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At least 415 records · Page 23Linked to original sources
PubMed1989

Activation of beta-adrenergic receptors causes stimulation of cyclic AMP, inhibition of inositol trisphosphate, and relaxation of bovine iris sphincter smooth muscle. Biochemical and functional interactions between the cyclic AMP and calcium signalling systems.

We have investigated the interactions between the cAMP and inositol 1,4,5-trisphosphate (IP3)-Ca2+ signalling systems in the bovine iris sphincter by measuring the effects of beta-adrenergic and cholinergic muscarinic agonists and antagonists on cAMP formation, IP3 accumulation and muscle contraction-relaxation. (1) Addition of 5 microM isoproterenol (ISO) or forskolin (5 microM) consistently produced stimulation of cAMP (540%), inhibition of IP3 (34%) and complete relaxation of the muscle. The ISO effects were dose-dependent, with EC50 values for cAMP formation, IP3 inhibition and muscle relaxation of 2.8 X 10(-7) M, 3.4 X 10(-7) M and 0.45 X 10(-7) M, respectively. (2) Timolol, a beta-adrenergic antagonist, inhibited the ISO effects in a dose-dependent manner, with IC50 values for cAMP formation, IP3 accumulation and muscle relaxation of 1.8 X 10(-5) M, 3.2 X 10(-5) M and 2.2 X 10(-5) M, respectively. (3) The effects of ISO (0.5 microM) were time-dependent, and they clearly indicate a temporal relationship between the agonist-induced stimulation of cAMP, inhibition of IP3, and relaxation of the muscle. Within 15 sec following the addition of ISO, there was a marked increase in the level of cAMP, a decrease in IP3, and this was accompanied by an equally rapid relaxation of the muscle. (4) Addition of carbachol (CCh) to iris sphincter pretreated with ISO decreased cAMP formation and reversed muscle relaxation to complete contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Transcriptional regulation of a plasminogen activator gene by cyclic AMP in a homologous cell-free system. Involvement of cyclic AMP-dependent protein kinase in transcriptional control.

We have developed a homologous cell-free transcription system using extracts from the porcine kidney cell line LLC-PK1 to study the molecular mechanisms by which cAMP regulates urokinase-type plasminogen activator (uPA) gene transcription. We demonstrated accurate initiation of transcription using a cloned fragment of the uPA gene as template. The in vitro transcription rate was stimulated by up to 10-fold by the addition of cAMP (greater than 10 microM). This effect of cAMP on the transcription was greater for closed circular than for linear templates. Furthermore, addition of the purified catalytic subunit of cAMP-dependent protein kinase stimulated the in vitro transcription in the absence of cAMP to levels 2-fold higher than those observed with cAMP. Addition of cAMP had no stimulatory effect on the transcription of the rat heme oxygenase gene promoter tested under identical conditions. HeLa whole cell extract by itself showed no stimulation of transcription of the uPA gene by cAMP. Results of reconstitution experiments using HeLa whole cell extracts and nuclear lysates from LLC-PK1 cells suggest the presence of putative cAMP regulatory factor(s) as well as general transcription factor(s) in the nucleus of LLC-PK1 cells. These results provide experimental evidence directly implicating cAMP-dependent protein kinase in the regulation of gene transcription.

Animals↗
PubMed1985

Pertussis toxin blocks both cyclic AMP-mediated and cyclic AMP-independent actions of somatostatin. Evidence for coupling of Ni to decreases in intracellular free calcium.

The neuropeptide somatostatin inhibits hormone release from GH4C1 pituitary cells via two mechanisms: inhibition of stimulated adenylate cyclase and a cAMP-independent process. To determine whether both mechanisms involve the guanyl nucleotide-binding protein Ni, we used pertussis toxin, which ADP-ribosylates Ni and thereby blocks its function. Pertussis toxin treatment of GH4C1 cells blocked somatostatin inhibition of both vasoactive intestinal peptide (VIP)-stimulated cAMP accumulation and prolactin secretion. In membranes prepared from toxin-treated cells, somatostatin inhibition of VIP-stimulated adenylate cyclase activity was reduced and 125I-Tyr1-somatostatin binding was decreased more than 95%. In contrast, pertussis toxin did not affect the biological actions or the membrane binding of thyrotropin-releasing hormone. These results indicate that ADP-ribosylated Ni cannot interact with occupied somatostatin receptors and that somatostatin inhibits VIP-stimulated adenylate cyclase via Ni. To investigate somatostatin's cAMP-independent mechanism, we used depolarizing concentrations of K+ to stimulate prolactin release without altering intracellular cAMP levels. Measurement of Quin-2 fluorescence showed that 11 mM K+ increased intracellular [Ca2+] within 5 s. Somatostatin caused an immediate, but transient, decrease in both basal and K+-elevated [Ca2+]. Consistent with these findings, somatostatin inhibited K+-stimulated prolactin release, also without affecting intracellular cAMP concentrations. Pertussis toxin blocked the somatostatin-induced reduction of [Ca2+]. Furthermore, the toxin antagonized somatostatin inhibition of K+-stimulated and VIP-stimulated secretion with the same potency (ED50 = 0.3 ng/ml). These results indicate that pertussis toxin acts at a common site to prevent somatostatin inhibition of both Ca2+- and cAMP-stimulated hormone release. Thus, Ni appears to be required for somatostatin to decrease both cAMP production and [Ca2+] and to inhibit the actions of secretagogues using either of these intracellular messengers.

Adenylate Cyclase Toxin↗
PubMed1985

Inhibition of cyclic AMP-dependent protein kinase-induced changes in the kinetic properties of hepatic pyruvate kinase by the specific cyclic AMP antagonist, the (Rp)-diastereomer of adenosine cyclic 3',5'-phosphorothioate.

The effects of the diastereomers of adenosine cyclic 3',5'-phosphorothioate, (Sp)- and (Rp)-cAMPS, on the kinetic properties of pyruvate kinase were studied in hepatocytes isolated from fed rats. Incubation of the cells with the cAMP-dependent protein kinase agonist, (Sp)-cAMPS, produced a concentration-dependent increase in S0.5 for phosphoenolpyruvate, but had no effect on Vmax. The (Sp)-cAMPS-treated enzyme was more sensitive to inhibition by alanine and ATP and, under the same conditions, was less responsive to activation by fructose-1,6-bisphosphate when assayed at a subsaturating phosphoenolpyruvate concentration. Incubation of the hepatocytes with only the cAMP-dependent protein kinase antagonist, (Rp)-cAMPS, produced no change in any kinetic parameters, but did suppress the (Sp)-cAMPS- or glucagon-induced increase in the S0.5 for phosphoenolpyruvate with IC50 values of 10 microM and 5 microM (Rp)-cAMPS. (Rp)-cAMPS is exerting an effect on the kinetic properties of pyruvate kinase through inhibition of cAMP-dependent protein kinase.

Adenosine Triphosphate↗