Distribution of myocardial beta-adrenoceptor subtypes and coupling to the adenylate cyclase in children with congenital heart disease and implications for treatment

In congestive heart failure, down-regulation of myocardial beta-adrenoceptors (beta-AR) due to an elevated sympathetic tone is well known. In infancy and childhood, heart failure is usually related to congenital heart disease (CHD). Therefore, 71 samples of right atrial tissue of infants and children with CHD undergoing cardiac surgery were studied for beta-adrenoceptor density and distribution of the beta 1-/beta 2-AR subtypes. In 49 cases, the coupling of the beta-AR to the adenylate cyclase (AC) was examined. In a further study of 19 myocardial samples, AC was selectively stimulated with beta 1- or beta 2-AR whereas the other subtype was blocked by an antagonist. The following results were obtained: (1) Infants and children with severe acyanotic or cyanotic CHD had severely reduced beta-AR densities. (2) In most of the cases, the beta-AR down regulation is beta 1-subtype selective, but in critically ill newborns with congenital aortic valve stenosis or transposition of the great arteries, there is additional significant beta 2-AR down-regulation. In Fallot patients treated with the beta-antagonist propranolol, a significant increased beta-AR number compared with untreated Fallot patients was found. (3) beta-Adrenoceptor reduction in CHD is correlated with elevated noradrenaline plasma levels, thus proving a sympathetic dysregulation. (4) In CHD with moderate hemodynamic load, beta 2-AR coupling to AC was markedly more efficient than beta 1-AR coupling. The small number of myocardial beta 2-AR produced most of the cyclic adenosine monophosphate. (5) In severe acyanotic and cyanotic CHD, a partial decoupling of the beta 2-AR to the AC occurred.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional activity and expression of the myocardial postreceptor adenylyl cyclase system in pressure overload hypertrophy in rat

OBJECTIVE: The aim of the present study was to investigate the functional regulation of the myocardial postreceptor adenylyl cyclase (AC) system in compensated left ventricular hypertrophy (LVH) and the effect of long-term angiotensin converting enzyme (ACE) inhibition. METHODS: Pressure overload LVH was induced in rats by supravalvular aortic banding for 12 weeks. At 12 weeks left ventricular function and inner diameters were analyzed by echocardiography of anesthetized animals, and responsiveness to forskolin (systolic developed pressure) was determined in isolated perfused hearts. Functional activities of AC and the stimulatory G protein Gs were measured as well as mRNA expression (quantitative slot blot analyses) of AC type V, isoforms of Gs alpha and Gi alpha 2. G protein alpha-subunits were also quantified by immunoblotting. Rats were treated with ramipril (Ram, 10 mg/kg per day p.o.) during weeks 7 to 12 to induce regression of LVH or with vehicle (Veh, tap water). RESULTS: Pressure overload induced severe LVH (3.2 +/- 0.09 g/kg in Veh vs. 1.8 +/- 0.03 in sham; P < 0.05) which was significantly reduced by ramipril (2.7 +/- 0.09; P < 0.05 vs. Veh). In-vivo left ventricular function and diameters were unchanged in LVH. In contrast, in hearts with LVH, responsiveness of left ventricles to forskolin was attenuated and basal, GTP gamma S and forskolin as well as manganese chloride-stimulated adenylyl cyclase activity was significantly downregulated by approximately 40% (basal 20.8 +/- 1.9 pmol cAMP/mg per min vs. 34.0 +/- 2.2 in sham; P < 0.01). However, no significant changes of AC type V mRNA were found in hypertrophied left ventricles. Functional activity of the stimulatory G protein Gs was reduced in LVH (48 +/- 7 pmol cAMP/mg per min in Veh vs. 68 +/- 3 in sham), whereas mRNA expression of long and short Gs alpha-isoforms was not altered and that of Gi alpha 2 was only slightly increased in ramipril-treated animals. Western analysis showed no significant differences of Gs alpha or Gi alpha 2 subunits. Long-term blockade of the renin-angiotensin system had no effect on the activity of the adenylyl cyclase system. CONCLUSIONS: Functional desensitization of adenylyl cyclase and stimulatory G protein occurred in rat adaptive LVH prior to the onset of severe left ventricular dysfunction which was not restored by ACE-inhibitor treatment. The desensitization seems not to be mediated by significant changes of mRNA expression of AC type V or abundance of regulatory G proteins.     

