Ventricular Membranes Research Articles

Ontogeny of positive inotropic responses to sympathomimetic agents and of myocardial adrenoceptors in rats

Positive inotropic efficacies (maximal increase in contractile force) and potencies of the alpha-adrenoceptor agonist methoxamine and beta-adrenoceptor agonist isoprenaline were determined on electrically driven (1 Hz) ventricular strips from rats aged 0.5, 1, 2, 3, 6, and 10 (adult) weeks. The inotropic response to methoxamine significantly decreased after 2 weeks of age. The inotropic potency of isoprenaline was slightly but significantly lower at all ages than at 0.5 weeks of age. Up to 2 weeks of age, the maximal inotropic effect of methoxamine was comparable with that of isoprenaline, thereafter it was but markedly less. Phenylephrine behaved like methoxamine, and noradrenaline like isoprenaline. The effect of methoxamine was antagonized by prazosin but not by propranolol; the reverse was true for isoprenaline. Injections at birth of triiodothyronine and dexamethasone exerted minimal effects on the inotropic responses to methoxamine and isoprenaline. Chemical sympathectomy with 6-hydroxydopamine caused supersensitivity to the inotropic effects of isoprenaline but produced subsensitivity to responses to methoxamine at 1 week; effects of methoxamine at 3 and 6 weeks of age were not altered by sympathectomy. No significant differences in alpha 1- or beta 1-adrenoceptor densities or affinities in ventricular membranes from 7-day-old and adult rats were found. It is concluded that the positive inotropic responses to sympathomimetic amines decline with age, the decline is most marked in the case of alpha 1-adrenoreceptor- mediated effects, and these changes do not appear to be due to a decrease in the number or affinity of alpha 1- and beta 1-adrenoceptors.

Evidence for a cooperation between adenosine A2 receptors and β1‐adrenoceptors on cardiac automaticity in the isolated right ventricle of the rat

1. The effects of the adenosine receptor agonists, 5'-N-ethyl-carboxamidoadenosine (NECA) and 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosin e (CGS-21680) on ventricular automaticity induced by a local injury in the isolated right ventricle of the rat were studied. 2. In concentrations ranging from 0.1 to 100 nM, NECA significantly increased ventricular automaticity. This effect was more reproducible when the adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) was present at 5 nM, a concentration that blocks A1 adenosine receptors. 3. The excitatory effect of NECA was not observed when DPCPX was present at a concentration of 10 microM, which antagonizes both A1 and A2 adenosine receptors, as well as when rats were reserpinized. 4. In reserpinized rats, NECA increased ventricular automaticity in the presence of isoprenaline and the beta 2-adrenoceptor antagonist, ICI-118,551, but not in the presence of the beta 1-adrenoceptor antagonists, bisoprolol or atenolol. 5. The A 2s-selective adenosine receptor agonist, CGS-21680 (0.1 nM-10 microM) was devoid of excitatory effect on ventricular automaticity. Binding studies of this compound to the rat ventricular membranes revealed that in the preparation there was no specific binding. 6. These results suggest that the excitatory effect of NECA on ectopic ventricular automaticity is dependent on endogenous catecholamines and is mediated by an A2 adenosine receptor which is in some way 'linked' to the beta 1-adrenoceptor. These A2 receptors do not appear to be of the A2a adenosine receptor subtype.

Radioimmunochemical Quantification of Giα in Right and Left Vehicles from Patients with Ischaemic and Dilated Cardiomyopathy and Predominant Left Ventricular Failure

