Rat Heart Membranes Research Articles

Вплив омега-3 поліненасичених жирних кислот на дихання та набухання мітохондрій серця при експериментальному цукровому діабеті

We studied the influence of omega-3 polyunsaturated fatty acids (PUFAs) on respiration, swelling of rat heart mitochondria and changes in rat heart fatty acid composition in tissue homogenate in rats with streptozotocin induced diabetes mellitus (diabetes), which was induced by single intraperitoneal administration of 55 mg/kg streptozotocin. We found that application of these acids increased parameters of active mitochondrial respiration V3 at 63.7%, controlled breathing V4 at 30.7% and the rate of phosphorylation by 18.9% in animals with experimental diabetes. We proved their ability to reduce swelling of mitochondria in heart at streptozotocin induced diabetes. In addition, we established changes of fatty acid composition of cell membranes in hearts under diabetic conditions with omega-3 PUFAs influence. The obtained results allow to conclude that the omega-3 PUFA have a positive effect on functional parameters of mitochondria due to stabilization cell membranes of rat heart with diabetes.

Physical Coupling Supports the Local Ca2+ Transfer between Sarcoplasmic Reticulum Subdomains and the Mitochondria in Heart Muscle

In many cell types, transfer of Ca(2+) released via ryanodine receptors (RyR) to the mitochondrial matrix is locally supported by high [Ca(2+)] microdomains at close contacts between the sarcoplasmic reticulum (SR) and mitochondria. Here we studied whether the close contacts were secured via direct physical coupling in cardiac muscle using isolated rat heart mitochondria (RHMs). "Immuno-organelle chemistry" revealed RyR2 and calsequestrin-positive SR particles associated with mitochondria in both crude and Percoll-purified "heavy" mitochondrial fractions (cRHM and pRHM), to a smaller extent in the latter one. Mitochondria-associated vesicles were also visualized by electron microscopy in the RHMs. Western blot analysis detected greatly reduced presence of SR markers (calsequestrin, SERCA2a, and phospholamban) in pRHM, suggesting that the mitochondria-associated particles represented a small subfraction of the SR. Fluorescence calcium imaging in rhod2-loaded cRHM revealed mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) responses to caffeine-induced Ca(2+) release that were prevented when thapsigargin was added to predeplete the SR or by mitochondrial Ca(2+) uptake inhibitors. Importantly, caffeine failed to increase [Ca(2+)] in the large volume of the incubation medium, suggesting that local Ca(2+) transfer between the SR particles and mitochondria mediated the [Ca(2+)](m) signal. Despite the substantially reduced SR presence, pRHM still displayed a caffeine-induced [Ca(2+)](m) rise comparable with the one recorded in cRHM. Thus, a relatively small fraction of the total SR is physically coupled and transfers Ca(2+) locally to the mitochondria in cardiac muscle. The transferred Ca(2+) stimulates dehydrogenase activity and affects mitochondrial membrane permeabilization, indicating the broad significance of the physical coupling in mitochondrial function.

Cardiac norepinephrine transporter protein expression is inversely correlated to chamber norepinephrine content

The cardiac neuronal norepinephrine (NE) transporter (NET) in sympathetic neurons is responsible for uptake of released NE from the neuroeffector junction. The purpose of this study was to assess the chamber distribution of cardiac NET protein measured using [(3)H]nisoxetine binding in rat heart membranes and to correlate NE content to NET amount. In whole mounts of atria, NET was colocalized in nerve fibers with tyrosine hydroxylase (TH) immunoreactivity. NE content expressed as micrograms NE per gram tissue was lowest in the ventricles; however, NET binding was significantly higher in the left ventricle than the right ventricle and atria (P < 0.05), resulting in a significant negative correlation (r(2) = 0.922; P < 0.05) of NET to NE content. The neurotoxin 6-hydroxydopamine, an NET substrate, reduced NE content more in the ventricles than the atria, demonstrating functional significance of high ventricular NET binding. In summary, there is a ventricular predominance of NET binding that corresponds to a high NE reuptake capacity in the ventricles, yet negatively correlates to tissue NE content.

