Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The positive inotropic action of the newer cardiotonic phosphodiesterase inhibitors such as indolidan, milrinone, and imazodan has been previously attributed to selective inhibition of cGMP-inhibitable Type IV (high affinity) cAMP phosphodiesterase activity. However, the subcellular binding site(s) for this class of compounds has not been defined. We have characterized the binding of [3H]LY186126, an analogue of indolidan, in subcellular fractions prepared from rabbit and sheep ventricular myocardium. Binding required magnesium ion and exhibited rapid association and dissociation kinetics. Specific binding (defined by ligand displacement with 5 microM indolidan) to enriched rabbit sarcoplasmic reticulum (SR) membrane vesicles was saturable (Bmax = 714 +/- 77 fmol/mg of protein) and of high affinity (Kd = 6.2 +/- 1.4 nM). Linear and nonlinear analyses of the binding isotherms fit a single-site model. Mixed SR preparations from sheep myocardium exhibited binding characteristics (Bmax = 944 +/- 115 fmol/mg; Kd = 8.5 +/- 2.3 nM) comparable to those of rabbit cardiac SR. Further subfractionation of sheep SR indicated that the binding sites were equally distributed between free (Bmax = 630 fmol/mg; Kd = 4.4 nM) and junctional SR (Bmax = 569 fmol/mg; Kd = 10.9 nM). Specific binding of [3H]LY186126 was also demonstrated in the cytosolic subfraction of rabbit myocardium that contained Type IV phosphodiesterase activity (Peak III from anion exchange chromatography). Competition for [3H] LY186126 binding studied in rabbit SR showed that, of the compounds tested, lixazinone (RS 82856) competed most effectively (IC50 = 0.030 +/- 0.008 nM), followed by indolidan (0.14 +/- 0.05 nM), cGMP (17.8 +/- 2.6 nM), milrinone (39.3 +/- 13.2 nM), and imazodan (192 +/- 73 nM). In contrast, rolipram, which does not inhibit SR-associated Type IV phosphodiesterase activity, was not effective at competing for [3H]LY186126 binding (IC50 greater than 30 microM). These results indicate that [3H]LY186126 has specific binding sites in myocardial subcellular fractions that contain cGMP-inhibitable Type IV (high affinity) cAMP phosphodiesterase activity.
Mol Pharmacol 1989 Aug
PMID:Analysis of the binding sites for the cardiotonic phosphodiesterase inhibitor [3H]LY186126 in ventricular myocardium. 250 59

Bradykinin is the prime initiator of pain and the key initial activator of the inflammatory response at the site of tissue injury. The subsequent transfer of nociceptive information (pain sensation) into the central nervous system is then mediated via afferent type C dorsal root ganglion neurons. A recently developed hybrid cell line, F-11, shows many qualities characteristic of these pain-sensitive cells. In these neuronal hybrids, we have found that bradykinin induces sequential elevation in the concentrations of several second messengers involved in neuronal activation, including inositol trisphosphate (6.5-fold), intracellular calcium (2.7-fold), and cyclic GMP (20.5-fold). Importantly, the production of these second messengers is potently inhibited by several novel bradykinin antagonists that possess no intrinsic agonist activity. The same relative rank order of potency of inhibition of bradykinin-induced second messenger production was achieved in the inositol trisphosphate, calcium, and cyclic GMP assay systems, suggesting strongly that all three messenger systems are being activated by the same bradykinin receptor. The most potent antagonist was D-Arg0-Hyp3-Thi5,8-D-Phe7-bradykinin, which inhibited in a competitive manner, with pA2 values, upon Schild plot analysis, in the nanomolar range. These potent bradykinin antagonists may be useful in the characterization of bradykinin receptors and in the clinical management of pain and inflammation.
Mol Pharmacol 1989 Jan
PMID:Bradykinin analogs antagonize bradykinin-induced second messenger production in a sensory neuron cell line. 253 66