An increase of Giα-related pertussis toxin substrates has been observed in the failing myocardium. In order to quantify the protein expression of Giα directly, we developed a fast radioimmunoassay using the iodinated synthetic peptide 125I-KENLKDCGLF. β-adrenoceptors were studied with 125I-cyanopindolol binding for comparison. Immunoblot experiments using recombinant G-protein α-subunits showed that DS4 immunostained the G-protein α-subunits with a rank order of potency rGiα1 = rGiα2>rGoα> >rGiα3. The G-protein α-subunits recognized by DS4 in human ventricular membranes co-migrated with rGiα1 and rGiα2. The radioimmunoassay had a sensitivity of 2.5 μg/ml transducin α with an interassay variation of less than 10%. The non-labelled peptide selectively competed with the myocardial 40 kDa membrane protein for binding to the antiserum DS4. Radioimmunochemical quantification of Giα from cardiac membranes showed that in left ventricular membranes (LV) from dilated cardiomyopathy (DCM), there was an increase of Giα by 138.5% when related to mg protein and 135% when related to 3H-ouabain binding sites as membrane marker. In LV from ischaemic cardiomyopathy (ICM), the increase was smaller (58.4%) when related to mg protein compared to the increase of Giα when related to 3 H-ouabain binding sites as membrane marker (155% v NF). In contrast, in the right ventricles (RV) there was no increase of Giα in ICM or DCM. The numbers of β-adrenoceptors were reduced in RV and LV of both, ICM and DCM. It is concluded that the radioimmunoassay may become an important tool for studying the expression of Giα-protein levels and changes thereof in pathological conditions. The amount of immunodetectable Giα-proteins is increased in failing LV due to DCM and ICM but not in RV, while β-adrenoceptor down-regulation occurred in RV and LV in both conditions. These findings might indicate that the liability of the LV but not of RV to express Giα-proteins may be increased in predominant LV heart failure. Alternatively, the underlying mechanism, e.g. sympathetic activation, amy be regulated locally in the failing heart producing different changes in adjacent chambers.

Histamine receptors mediating a positive inotropic effect in guinea pig and rabbit ventricular myocardium: distribution of the receptors and their possible intracellular coupling processes.

The difference in histamine receptor subtypes that are involved in the positive inotropic effect of histamine in guinea pig and rabbit ventricular myocardium was analytically characterized. In guinea pig papillary muscles, the positive inotropic effect of histamine was antagonized by cimetidine but not by mepyramine. The converse was true in rabbit papillary muscles. However, histamine evoked a positive inotropic effect through H1- and H2-receptors after blockade of H2- and H1-receptors in guinea pig and rabbit papillary muscles, respectively. Adenylate cyclase was significantly activated by histamine via H2-receptors in guinea pig but not in rabbit myocardial ventricular membranes. Accumulation of [3H]inositol monophosphate in ventricular strips prelabeled with myo-[3H]inositol was increased by histamine via H1-receptors to a similar extent in rabbits and guinea pigs. Radioligand binding experiments with [3H]mepyramine and [3H]tiotidine showed an increased number of H1-receptors and a decreased number of H2-receptors in guinea pig compared with rabbit ventricular myocardium. These results suggest that the positive inotropic effects of histamine are dominated by an H1-receptor-mediated effect in rabbits and by an H2-receptor-mediated one in guinea pig ventricular myocardium, and the positive inotropic effect manifested by one subtype apparently restricts the expression of the positive inotropic effect mediated by the other subtype. This species difference is not due to a difference in densities of the receptor subtypes, but may be partly related to a difference in the extents of coupling of H2-receptors to adenylate cyclase.

Impaired expression of G sα protein mRNA in rat ventricular myocardium with aging

We have recently reported that the responsiveness of adrenoceptors is decreased with aging in rat ventricular myocardium. Thus, the current study determined aging-dependent changes in: (a) characteristics of myocardial G proteins as determined by Western blot analysis; (b) steady-state levels of G protein mRNA as determined by Northern blot analysis; and (c) the inotropic response to isoproterenol, a β-adrenoceptor agonist. Cardiac preparations were isolated from male Wister rats of 6 (adult) and 24 (old) months old. Compared with adults, aging decreased the combined level of the three G sα subunits (45, 47 and 52 kDa) by a total of 23% in ventricular membrane preparations. In contrast, levels of G iα (40/41 kDa), G qα (42 kDa), G oα (39 kDa) and G commonβ (35/36 kDa) immunoreactivity were not affected by aging in the same membrane preparations. In ventricular myocardium, steady-state levels of G sα mRNA (1.9 kb) decreased by 20–28% between 6 and 24 months of age with no change in G iα mRNA (2.4 kb). An aging-associated decline in β-adrenergic stimulation was observed in the maximum positive inotropic effect elicited by isoproterenol in the presence of prazosin in left papillary muscles, with no change in ED 50 values. These results suggest that age-related changes in cardiac excitation and contraction coupling following β-adrenoceptor stimulation are mediated, at least in part, by G sα protein dysfunction.

