Biomedical subjects

P V Sulakhe

Publications and source records attributed to P V Sulakhe.

At least 19 recordsLinked to original sources

PubMed1997

Altered responsiveness of guanylyl cyclase to nitric oxide following treatment of cardiomyocytes with S-nitroso-D,L-acetylpenicillamine and sodium nitroprusside.

The stimulation of cardiomyocyte guanylyl cyclase by nitric oxide (NO)-donor drugs was examined before and after exposure of these cells to the NO-donor drugs: S-nitroso-d,l-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP). Short- (2-hr) and long-term (24-hr) exposure attenuated the maximal stimulation of GC by either SNAP or SNP by up to 80% ("desensitization"). However this "desensitization" of the myocardial GC was atypical in nature in that the reduction in maximal NO-stimulated GC activity was associated with an increase in the affinity of the GC towards either NO-donor, a finding not as yet reported. There was also evidence of "cross-desensitization" of GC (e.g., SNAP exposure decreasing the stimulatory effect of SNP). Further, this is the first time that SNAP-induced desensitization of GC has been observed.

Animals↗

PubMed1997

Sarcoplasmic reticulum Ca(2+)-pump dysfunction in rat cardiomyocytes briefly exposed to hydroxyl radicals.

The effects of hydroxyl radical exposure of intact cardiomyocytes on sarcoplasmic reticulum (SR) function were investigated. For this purpose, isolated rat heart myocytes were exposed briefly (1 min) to the hydroxyl radical generating system (H2O2/FeCl2 or FeSO4) or 5-5'-dithiobis-nitrobenzoic acid (DTNB), a sulfhydryl oxidizing reagent, and following this a SR-enriched fraction was isolated. Marked decreases in the SR calcium uptake activities were seen in the myocytes exposed to either the hydroxyl radical-generating system or DTNB. The exposure of myocytes to the hydroxyl radical, but not DTNB, markedly increased the amount of malonyldialdehyde (MDA) in the subsequently isolated SR. Total sulfhydryl group content in SR was decreased by exposure of myocytes to DTNB. Further, there was a significant decrease in [3H]-NEM binding to SR isolated from the hydoxyl radical-treated myocytes indicating that sulfhydryl groups are affected (oxidized). Both mannitol and catalase were found to offer complete protection against the inhibitory effect of peroxide +/- iron on calcium uptake. Also the above-mentioned alterations in both MDA and sulfhydryl group content were prevented by mannitol and catalase. Exogenously added cyclic AMP-dependent protein kinase (A-PK) or calmodulin (CAM) increased SR calcium uptake activity. In the SR isolated from the treated myocytes, the stimulatory effects of A-PK and CAM were also seen, although under all assay conditions calcium uptakes were of lower magnitude. The findings are consistent with the view that the damaging effect of the hydroxyl radical and DTNB on the functioning of SR occurs rapidly in the intact cardiomyocytes. The hydroxyl radical-provoked damage involves both protein sulfhydryl and lipid oxidation.

Animals↗

PubMed1997

Protein phosphorylation in rat cardiac microsomes: effects of inhibitors of protein kinase A and of phosphatases.

