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C J Homcy

Publications and source records attributed to C J Homcy.

15 recordsLinked to original sources
PubMed1992

Cloning and characterization of a sixth adenylyl cyclase isoform: types V and VI constitute a subgroup within the mammalian adenylyl cyclase family.

A sixth member of the mammalian adenylyl cyclase family has been isolated from a canine cardiac cDNA library. This isoform is more highly homologous to type V than to the other adenylyl cyclase types; sequence similarity is apparent even in the transmembrane regions where the greatest divergence among the types exists. Type VI mRNA expression is most abundant in heart and brain; however, unlike type V, a low level of expression is also observed in a variety of other tissues examined. Type VI adenylyl cyclase can be stimulated by NaF, guanosine 5'-[gamma-thio]triphosphate, and forskolin but not by Ca2+/calmodulin, whereas it is inhibited by adenosine and its analogues. Comparison of both their structural and biochemical properties suggests that types V and VI constitute a distinct subgroup of the mammalian adenylyl cyclase family.

Adenosine↗
PubMed1992

Isolation and characterization of a novel cardiac adenylylcyclase cDNA.

A novel adenylylcyclase cDNA (type V) was isolated from a canine heart cDNA library. Northern blotting indicates that the expression of this message is most abundant in heart with a lesser amount in brain but is absent in a variety of other tissues including lung, kidney, skeletal muscle, lymphocyte, and testis. The putative protein product predicted from the cDNA sequence has the motif of tandem six-transmembrane spans separated by a large hydrophilic cytoplasmic loop as seen in other members of the adenylylcyclase family. When this protein is expressed using a CMT cell transient expression system, the adenylylcyclase activity was stimulated by NaF, GTP gamma S, and forskolin, but not by calmodulin. The activity was inhibited in a concentration-dependent manner with either P-site active agents such as adenosine or in the presence of calcium. These data indicate that the protein encoded by this cDNA is adenylylcyclase with the biochemical features characteristic of the cardiac isoform.

Adenosine↗
PubMed1992

Specific receptor-guanine nucleotide binding protein interaction mediates the release of endothelium-derived relaxing factor.

High affinity agonist-binding (HAB) sites are formed from specific receptor interaction with guanine nucleotide-binding (Gi) proteins. To determine whether the release of endothelium-derived relaxing factor (EDRF) is regulated by specific receptor-Gi protein coupling, we treated bovine aortic endothelial cells with 100 ng/ml pertussis toxin (PTX) for 16 hours to effect receptor-Gi protein uncoupling. The degree of receptor uncoupling as measured by the loss of HAB sites for the alpha 2-adrenergic receptor and bradykinin receptor was assessed by radioligand binding studies using partially purified bovine aortic endothelial cell membranes. The release of EDRF in response to UK14304 (an alpha 2-adrenergic receptor agonist) and bradykinin stimulation was measured with a bioassay apparatus. The Gi protein isoforms were characterized by Western blotting, and complete ADP-ribosylation of these proteins was confirmed by PTX-catalyzed [32P]NAD ribosylation. PTX produced a greater inhibition of EDRF release via the alpha 2-adrenergic receptor pathway compared with the bradykinin receptor pathway (80% versus 46%, p less than 0.01). This corresponded to the loss of HAB sites from the alpha 2-adrenergic receptor and bradykinin receptor pathway (72% versus 46%, p less than 0.01) as compared with complete loss of both HAB sites in the presence of GppNHp (0.1 mM). Since loss of HAB sites from PTX-mediated receptor uncoupling parallels the inhibition of EDRF release, these data suggest that Gi proteins contribute to a greater proportion of HAB sites derived from alpha 2-adrenergic receptor rather than bradykinin receptor interaction and that the inhibition of EDRF release by PTX is mainly due to the loss of these HAB sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Isolation of rat genomic clones encoding subtypes of the alpha 2-adrenergic receptor. Identification of a unique receptor subtype.