Cardiac desensitization to adenosine analogues after prolonged R-PIA infusion in vivo.

To determine the effects of chronic in vivo stimulation of adenosine receptors, R-(-)-N6-(2-phenylisopropyl)adenosine (R-PIA), a selective A1 receptor agonist, was administered to rats as a continuous 7-day infusion (200 nmol/h). Inotropic and chronotropic responses of isolated atria to adenosine receptor agonists were markedly desensitized compared with the responses of atria from age-matched control animals. Carbachol's negative chronotropic effect was also attenuated, indicating a heterologous mode of desensitization. Antagonist radioligand binding assays indicated a 52% reduction in A1 adenosine receptor maximum binding, and competition binding assays revealed a significant loss of G protein-coupled high-affinity A1 receptors in atria from R-PIA-treated rats. Inhibitory G proteins (Gi) were significantly reduced, as quantified by immunoblot analysis, with no change in the amount of stimulatory G proteins. Ventricular membranes from R-PIA rats showed loss of Gi and uncoupling of A1 receptors, without a significant change in A1 receptor density. Thus chronic R-PIA infusion desensitized rat atrial muscle to the effects of adenosine receptor agonists via several regulatory adaptations, including downregulation of A1 adenosine receptors, uncoupling of A1 receptors from their associated G proteins, and loss of Gi proteins.

Rat angiotensin II (type 1A) receptor mRNA regulation and subtype expression in myocardial growth and hypertrophy.

Two subtypes of angiotensin II (Ang II) receptors (AT1 and AT2) are distinguished by using the respective specific antagonists. In the present study, we report the regulation of cardiac AT1 type A (AT1A) receptor mRNA levels and the expression pattern of AT1 and AT2 receptors in the growth of the heart and the development and regression of cardiac hypertrophy. The ventricular AT1A mRNA level and the density of Ang II receptors at the neonatal period were significantly increased (3.5-fold and 2.5-fold, respectively) and then downregulated with maturation. The cardiac hypertrophy established in spontaneously hypertensive rats or two-kidney one-clip renovascular hypertensive rats resulted in substantial increases in ventricular AT1A mRNA levels (threefold) and Ang II receptor densities (twofold) as compared with those in respective control rats, whereas the receptor affinity was similar. The proportion of AT1 and AT2 subtypes in the specific Ang II binding in ventricular membranes prepared from normal adult rats was nearly equal. This proportion did not change significantly in the development of myocardial hypertrophy. The regression of cardiac hypertrophy by the normalization of elevated blood pressure completely reversed the increased levels of AT1A mRNA and the receptor density to the control level. Thus, AT1 and AT2 receptors are present in rat ventricular myocardium, and their expression is developmentally regulated and upregulated in response to hypertrophic change. Ang II action exerted through the increased number of Ang II receptors may contribute to the growth of the heart and thus to the maintenance of established hypertrophy as one of the hormones involved in hypertrophy development.

Decreased density of mesenteric arteries but not of myocardial endothelin receptors and function in rats with chronic ischemic heart failure.