Chronic L-arginine treatment increases cardiac cyclic guanosine 5'-monophosphate in rats with aortic stenosis: effects on left ventricular mass and beta-adrenergic contractile reserve

OBJECTIVES: We tested the hypothesis that nitric oxide (NO) cyclic guanosine 5'-monophosphate (GMP) signaling is deficient in pressure overload hypertrophy due to ascending aortic stenosis, and that long-term L-arginine treatment will increase cardiac cyclic GMP production and modify left ventricular (LV) pressure overload hypertrophy and beta-adrenergic contractile response. BACKGROUND: Nitric oxide cyclic GMP signaling is postulated to depress vascular growth, but its effects on cardiac hypertrophic growth are controversial. METHODS: Forty control rats and 40 rats with aortic stenosis left ventricular hypertrophy ([LVH] group) were randomized to receive either L-arginine (0.40 g/kg/day) or no drug for 6 weeks. RESULTS: The dose of L-arginine did not alter systemic blood pressure. Animals with LVH had similar LV constitutive nitric oxide synthase (cNOS) mRNA and protein levels, and LV cyclic GMP levels as compared with age-matched controls. In rats with LVH L-arginine treatment led to a 35% increase in cNOS protein levels (p = 0.09 vs untreated animals with LVH) and a 1.7-fold increase in LV cyclic GMP levels (p < 0.05 vs untreated animals with LVH). However, L-arginine treatment did not suppress LVH in the animals with aortic stenosis. In contrast, in vivo LV systolic pressure was depressed in L-arginine treated versus untreated rats with LVH (163 +/- 16 vs 198 +/- 10 mm Hg, p < 0.05). In addition, the contractile response to isoproterenol was blunted in both isolated intact hearts and isolated myocytes from L-arginine treated rats with LVH compared with untreated rats with LVH. This effect was mediated by a blunted increase in peak systolic intracellular calcium in response to beta-adrenergic stimulation. CONCLUSIONS: Left ventricular hypertrophy due to chronic mechanical systolic pressure overload is not characterized by a deficiency of LV cNOS and cyclic GMP levels. In rats with aortic stenosis, L-arginine treatment increased cardiac levels of cyclic GMP, but it did not modify cardiac mass in rats with aortic stenosis. However, long-term stimulation of NO-cyclic GMP signaling depressed in vivo LV systolic function in LVH rats and markedly blunted the contractile response to beta-adrenergic stimulation.     

Inter and intra-species-related differences in the regulation of the cardiac autonomic system

The heart rate response to isoproterenol (HR-Iso), density and affinity (kd) of beta-adrenergic (beta-AR) and muscarinic (M2) receptors were compared among three rodents with different generation-life histories of confinement and of high altitude exposure. The European guinea pig (Cavia porcellus) (EGp), a laboratory animal that arrived in Europe after the Spanish Conquest of South America and the Peruvian guinea pig (C. porcellus) (PGp), a semi-wild animal that came from the altiplano to sea level at least 25 generations ago, were used for intra-species comparison. Wistar rats (WR) were used for inter-species comparison as representative of a typical sea level laboratory animal. The HR-Iso was lower in EGp than in the PGp. The PGp showed the highest beta-AR density (P < 0.0005) and the highest beta-AR kd values (P < 0.0005) when compared to both EGp and WR groups (beta-AR Bmax (fmol mg-1 prot), WR, 19 +/- 4; Egp, 34 +/- 10; PGp, 74 +/- 15. beta-AR kd (pM), WR, 24 +/- 10; Egp, 17 +/- 7; PGp, 39 +/- 14). In contrast, PGp showed lower M2 receptor density values than the EGp (P < 0.0005). The WR had the highest M2 receptor densities (M2 Bmax (fmol mg-1 prot), WR, 188 +/- 15; Egp, 147 +/- 9; PGp, 118 +/- 6 and M2 kd (pM), WR, 65 +/- 12; Egp, 67 +/- 6; PGp, 92 +/- 2). The inter and intra-species differences found may be related to their respective history of confinement rather than to their history of exposure to high altitude.     

