that estrogen influences the cell cycle of yeast in the early G1 phase by controlling the level of cAMP through the increase of adenylate cyclase mRNA.     

Neuropeptide Y, an orexigenic hormone, regulates phagocytic activity of lizard splenic phagocytes

Present in vitro study in the wall lizard Hemidactylus flaviviridis, for the first time in ectothermic vertebrates, demonstrated the immunoregulatory role of neuropeptide Y (NPY) and its receptor-coupled downstream signaling cascade. NPY inhibited the percentage phagocytosis and phagocytic index of splenic phagocytes. The inhibitory effect of NPY on phagocytosis was completely antagonized by Y₂ and Y₅ receptor antagonists. This suggests that NPY mediated its effect on phagocytosis through Y₂ and Y₅ receptors. Further, NPY receptor-coupled downstream signaling cascade for NPY effect on phagocytosis was explored using the inhibitors of adenylate cyclase (SQ 22536) and protein kinase A (H-89). The SQ 22536/H-89 in a concentration-related manner decreased the inhibitory effect of NPY on phagocytosis. Further, an increase in intracellular cAMP level was observed in response to NPY. Taken together, it can be concluded that NPY via Y₂ and Y₅ receptor-coupled AC-cAMP-PKA pathway downregulated the phagocytic activity of lizard splenic phagocytes.     

Cyclooxygenase-2 and adenylate cyclase/protein kinase A signaling pathway enhances angiogenesis through induction of vascular endothelial growth factor in rat sponge implants

Angiogenesis is reportedly enhanced by prostaglandins (PGs). In the present experiment, we tested whether or not COX-2 and adenylate cyclase/protein kinase A (AC/PKA)-dependent VEGF induction enhanced angiogenesis in this model. Angiogenesis was enhanced by topical injection of human recombinant basic fibroblast growth factor (bFGF). The enhanced angiogenesis by bFGF was inhibited by indomethacin or selective COX-2 inhibitors, NS398, nimesulide, and JTE-522. Topical daily injections of 8-bromo-cAMP enhanced angiogenesis in a dose-dependent manner. Forskolin, an activator of AC, also facilitated angiogenesis in a dose-dependent manner, as did amrinone, an inhibitor of phosphodiesterase. VEGF induction was confirmed by the increased levels in the fluids in the sponge matrix after topical injection of 8-bromo-cAMP. Immunohistochemical investigation further revealed the VEGF-expressed cells in the sponge granulation tissues to be fibroblasts, and the intensity of positive reactions was enhanced by bFGF, 8-bromo-cAMP, forskolin, and amrinone. Angiogenesis was inhibited by indometacin or selective COX-2 inhibitors, NS-398, nimesulide, and JTE-522. In addition, angiogenesis without topical injections of the above compounds was also suppressed by SQ22,536, an inhibitor for AC. or H-89, an inhibitor for PKA, with concomitant reductions in VEGF levels. Daily topical injections of neutralizing antibody or anti-sense oligonucleotide against VEGF significantly suppressed angiogenesis. These results suggested that COX-2 and AC/PKA-dependent induction of VEGF certainly enhanced angiogenesis, and that pharmacological tools for controlling this signaling pathway may be able to facilitate the management of conditions involving angiogenesis.     

Actions of vasopressin and isoprenaline on the ionic transport across the isolated frog skin in the presence and the absence of adenyl cyclase inhibitors MDL12330A and SQ22536.

1. The effects of both adenyl cyclase inhibitors (MDL12330A and SQ22536) have been studied on the ionic transport induced by vasopressin and isoprenaline across the frog skin. 2. MDL12330A inhibits the vasopressin action on the short-circuit current (SCC), confirming that this effect is cAMP-mediated. 3. On the other hand, isoprenaline action on the SCC is unaffected by MDL12330A. However, this lack of effect is not a sufficient argument against the role of cAMP in this action; in fact, as MDL12330A is also an inhibitor of cAMP phosphodiesterase, this action could mask the inhibitory effect of the drug on adenyl cyclase. 4. By using the other adenyl cyclase inhibitor (SQ22536), probably deprived of effect on the cAMP phosphodiesterase, we obtained a strong inhibition of isoprenaline action on the SCC. Thus we conclude that the actions of isoprenaline on the ionic transport across the frog skin are also cAMP-mediated.