1. Recent data have clearly shown the existence of specific receptor binding sites for atrial natriuretic factors (ANF) or polypeptides in mammalian brain tissues. 2. Ligand selectivity pattern and coupling to cGMP production suggest that brain ANF sites are similar to high-affinity/low-capacity sites found in various peripheral tissues (kidney, adrenal gland, blood vessels). These brain ANF sites possibly are of the B-ANP subtype. 3. High densities of ANF binding sites are found especially in areas of the central nervous system associated with the control of various cardiovascular parameters (such as the subfornical organ and area postrema). However, high densities of sites are also present in other regions such as the hippocampus, cerebellum, and thalamus in the brain of certain mammalian species, suggesting that brain ANF could act as a neuromodulator of noncardiovascular functions. 4. The density of brain ANF binding sites is modified in certain animal models of cardiovascular disorders and during postnatal ontogeny, demonstrating the plasticity of these sites in the central nervous system (CNS). 5. Specific ANF binding sites are also found in various other CNS-associated tissues such as the eye, pituitary gland, and adrenal medulla. In these tissues ANF appears to act as a modulator of fluid production and hormone release. 6. Thus, ANF-like peptides and ANF receptor sites are present in brain and various peripheral tissues, demonstrating the existence of a family of brain/heart peptides.
Cell Mol Neurobiol 1989 Mar
PMID:Receptor sites for atrial natriuretic factors in brain and associated structures: an overview. 254 Sep 11

We have characterized two atrial natriuretic factor (ANF) receptor subtypes, designated ANF-R1 and ANF-R2, in two established cell lines that express exclusively one receptor subtype. The ANF-R1 receptor is selectively expressed by the kidney epithelial cell line LLC-PK1. It is a 130-kDa protein that has a much higher affinity for the biologically active forms of ANF than for its metabolites. The binding of ANF to this subtype is potentiated by amiloride and by divalent cations. The activation of the ANF-R1 receptor leads to an accumulation of cyclic GMP that is only partially inhibited by methylene blue. The ANF-R2 receptor, which is expressed selectively by the fibroblast cell line NIH-3T3, is a 130-kDa protein composed of two disulfide-linked subunits of 64-kDa. Activation of this subtype by saturating concentrations of ANF does not appear to elicit cyclic GMP production. However, supraphysiological concentrations of ANF induce a nonsaturable accumulation of cyclic GMP with an apparent ED50 in the high micromolar range. In contrast to the ANF-R1 subtype, the stimulation of cyclic GMP production is completely abolished by methylene blue. This subtype recognizes the active forms of ANF as well as its metabolites, and the binding is insensitive to amiloride and is decreased by divalent cations. These two cell lines can serve as models for studying the differential regulatory properties of ANF-R1 and ANF-R2 subtypes. In addition, we have also characterized the two ANF receptor subtypes in rat kidney glomeruli, where they show the same structure and pharmacological characteristics as in the two model cell lines.
Mol Pharmacol 1989 May
PMID:Distinct properties of atrial natriuretic factor receptor subpopulations in epithelial and fibroblast cell lines. 254 56

The metabolic effects of atrial natriuretic peptide (ANP) have not been widely investigated. Since adipocyte cells represent a model system extensively used to examine the metabolic actions of many peptide hormones, we sought to establish whether ANP could bind to adipocyte membranes, alter cyclic nucleotide metabolism, and affect spontaneous or hormone-stimulated lipolysis. Using in vitro autoradiographic techniques, radiolabelled ANP was found to bind specifically to mammary gland fat cells. Additionally, endogenous ANP-like immunoreactivity could be localized in the plasma membrane compartment and cytoplasmic matrix of fat cells, but not in fat vacuoles. [125I]ANP bound to single high affinity sites (Kd = 0.72 nM) in fat cell membranes. The binding was rapid (equilibrium within 1 min at 25 degrees C) and specific. The atrial peptide was capable of stimulating a time- and concentration-dependent increase in cGMP accumulation in isolated adipocytes, but had no effect on spontaneous or stimulated [-)-isoproterenol, ACTH, forskolin) cAMP formation. ANP did not alter the increase in glycerol production stimulated by l-epinephrine in isolated fat cells. While i.v. infusion of ANP stimulated a marked increase in circulating levels of cGMP, the atrial peptide did not alter plasma triglyceride levels. These data demonstrate the presence of specific ANP binding sites on adipocyte membranes and internalization of ANP-associated immunoreactivity. These receptors are biochemically functional given the ability of ANP to augment cGMP formation. The peptide, however, does not exert an action on adipocyte lipolysis. Adipocytes, therefore, represent an ANP target tissue in which the physiological action of the peptide is yet to be defined.
Mol Cell Endocrinol 1989 Mar
PMID:Immunocytochemical localization, binding, and effects of atrial natriuretic peptide in rat adipocytes. 254 86