alpha 2-Adrenergic receptors (alpha 2-AR) exist as subtypes that are expressed in a tissue-specific manner and differ in 1) their ligand recognition properties, 2) their extent of receptor protein glycosylation, and possible 3) their mechanism of signal transduction. Genomic or cDNA clones encoding three receptor subtypes have been characterized; however, both functional and radioligand binding studies in rodents suggest the existence of a fourth receptor subtype. To isolate the rat genes encoding receptor subtypes we screened a rat genomic library with an oligonucleotide probe encompassing the third membrane span of the human C-4 alpha 2-AR. Two intronless rat genes were isolated that encode distinct receptor subtypes (RG10, RG20). RG10 and RG20 encode proteins of 458 and 450 amino acids, respectively, that are 56% homologous and possess the structural features expected of this class of membrane-bound receptors. RG10 identifies a mRNA species of approximately 2500 nucleotides that is found primarily in brain, whereas RG20 identifies a larger mRNA species (approximately 4000 nucleotides) that is found in several tissues including brain, kidney, and salivary gland. RG10 is 88% homologous to the human C-4 alpha 2-AR and exhibits similar binding properties ( [3H]rauwolscine KD = 0.7 +/- 0.3 nM) as determined following transient expression of the receptor in COS-1 cells. RG20 exhibits ligand binding properties distinct from the three receptor subtypes identified by molecular cloning. Saturation binding studies indicate an affinity constant of 15 +/- 1.2 nM for the alpha 2-AR antagonist [3H]rauwolscine, a value 6-20 times higher than that observed for the three cloned receptor subtypes. In competition binding studies the potency order of competing ligands for RG20 is phentolamine greater than idazoxan greater than yohimbine greater than rauwolscine greater than prazosin. Of the three previously cloned alpha 2-AR, RG20 is most closely related to the human C-10 alpha 2-AR (89% homology) and is also capable of mediating adenylylcyclase inhibition as determined following its stable expression in NIH-3T3 fibroblasts. However, in contrast to RG20, [3H] rauwolscine exhibits a KD of 2 nM for the C-10 receptor, and the potency order for competing ligands is rauwolscine greater than or equal to yohimbine greater than idazoxan greater than phentolamine greater than prazosin. RG20 and C-10 are also distinguished by their affinity for SKF-10478 (RG20 Ki = 531 nM, C-10 Ki = 101 nM), a compound that may functionally distinguish pre- and postsynaptic alpha 2-AR. These data suggest that RG20 represents a fourth alpha 2-AR subtype distinct from the known alpha 2A-C receptor subtypes.

Adenylyl Cyclases↗
PubMed1991

The beta-adrenergic signaling pathway in the heart.

Postsynaptic defects in this important pathway for regulating heart rate and contractile force may contribute to the etiology and pathogenesis of congestive cardiac failure. Such defects may involve the beta-adrenergic receptor, its associated G protein, or adenylate cyclase. Current knowledge about these components of transmembrane cardiac signaling is reviewed.

Adenylyl Cyclases↗
PubMed1991

Inotropic response to norepinephrine is augmented early and maintained late in conscious dogs with perinephritic hypertension.

We studied the inotropic responses to intravenous infusions of norepinephrine in nine conscious chronically instrumented dogs before and early (2-4 weeks) in the development of perinephritic hypertension; seven conscious dogs were studied later (approximately 14 weeks), during a more stable phase of hypertension. perinephritic hypertension was associated with a 24% increase in left ventricular (LV) mass during developing hypertension; no further increase was seen during the stable hypertension phase. LV end-systolic stress was increased early (p less than 0.01) but was normalized later. The LV end-systolic stress-volume relation demonstrated an enhanced contractile response to norepinephrine during developing hypertension, which returned toward control later in the course of stable hypertension. The LV dP/dt responses to norepinephrine (0.4 microgram/kg/min) were significantly greater during developing hypertension (7,509 +/- 337 mm Hg/sec, p less than 0.05) compared with the control period (4,737 +/- 286 mm Hg/sec) and returned toward the control value during stable hypertension (5,168 +/- 465 mm Hg/sec). The enhanced inotropic responses to norepinephrine in developing hypertension were preserved in the presence of ganglionic blockade, suggesting that the augmentation was not mediated via reflex mechanisms. These physiological responses were associated with an increase in beta-adrenergic receptor density, but no significant change in basal or maximal adenylate cyclase stimulation occurred during developing hypertension. Thus, in contrast to prior studies in anesthetized animals, the inotropic response to beta-adrenergic stimulation is not depressed in conscious dogs but is enhanced selectively during the development of hypertension and maintained during stable hypertension.