The phosphorylation of rat cardiac microsomal proteins was investigated with special attention to the effects of okadaic acid (an inhibitor of protein phosphatases), inhibitor 2 of protein phosphatase 1 and inhibitor of cyclic AMP-dependent protein kinase (protein kinase A). The results showed that okadaic acid (5 microM) modestly but reproducibly augmented the protein kinase A-catalyzed phospholamban (PLN) phosphorylation, although exerted little effect on the calcium/calmodulin kinase-catalyzed PLN phosphorylation. Microsomes contained three other substrates (M(r) 23, 19 and 17 kDa) that were phosphorylated by protein kinase A but not by calcium/calmodulin kinase. The protein kinase A-catalyzed phosphorylation of these three substrates was markedly (2-3 fold) increased by 5 microM okadaic acid. Calmodulin was found to antagonize the action of okadaic acid on such phosphorylation. Protein kinase A inhibitor was found to decrease the protein kinase A-catalyzed phosphorylation of microsomal polypeptides. Unexpectedly, inhibitor 2 was also found to markedly decrease protein kinase A-catalyzed phosphorylation of phospholamban as well these other microsomal substrates. These results are consistent with the views that protein phosphatase 1 is capable of dephosphorylating membrane-associated phospholamban when it is phosphorylated by protein kinase A, but not by calcium/calmodulin kinase, and that under certain conditions, calcium/calmodulin-stimulated protein phosphatase (protein phosphatase 2B) is also able to dephosphorylate PLN phosphorylated by protein kinase A. Additionally, the observations show that protein phosphatase 1 is extremely active against the three protein kinase A substrates (M(r) 23, 19 and 17 kDa) that were present in the isolated microsomes and whose state of phosphorylation was particularly affected in the presence of dimethylsulfoxide. Protein phosphatase 2B is also capable of dephosphorylating these three substrates.

Adenosine Triphosphatases↗

PubMed1997

Phosphorylation of inhibitory subunit of troponin and phospholamban in rat cardiomyocytes: modulation by exposure of cardiomyocytes to hydroxyl radicals and sulfhydryl group reagents.

Myocytes were isolated from rat heart ventricles and then incubated with [32P]-sodium phosphate to label intracellular ATP stores. Incubations of the [32P]-labelled cardiomyocytes with a beta-adrenoceptor agonist isoproterenol (10 microM) and with a plant diterpene forskolin (100 microM) which directly stimulates adenylyl cyclase increased the phosphorylation of an inhibitory subunit of troponin (TN-I) and phospholamban (PLN). Brief exposure (1 min) of labelled myocytes to the hydroxyl radical generating system (H2O2 plus FeCl2) decreased markedly the stimulatory action of isoproterenol and forskolin on TN-I and PLN phosphorylation. Similar exposure of myocytes to 5-5'-dithiobis-nitrobenzoic acid (DTNB) a sulfhydryl oxidizing reagent exerted little inhibitory effect on the isoproterenol or forskolin stimulated TN-I and PLN phosphorylation. In contrast exposure of myocytes to low concentrations (< 50 microM) of N-ethylmaleimide (NEM) a sulfhydryl alkylating reagent augmented the stimulatory effect of isoproterenol on TN-I and PLN phosphorylation. The results further showed that brief treatment of myocytes to H2O2 plus FeCl2 markedly decreased isoproterenol-, but not forskolin-, stimulated cyclic AMP accumulation in the myocytes. The stimulatory action of NEM on the isoproterenol-stimulated TN-I and PLN phosphorylation appeared related to greater increase in the isoproterenol-stimulated cyclic AMP accumulation in the NEM-treated cardiomyocytes. The results are consistent with the postulate that hydroxyl radical exposure of cardiomyocytes blunts the beta-adrenoceptor-mediated stimulation of adenylyl cyclase leading to decreased phosphorylation of TN-I and PLN and imply that such alterations account in part the reported depressed rate of relaxation of the myocardium exposed to oxygen free radicals.

Adenosine Triphosphatases↗

PubMed1997

Differential nature of cross-talk among three G-coupled receptors regulating adenylyl cyclase in rat cardiomyocytes chronically exposed to receptor agonists.