Evaluation of radioiodinated (2S,αS)-2-(α-(2-iodophenoxy)benzyl)morpholine as a radioligand for imaging of norepinephrine transporter in the heart

The norepinephrine transporter (NET) is located presynaptically on noradrenergic nerve terminals and plays a critical role in the regulation of the synaptic norepinephrine (NE) concentration via the reuptake of NE. Changes in NET have been recently reported in several cardiac failures. Therefore, a NET-specific radioligand is useful for in vivo assessment of changes in NET density in various cardiac disorders. Recently, we developed a radioiodinated reboxetine analogue, (2S,alphaS)-2-(alpha-(2-iodophenoxy)benzyl)morpholine ((S,S)-IPBM), for NET imaging. In the current study, we assessed the applicability of radioiodinated (S,S)-IPBM to NET imaging in the heart. The NET affinity and selectivity were measured from the ability to displace specific [3H]nisoxetine and (S,S)-[125 I]IPBM binding to rat heart membrane, respectively. To evaluate the distribution of (S,S)-[125 I]IPBM in vivo, biodistribution experiment was performed in rats. With the use of several monoamine transporter binding agents, pharmacological blocking experiments were performed in rats. In vitro binding assays showed that the affinity of (S,S)-IPBM to NET was similar to those of the well-known NET-specific binding agents, nisoxetine and desipramine. Furthermore, (S,S)-[125 I]IPBM binding was inhibited by nisoxetine and desipramine, but not by dopamine or serotonin transporter binding agents. These data indicated that (S,S)-IPBM had high affinity and selectivity for NET in vitro. Biodistribution studies in rats showed rapid and high uptake of (S,S)-[125 I]IPBM by the heart and rapid clearance from the blood. The heart-to-blood ratio was 31.9 at 180 min after the injection. The administration of nisoxetine and desipramine decreased (S,S)-[125 I]IPBM accumulation in the heart, but injection of fluoxetine and GBR12909 had little influence. Radioiodinated (S,S)-IPBM is a potential radioligand for NET imaging in the heart.

Artificial rearing with docosahexaenoic acid and n-6 docosapentaenoic acid alters rat tissue fatty acid composition

Docosahexaenoic acid (DHA; 22:6n-3) and n-6 docosapentaenoic acid (DPAn-6; 22:5n-6) are components of enriched animal feed and oil derived from Schizochytrium species microalgae. A one generation, artificial rearing model from day 2 after birth onward (AR) and a dam-reared control group (DAM) were used to examine DPAn-6 feeding on the fatty acid composition of various rat tissues at 15 weeks of age. Four AR diets were based on an n-3 fatty acid-deficient, 18:2n-6-based artificial milk with 22:6n-3 and/or 22:5n-6 added: AR-LA, AR-DHA, AR-DPAn-6, and AR-DHA+DPAn-6. The 22:6n-3 levels for the DAM, AR-DHA, and AR-DHA+DPAn-6 groups tended to be similar and higher than in the AR-LA and AR-DPAn-6 groups. The levels of 22:5n-6 tended to be higher only in the absence of dietary 22:6n-3. Adipose levels of 22:5n-6 was the only exception, as 22:5n-6 was significantly higher in AR-DHA+DPAn-6 than was observed in either the DAM or the AR-DHA group. There were no differences in 20:4n-6 levels within the tissues examined. In conclusion, 22:5n-6 replaces 22:6n-3 in the absence of 22:6n-3 only and does not appear to compete with 22:6n-3 in the presence of dietary 22:6n-3, suggesting that oils containing 22:5n-6 and 22:6n-3 may be a good dietary source of 22:6n-3.