Mesenteric artery and cardiac ventricular endothelin receptors and endothelin-1-induced pressor responses were studied in normal rats and rats with chronic congestive heart failure induced by myocardial ischemia (4 weeks after coronary artery ligation). In mesenteric arteries of rats with chronic ischemic heart failure, endothelin receptor density was significantly decreased by 59%, whereas the dissociation constant was increased 2.8-fold, as compared with controls. There were, however, no changes in endothelin-receptor density or the dissociation constant in cardiac ventricular membrane preparations from rats with congestive heart failure as compared with controls. In pithed rats with congestive heart failure there was a reduced pressor response to a bolus injection of endothelin-1 (800 pmole/kg body weight), while the vasodilatory response was unaltered as compared with sham-operated controls. These results demonstrate that there is a decreased vascular endothelin-receptor function due to a down-regulated endothelin receptor. The in vivo data indicate that this is due to impaired endothelin A but not endothelin B receptor function. Thus, there is an impaired arterial but not cardiac ventricular endothelin receptor-mediated signalling system in the rat with chronic ischemic heart failure.

α1-Adrenoceptor Subtypes and Inositol Phosphates Production in Heart Ventricles of Spontaneously Hypertensive Rats

To study potential cardiac alpha 1-adrenoceptor changes in the stage of established hypertension, [3H]prazosin binding characteristics and norepinephrine (NE) stimulated production of inositol phosphates in the ventricular tissue of spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto control rats (WKY) were determined. The maximum number of cardiac [3H]prazosin binding sites was significantly reduced in SHR as compared with WKY rats, but the Kd values were similar in both groups of animals. Distribution of the [3H]prazosin-labeled high- and low-affinity binding sites for WB 4101 was similar in SHR and WKY rats. Pretreatment of ventricular heart membranes with the irreversible alpha 1B-adrenoceptor-selective antagonist chloroethylclonidine resulted in the same percentage of loss of specific [3H]prazosin binding in both groups of rats. Together, these results showed a similar ratio of alpha and alpha 1B-adrenoceptor subtypes in SHR and WKY rats. In addition, [3H]inositol phosphate accumulation in response to cardiac alpha 1-adrenoceptor stimulation by NE was significantly reduced in SHR as compared with WKY rats although the EC50 values were not significantly different in the two strains of animals. Thus, we conclude that in an established stage of hypertension there is a specific loss in the number of cardiac alpha 1-adrenoceptors with consequently reduced inositol phosphate turnover. These results are compatible with the reported decrease in the positive inotropic effect mediated through myocardial alpha 1-adrenoceptors in spontaneous hypertension.

Subtype selective regulation of coupling of rat cardiac ß adrenoceptors to adenylate cyclase

There is now evidence from human studies to suggest that cardiac ß-adrenoceptor density and coupling to adenylate cyclase may be regulated in a subtype selective fashion. An animal model was used to investigate this further. Rats were infused for 6 days with the non-selective full agonist isoprenaline (n = 6) or the ß 1-selective partial agonist xamoterol (n = 6) with sham operated rats (n = 6) for control. ß-Adrenoceptor subtype density and coupling to adenylate cyclase were determined in left ventricular membranes. Isoprenaline infusion downregulated both ß 1- (30%) and ß 2- (63%) adrenoceptor subtypes with associated reduction in adenylate cyclase stimulation through both subtypes. Xamoterol did not downregulate either subtype, but selectively uncoupled ß 1-adrenoceptors. ß 1- and total ß-adrenoceptor-mediated stimulation of adenylate cyclase was reduced less by xamoterol than isoprenaline. We conclude that coupling of rat cardiac ß-adrenoceptors can be regulated in a subtype selective fashion and that the partial agonist xamoterol does not dosensitise ß-adrenoceptor mediated stimulation of adenylate cyclase to the same extent as the full agonist isoprenaline.

Localization of a functional autoimmune epitope on the muscarinic acetylcholine receptor-2 in patients with idiopathic dilated cardiomyopathy.