Chronic exposure of cells to cognate agonists has been established to cause homologous desensitization of G protein-coupled receptors. In this work, we show that exposure of adult rat cardiomyocytes to isoproterenol (ISO) for 24 h led to the desensitization of beta-adrenoceptor (beta-AR) coupled adenylyl cyclase (AC) activity, which was associated with an increased inhibition of AC by M2-muscarinic receptor (MR) agonist, carbachol (Cch), and a decreased inhibition of AC by A1-adenosine receptor (AdR) agonist, N6-phenylisopropyladenosine (R-PIA). Chronic exposure of cells to Cch caused the desensitization of M2-MR-coupled AC, decreased the inhibitory action of R-PIA on AC and increased ISO-stimulated AC, while chronic exposure to R-PIA caused the desensitization of A1-AdR-coupled AC and modestly increased ISO-stimulated AC without any significant effect on Cch inhibition of the enzyme. Thus, chronic exposure of cardiomyocytes revealed for the first time a more complex and differential nature of cross-talk among the three major G-coupled receptors in modulating AC.

Adenosine↗

PubMed1996

Enhanced forebrain nitric oxide synthase activity in epileptic fowl.

Nitric oxide synthase (NOS) activity was examined in forebrain, cerebellum and optic lobes of adult domestic fowl, having a hereditary primary generalized convulsive disorder. NOS was approximately 2-fold higher in only the forebrain of adult epileptic fowl compared to non-epileptic (carrier) hatchmates. A significant increase in NOS was also evident in forebrains of 1-day-old epileptic chicks. Ca(2+)-dependency experiments confirmed that these increments were principally due to type I NOS (NOS-I). Induction of convulsions by intermittent photic stimulation did not affect pre-existing forebrain NOS-I activity. The present data suggest that an enhanced NO signaling may ensue in selected regions of the brain as an adaptive response to hereditary epileptogenesis.

Animals↗

PubMed1996

Expressed alpha 1-adrenoceptors in adult rat brown adipocytes are primarily of alpha 1A subtype.

The main objective of this study was to characterize the alpha 1-adrenoceptors expressed in adult rat brown adipocytes. For this purpose, membrane fractions were prepared from brown adipose tissue as well as from isolated brown adipocytes. The following are major findings: (i) BAT membranes were considerably enriched in alpha 1-adrenoceptors (specific [3H]prazosin binding, Bmax, 79.49 +/- 16.77 fmol/mg protein; KD, 0.24 +/- 0.04 nM); (ii) among the cells that comprise brown adipose tissue, brown adipocytes were enriched in alpha 1-adrenoceptors; (iii) > 95% of total alpha 1-adrenoceptors were resistant to inactivation by 20 microM chloroethylclonidine, which readily and essentially completely inactivated alpha 1B-adrenoceptors in rat liver membranes; (iv) brown adipose tissue membrane alpha 1-adrenoceptors showed high affinity towards 5-methyl urapidil (KD 7.23 +/- 2.49 nM) and WB 4101 (KD 0.66 +/- 0.30 nM) and low affinity towards BMY 7378 (KD 0.34 +/- 0.03 microM); essentially similar affinities for these drugs were seen for membranes prepared from brown adipocytes; and (v) EBDA/LIGAND analysis of 5-methyl urapidil, WB 4101, and BMY 7378 competition curves revealed the presence of a single binding site for these drugs. Recent work has documented that 5-methyl urapidil and WB 4101 interact with high affinity with alpha 1A-adrenoceptors, while BMY 7378 interacts with high affinity with alpha 1D-adrenoceptors. Taken together, these findings are consistent with the view that alpha 1-adrenoceptors expressed in adult rat BAT are mainly of the alpha 1A subtype.

Adipocytes↗

PubMed1995

M1 muscarinic receptors heterologously expressed in cardiac myocytes mediate Ras-dependent changes in gene expression.