The Glycolytic Enzymes, Glyceraldehyde-3-phosphate Dehydrogenase, Triose-phosphate Isomerase, and Pyruvate Kinase Are Components of the KATP Channel Macromolecular Complex and Regulate Its Function

The regulation of ATP-sensitive potassium (K(ATP)) channel activity is complex and a multitude of factors determine their open probability. Physiologically and pathophysiologically, the most important of these are intracellular nucleotides, with a long-recognized role for glycolytically derived ATP in regulating channel activity. To identify novel regulatory subunits of the K(ATP) channel complex, we performed a two-hybrid protein-protein interaction screen, using as bait the mouse Kir6.2 C terminus. Screening a rat heart cDNA library, we identified two potential interacting proteins to be the glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triose-phosphate isomerase. The veracity of interaction was verified by co-immunoprecipitation techniques in transfected mammalian cells. We additionally demonstrated that pyruvate kinase also interacts with Kir6.2 subunits. The physiological relevance of these interactions is illustrated by the demonstration that native Kir6.2 protein similarly interact with GAPDH and pyruvate kinase in rat heart membrane fractions and that Kir6.2 protein co-localize with these glycolytic enzymes in rat ventricular myocytes. The functional relevance of our findings is demonstrated by the ability of GAPDH or pyruvate kinase substrates to directly block the K(ATP) channel under patch clamp recording conditions. Taken together, our data provide direct evidence for the concept that key enzymes involved in glycolytic ATP production are part of a multisubunit K(ATP) channel protein complex. Our data are consistent with the concept that the activity of these enzymes (possibly by ATP formation in the immediate intracellular microenvironment of this macromolecular K(ATP) channel complex) causes channel closure.

Des-aspartate-angiotensin I and angiotensin AT 1 receptors in rat cardiac ventricles

The binding of 125I-[Sar 1,Ile 8]angiotensin II and 125I-angiotensin II to ventricular membranes of rat heart was studied. Displacement of bound 125I-[Sar 1,Ile 8]angiotensin II by its cold equivalents, angiotensin I, angiotensin II, angiotensin III, des-aspartate-angiotensin I, losartan, PD123319 and CGP42112B supports the presence of the AT 1 and the near absence of the AT 2 angiotensin receptor in adult rat ventricle. The presence of binding sites for des-aspartate-angiotensin I could account for its reported cardioprotective actions. Binding of 125I-angiotensin II but not that of 125I-[Sar 1,Ile 8]angiotensin II was partially displaced by GppNHp suggesting that a portion of the receptor population was in the active state with dissociated G-protein. Saturation experiments carried out in the absence and presence of 1 mM GppNHp showed similar magnitude of decrease in the number of receptors (Bmax from 26.2 ± 1.3 to 15.7 ± 1.1 fmol/mg protein) in [ 125I]-angiotensin II binding. However, the guanine nucleotide had no effect on the binding of 125I-[Sar 1,Ile 8]angiotensin II as has also been reported elsewhere, and may suggest that Sar 1-Ile 8-angiotensin II, being a partial agonist, binds to both the G-protein coupled and uncoupled states of the angiotensin receptors. The present study demonstrates that des-aspartate-angiotensin I binds to angiotensin receptors in the heart, and provides further evidence for its involvement in the pathophysiology of the organ.

Beta-adrenergic regulation requires direct anchoring of PKA to cardiac CaV1.2 channels via a leucine zipper interaction with A kinase-anchoring protein 15.

Activation of beta-adrenergic receptors and consequent phosphorylation by cAMP-dependent protein kinase A (PKA) greatly increases the L-type Ca2+ current through CaV1.2 channels in isolated cardiac myocytes. A kinase-anchoring protein 15 (AKAP15) coimmunoprecipitates with CaV1.2 channels isolated from rat heart membrane extracts and transfected cells, and it colocalizes with CaV1.2 channels and PKA in the transverse tubules of isolated ventricular myocytes. Site-directed mutagenesis studies reveal that AKAP15 directly interacts with the distal C terminus of the cardiac CaV1.2 channel via a leucine zipper-like motif. Disruption of PKA anchoring to CaV1.2 channels via AKAP15 using competing peptides markedly inhibits the beta-adrenergic regulation of CaV1.2 channels via the PKA pathway in ventricular myocytes. These results identify a conserved leucine zipper motif in the C terminus of the CaV1 family of Ca2+ channels that directly anchors an AKAP15-PKA signaling complex to ensure rapid and efficient regulation of L-type Ca2+ currents in response to beta-adrenergic stimulation and local increases in cAMP.

