Search PubMed⌕ Search

Biomedical subjects

C J Homcy

Publications and source records attributed to C J Homcy.

At least 37 records · Page 2Linked to original sources
PubMed1995

Overexpression of Gs alpha protein in the hearts of transgenic mice.

Alterations in beta-adrenergic receptor-Gs-adenylyl cyclase coupling underlie the reduced catecholamine responsiveness that is a hallmark of human and animal models of heart failure. To study the effect of altered expression of Gs alpha, we overexpressed the short isoform of Gs alpha in the hearts of transgenic mice, using a rat alpha-myosin heavy chain promoter. Gs alpha mRNA levels were increased selectively in the hearts of transgenic mice, with a level 38 times the control. Despite this marked increase in mRNA, Western blotting identified only a 2.8-fold increase in the content of the Gs alpha short isoform, whereas Gs activity was increased by 88%. The discrepancy between Gs alpha mRNA and Gs alpha protein levels suggests that the membrane content of Gs alpha is posttranscriptionally regulated. The steady-state adenylyl cyclase catalytic activity was not altered under either basal or stimulated conditions (GTP + isoproterenol, GTP gamma S, NaF, or forskolin). However, progress curve studies did show a significant decrease in the lag period necessary for GppNHp to stimulate adenylyl cyclase activity. Furthermore, the relative number of beta-adrenergic receptors binding agonist with high affinity was significantly increased. Our data demonstrate that a relatively small increase in the amount of the coupling protein Gs alpha can modify the rate of catalyst activation and the formation of agonist high affinity receptors.

Adenylyl Cyclases↗
PubMed1994

A novel peptide inhibitor of adenylyl cyclase (AC). A peptide from type V AC directly inhibits AC catalytic activity.

Peptides derived from various regions of type V adenylyl cyclase (AC) were studied to determine their effect on AC catalytic activity. Out of 10 examined, only one peptide, peptide 2 (Lys425-Cys444), significantly inhibited both basal and stimulated (forskolin or GTP gamma S (guanosine 5'-O-thiotriphosphate)) type V AC catalytic activity overexpressed in CMT cells. The sequence of this peptide was taken from the first cytoplasmic domain of type V AC, which has a high sequence homology to other AC isoforms. Competition studies performed between the peptide and the substrate ATP showed that the inhibition was noncompetitive. Mutation or truncation of the peptide designed to destroy its secondary structure totally negated the inhibitory effect. This peptide also inhibited the catalytic activity of purified types II and V AC, as well as that of various cells, including S49 cyc- cells. Our data indicate that the peptide directly interacts with AC to inhibit catalytic activity; this provides new information regarding regions of the enzyme involved in its catalytic activation.

Adenylyl Cyclase Inhibitors↗
PubMed1994

Differential activation of adenylyl cyclase by protein kinase C isoenzymes.

Cyclic AMP production within cells is altered upon protein kinase C (PKC) activation; however, whether PKC directly modulates adenylyl cyclase (AC) catalytic activity has been controversial. Molecular studies have elucidated the existence of multiple PKC isoenzymes although the functional role of this diversity is not clear. Using purified PKC and AC isoenzymes, we demonstrate that PKC zeta directly phosphorylates type VAC, leading to an approximate 20-fold increase in its catalytic activity, a significantly larger enhancement than that achieved with forskolin (approximately 5-fold), the most potent activator of AC. When forskolin and PKC phosphorylation are combined, type V AC catalytic activity is increased 100-fold over basal levels. The two PKC isoenzymes (alpha and zeta) are additive in their capacity to activate AC, although PKC alpha is less potent than PKC zeta. Our data indicate that PKC can directly and potently regulate AC activity in an isoenzyme-specific manner, suggesting that direct cross-talk plays a major role in coordinating the activity of these two principal signal transduction pathways.

Adenylyl Cyclases↗
PubMed1994

Mammalian adenylyl cyclase family members are randomly located on different chromosomes.

Molecular cloning studies have elucidated the presence of multiple isoforms of mammalian adenylyl cyclase. So far, six different isoforms (I to VI) have been fully characterized. Comparison of their structural and biochemical characteristics suggests that the mammalian adenylyl cyclase family can be classified into four subfamilies: type I, type III, type II/IV, and type V/VI. We have determined the chromosomal localization of these genes. Type I gene was assigned to chromosome 7, type III to chromosome 2, types II and IV to chromosomes 5 and 14, and types V and VI to chromosomes 3 and 12. Our results indicate that the different adenylyl cyclase isoforms, even within the same subfamily, are distributed randomly in the genome, in contrast to the chromosomal organization of other components within the same signaling pathway, such as catecholamine receptors and G proteins.