Stimulation of alpha 1-adrenergic receptors in neonatal ventricular cardiomyocytes induces hypertrophic changes including activation of the atrial natriuretic factor (ANF) gene. This receptor couples to Gq to activate phospholipase C (PLC) and protein kinase C, which have been implicated as mediators of the hypertrophic response. To directly determine whether receptor coupling to Gq/PLC is sufficient to induce ANF expression, we expressed wild-type and chimeric muscarinic cholinergic receptors (mAChRs) with altered G-protein coupling properties in cardiac myocytes and examined their ability to activate an ANF promoter/luciferase reporter gene. The cholinergic agonist carbachol failed to induce transcriptional activation of the ANF reporter gene through endogenous Gi-linked M2mAChRs or in cells transfected with M2mAChRs. In contrast, in cells transfected with M1mAChRs, which effectively couple to Gq/PLC, carbachol increased ANF reporter gene expression 10-fold and also increased ANF protein, as determined by immunofluorescence. Carbachol-mediated ANF gene expression was inhibited by the mAChR antagonist pirenzepine with a Ki value characteristic of an M1mAChR. Studies using chimeric M1- and M2mAChRs demonstrated that the N-terminal 21 amino acids of the third intracellular loop of the M1mAChR were required for receptor coupling to ANF gene expression. This region, previously shown to specify receptor coupling to Gq/PLC, also conferred partial activity to a chimeric M2 receptor. We further demonstrated that M1mAChR coupling to ANF gene expression was Ras-dependent since co-expression of dominant-interfering Ala-15 Ras inhibited M1mAChR-induced ANF expression by 60%. In contrast, ANF expression induced by the chimeric M2 receptor was not blocked by dominant-interfering Ras. We suggest that receptor coupling to Gq/PLC is sufficient to induce ANF expression and that a Ras-dependent pathway contributes additional signals required for maximal M1mAChR-mediated ANF gene expression.

Animals↗

PubMed1991

Divalent cation-sensitive antagonist binding to heart muscarinic receptors.

1. The binding of (-) [3H]quinuclidinylbenzilate (QNB), a potent muscarinic antagonist, to cardiac muscarinic receptors was examined in washed particles and microsomes isolated from rat heart atria. 2. Addition of divalent cations (Mg2+, Mn2+ and Ca2+) to the binding assays increased (6 to 8-fold) the antagonist binding to microsomes but exerted little or only weak (up to 35%) stimulation of the antagonist binding to washed particles. 3. Mg2+ ions present only in the dilution and washing buffer prior to rapid filtration of the binding reaction were capable of markedly increasing the antagonist binding to microsomes but not to washed particles. 4. Such differential action of Mg2+ on antagonist binding to subcellular fractions was seen when fractions were either isolated and or incubated in media containing either 10 or 50 mM imidazole-Cl, Tris-phosphate or Tris-Cl buffer, each at pH 7.5. While the modulatory effect of Mg2+ was not seen in Na-K-phosphate buffer, pH 7.5, the antagonist binding per se was much higher in Na-K-phosphate buffer compared to other buffers.

Animals↗

PubMed1991

Vasopressin (V1) receptor characteristics in rat aortic smooth muscle cells.

We report saturable, high-affinity, specific, reversible binding sites for both [3H]arginine vasopressin ([3H]AVP) and d(CH2)5Tyr(Me)-[3H]AVP, a V1-selective antagonist, in cultured smooth muscle cells obtained from rat aorta (RA) and rat mesenteric artery (RMA). Specific binding of [3H]AVP had the following characteristics in adherent monolayers of RA and RMA smooth muscle cells: dissociation constant (KD) = 1.42 and 1.23 nM and maximal binding capacity (Bmax) = 9,500 and 29,910 sites/cell, respectively. Lower KD and higher Bmax values were detected for 3H-labeled V1 antagonist binding to both types of cells. Complete dissociation of [3H]-AVP binding to RA cells occurred in a biphasic manner with two rate constants of dissociation, suggesting high- and low-affinity states of the binding site for the agonist interaction. Partial dissociation of the antagonist-specific binding occurred, and it was monophasic, suggesting interaction of the 3H-labeled V1 antagonist radioligand to the high-affinity binding state. Inhibition constant (Ki) values determined by competitive inhibition of [3H]AVP binding to RA cells by a series of AVP-related peptide analogues and antagonists were consistent with the saturation data. AVP in a concentration-dependent manner induced the accumulation of inositol phosphates [mean effective concentration (EC50) 1 nM] in the adherent RA cells. The free cytosolic Ca2+ level in the dispersed RA smooth muscle cells was increased by AVP (EC50 8.1 nM). Pretreatment with the V1 antagonist abolished these AVP-evoked responses. The data support the conclusion that the agonist binding occurs at a homogeneous population of V1-subtype receptors in the high-affinity (KD = approximately 1 nM) state and that these receptors are functionally coupled to phospholipase C.