A new norepinephrine transporter imaging agent for cardiac sympathetic nervous function imaging: radioiodinated (R)- N-methyl-3-(2-iodophenoxy)-3-phenylpropanamine

Changes of the cardiac norepinephrine transporter (NET) have been reported in several cardiac failures. (R)- N-methyl-3-(2-iodophenoxy)-3-phenylpropanamine (MIPP), the 3-phenoxy-3-phenylpropylamine analogue iodinated at the 2-position of the phenoxy ring, was synthesized and evaluated as a potential radiopharmaceutical for investigating the cardiac norepinephrine transporter by single photon emission computed tomography (SPECT). (R)-[ 125I]MIPP was synthesized via a halogen exchange reaction under no-carrier-added conditions and purified by high-performance liquid chromatography (HPLC) with high radiochemical yield (60%) and high radiochemical purity (> 98%). The binding affinity of (R)-MIPP for cardiac NET was measured in terms of the displacement of [ 3H]desipramine and (R)-[ 125I]MIPP from binding sites in rat heart membranes. The binding data revealed that the affinity of (R)-MIPP was 5 times that of nisoxetine which is a selective NET inhibitor. In biodistribution studies, (R)-[ 125I]MIPP showed a high uptake followed by rapid clearance in the heart. (R)-[ 125I]MIPP binding sites were saturable and the administration of nisoxetine and desipramine, selective NET inhibitors, decreased the cardiac accumulation of (R)-[ 125I]MIPP. These results suggested that (R)-[ 123I]MIPP may be an useful radiopharmaceutical for imaging cardiac sympathetic nervous functions.

Assessment of affinities and dissociation potencies of several 5-HT2 antagonists to and from M2 muscarinic receptor in rat heart membranes.

The aim of the present study was to investigate the binding affinities and dissociation potencies of several 5-HT(2) antagonists in M(2) muscarinic receptor of rat heart membranes using [(3)H]QNB as a radioligand. The 5-HT(2) antagonists used in this study were sarpogrelate, ketanserin and cyproheptadine. The results showed that sarpogrelate and ketanserin had very weak binding affinities to M(2) muscarinic receptor, whereas cyproheptadine had higher binding affinity to this receptor than the muscarinic receptor antagonist, atropine. All of these three 5-HT(2) antagonists as well as muscarinic receptor antagonists (atropine and pirenzepine) were readily dissociated from M(2) muscarinic receptor in rat heart membranes after washing. Therefore, the findings of the present investigation suggest that the dissociation potencies of neither 5-HT(2) antagonists nor muscarinic antagonists used correlate with their binding affinities to M(2) muscarinic receptors in rat heart.

S-nitrosothiol detection in isolated perfused rat heart.

Nitrogen monoxide (NO) has important cardiovascular actions, and it has been suggested that they may be partly mediated by the reaction with protein sulfhydryl groups to produce S-nitrosothiols. In this work we describe and test a method that allows S-nitrosothiol detection in crude membrane preparations obtained from isolated perfused rat hearts. Isolated rat hearts were perfused under control conditions or in the presence of the NO donors SIN-1 and isosorbide dinitrate. Additional hearts were subjected to 10-20 min of ischemia followed or not by 10-20 min of reperfusion. At the end of perfusion a crude membrane fraction was prepared, and S-nitrosothiol concentration was assayed fluorometrically, on the basis of 2,3-naphthotriazole production from 2,3-diaminonaphthylene. The sensitivity of the method, as evaluated using S-nitrosoalbumin, was on the order of 1-2 pmol/mg of protein. S-nitrosothiols were undetectable under control conditions, as well as after ischemia or ischemia-reperfusion. On the other hand, significant S-nitrosothiol formation was observed after infusion of SIN-1 or isosorbide dinitrate (26.4 +/- 7.4 and 19.9 +/- 5.6 pmol per mg of protein, respectively). In conclusion, S-nitrosothiol production was observed in rat heart membranes after exposure to NO donors, while S-nitrosothiol concentration was below the sensitivity limits of the assay either under baseline conditions or after acute ischemia and reperfusion.