Adenylyl Cyclases↗
PubMed1994

Changes in type VI adenylyl cyclase isoform expression correlate with a decreased capacity for cAMP generation in the aging ventricle.

We investigated the developmental regulation of the beta-adrenergic receptor-Gs-adenylyl cyclase pathway in myocardial membranes from fetal, neonatal, adult, and mature adult rats by measuring the density of the beta-adrenergic receptor and the activities of the stimulatory guanine nucleotide-binding protein Gs and the adenylyl cyclase enzyme. Total beta-adrenergic receptor content (in femtomoles per milligram protein) was greatest in the fetal (124.4 +/- 20.5 fmol/mg) and neonatal (122.3 +/- 16.1 fmol/mg) stages and gradually decreased in the adult (90.9 +/- 8.0 fmol/mg) and mature adult (70.0 +/- 9.6 fmol/mg) stages. An equivalent pattern was seen for adenylyl cyclase activity: the basal activity of the effector enzyme or that measured in the presence of 0.1 mmol/L isoproterenol with 0.1 mmol/L Gpp(NH)p, 10 mmol/L NaF, or 0.05 mmol/L forskolin was greater in the fetus and the neonate than in the adult and the mature adult. These data suggested that decreased stimulation of the catalytic unit by Gs could be the underlying cause of diminished adenylyl cyclase activity with aging. However, quantification of Gs by reconstitution into S49 cyc- membranes (in picomoles cAMP per microgram for 10 minutes) demonstrated no significant decrease during development from fetus (1.55 +/- 0.1 pmol/microgram) to neonate (1.9 +/- 0.5 pmol/microgram) and subsequent aging to adult (2.6 +/- 0.2 pmol/micrograms) and mature adult (1.9 +/- 0.2 pmol/microgram). When Northern blot analysis was used to characterize the relative amounts of mRNA coding for Gs alpha, no significant differences were seen among the developmental stages studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1994

Downregulation of adenylylcyclase types V and VI mRNA levels in pacing-induced heart failure in dogs.

We have shown that the heart expresses two distinct forms of adenylylcyclase mRNA, types V and VI. In this study we have characterized the expression of these two mRNA species in heart failure generated by overdrive pacing at a rate of 240 beats/min. After 4 wk, left ventricular end-diastolic pressure and heart rate increased significantly with the appearance of signs of heart failure, i.e., edema, ascites, and exercise intolerance. Basal as well as forskolin-stimulated adenylylcyclase activities decreased significantly, which was accompanied by a reduction in the steady state mRNA levels of adenylylcyclase types V and VI. These data suggest that in this model of cardiomyopathy, the downregulation of adenylylcyclase catalytic activity results, at least in part, from a reduction in the steady state levels of types V and VI adenylylcyclase mRNA levels.

Adenylyl Cyclases↗
PubMed1993

In vivo generation of an adenylylcyclase isoform with a half-molecule motif.

A truncated form of adenylylcyclase (type V-alpha) has been cloned from a cardiac cDNA library. It constitutes a half-molecule of type V adenylylcyclase diverging at the end of the first cytoplasmic loop. Northern blotting study has revealed the presence of such a mRNA species (approximately 3.5 kilobases in size) in the heart. Genomic sequence analysis has revealed that type V-alpha is generated via usage of a polyadenylation signal located within an intronic sequence of type V adenylylcyclase gene. When type V-alpha is co-expressed with an artificially generated half-molecule constituting the latter half of type V adenylylcyclase, the catalytic activity in transfected cell membranes is significantly higher than that of controls. However, when either alone is overexpressed, no significant increase in catalytic activity results. These results indicate that a half-molecule of adenylylcyclase, i.e. a protein containing six-transmembrane spans followed by a single cytoplasmic domain, can be generated in vivo, but catalytic activity is lacking unless heterodimerization can occur. This finding identifies another potential mechanism for generating diversity within this enzyme family.

Adenylyl Cyclases↗
PubMed1993

Regulation of angiotensin II receptors on ventricular myocytes after myocardial infarction in rats.