Angiotensin Receptor Antagonists↗

PubMed1990

Muscarinic acetylcholine receptors in the sino-atrial node and right atrium of bovine heart.

Muscarinic acetylcholine receptors were identified by the specific binding of [H](-)quinuclidinylbenzilate [( 3H](-)QNB) and [3H]oxotremorine-M [( 3H]Oxo-M), to membranes isolated from the sino-atrial (SA) node and right atrium (RA) of bovine heart. The density of [3H](-)QNB binding sites was greater in the SA node compared to the RA. Specific [3H](-)QNB binding was saturable and occurred to a single population of binding sites in both regions. The binding of antagonists, as assessed by competition with [3H](-)QNB, also occurred to a single population of sites; the binding affinities of all antagonists were similar in either region. Agonist competition curves, except for McN-A-343, were complex and computer analyses indicated that agonists bound to at least two populations of binding sites that differed in affinity. The proportion of high-affinity agonist binding sites was consistently greater in the SA nodal, relative to the RA membranes, while the affinity of the high-affinity agonist binding sites to a given agonist was essentially similar in either region. The high-affinity binding of [3H]Oxo-M was saturable and occurred to a single population of sites. The maximal binding of [3H]Oxo-M in the SA nodal membranes was higher than in the RA membranes. Guanine nucleotides and N-ethylmaleimide (NEM) markedly decreased [3H]Oxo-M binding; NEM did not appear to influence guanine nucleotide-dependent decrease in [3H]Oxo-M binding. Phospholipase A2 decreased both [3H](-)QNB and [3H]Oxo-M specific binding, the latter being affected to a greater extent. Phospholipase C also decreased [3H](-)QNB and [3H]Oxo-M binding, although to a lesser degree compared to phospholipase A2. Either lipase, however, increased the guanine nucleotide-sensitive agonist binding. Analysis of [3H](-)QNB binding to microsomal subfractions showed that binding sites were enriched in the light plasma membrane fractions that were also enriched in pertussis toxin sensitive guanine nucleotide binding proteins.

Animals↗

PubMed1990

Effects of sulfhydryl agents, trifluoperazine, phosphatase inhibitors and tryptic proteolysis on calcineurin isolated from bovine cerebral cortex.

Calcineurin was discovered as an inhibitor of calmodulin stimulated cyclic AMP phosphodiesterase and its ability to act as a calmodulin binding protein largely explains its inhibitory action on calmodulin regulated enzymes. Recent studies establish calcineurin as the enzyme protein phosphatase whose activity is regulated by calmodulin and a variety of divalent metals. In this work, we have investigated the effects of several agents including sulfhydryl agents, trifluoperazine (a calmodulin antagonist), PPi, NaF and orthovanadate and of tryptic proteolysis on the calcineurin inhibition of cyclic AMP phosphodiesterase (called inhibitory activity) and on protein phosphatase activity. Inhibitors for sulfhydryl groups (pHMB, NEM) inhibited phosphatase activity without any effect on the inhibitory activity. Dithioerythritol completely reversed the inhibition by pHMB. Limited proteolysis of calcineurin caused an activation of basal phosphatase activity with a complete loss of inhibitory activity. Phosphatase activity of the proteolyzed calcineurin was not stimulated by calmodulin. The presence of calmodulin along with calcineurin during tryptic digestion appeared to preserve the stimulation of phosphatase by Ca2(+)-calmodulin. [3H]-Trifluoperazine (TFP) was found to be incorporated irreversibly into calcineurin in the presence of ultraviolet light. This incorporation was evident into the A and B subunits of calcineurin. TFP-caused a decrease in the phosphatase activity and an increase in its inhibitory activity. [3H]-TFP incorporation into the A subunit was drastically decreased in the proteolyzed calcineurin. This was also true when the [3H]-TFP incorporated calcineurin was subjected to tryptic proteolysis. The incorporation into the B unit was essentially unaffected in the trypsinized calcineurin. Phosphatase activity was inhibited by orthovanadate, NaF, PPi, and EDTA. Inhibitions by these compounds were more pronounced when the phosphatase was determined in the presence of Ca2(+)-calmodulin than in their absence.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1990