A peptide corresponding to the sequence 169-193 of the second extracellular loop of the human muscarinic acetylcholine receptor-2 was used as an antigen to screen sera from patients with idiopathic dilated cardiomyopathy (DCM, n = 36) and healthy blood donors (HBD, n = 40). The sera from 14 patients with DCM (38.8%) and 3 HBD (7.5%) recognized the muscarinic receptor peptide at dilutions varying from 1:20 to 1:160 in ELISA. A highly significant correlation (P = 0.006) was found between the presence of antimuscarinic receptor-2 autoantibodies and anti-beta-adrenoceptor-1 autoantibodies in the patients' sera. Affinity-purified autoantibodies from positive sera of patients with DCM recognized on the electrotransferred protein of rat ventricular membrane a major band of about 80 kD. Incubation of autoantibodies with membrane resulted not only in a decrease in the maximal binding sites (Bmax) but also in an increase in Kd of radioligand binding in a concentration-dependent manner. This suggests a mixed-type of inhibition. Moreover, preincubation with atropine abolished the inhibitory effect of autoantibodies on the receptor binding whereas carbachol appeared to have no effect on the activity of the autoantibodies. These data define a subgroup of patients with idiopathic DCM who have in their sera functionally active autoantibodies against muscarinic receptor-2.

Cardiac alpha 1-adrenoceptors stimulate a high-affinity GTPase activity in sarcolemmal membranes from rabbit atrial and ventricular myocytes.

The interaction between cardiac alpha 1-adrenoceptors and GTP-binding regulatory proteins was characterized in isolated rabbit cardiac myocytes (thereby avoiding interference by other cell types present in the myocardium) by examining the alpha 1-adrenergic stimulation of GTPase activity in sarcolemma-enriched membrane fractions. Stimulation of membrane-associated GTPase activity in both atrial and ventricular myocyte preparations by the alpha 1-adrenergic agonists 1-noradrenaline and methoxamine (in the presence of propranolol) was observed to be both linear with time and saturable. alpha 1-adrenergic stimulation did not change the Km for GTP (0.14-0.21 microM), but increased the Vmax by 39% and 72% above basal levels in atrial and ventricular membranes, respectively. Stimulation of GTPase activity by alpha 1-agonists occurred in a concentration-dependent fashion and was blocked in the presence of the alpha-adrenoceptor antagonists phentolamine and prazosin, but not yohimbine. Prior treatment of myocytes with pertussis toxin had no effect on the alpha 1-adrenergic stimulation of GTPase activity, but inhibited stimulation by muscarinic-receptor activation with carbachol. Finally, photoaffinity labelling of an approximately 75-kDa membrane-bound protein with [alpha-32P]GTP was enhanced in the presence of the alpha 1-agonist methoxamine and abolished by addition of excess non-labelled GTP, suggesting that this GTP-binding protein may interact with cardiac alpha 1-adrenoceptors; a similar GTP-binding protein which may be coupled to alpha 1-adrenoceptors has been reported in rat liver plasma membranes (Im, M. J. & Graham, R. M. (1990) J. Biol. Chem. 265, 18944-18951).

Cardiac β-adrenoceptors, G-proteins and adenylate cyclase regulation during myocardial hypertrophy

The role of the β-adrenoceptor-G-protein-adenylate cyclase system in the pathogenesis of cardiac hypertrophy was studied. We have used a minipig model of pressure-overload cardiac hypertrophy secondary to aortic banding. Four groups of five animals were used: minipigs made hypertrophic were evaluated 2 months (CH2 group) and 9 months (CH9 group) later and compared to controls (C2 and C9 groups, respectively). A decrease in β-adrenergic receptor density and an increase in antagonist affinity were shown in left ventricular membranes of hypertrophied animals compared with controls. In both groups, CH2 and CH9, an increase in EC 50 for isopreterenol-stimulated adenylate cyclase activity, an increase in forskolin-stimulated adenylate cyclase activity and a diminished inhibition by carbachol of isoproterenol-stimulated adenylate cyclase were observed. In contrast, fluoride-stimulated adenylate cyclase activity was markedly increased only in the end stage of hypertrophy. α s-cholera toxin-catalysed ADP-ribosylation is increased in early hypertrophy and then decreases with late hypertrophy and a similar pattern is observed with α O pertussis toxin-catalysed ADP-ribosylation, whereas α i-ADP-ribosylation remains unchanged. Tissue content of G s-, G i- and G 0-proteins, as assessed by specific antibodies, was found unchanged in CH9 and CH2 groups when compared with that in C9 and C2 control groups, respectively. Modifications in G s functional activity in later hypertrophic stages, expressed as alterations in cholera toxin ADP-ribosylation and adenylate cyclase fluoride responsiveness, may be important in the pathogenesis of decompensation from compensated hypertrophy to cardiac failure.