Targeted disruption of the mouse beta1-adrenergic receptor gene: developmental and cardiovascular effects

At least three distinct beta-adrenergic receptor (beta-AR) subtypes exist in mammals. These receptors modulate a wide variety of processes, from development and behavior, to cardiac function, metabolism, and smooth muscle tone. To understand the roles that individual beta-AR subtypes play in these processes, we have used the technique of gene targeting to create homozygous beta 1-AR null mutants (beta 1-AR -/-) in mice. The majority of beta 1-AR -/- mice die prenatally, and the penetrance of lethality shows strain dependence. Beta l-AR -/- mice that do survive to adulthood appear normal, but lack the chronotropic and inotropic responses seen in wild-type mice when beta-AR agonists such as isoproterenol are administered. Moreover, this lack of responsiveness is accompanied by markedly reduced stimulation of adenylate cyclase in cardiac membranes from beta 1-AR -/- mice. These findings occur despite persistent cardiac beta 2-AR expression, demonstrating the importance of beta 1-ARs for proper mouse development and cardiac function, while highlighting functional differences between beta-AR subtypes.     

Characterization of beta-adrenergic receptors of freshly isolated astrocytes and neurons from rat brain

The binding and characteristics of rat brain beta-adrenergic receptors (beta-AR) isolated from astrocytes and neurons were investigated. Equilibrium binding experiments demonstrated that beta-AR were more concentrated on astrocytes than on neurons isolated from forebrain, cerebral cortex and cerebellum. Inhibition experiments revealed that beta 1-AR and beta 2-AR were present in the two cell types. Isoproterenol revealed two interchangeable states of high and low affinity binding to both beta 1- and beta 2-AR in neurons. The high affinity binding sites were sensitive to guanylylimidodiphosphate (GppNHp). Similar results were found with other beta-AR agonists but not with salbutamol and salmeterol which recognized both affinity states of the neuronal beta 2-AR but only the low affinity state of beta 1-AR. In astrocytes only the low affinity state of beta-AR was observed.     

Agonist interactions with chimeric and mutant beta1- and beta3-adrenergic receptors: involvement of the seventh transmembrane region in conferring subtype specificity

beta1- and beta3-adrenergic receptors (AR) are the predominant beta-AR subtypes in adipocytes, and analysis of native and recombinant beta-AR has revealed several pharmacological and biochemical differences between these subtypes. This study used chimeric and mutated rat beta-AR expressed in Chinese hamster ovary cells to examine the basis of certain characteristic differences in the agonist properties of catecholamines and prototypic beta3-AR agonists. The exchange of sequence beyond transmembrane (TM) region 6 between the beta-AR subtypes had dramatic and reciprocal effects on the affinity and efficacy of the prototypic beta3-AR agonists BRL 37,344 and CL 316,243, without affecting the interactions with catecholamines. Mutation of Phe350 and Phe351 in TM7 of the beta1-AR to Ala and Leu found in the beta3-AR was sufficient to allow activation by prototypic beta3-AR agonists. Interestingly, this mutation did not affect catecholamine action and it did not impair the ability of propranolol to block the actions of isoproterenol or the selective beta3-AR agonists. beta1-AR containing beta3-AR sequence from predicted TM5 through TM6 exhibited reduced affinity for catecholamines without altering agonist potency, suggesting enhanced coupling efficiency. Inclusion of the homologous beta1-AR sequence in the beta3-AR, however, did not produce reciprocal effects. These results are the first to define a major determinant of beta3-AR subtype-selective agonism in TM7 and demonstrate that the determinants of selective phenethanolamines, catecholamines, and propranolol action are distinct.     

Regulation of the mRNA-binding protein AUF1 by activation of the beta-adrenergic receptor signal transduction pathway

In both cell culture based model systems and in the failing human heart, beta-adrenergic receptors ( beta-AR) undergo agonist-mediated down-regulation. This decrease correlates closely with down-regulation of its mRNA, an effect regulated in part by changes in mRNA stability. Regulation of mRNA stability has been associated with mRNA-binding proteins that recognize A + U-rich elements within the 3'-untranslated regions of many mRNAs encoding proto-oncogene and cytokine mRNAs. We demonstrate here that the mRNA-binding protein, AUF1, is present in both human heart and in hamster DDT1-MF2 smooth muscle cells and that its abundance is regulated by beta-AR agonist stimulation. In human heart, AUF1 mRNA and protein was significantly increased in individuals with myocardial failure, a condition associated with increases in the beta-adrenergic receptor agonist norepinephrine. In the same hearts, there was a significant decrease (approximately 50%) in the abundance of beta1-AR mRNA and protein. In DDT1-MF2 cells, where agonist-mediated destabilization of beta2-AR mRNA was first described, exposure to beta-AR agonist resulted in a significant increase in AUF1 mRNA and protein (approximately 100%). Conversely, agonist exposure significantly decreased (approximately 40%) beta2-adrenergic receptor mRNA abundance. Last, we demonstrate that AUF1 can be immunoprecipitated from polysome-derived proteins following UV cross-linking to the 3'-untranslated region of the human beta1-AR mRNA and that purified, recombinant p37AUF1 protein also binds to beta1-AR 3'-untranslated region mRNA.     