Divalent cation effects on calcineurin phosphatase: differential involvement of hydrophobic and metal binding domains in the regulation of the enzyme activity.

The effects of divalent metals, metal chelators (EDTA, EGTA) and sodium dodecyl sulfate were investigated on the phosphatase activity of isolated bovine brain calcineurin assayed in the absence (called intrinsic) and presence of calmodulin. Intrinsic phosphatase was increased by Mn2+, was unaffected by Mg2+, Ca2+, and Ba2+, and was markedly inhibited by Ni2+, Fe2+, Zn2+ and Cu2+. When assayed in the presence of calmodulin, many divalent metals (Ni2+, Zn2+, Pb2+, Cd2+), besides Mn2+, increased modestly the phosphatase activity at low concentrations (10-100 microM) and inhibited it markedly at high concentrations. Ca2(+)-calmodulin stimulated phosphatase activity was antagonized by Ni2+, Zn2+, Fe2+, Cu2+, Pb2+, at low concentrations (50 microM), and by Ba2+, Cd2+ at slightly higher concentrations (greater than 100 microM); Mn2+ and Co2+ (50 microM to 1 mM) in fact augmented it. EDTA and EGTA in a concentration and time dependent fashion inhibited the intrinsic phosphatase activity, particularly that of trypsinized calcineurin. SDS in low concentrations (0.005%) augmented the phosphatase activity and inhibited it at high concentrations. Mn2+ (+/- calmodulin) and Ca2+ only with calmodulin present increased the phosphatase activity assayed with low concentrations of SDS. The EDTA dependent inhibition of intrinsic phosphatase was almost abolished in assays containing SDS. Prior exposure of calcineurin to Mn2+ led to a high activity conformation state of calcineurin that was 'long-lived' or 'pseudo-irreversible'. Such Mn2(+)-activated state of calcineurin exhibited no discernible change in the affinity towards myelin basic protein or its inhibition by trifluoperazine. At alkaline pH, Mg2+ supported the intrinsic phosphatase activity, although to a lesser degree than Mn2+. The latter cation, compared to Mg2+ and Ni2+, was also a more powerful stimulator of the calcineurin phosphatase assayed with histone (III-S) and myosin light chain as substrates.

Animals↗

PubMed1990

Pertussis toxin-sensitive G-proteins in the sino-atrial node and right atrium of bovine heart.

Three apparently distinct pertussis toxin (PTX)-sensitive substrates, with Mrs of 39, 40 and 41 kDa, were identified in membranes prepared from the sino-atrial (SA) node and right atrium of bovine heart. Based on their biochemical characterization, the effects of guanine nucleotides/MgCl2 on their PTX-catalyzed [32P]ADP ribosylation, and the PTX-induced decrease in radiolabelled agonist high-affinity binding to muscarinic acetylcholine receptors present in these membranes, we tentatively identify these proteins as the alpha-subunits of the G0 and Gi subtypes of G-proteins. These results indicate that PTX alters the G-protein modulation of SA nodal and atrial muscarinic acetylcholine receptors by disrupting at least one of a group of PTX-sensitive G-proteins present in these tissues.