An Enzymatic Fluorometric Assay for Adenylate Cyclase Activity

Adenylate cyclase, the catalytic protein that converts ATP to cAMP, plays a fundamental role in adrenergic signal transduction. Adenylate cyclase activity (pmol cAMP/mg/min) is generally assayed by measuring radiolabeled cAMP generated from [α- 32P]ATP. Although sensitive, the radioactive approach is costly and time consuming. Given safety and environmental concerns, we developed a highly sensitive fluorometric assay for adenylate cyclase activity. This assay depends upon the breakdown of cAMP by phosphodiesterase to AMP, and the subsequent stimulation by AMP of glycogen phosphorylase a. Radioactive and fluorescence methods were compared using the same ventricular membrane preparations from five different rabbit hearts. Theophylline, used in the fluorometric assay, increased basal adenylate cyclase activity. However, adenylate cyclase kinetics, the dose response to isoproterenol, and the "fold" stimulation (agonist stimulated/basal adenylate cyclase activity) after isoproterenol (10 −6M), guanylyl-5′-imidodiphosphate (GppNHp) (10 −4M), and NaF (10 −2M) were nearly identical with both methods. Adenylate cyclase activity can be measured with the fluorometric assay in samples as small as 10 μg of membrane protein. In summary, this new fluorometric assay is highly sensitive, safer, less costly, and less time consuming than radioactive assays for adenylate cyclase activity.

Down-regulation of β-adrenoceptors and loss of Gsα subunit levels in ventricular myocardium of rats treated with isoproterenol

We investigated the influence of chronic β-adrenergic stimulation on the β-adrenoceptor-G protein-adenylyl cyclase system in rat ventricular myocardium. The rats received twice-daily injections of 4 mg/kg isoproterenol (ISO) alone or with 8 mg/kg propranolol (PROP) for 4 days. The ISO (10 μM)-induced increase in tissue cAMP production was lower (50%) after chronic ISO treatment than in control myocardium. The β-adrenoceptor density decreased by 43% in ventricular membranes from ISO-treated rats. Immunoblotting techniques using specific antibodies against G proteins revealed that ventricular myocardium contains three Gsα subunit isoforms of 45, 47 and 52kDa. ISO treatment decreased levels of the three Gsα subunits by a total of 40%, while no change in Giα (40/41kDa) and Gcommonβ (35/36kDa) levels were found in the same membrane preparations. The antagonist PROP almost totally blocked the effects of ISO treatment on cAMP, β-adrenoceptors and Gsα subunits. These results suggest that chronic β-adrenergic stimulation causes not only down-regulation of β-adcenoceptors, but also loss of Gsα subunit levels in rat ventricular myocardium.

Transcriptional regulation of left ventricular beta-adrenergic receptors during chronic hypoxia.