Angiotensin-converting enzyme inhibition prolongs survival and modifies the transition to heart failure in rats with pressure overload hypertrophy due to ascending aortic stenosis

BACKGROUND: We tested the hypotheses that long-term administration of the angiotensin-converting enzyme (ACE) inhibitor fosinopril will regress hypertrophy, modify the transition to heart failure, and prolong survival in rats with chronic left ventricular (LV) pressure overload due to ascending aortic stenosis. METHODS AND RESULTS: Aortic stenosis was created in weanling male Wistar rats by a stainless steel clip placed on the ascending aorta. Age-matched control animals underwent a sham operation (Sham group, n = 57). Six weeks after surgery, rats with aortic stenosis were randomized to receive either oral fosinopril 50 mg.kg-1.d-1 (Fos/LVH group, n = 38) or no drug (LVH group, n = 36) for 15 weeks. Pilot studies confirmed that this dosage produced significant inhibition of LV tissue ACE in vivo. Animals were monitored daily, and survival during the 15-week treatment period was assessed by actuarial analysis. At 15 weeks, in vivo LV systolic and diastolic pressures and heart rate were measured. To assess contractile function, the force-calcium relation was evaluated by use of the isovolumic buffer-perfused, balloon-in-LV heart preparation at comparable coronary flow rates per gram LV weight. Quantitative morphometry was performed. Mortality during the 15-week trial was significantly less in the Fos/LVH group than in the LVH group (3% versus 31%, P < .005). No deaths occurred in the Sham group. In vivo LV systolic pressure was similar between Fos/LVH and LVH hearts (223 +/- 10 versus 232 +/- 9 mm Hg) and significantly higher than the Sham group (99 +/- 3 mm Hg, P < .05). In vivo LV diastolic pressure was significantly lower in Fos/LVH hearts than in LVH hearts (10 +/- 2 versus 15 +/- 2 mm Hg), and both were significantly higher than in the Sham group (5 +/- 1 mm Hg, P < .05). Heart rate was similar among all groups. Despite equivalent elevation of LV systolic pressure, fosinopril resulted in regression of myocyte hypertrophy in Fos/LVH versus LVH (myocyte cell width, 14.8 +/- 0.5 versus 20.8 +/- 2.2 microns, P < .05) to normal levels (Sham, 16.3 +/- 0.9 microns). Quantitative morphometry demonstrated that the regression of LV myocyte hypertrophy in the Fos/LVH group was associated with a relative increase in the fractional volume of fibrillar collagen and noncollagen interstitium. In the isolated heart experiments, LV systolic developed pressure relative to perfusate [Ca2+] was significantly higher in Fos/LVH hearts than in LVH hearts. The improvement in systolic function was not related to any difference in myocardial high-energy phosphate levels, since LV ATP and creatine phosphate levels were similar in Fos/LVH and LVH hearts. CONCLUSIONS: In rats with ascending aortic stenosis, chronic ACE inhibition with fosinopril improved survival, decreased the extent of LV hypertrophy, and improved cardiac function despite persistent elevation of LV systolic pressure. The favorable effects of fosinopril may be related in part to inhibition of the effects of cardiac ACE on myocyte hypertrophy rather than to systemic hemodynamic mechanisms.     

Physiological consequences of beta-adrenergic receptor disruption

Activation of beta-adrenergic receptors (beta-ARs) in vivo is an important means by which animals regulate cardiac performance, vascular tone, lipid and carbohydrate metabolism, and behavior. The advent of targeted gene disruption in mice has led to significant advances in our understanding of the role that beta-AR subtypes play in these processes, and this technique has become an important tool for the study of G protein coupled receptors in general. To date, targeted disruption of both beta1- and beta3-ARs in mice has been reported. Mice lacking beta1-ARs are unresponsive to cardiac beta-AR stimulation, suggesting that neither beta2- nor beta3-ARs couple to inotropic or chronotropic responses in the mouse. Conversely, mice lacking beta3-ARs retain at least some adipose beta-AR responsiveness through remaining beta1- and beta2-ARs, suggesting that all three beta-AR subtypes mediate similar functions in this tissue. While these knockout models have been extremely valuable tools for revealing the roles that individual beta-ARs play in whole animal physiology, it is also useful to integrate the results of experiments derived from either transgenic overexpression of beta-ARs or purely pharmacological approaches to the study of beta-AR function in order to create a comprehensive model of beta-AR function in vivo.     