Clonidine displacement from type 1 imidazoline receptor by p-aminobenzamidine, the prototype of trypsin-like serine protease inhibitors.

p-Aminobenzamidine inhibits competitively the catalytic activity of enzymes that recognize preferentially the L-arginyl side chain and related structures. Notably, p-aminobenzamidine is considered as the prototype of trypsin-like serine protease inhibitors. Furthermore, p-aminobenzamidine inhibits the catalytic activity of nitric oxide synthase type I and type II as well as copper amine oxidase. Taking into account the structural similarity between p-aminobenzamidine, agmatine (the putative endogenous ligand of the membrane type 1 imidazoline receptor (I1-R)), and N-amidino-2-hydroxypyrrolidine (the product of agmatine oxidation by copper amine oxidase), the [3H]clonidine displacement from I1-R in rat heart membranes by p-aminobenzamidine was investigated. p-Aminobenzamidine is as effective as agmatine and N-amidino-2-hydroxypyrrolidine and more effective than the antihypertensive drug clonidine to displace [3H]clonidine from I1-R. Therefore, trypsin-like serine protease inhibitors structurally related to p-aminobenzamidine should be administrated under careful control.

Influence of silymarin and its flavonolignans on doxorubicin-iron induced lipid peroxidation in rat heart microsomes and mitochondria in comparison with quercetin.

Silymarin, an extract of Silybum marianum seeds, and the constituent flavonolignans silybin, silydianin and silychristin, as well as the flavonol quercetin, protected rat heart microsomes and mitochondria against iron-dependent doxorubicin induced lipid peroxidation. Quercetin was found to be more potent than either silymarin or its three constituents, whose cytoprotectivity was comparable. The radical scavenging activity of the compounds was investigated using a DPPH colour reduction assay and cyclic voltametry to assess their antioxidant activities. In contrast to quercetin, silybin, silydianin and silychristin did not chelate iron in aqueous solution. The results suggest that silymarin may prevent doxorubicin-mediated damage to rat heart membrane primarily through a free radical scavenging mechanism.

Inward rectifier potassium channel Kir2.2 is associated with synapse-associated protein SAP97.

The strong inwardly rectifying potassium channels Kir2.x are involved in maintenance and control of cell excitability. Recent studies reveal that the function and localization of ion channels are regulated by interactions with members of the membrane-associated guanylate kinase (MAGUK) protein family. To identify novel interacting MAGUK family members, we constructed GST-fusion proteins with the C termini of Kir2.1, Kir2.2 and Kir2.3. GST affinity-pulldown assays from solubilized rat cerebellum and heart membrane proteins revealed an interaction between all three Kir2.x C-terminal fusion proteins and the MAGUK protein synapse-associated protein 97 (SAP97). A truncated form of the C-terminal GST-Kir2.2 fusion protein indicated that the last three amino acids (S-E-I) are essential for association with SAP97. Affinity interactions using GST-fusion proteins containing the modular domains of SAP97 demonstrate that the second PSD-95/Dlg/ZO-1 (PDZ) domain is sufficient for interaction with Kir2.2. Coimmunoprecipitations demonstrated that endogenous Kir2.2 associates with SAP97 in rat cerebellum and heart. Additionally, phosphorylation of the Kir2.2 C terminus by protein kinase A inhibited the association with SAP97. In rat cardiac ventricular myocytes, Kir2.2 and SAP97 colocalized in striated bands corresponding to T-tubules. In rat cerebellum, Kir2.2 was present in a punctate pattern along SAP97-positive processes of Bergmann glia in the molecular layer, and colocalized with astrocytes and granule cells in the granule cell layer. These results identify a direct association of Kir2.1, Kir2.2 and Kir2.3 with the MAGUK family member SAP97 that may form part of a macromolecular signaling complex in many different tissues.