Adenosine Diphosphate↗

PubMed1990

MgCl2-sensitive and Gpp(NH)p-sensitive antagonist binding states of rat heart muscarinic receptors: preferential detection at ambient temperature assay and location in two subcellular fractions.

Some novel observations dealing with antagonist binding to cardiac particulate muscarinic receptors are described. Gpp(NH)p increased (2-3 fold) the specific binding of [3H]-QNB or [3H]-NMS, both potent muscarinic antagonists, to washed particles (WP), but not microsomes (MIC), when the binding was conducted at 30 degrees C. Magnesium, on the other hand, increased (2-3 fold) the binding of these antagonists to MIC, but not to WP, under the same condition. The treatment of subcellular fractions with 0.2 mM N-ethylmaleimide (NEM), a sulfhydryl reagent, failed to significantly modify the respective stimulatory actions of either Gpp(NH)p on WP binding or of magnesium on MIC binding of these antagonists; treatment with dithiothreitol (1 mM) was also ineffective in this regard. Gpp(NH)p decreased Kd (WP) while magnesium increased Kd (MIC) for [3H]-QNB. Repeated freezing/thawing of isolated subcellular fractions abolished the stimulatory effect of magnesium on antagonist binding to MIC but not of Gpp(NH)p on WP antagonist binding; the freeze/thaw procedure per se increased MIC binding but not WP binding of these antagonists. When the binding was conducted at 4 degrees C (24 hr), the stimulatory effect of Gpp(NH)p on [3H]-QNB binding was enhanced (6-fold) in the case of WP and was detectable (80%) in the case of MIC. Under this condition, the stimulatory effect of magnesium on [3H]-QNB binding was also enhanced (5-fold) in the case of MIC and became evident (200%) in the case of WP. The results of this work support the following views: (a) antagonist-occupied cardiac muscarinic receptors are capable of interaction with guanine nucleotide binding proteins (G protein like Gi, Go) and such interaction influences antagonist binding properties (e.g. increased affinity) of the cardiac membrane-associated muscarinic receptors (b) magnesium influences (decreased affinity) antagonist binding properties by interacting with multiple sites of which some are likely associated with components other than G proteins of the particulate fractions (c) a pool of NEM-sensitive sulfhydryls involved in the regulation of Gpp(NH)p-sensitive agonist binding to cardiac muscarinic receptors is not involved in the regulation by either Gpp(NH)p or magnesium of antagonist binding in these subcellular fractions and (d) membrane fluidity and microenvironment surrounding the receptor and G proteins contribute to the actions of Gpp(NH)p and magnesium on antagonist binding.

Animals↗

PubMed1990

Muscarinic cholinergic receptor mediated inhibitory transduction of adenylate cyclase activity in subcellular fractions from rat heart: improved detection in sodium phosphate buffer.

Cholinergic inhibition of myocardial adenylate cyclase activity in cell-free fractions has been known for many years, although the reported degrees of inhibition have been rather modest (20-30%), notably in rat heart fractions. The present study conducted with rat heart subcellular fractions document following major findings: (1) Myocardial adenylate cyclase activity and notably its cholinergic inhibition in cell-free fractions are notoriously labile to storage at 4 degrees C whereas its stimulation by beta adrenergic receptor agonists or forskolin are reasonably well preserved during storage. (2) Among four buffers (Tris, glycylglycine, imidazole and sodium phosphate) examined, sodium phosphate buffer afforded the best preservation of cholinergic inhibitory response of adenylate cyclase. (3) The commonly used biochemical buffers, notably imidazole, exerted deleterious effect on the cholinergic inhibition of myocardial adenylate cyclase such that it was considerably attenuated or barely detectable; this explains, in part, the reported poor inhibition of myocardial enzyme by others. (4) Imidazole buffer, on the other hand, augmented beta adrenergic and forskolin stimulated adenylate cyclase activity. The likely significance of these findings is discussed from consideration that the observed differential influence of buffers results from differential actions on the interactions between the components (receptor/coupling G proteins/catalyst) comprising autonomic receptor coupled adenylate cyclase system in rat heart.