Animals↗
PubMed1991

Decreased Gs alpha mRNA levels accompany the fall in Gs and adenylyl cyclase activities in compensated left ventricular hypertrophy. In heart failure, only the impairment in adenylyl cyclase activation progresses.

We have previously reported that there is a global reduction in adenylyl cyclase associated with a decrement in Gs functional activity in cardiac sarcolemma from animals with pressure overload-induced hypertrophy and heart failure. This study was performed to determine whether hypertrophy alone in the absence of heart failure is sufficient to promote these changes and whether the superimposition of heart failure intensified these changes. Basal and stimulated adenylyl cyclase and Gs activity, as determined in the S49 cyc- reconstitution assay, were measured in sarcolemma from normal (NL), left ventricular hypertrophy (LVH) and heart failure (HF) animals. Simultaneously, we measured the mRNA level encoding for the Gs alpha subunit. These studies indicate that Gs activity and Gs alpha mRNA are decreased by approximately 30% both in the failing heart and even in the heart with compensated hypertrophy before heart failure develops (Gs activity, pmol cyclic AMP/10 min per microgram, NL 4.2 +/- 0.4, LVH 3.0 +/- 0.2, HF 3.2 +/- 0.3; Gs alpha mRNA, pg/10 micrograms RNA, NL 131 +/- 9.0, LVH 104 +/- 7.4, HF 97.4 +/- 9.1; P less than 0.05 as compared with NL for LVH and HF). Accompanying this decrement in Gs activity is a fall in adenylyl cyclase, both basal and stimulated. However, we also identified a further decrease in adenylyl cyclase without any additional change in Gs or in its alpha subunit mRNA level. This is seen only in the sarcolemma from animals with heart failure as compared with those with compensated LV hypertrophy (e.g., NaF-stimulated activity, pmol cyclic AMP/min per mg, NL 420.2 +/- 17.5, LVH 347.1 +/- 29.6, HF 244.2 +/- 27.3; P less than 0.05 compared with NL for LVH and HF, P less than 0.05 compared with LVH for HF). In summary, these studies indicate that both Gs and adenylyl cyclase activities fall in parallel with the development of LV hypertrophy followed by a further decrement in adenylyl cyclase, independent of Gs, in the setting of heart failure.

Adenylyl Cyclases↗
PubMed1990

Alternative promoter and 5' exon generate a novel Gs alpha mRNA.

Several species of mRNA have been shown to encode the alpha subunit of the stimulatory GTP-binding regulatory protein, Gs alpha. The various Gs alpha mRNAs are generated through alternative splicing of a single precursor RNA and through the use of alternative acceptor splice sites. We now report the existence of a Gs alpha mRNA that uses a previously unidentified promoter and leading exon (termed exon 1'). In both the canine and human Gs alpha genes, exon 1' is located 2.5 kilobases 5' of exon 1. Exon 1' does not contribute an in-frame ATG, and thus its mRNA encodes a truncated form of Gs alpha. Initiation of translation is predicted to begin at an AUG in exon 2, as demonstrated both by in vitro translation and COS cell expression studies.

Animals↗
PubMed1990

Binding of tissue-type plasminogen activator with human endothelial cell monolayers. Characterization of the high affinity interaction with plasminogen activator inhibitor-1.