Pancreastatin, a chromogranin A-derived peptide, activates Gα 16 and phospholipase C-β 2 by interacting with specific receptors in rat heart membranes

Pancreastatin (PST) is one of the chromogranin A (CGA)-derived peptides with known biological activity. It has a general inhibitory effect on secretion in many exocrine and endocrine systems including the heart atrium. Besides, a role of PST as a counter-regulatory peptide of insulin action has been proposed in the light of its effects on glucose and lipid metabolism in the liver and adipose tissue, where receptors and signaling have been described. Gα q/11 pathway seems to mediate PST action. Since PST has been shown to function as a typical calcium-dependent hormone, and increased plasma levels have been found in essential hypertension correlating with catecholamines, we sought to study its possible interaction and signaling in heart membranes. Here, we are characterizing specific PST binding sites and signaling in rat heart membranes. We have found that PST receptor has a K d of 0.5 nM and a B max of 34 fmol/mg of protein. The PST binding is inhibited by guanine nucleotides, suggesting the functional coupling of the receptor with GTP binding proteins (G proteins). Moreover, PST dose-dependently increases GTP binding to rat heart membranes. Finally, we have studied PST signaling-effector system by measuring phospholipase C (PLC) activity using blocking antibodies against different G proteins and PLC isoforms. We have found that PST stimulates PLCβ 2>PLCβ 1>PLCβ 3 by activating Gα 16 in rat heart membranes. These data suggest that PST may modulate the cardiac function.

6beta-Acyloxy(nor)tropanes: affinities for antagonist/agonist binding sites on transfected and native muscarinic receptors.

A series of esters of 6beta-hydroxynortropane and the N-methyl analogue 6beta-tropanol were synthesized and screened versus binding of an antagonist (quinuclidinyl benzilate) and an agonist (oxotremorine-M) at sites on human m(1)-, m(2)-, m(3)-, and m(4)-muscarinic receptors in transfected cell membranes and on native M(1)-muscarinic receptors in rat brain membranes and native M(2)-muscarinic receptors in rat heart membranes. Most 6beta-acyloxy(nor)tropanes had higher affinity versus oxotremorine-M binding compared to quinuclidinyl benzilate binding at transfected m(1)- and native M(1)-receptors, indicative of agonist activity. 6beta-Acetoxynortropane had K(i) values versus oxotremorine-M binding at m(1)-, m(2)-, and m(4)-receptors ranging from 4 to 7 nM. N-Methylation reduced affinity greatly as did increasing the size of the acyl moiety. The affinity of 6beta-benzoyloxynortropane and other analogues with larger acyl moieties was little affected by N-methylation or in some cases was increased. 6beta-Acyloxy(nor)tropanes and classical muscarinic agonists, such as muscarine and oxotremorine, had higher affinity versus oxotremorine-M binding compared to quinuclidinyl benzilate binding at native M(2)-muscarinic receptors of heart, but not at transfected m(2)-muscarinic receptors. Antagonist/agonist binding ratios were not obtained for transfected m(3)-receptors, since significant oxotremorine-M binding could not be detected. 6beta-Acyloxy(nor)tropane, two other (nor)tropanes, and the classical muscarinic agonists had higher affinity versus agonist binding compared to antagonist binding for transfected m(4)-receptors. The antagonist/agonist binding ratio method is clearly not always reliable for predicting agonist activity at muscarinic receptors.

Expression of Distinct ERG Proteins in Rat, Mouse, and Human Heart: RELATION TO FUNCTIONAL IKr CHANNELS

One form of inherited long QT syndrome, LQT2, results from mutations in HERG1, the human ether-a-go-go-related gene, which encodes a voltage-gated K(+) channel alpha subunit. Heterologous expression of HERG1 gives rise to K(+) currents that are similar (but not identical) to the rapid component of delayed rectification, I(Kr), in cardiac myocytes. In addition, N-terminal splice variants of HERG1 and MERG1 (mouse ERG1) referred to as HERG1b and MERG1b have been cloned and suggested to play roles in the generation of functional I(Kr) channels. In the experiments here, antibodies generated against HERG1 were used to examine ERG1 protein expression in heart and in brain. In Western blots of extracts of QT-6 cells expressing HERG1, MERG1, or RERG1 (rat ERG1) probed with antibodies targeted against the C terminus of HERG1, a single 155-kDa protein is identified, whereas a 95-kDa band is evident in blots of extracts from cells expressing MERG1b or HERG1b. In immunoblots of fractionated rat (and mouse) brain and heart membrane proteins, however, two prominent high molecular mass proteins of 165 and 205 kDa were detected. Following treatment with glycopeptidase F, the 165- and 205-kDa proteins were replaced by two new bands at 175 and 130 kDa, suggesting that ERG1 is differentially glycosylated in rat/mouse brain and heart. In human heart, a single HERG1 protein with an apparent molecular mass of 145 kDa is evident. In rats, ERG1 protein (and I(Kr)) expression is higher in atria than ventricles, whereas in humans, HERG1 expression is higher in ventricular, than atrial, tissue. Taken together, these results suggest that the N-terminal alternatively spliced variants of ERG1 (i.e. ERG1b) are not expressed at the protein level in rat, mouse, or human heart and that these variants do not, therefore, play roles in the generation of functional cardiac I(Kr) channels.