Modulation of renin synthesis by lipoxygenase products has been studied in cultured human mesangial cells under basal conditions and in the presence of prostaglandin (PG) E2. Total renin and cyclic AMP productions were stimulated in a dose-dependent manner (0.1-10 microM) by PGE2. The stimulatory effect of PGE2 on renin production was inhibited by 12-hydroxyeicosatetraenoic acid (12-HETE) between 0.1 and 100 nM. Extracellular and intracellular renin were affected similarly. Neither basal and PGE2-dependent cyclic AMP nor basal cyclic GMP productions were modified. 15-Hydroxyeicosatetraenoic acid (15-HPETE), 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and 15-hydroperoxyeicosatetraenoic acid (15-HPETE) had the same effects as 12-HETE. Intracellular calcium concentration was not modified in the presence of 12-HETE. Since oleyl-2-acetylglycerol (OAG), an analog of diacylglycerol, also inhibited PGE2-stimulated renin production, it is hypothesized that the effect of the lipoxygenase products is mediated via protein kinase C stimulation.
Mol Cell Endocrinol 1989 Apr
PMID:Modulation of renin synthesis by lipoxygenase products in cultured human mesangial cells. 254 91

Cytosolic and particulate Type IV (high-affinity) cAMP phosphodiesterase (PDE) activities were isolated from the ventricular myocardium of newborn (NB; 24 to 48 h), immature (IM; 14 to 16 days) and adult (AD; 6 to 8 months) rabbits. Cytosolic activity from each age group was resolved into three distinct peaks of activity by DEAE cellulose anion exchange chromatography. Type IV PDE activity was identified as a predominant activity in the cytosolic peak III activity in all three age groups when measured with 0.25 microM cAMP as substrate. A particulate Type IV PDE activity was associated with the sarcoplasmic reticulum (SR) fractions in each age group. No significant age-related changes in the affinity of the particulate enzyme for cAMP (apparent Km = 0.3 to 0.5 microM) were evident, but the Vmax for this SR-associated activity increased from 553 +/- 7 pmol/min/mg in the NB to 725 +/- 9 pmol/min/mg in the IM and 2450 +/- 33 pmol/min/mg in the AD. In each age group, milrinone, imazodan, piroximone and indolidan were more potent inhibitors of the SR-associated activity as compared with the cytosolic peak III activity. In contrast, RO 20-1724 and rolipram were relatively more selective inhibitors of the cytosolic peak III activity. Age-related differences in the sensitivity of type IV PDE to inhibition was dependent upon the selectivity of the inhibitor and the subcellular enzymic distribution. Cytosolic peak III PDE activity was further resolved by gel filtration chromatography into two peaks. Hydrolysis of cAMP by the higher molecular weight peak was inhibitable by cGMP (IC50 = 0.25 +/- 0.07 microM in NB and 0.07 +/- 0.01 microM in AD) whereas the lower molecular weight peak activity was relatively insensitive to inhibition by cGMP (IC50 greater than 100 microM). The lower molecular weight peak constituted a relatively greater proportion of the total peak III activity in the NB as compared to the AD. Analysis of the kinetics of cGMP inhibition of high-affinity cAMP hydrolysis was consistent with the presence of a greater number of high-affinity (presumably drug-sensitive) binding sites in the SR-associated activity as compared to the cytosolic peak III activity in both NB and AD. These results support the hypothesis that the cGMP-inhibitable Type IV PDE activity may be the primary site of action for certain newer cardiotonic drugs. Differences in drug action in young versus adult myocardium may be related to the selectivity of the cardiotonic drugs for this specific isozyme and its lower specific activity during the early stages of maturation.
J Mol Cell Cardiol 1989 May
PMID:Subcellular distribution of high-affinity type IV cyclic AMP phosphodiesterase activities in rabbit ventricular myocardium: relations to post-natal maturation. 255 Jun 53