The formation and release of covalent complexes between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) limits the application of equilibrium radioligand binding analysis to characterize the interaction between t-PA and human umbilical vein endothelial cell (HUVEC) monolayers. To avoid this difficulty, we used a recombinant mutant of t-PA, S478A rt-PA, in which alanine has been substituted for the active-site serine. Although the mutant is incapable of covalently reacting with PAI-1, 125I-labeled S478A rt-PA binding to HUVEC monolayers is specific and reversible and is characterized by a high affinity (Kd of 1.5 nM) and a large number of sites (1.5 x 10(6)/cell). This binding was shown to occur through noncovalent interaction with PAI-1 in the HUVEC monolayer by the fact that a monoclonal anti-PAI-1 antibody (MA-7D4) completely blocked S478A rt-PA binding. Two solution-phase assays with recombinant PAI-1 (rPAI-1) confirmed this noncovalent interaction: complexes between 125I-S478A rt-PA and rPAI-1 could be isolated by immunoprecipitation with anti-PAI-1 antibodies, and S478A rt-PA competed with rt-PA for inactivation by rPAI-1. In contrast diisopropylphosphate rt-PA (in which the active site serine is chemically modified) showed minimal binding to HUVEC monolayers, as a result of impaired interaction with PAI-1, in the two assays. Thus, both wild-type rt-PA and S478A rt-PA interact with the HUVEC monolayer through PAI-1. With rt-PA this results in the formation of covalent rt-PA.PAI-1 complexes that are released from the monolayer into the supernatant. With S478A rt-PA this results in the formation of noncovalent complexes that remain associated with the HUVEC monolayer, thereby identifying a large pool of reactive PAI-1 molecules in the monolayer.

Alanine↗
PubMed1990

Phosphorylation state of pro-atrial natriuretic factor in rat atrial secretory granules.

Recent studies have demonstrated that phosphorylation of atrial natriuretic factor (ANF) (99-126) in vitro modulates the bioactivity of this hormone. The potential physiological relevance of this observation was revealed in latter studies showing that endogenous proANF can be 32PO4-biosynthetically labeled by primary cultured atrial myocytes and by atrial appendage explants. The site and extent of proANF phosphorylation were different, however, in these two model systems. Whereas proANF extracted from atrial explants was phosphorylated on the bioactive, carboxy (C)-terminal portion of the molecule [ANF(99-126)], cultured atrial myocytes phosphorylated proANF on the amino (N)-terminal portion of the prohormone molecule [ANF(1-98)]. It was the goal of this study, therefore, to determine whether the bioactive region of proANF, ANF(99-126), is phosphorylated in vivo. ProANF was obtained by acid extraction of isolated rat atrial secretory granules followed by purification using reverse phase-HPLC. Analysis of purified 125I-labeled proANF by isoelectric focusing (IEF) revealed two bands with isoelectric points of 5.3 and 5.0. The more acidic band comigrated on IEF gels with 32PO4-biosynthetically labeled proANF obtained from primary cultures of atrial myocytes, suggesting that this species of proANF represented endogenously phosphorylated proANF. The more acidic band accounted for only 15-25% of the total proANF found in the mature atrial secretory granule. The phosphorylation state of ANF(99-126) produced by thrombin cleavage of secretory granule proANF was examined using three complementary methods: 1) cation-exchange HPLC, 2) amino-terminal amino acid sequence analysis and 3) anti-ANF(99-105) antibody immunoreactivity. Evidence from these three independent approaches indicated that proANF is not phosphorylated on the C-terminal portion of the molecule in vivo. Therefore, phosphorylation is not a physiological regulator of ANF(99-126) bioactivity.

Animals↗
PubMed1979

Atrial flutter with exit block.

The mechanism of atrial flutter is controversial. A 76-year-old woman with rheumatic heart disease was referred to our clinic with an unusual rhythm disturbance which initially appeared to be classic atrial flutter at a rate of 300 beats/min. Later tracings, however, demonstrated a rate exactly one-half that of the earlier ECGs, with an identical p-wave morphology and vector. This latter rhythm also behaved in a manner expected for a flutter mechanism in that both spontaneously and with carotid pressure high-degree atrioventricular block occurred without alteration of the underlying atrial mechanism. Finally, the two rates interchanged spontaneously over several days without any significant interval changes in medical therapy. These findings were initially explained as probable digoxin toxicity. The underlying mechanism, however, was more likely atrial flutter with exit block and in this patient may have represented another facet of her sick sinus syndrome. This unusual phenomenon is discussed in terms of previous reports and possible implications for the mechanism of atrial flutter.

Aged↗