beta-Adrenergic receptor downregulation is the end result of cellular adaptation to prolonged agonist exposure. The factors mediating receptor downregulation include receptor phosphorylation, receptor movement from the plasma membrane to intracellular sites, and alterations in nascent receptor synthesis. We have previously demonstrated a downregulation of the left ventricular beta-receptor during chronic hypoxia in vivo. To determine the mechanism of this downregulation, we produced chronic hypoxia in seven newborn lambs by creating right ventricular outflow obstruction and an atrial septal defect. Oxygen saturation was reduced to 65-74% for 2 weeks. Six lambs served as normoxic controls. Sarcolemmal membrane and cytosolic fractions were prepared from left ventricular free wall samples. beta-Receptor density in each fraction was determined with the radioligand [125I]iodocyanopindolol. Steady-state levels of beta-receptor mRNA were determined by Northern blot analysis using a beta 1-adrenergic receptor cDNA probe. During chronic hypoxia, left ventricular membrane beta-adrenergic receptor density decreased by 55% (153 +/- 28 fmol/mg for hypoxic lambs versus 342 +/- 79 fmol/mg for control lambs, p < 0.05). There was no corresponding increase in beta-receptor density in the cytosolic fraction (23 +/- 3 fmol/mg for hypoxic lambs versus 33 +/- 9 fmol/mg for control lambs, p = NS), nor was there a significant change in the ratio of beta 1-receptor/beta 2-receptor subtypes as assessed by radioligand binding (beta 1 subtype, 84.1 +/- 10.1% for hypoxic lambs versus 93.2 +/- 8.8% for control lambs; p = NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac beta-adrenoceptor changes in experimental hyperthyroidism in dogs.

1. Triiodothyronine (T3; 1.0 mg/kg per day subcutaneously) was administered to 10 dogs for 14 days; 10 saline-treated dogs served as controls. T3-treated dogs showed the expected physiological responses of hyperthyroidism; further, chronotropic responses to isoprenaline in vivo were significantly increased in T3-treated dogs. 2. Beta-adrenoceptor subtype density was measured in membrane preparations by displacement of 125I-iodocyanopindolol binding by the selective beta 2-adrenoceptor antagonist, ICI 118, 551. T3 treatment led to a 93% increase in right atrial beta 1-adrenoceptor density and a 141% increase in left ventricular beta 1-adrenoceptor density; beta 2-adrenoceptor densities in right atrial, left ventricular and lung membranes were unchanged. 3. T3-treatment did not change basal or maximally stimulated adenylate cyclase activities in left ventricular membranes. 4. Thus, the cardiovascular changes in experimental hyperthyroidism in dogs were accompanied by an increased chronotropic response in vivo to isoprenaline and an increased beta 1-adrenoceptor density in atrial and ventricular membranes. However, there was no corresponding change in basal or maximal responsiveness of adenylate cyclase in ventricular membranes.

Transient elevation of cardiac beta-adrenoceptor responsiveness and receptor number in the streptozotocin-diabetic rat.

1. The effects on cardiac responsiveness of diabetes of up to 12 weeks duration has been examined in streptozotocin-pretreated rats. 2. Two weeks of diabetes resulted in a supersensitivity of isolated left atria and papillary muscles to isoprenaline, which was associated with an increase in the density of ventricular [3H]-dihydroalprenolol binding sites. 3. The beta-adrenoceptor supersensitivity was still evident in both tissues after 4 weeks of diabetes, but in left atria the supersensitivity was reduced compared with that observed at 2 weeks, while for papillary muscles it was greater than at 2 weeks. 4. Following 12 weeks of diabetes, responses of papillary muscles to isoprenaline were similar to controls, while beta-mediated responses of left atria were significantly depressed. 5. The alpha-adrenoceptor-mediated responses of cardiac tissues to phenylephrine were similar to controls following diabetes of 2 or 4 weeks duration. At 12 weeks, however, papillary muscle alpha-adrenoceptor-mediated responses were enhanced. The change in ventricular responsiveness to phenylephrine was not accompanied by any change in [3H]-prazosin binding to ventricular membranes. 6. The results demonstrate a transient elevation of cardiac beta-adrenoceptor sensitivity and receptor density during acute diabetes and illustrate the time- and tissue-dependence of diabetes-induced changes in cardiac adrenoceptor sensitivity.