Adenylyl Cyclases↗

PubMed1990

Two distinct, divalent cation-sensitive, antagonist binding states of heart muscarinic receptors: differential modulation by guanine nucleotide.

1. The binding of [3H]quinuclidinylbenzilate (QNB), a muscarinic antagonist, to cardiac muscarinic receptors was investigated in two subcellular fractions (washed particles and microsomes) isolated from rat heart atria and ventricles. 2. 5'-guanylylimidodiphosphate (Gpp(NH)p, 0.1 mM), increased (2-3-fold) the binding to washed particles, but not to microsomes, whereas Mg2+ (1-20 mM) increased (up to 5-fold) the binding to microsomes, but not to washed particles. Gpp(NH)p modestly increased the affinity while Mg2+ decreased the affinity towards the radiolabelled antagonist. 3. Treatment with N-ethylmaleimide (NEM, 2 mM) increased the antagonist binding to either fraction. The stimulatory effect of Gpp(NH)p was not evident while that of Mg2+ survived in the NEM-treated fractions. 4. The treatment of fractions with divalent cations chelators (EDTA, EGTA; 10 mM) augmented the stimulatory effect of Mg2+ on [3H]QNB binding to microsomes while that of Gpp(NH)p on the washed particle [3H]QNB binding was decreased. Such treatment further revealed an inhibitory action (about 40%) of Mg2+ on the washed particle binding.

Animals↗

PubMed1990

Calcium antagonizes the magnesium-induced high affinity state of the hepatic vasopressin receptor for the agonist interaction.

1. The present study describes the role of Ca2+ in the regulation of the hepatic vasopressin V1 receptor. With low concentrations of Ca2+, there was a small increase in [3H]-arginine vasopressin [( 3H]-AVP) binding, but above 10 mM, Ca2+ decreased the binding of this agonist. In contrast, low concentrations of Mg2+ were associated with a dramatic concentration-dependent increase in [3H]-AVP binding, reaching a maximal effect of 650% above control at concentrations ranging between 1-5 mM. At higher concentrations of Mg2+, the stimulatory effect of this cation was less pronounced, falling to 210% of control at 100 mM Mg2+. Strikingly, Ca2(+)-inhibited the stimulatory effect of Mg2+ in a concentration-dependent fashion. 2. Saturation binding data revealed that Ca2+ (2 to 10 mM) per se promotes the high affinity conformation of the V1 receptor for the agonist binding with the KD decreased from a control value of 2.3 nM to 0.5 nM in the presence of 10 mM Ca2+. This effect was attenuated with an increase in Ca2+ above 10 mM. With an increase in Ca2+ to 20 mM, however, the Bmax for [3H]-AVP binding was decreased. Ca2+ also decreased the high affinity/high capacity state (KD 100 pM) of the receptor induced by 1 mM Mg2+ for agonist interaction. 3. [3H]-V1 antagonist binding was inhibited by both Ca2+ and Mg2+. The IC50 values (mean +/- s.e. mean) for Ca2+ and Mg2+ were 32 +/- 8 and 53 +/- 9 mM respectively. Maximal inhibition achieved at 100 mM was 29% for Ca2+ and 42% for Mg2+. Both cations decreased the affinity and increased the capacity of the V1 receptor for the antagonist. 4. The results suggest that the divalent metal ion binding site(s) modulated by Mg2 + is also accessible to Ca2 +. Although Ca2 + opposes the powerful stimulatory effects of Mg2 + on agonist binding, the effects of Ca2+ and Mg2 + on the B,,x of [3H]-AVP binding were different, suggesting that the divalent cations may bind to two different sites, thereby regulating the affinity and the capacity characteristics of the V1 receptor.

Animals↗