To determine the effects of acute myocardial infarction on the regulation of angiotensin II (Ang II) receptors and contractile performance of left and right ventricular myocytes, coronary artery ligation was surgically induced in rats, and Ang II receptor density and affinity and the mechanical properties of surviving muscle cells were examined 1 week later. Physiological determinations of cardiac pump function revealed the presence of ventricular failure, which was associated at the cellular level with a depression in the velocity of myocyte shortening and relengthening, a prolongation of time to peak shortening, and a reduction in the extent of cell shortening. These abnormalities in single-cell function were more prominent in left than in right ventricular myocytes. Cellular hypertrophy was documented by increases in cell length and width, which were also greater in the spared myocytes of the infarcted left ventricle. Reactive hypertrophy was accompanied by a 1.84- and 1.85-fold increase in the density of Ang II receptors on left and right myocytes, respectively. On the other hand, the affinity of Ang II receptors for the radiolabeled antagonist was not altered. However, Ang II-stimulated phosphoinositol turnover was enhanced by 3.7- and 2.5-fold in left and right myocytes, respectively, after infarction. Ventricular myocytes were found to possess the AT1 receptor subtype exclusively. In conclusion, myocardial infarction leads to impairment in the contractile behavior of the remaining cells and to the activation of Ang II receptors and effector pathway associated with these receptors, which may be involved in the reactive growth adaptation of the viable myocytes.

Angiotensin Receptor Antagonists↗
PubMed1993

Myocardial beta-adrenergic receptor function during the development of pacing-induced heart failure.

The development of pacing-induced heart failure was studied in chronically instrumented, conscious dogs paced at a rate of 240 beats/min for 1 d (n = 6), 1 wk (n = 6), and 3-4 wk (n = 7). Left ventricular (LV) dP/dt was decreased (P < 0.0125) at 1 d, LV end-diastolic pressure and heart rate were increased (P < 0.0125) at 1 wk, but clinical signs of heart failure were only observed after 3-4 wk of pacing. Plasma norepinephrine rose (P < 0.0125) after 1 d of pacing, whereas LV norepinephrine was reduced (P < 0.0125) only after 3-4 wk of pacing. Both the fraction of beta-adrenergic receptors binding agonist with high affinity and adenylyl cyclase activity decreased (P < 0.0125) after 1 d of pacing. Total beta-adrenergic receptor density was not changed at any time point, but beta 1-adrenergic receptor density was decreased (P < 0.0125) after 1 wk. The functional activity of the guanine nucleotide binding protein, Gs, was not reduced, but the Gi alpha 2 isoform of the alpha subunit of the GTP-inhibitory protein rose after 3-4 wk of pacing. Thus, myocardial beta-adrenergic signal transduction undergoes change shortly (1d) after the initiation of pacing, before heart failure develops. The mechanism of beta-adrenergic receptor dysfunction in pacing-induced heart failure is characterized initially by elevated plasma levels of catecholamines, uncoupling of beta-adrenergic receptors, and a defect in the adenylyl cyclase catalytic unit. Selective down-regulation of beta 1-adrenergic receptors, increases in Gi alpha 2, and decreases in myocardial catecholamine levels occur as later events.

Adenylyl Cyclases↗
PubMed1993

The G proteins of the G alpha i and G alpha q family couple the bradykinin receptor to the release of endothelium-derived relaxing factor.

Bradykinin stimulates diverse functions in endothelial cells including the release of endothelium-derived relaxing factor (EDRF). Little is known, however, regarding the identity of the G protein(s) involved. Here we demonstrate that G proteins of the G alpha i and G alpha q family are coupled to the bradykinin receptor (BKR) in bovine aortic endothelial cells by using specific antisera directed against the COOH-terminal region of G alpha i2 (P4), G alpha i3 (EC), and G alpha q (QL). These antisera are specific since their effects are blocked by the decapeptides from which they were derived. The degree of receptor-G protein coupling was assessed by the formation of high affinity agonist binding sites (HABS) and GTP hydrolysis. In a concentration-dependent manner, the QL antisera reduced HABS and GTPase activity by 65 and 60%, respectively, and effectively abolished them in membranes from pertussis toxin-treated cells. The combination of P4 and EC antisera produced a loss of HABS (41%) and GTPase activity (40%) comparable to the effects of pertussis toxin. These findings indicate that G alpha i and G alpha q proteins mediate the cellular responses to bradykinin in bovine aortic endothelial cells and support the observation that bradykinin-stimulated EDRF release is relatively insensitive to pertussis toxin.

Amino Acid Sequence↗
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↗