Existence of PAF receptors in human platelets and human lung tissue but not in the human myocardium

The aim of this study was to investigate whether platelet-activating factor (PAF), PAF receptors, and PAF receptor-mediated effects in the human myocardium play a role in cardiac depression during anaphylaxis or septic shock. The effects of PAF, the biologically inactive derivative lyso-PAF, and the specific PAF antagonist WEB 2086 were studied in human myocardial tissue, in human coronary arteries, in human platelets, and in human lung tissue. PAF (C 16-PAF, C 18-PAF; 0.000001 to 1 μmol/L) had no effect on isometric force of contraction of electrically driven right atrial trabeculae (patients undergoing aortocoronary bypass surgery) and left ventricular papillary muscle strips (mitral valve replacement). PAF (0.2 μmol/L) did not influence the concentration-response curve of either the β-adrenoceptor agonist isoprenaline (ISO, 0.0001 to 1 μmol/L) or the m-cholinoceptor agonist carbachol (CARB, 0.0001 to 10 μmol/L). The effectiveness (ISO +4.7 ± 0.7 mN, PAF + ISO + 4.3 ± 0.44 mN, CARB −2.7 ± 1.06 mN; PAF + CARB −2.6 ± 0.52 mN) and the potency—as indicated by the EC 50 values—of both isoprenaline and carbachol were identical with and without pretreatment with PAF (0.2 μmol/L). PAF at concentrations of 0.000001 to 10 μmol/L exerted no effect on force of contraction either precontracted (prostaglandin F 2α, 0.3 μmol/L) or unprecontracted in human coronary artery rings. Histamine (0.01 to 100 μmol/L) and noradrenalin (0.001 to 30 μmol/L) initiated concentration-dependent contraction in human coronary artery rings (EC 50: histamine, 1.86 μmol/L; noradrenalin, 0.69 μmol/L). At lower concentrations (PAF, 0.01 μmol/L) PAF produced complete aggregation of human platelets. In human platelet membranes and lung membranes, 3H-WEB 2086 exhibited saturable high-affinity binding (K D 14.4 nmol/L and 14.3 nmol/L). The maximal binding capacity was 292 fmol/mg protein and 268 fmol/mg protein, respectively. In displacement experiments PAF (0.01 to 10000 nmol/L) and WEB 2086 (0.01 to 10000 nmol/L), but not lyso-PAF, completely displaced 3H-WEB 2086 from its binding sites on human platelet and lung membranes. In contrast, neither in left ventricular membranes nor in right atrial membranes was specific binding of 3H-WEB 2086 observed. These results suggest that there are neither specific PAF receptors nor direct PAF-mediated actions in human myocardial tissue or human coronary artery rings. The effects of PAF on myocardial function may be due to the activation of mediators (e.g., histamine).

Altered renal dopamine receptors during development of cardiac hypertrophy.

The characteristics of dopamine receptors were studied in heart and kidney using the radiolabeled receptor assay of [3H]spiperone during the development of cardiac hypertrophy. Male Sprague-Dawley rats (175-200 g) underwent abdominal aortic constriction above the renal arteries and were studied 3, 14, and 28 days thereafter. Sham-operated animals without aortic constriction were used as control. Although the ratio of left ventricular weight to total body weight was significantly increased 14 and 28 days after aortic constriction in animals, [3H]spiperone binding in left ventricular membrane was increased as early as 3 days after aortic constriction. At 14 days, the binding was still elevated and, by 28 days, it returned to control values. In contrast, membranes obtained from kidney cortex showed an elevation of [3H]spiperone binding only at 28 days after aortic constriction; at 3 days the binding values were decreased. A reciprocal correlation was found between the number of dopamine receptors and the activity of Na(+)-K(+)-ATPase at 28 days of aortic constriction; the enzyme activity, as measured by the release of 32Pi from [gamma-32P]ATP, was decreased in kidney cortex. Autoradiographic data also showed an increased number of dopamine receptors in kidney at 28 days after abdominal aortic constriction. These results suggest that the dopamine receptor is increased very early in heart in response to pressure overload as a result of a compensatory response to maintain an optimal left ventricular output. Kidney dopamine receptors are triggered at a later stage possibly to maintain fluid homeostasis secondary to the cardiac hypertrophic process.