cyclic-gmp and protein-kinase-inhibitor-peptide

We have investigated whether the activation of cAMP- and cGMP-dependent pathways modifies the properties of voltage-dependent Ba(2+) currents (I(Ba)) recorded from guinea-pig gastric myocytes using patch-clamp techniques. All experiments were carried on single smooth muscle cells, dispersed from the circular layer of the guinea-pig gastric antrum. Both dibutyryl cAMP (db-cAMP, 0.1-1 mM), a membrane-permeable ester of cAMP, and isoproterenol, a selective beta-stimulant, inhibited I(Ba) in a concentration-dependent manner. Forskolin, but not dideoxy-forskolin, an inactive isomer of forskolin, inhibited the peak amplitude of I(Ba). In the presence of either Rp-cAMP or the PKA (cAMP-dependent protein kinase) inhibitor peptide 5-24 (PKA-IP), neither forskolin nor db-cAMP inhibited I(Ba). After establishing a conventional whole-cell recording, the peak amplitude of I(Ba) gradually decreased when the catalytic subunit of PKA was included in the pipette. The further application of Rp-cAMP reversibly enhanced I(Ba). Sodium nitroprusside (0.1-1 mM) and 8-Br-cGMP (0.1-1 mM) also inhibited I(Ba) in a concentration-dependent manner. The inhibitory effects of forskolin or db-cAMP on I(Ba) were not significantly changed by pretreatment with a cGMP-dependent protein kinase (PKG) inhibitor. Similarly, the inhibitory actions of 8-Br-cGMP on I(Ba) were not modified by PKA-IP. The membrane-permeable cyclic nucleotides db-cAMP and 8-Br-cGMP caused little shift of the voltage dependence of the steady-state inactivation and reactivation curves. Neither of the membrane-permeable cyclic nucleotides db-cAMP or 8-Br-cGMP had additive inhibitory effects on I(Ba). These results indicate that two distinct cyclic nucleotide-dependent pathways are present in the guinea-pig gastric antrum, and that both inhibited I(Ba) in an independent manner.

Topics: Animals; Barium; Bucladesine; Calcium Channels, L-Type; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Female; Guinea Pigs; In Vitro Techniques; Isoproterenol; Male; Membrane Potentials; Myocytes, Smooth Muscle; Peptides; Pyloric Antrum; Thionucleotides

In the anterior pituitary, cGMP is produced in response to a number of stimuli, but intracellular events distal to cGMP production are obscure. Since cGMP-dependent protein kinase (PKG) is a major effector of cGMP actions in other tissues we have determined whether PKG and its specific substrates might be present and responsive to external signals in the ovine anterior pituitary. Photoaffinity labelling with [32P]cGMP revealed a specific 78 kDa protein in ovine anterior pituitary that comigrated with purified bovine lung PKG-I. PKG in protein extracts from anterior pituitary or cultured anterior pituitary cells was enriched by DEAE ion-exchange chromatography and assayed for activity. Both tissue and cultured cells had a relatively high PKG activity by comparison with aortic smooth muscle (known high activity) and brain (known low activity). Subcellular distribution studies showed that in anterior pituitary, aortic and brain, PKG activity was present in both cytosol and triton-extracted membrane fractions, while in platelets the activity was associated with only the membrane fraction. To determine if this PKG might be responsive to extracellular signals an activity ratio assay was used. Incubation of cultured cells with atrial natriuretic peptide (ANP) and sodium nitroprusside, activators of membrane and cytosolic guanylate cyclases respectively, increased the activity of PKG. To determine events distal to PKG activation, a search for potential substrates of PKG was performed. Few substrates were detectable upon addition of purified PKG to tissue lysates due to the high background activity of endogenous protein kinases in the anterior pituitary. However, 19 substrates of PKG were detected in heat-stable and 14 in acid-soluble protein extracts of the anterior pituitary, in which background phosphorylation was almost abolished. After partial purification through Q-Sepharose ion-exchange chromatography some of these proteins were preferentially phosphorylated by addition of PKG-I, while the others were additionally substrates of exogenous cAMP-dependent protein kinase (PKA) or Ca2+ and phospholipid-dependent protein kinase (PKC). A 132-kDa substrate showed an identical phosphopeptide map to a PKG substrate previously described in vascular smooth muscle and platelets. These data demonstrate for the first time the presence of functional PKG activity and multiple PKG substrates in the anterior pituitary where they may play a role in mediating the intracellular

Topics: Affinity Labels; Amino Acid Sequence; Animals; Calcium-Binding Proteins; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Molecular Sequence Data; Peptide Mapping; Peptides; Phosphoproteins; Phosphorus Radioisotopes; Photochemistry; Pituitary Gland, Anterior; Protein Kinase C; Protein Kinases; Sheep; Substrate Specificity

Affinities of the catalytic subunit (C1) of Saccharomyces cerevisiae cAMP-dependent protein kinase and of mammalian cGMP-dependent protein kinase were determined for the protein kinase inhibitor (PKI) peptide PKI(6-22)amide and seven analogues. These analogues contained structural alterations in the N-terminal alpha-helix, the C-terminal pseudosubstrate portion, or the central connecting region of the PKI peptide. In all cases, the PKI peptides were appreciably less active as inhibitors of yeast C1 than of mammalian C alpha subunit. Ki values ranged from 5- to 290-fold higher for the yeast enzyme than for its mammalian counterpart. Consistent with these results, yeast C1 exhibited a higher Km for the peptide substrate Kemptide. All of the PKI peptides were even less active against the mammalian cGMP-dependent protein kinase than toward yeast cAMP-dependent protein kinase, and Kemptide was a poorer substrate for the former enzyme. Alignment of amino acid sequences of these homologous protein kinases around residues in the active site of mammalian C alpha subunit known to interact with determinants in the PKI peptide [Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N-h, Taylor, S. S., & Sowadski, J. M. (1991) Science 253, 414-420] provides a structural basis for the inherently lower affinities of yeast C1 and cGMP-dependent protein kinase for binding peptide inhibitors and substrates. Both yeast cAMP-dependent and mammalian cGMP-dependent protein kinases are missing two of the three acidic residues that interact with arginine-18 in the pseudosubstrate portion of PKI. Further, the cGMP-dependent protein kinase appears to completely lack the hydrophobic/aromatic pocket that recognizes the important phenylalanine-10 residue in the N-terminus of the PKI peptide, and binding of the inhibitor by the yeast protein kinase at this site appears to be partially compromised.

Topics: Amino Acid Sequence; Animals; Binding Sites; Cattle; Cyclic AMP; Cyclic GMP; Kinetics; Molecular Sequence Data; Myocardium; Oligopeptides; Peptide Fragments; Peptides; Protein Kinase Inhibitors; Protein Kinases; Saccharomyces cerevisiae; Substrate Specificity