Primary cultures of anterior and intermediate pituitary tissues were monitored immunocytochemically for the presence of endocrine and nonendocrine cells and simultaneously tested for their ability to produce cyclic GMP in response to atrial natriuretic factor (ANF). Cells cultured for 3 days and 6 days, in which nonendocrine (vimentin-positive) cells were found to rapidly overgrow the endocrine cells, showed a dramatic elevation in cyclic GMP production stimulated by ANF, with maximum stimulation 300-700% that seen in 1-day cultured cells. Also, ANF-induced accumulation of cyclic GMP in an enriched population of vimentin-positive cells appeared to closely match that triggered in a 3-day culture of anterior pituitary cells, emphasizing the major role played by nonendocrine cells and their ability to synthesize cyclic GMP. In contrast, in the homogeneous population of tumor corticotrophs AtT-20, there was a close relationship between cyclic GMP formation and cell density. It thus appears that contamination of primary cultures of anterior and intermediate pituitary tissues by proliferating nonendocrine cells (mainly fibroblasts), in which ANF-induced accumulation of cyclic GMP may be confused with that of the very secretory cells, leads to overestimation and masking of guanylate cyclase activity of endocrine cells.
Mol Cell Endocrinol 1989 Jul
PMID:Stimulation by atrial natriuretic factor of cyclic GMP production in cultured anterior and intermediate pituitary tissues: evidence for a major contribution of proliferating nonendocrine cells. 255 58

Oscillation of cyclic AMP and in the activity ratio of cyclic AMP-dependent protein kinase and of glycogen phosphorylase with the cardiac cycle were demonstrated in the canine heart in situ. For tissue sampling an ECG (R-wave)-triggered, automatically working push-freeze-drill apparatus was developed which allows intraventricular cryobiopsies from the left ventricular muscle of anaesthetized open-chest dogs. The nucleotide cyclic AMP oscillated with the cardiac cycle during normal working condition, the higher cyclic AMP level occuring during systole. Cyclic GMP was assayed to be without oscillatory changes during the contraction-relaxation cycle. The rise in the activity ratio of protein kinase was found to coincide with the maximum in the level of cyclic AMP. Propranolol pretreatment prevents the transient in the level of the nucleotide as well as in the activity ratio of the kinase indicating i) a causal relationship between these changes and ii) a neurohumoral, beat-to-beat regulation by catecholamines released from the sympathetic nerve endings within the heart. Contrary the activity ratio of phosphorylase retains its transient changes during the cardiac cycle in the presence of propranolol, indicating a Ca-mediated activation of phosphorlase kinase during the contraction process.
Mol Cell Biochem 1989 Sep 07
PMID:Transient changes in cyclic AMP and in the enzymic activity of protein kinase and phosphorylase during the cardiac cycle in the canine myocardium and the effect of propranolol. 255 24

Few high affinity inhibitors of the photoreceptor phosphodiesterases have been identified. We show here that dipyridamole and M&B 22,948 (Zaprinast), potent inhibitors of the cGMP-binding, cGMP-specific phosphodiesterase (PDE), also inhibit trypsin- or transducin-activated bovine rod and cone photoreceptor phosphodiesterases at submicromolar concentrations. Dixon plots demonstrated that the inhibition of trypsin-activated rod PDE was competitive, with Ki values of 140 nM for M&B 22,948 and 380 nM for dipyridamole. Both of these drugs were much more potent than other PDE inhibitors, including isobutylmethylxanthine (IBMX). These results reinforce the suggestion that the photoreceptor and the cGMP-binding, cGMP-specific PDE are closely related. In addition, the high affinity and selectivity of these agents should make them useful for probing the regulation and function of PDE in the photoreceptor. At low substrate concentrations, the effects of these drugs on basal unactivated PDE activity were similar to those seen with trypsin- or transducin-activated PDE. At millimolar substrate concentrations, however, the effects of the drugs were biphasic; PDE activity was stimulated at drug concentrations from 1 to 10 microM and inhibited at higher concentrations. Stimulation was not observed with IBMX. This stimulation of activity apparently was not an allosteric effect caused by direct binding of the dipyridamole and M&B 22,948 to the high affinity noncatalytic cGMP binding sites on the PDEs; whereas no cooperativity of cGMP binding to this site has been demonstrated, the drugs actually stimulated the binding of low concentrations of cGMP to this site. In addition, whereas preincubation with cGMP and cGMP analogs blocked the stimulation exerted by the drugs, they did so only at much higher concentrations than those necessary for saturation of the high affinity noncatalytic cGMP site. Because the stimulation can only be seen at higher substrate levels than are thought to exist in the photoreceptor, only the inhibitory effects of the drugs are likely to be pharmacologically relevant. However, the stimulation exerted by these drugs may point to a hitherto unknown allosteric interaction between the catalytic and regulatory sites on the PDE or to a previously unrecognized regulatory site.
Mol Pharmacol 1989 Nov
PMID:Inhibition and stimulation of photoreceptor phosphodiesterases by dipyridamole and M&B 22,948. 255 75


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