Clinical spectrum of nonmenstrual toxic shock syndrome (TSS): comparison with menstrual TSS by multivariate discriminant analyses

1. The present experiments were undertaken in order to characterize further the apparently irreversible inhibition of the contraction of depolarized rat aorta caused by lacidipine, a 1,4-dihydropyridine calcium antagonist. 2. We studied the effect of lacidipine on contraction evoked by 100 mM KCl solution in rat aorta, treated by N omega-nitro-L-arginine (0.1 mM), an inhibitor of nitric oxide (NO) synthesis. We compared the effect of prolonged depolarization on lacidipine and (+)-isradipine inhibition and the reversal of this inhibition after washout in the absence of dihydropyridines. Assuming that the onset of lacidipine-evoked inhibition was a pseudo-first order association kinetics, we estimated the dissociation rate constant (k-1 = 0.031 min-1), the association rate constant (k1 = 2.70 x 10(8) M-1 min-1) and the dissociation constant (KD = k-1/k1 = 115 pM) which was close to the IC50 value in steady-state conditions (160 pM). 3. The inhibitory effects of lacidipine and (+)-isradipine on rat aorta contraction were reversibly enhanced after preincubation with the drug in a 40 mM KCl-solution. Washout with drug-free 40 mM K(+)-depolarizing solution reversed inhibition in the (+)-isradipine-treated preparations, but not in the lacidipine-treated ones. 4. Radioligand binding studies were performed with [3H]-lacidipine and [3H]-isradipine in microsomes from rat aorta and rat ileum. Both ligands bound to a homogeneous population of binding sites (for[3H]-lacidipine: KD = 23 +/- 2.6 pM, Bmax = 380 +/- 21 fmol mg-1 protein in membranes from aorta; KD =23 +/- 3.1 pM, Bmax = 790 +/- 60 fmol mg-1 protein in membranes from ileum; for [3H]-isradipine:KD = 140 +/- 46 pM, Bmax = 350 +/- 64 fmol mg-1 protein in membrane from aorta; KD = 68 +/- 14 pM,Bmax = 760 +/- 75 fmol mg-1 protein in membranes from ileum). After isotopic dilution, [3H]-lacidipine and [3H]-isradipine dissociated according to a monoexponential kinetics. In membranes from ileum, the calculated dissociation rate constants (kappa_ 1) were 0.0257 min-1 and 0.0595 min-1, for [3H]-lacidipine and[3H]-isradipine, respectively.5. The non specific binding of [3H]-lacidipine and [3H]-isradipine, was measured in intact rat aorta preparations incubated under the conditions of the functional experiments, in the presence of nifedipine(1 microM). After incubation with [3H]-lacidipine 77.6 +/- 1.9 pM for 2 h the concentration of drug in the tissue was 15.15 +/- 1.18 fmol mg-1 w.wt. and still amounted to 7.24 +/- 0.61 fmol mg-1 w.wt. after 3.5 h washout in drug-free solution. After incubation with [3H]-isradipine 47.2 +/- 0.4 pM for 2 h it was 2.26 +/-0.07 fmol mg-1 w.wt. and was undetectable after 3.5 h washout in a drug-free solution.6. It is concluded that lacidipine interacts reversibly with dihydropyridine binding sites and that the apparent irreversible inhibition of contraction in depolarized preparations could be related to a nonspecific binding in a tissue compartment different from the plasma membrane.     

Increased density of I2-imidazoline receptors in human glioblastomas

A glial location has been proposed for the non-adrenoceptor [3H]idazoxan binding site termed the I2-imidazoline receptor. The specific binding of [3H]idazoxan in the presence of (-)adrenaline was measured in membranes from excised human glioblastomas (n = 6), meningiomas (n = 6) and normal brains (n = 6). The pharmacological profile of the [3H]idazoxan binding in astrocytic tumours was similar to that in normal brain, compatible with the presence of I2-imidazoline receptors. There was a higher density of I2-imidazoline receptors in astrocytic tumours (Bmax = 266 +/- 18 fmol mg-1 protein; p < 0.001) than in normal brain (Bmax = 54 +/- 4 fmol mg-1 protein), with no differences in affinity values. Almost no [3H]idazoxan-specific binding was shown in meningiomas. The results suggest that I2-imidazoline receptors may be a selective marker for glial tumours in the evaluation of intracranial neoplasms.     