The influence of isatin on guanylyl cyclase of rat heart membranes

The influence of indole-2,3 dione (isatin) on particulate guanylyl cyclase (GC) from rat heart membranes was investigated in the presence of adenylylimidodiphosphate (AMP-PNP). The latter activated GC in a concentration-dependent manner and 100 μM isatin abolished this effect. The IC 50 value, 2 μM, for the inhibition of stimulation of GC induced by 50 μM AMP-PNP, was close to the upper physiological level of isatin. These results indicate that isatin may interact with GC independently of its regulation by natriuretic peptides.

In vitro characterisation of the muscarinic receptor partial agonist, sabcomeline, in rat cortical and heart membranes

We have investigated the pharmacology of the functionally selective muscarinic M 1 receptor partial agonist, sabcomeline [SB-202026 ( R-( Z)-(+)-α-(methoxyamino)-1-azabicyclo[2.2.2] octane-3-acetonitrile)], in rat cortex and heart using radioligand binding and functional studies. The Quinuclidinyl benzilate/Oxotremorine-M acetate ratio from radioligand binding studies suggested that sabcomeline and xanomeline [3(3-hexyloxy-1,25-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine] are muscarinic receptor partial agonists in cortical and heart membranes. In [ 35 S ]GTPγS binding studies in rat cortex, carbachol stimulated binding via muscarinic M 2/M 4 receptors which could be blocked by sabcomeline with a p A 2 of 7.2. In rat heart membranes, carbachol also stimulated [ 35 S ]GTPγS binding studies through muscarinic M 2 receptors. Sabcomeline caused a small stimulation of basal [ 35 S ]GTPγS binding in both rat and heart tissues. Sabcomeline did not stimulate phosphoinositide hydrolysis in rat cortical slices, but did block the muscarinic M 1 receptor-mediated response caused by carbachol with apparent p K b of 6.9. Xanomeline and milameline also had no effect on phosphoinositide hydrolysis up to 100 μM. In adenylyl cyclase studies in rat atria, sabcomeline inhibited forskolin-stimulated adenylyl cyclase activity to a similar extent to that of carbachol, xanomeline and milameline. The present study, using the techniques of radioligand binding, supports previous publications which have claimed that sabcomeline is a muscarinic receptor partial agonist. As expected, this study shows that the functional actions of this compound at muscarinic receptor subtypes and in different tissues will depend on receptor reserve.

Efficacy of isatin analogues as antagonists of rat brain and heart atrial natriuretic peptide receptors coupled to particulate guanylyl cyclase

Isatin is an endogenous indole and an inhibitor of atrial natriuretic peptide (ANP) receptors coupled with particulate guanylyl cyclase (GC). In this study, several isatin analogues were tested as inhibitors of ANP-stimulated GC in rat brain and heart membranes. None of these analogues affected activity in the absence of ANP, or stimulated ANP-induced activity. In both tissues, some 5-substituted isatins (5-hydroxyisatin, 5-methylisatin, and 5-aminoisatin) exhibited more effective inhibitory activity than isatin itself, with ic 50 values in the range 1.3–20 μM. The efficacy of other analogues varied and was not consistent between the two tissues, raising the possibility of receptor heterogeneity and relative selectivity of inhibition. Some substituted isatins may have a role as pharmacological tools for investigating the physiological roles of natriuretic peptides and their receptors.