Differential regulation of cardiac angiotensin converting enzyme binding sites and AT1 receptor density in the failing human heart

BACKGROUND: The regulation and interaction of ACE and the angiotensin II (Ang II) type I (AT1) receptor in the failing human heart are not understood. METHODS AND RESULTS: Radioligand binding with 3H-ramiprilat was used to measure ACE protein in membrane preparations of hearts obtained from 36 subjects with idiopathic dilated cardiomyopathy (IDC), 8 subjects with primary pulmonary hypertension (PPH), and 32 organ donors with normal cardiac function (NF hearts). 125I-Ang II formation was measured in a subset of hearts. Saralasin (125I-(Sar1,Ile8)-Ang II) was used to measure total Ang II receptor density. AT1 and AT2 receptor binding were determined with the AT1 receptor antagonist losartan. Maximal ACE binding (Bmax) was 578+/-47 fmol/mg in IDC left ventricle (LV), 713+/-97 fmol/mg in PPH LV, and 325+/-27 fmol/mg in NF LV (P<0.001, IDC or PPH versus NF). In IDC, PPH, and NF right ventricles (RV), ACE Bmax was 737+/-78, 638+/-137, and 422+/-49 fmol/mg, respectively (P=0.02, IDC versus NF; P=0.08, PPH versus NF). 125I-Ang II formation correlated with ACE binding sites (r=0.60, P=0.00005). There was selective downregulation of the AT1 receptor subtype in failing PPH ventricles: 6.41+/-1.23 fmol/mg in PPH LV, 2.37+/-0.50 fmol/mg in PPH RV, 5.38+/-0.53 fmol/mg in NF LV, and 7.30+/-1.10 fmol/mg in NF RV (P=0.01, PPH RV versus PPH LV; P=0.0006, PPH RV versus NF RV). CONCLUSIONS: ACE binding sites are increased in both failing IDC and nonfailing PPH ventricles. In PPH hearts, the AT1 receptor is downregulated only in the failing RV.     

Profile of the DNA recognition site of the archaeal homing endonuclease I-DmoI

1. Long-term treatment with beta 2-adrenoceptor agonists can lead to a decreased therapeutic efficacy of bronchodilatation in patients with obstructive pulmonary disease. In order to examine whether or not this is due to beta-adrenoceptor desensitization, human bronchial muscle relaxation was studied in isolated bronchial rings after pretreatment with beta 2-adrenoceptor agonists. Additionally, the influence of pretreatment with dexamethasone on desensitization was studied. 2. The effect of beta 2-agonist incubation alone and after coincubation with dexamethasone on density and affinity of beta-adrenoceptors was investigated by radioligand binding experiments. 3. In human isolated bronchi, isoprenaline induces a time- and concentration-dependent beta-adrenoceptor desensitization as judged from maximal reduction in potency by a factor of 7 and reduction of 73 +/- 4% in efficacy of isoprenaline to relax human bronchial smooth muscle. 4. After an incubation period of 60 min with 100 mumol l-1 terbutaline, a significant decline in its relaxing efficacy (81 +/- 8%) and potency (by a factor 5.5) occurred. 5. Incubation with 30 mumol l-1 isoprenaline for 60 min did not impair the maximal effect of a subsequent aminophylline response but led to an increase in potency (factor 4.4). 6. Coincubation of dexamethasone with isoprenaline (120 min; 30 mumol l-1) preserved the effect of isoprenaline on relaxation (129 +/- 15%). 7. In radioligand binding experiments, pretreatment of lung tissue for 60 min with isoprenaline (30 mumol l-1) resulted in a decrease in beta-adrenoceptor binding sites (Bmax) to 64 +/- 1.6% (P < 0.05), while the antagonist affinity (KD) for [3H]-CGP-12177 remained unchanged. 8. In contrast, radioligand binding studies on lung tissue pretreated with either dexamethasone (30 mumol l-1) or isoprenaline (30 mumol l-1) plus dexamethasone (30 mumol l-1) for 120 min did not lead to a significant change of Bmax (160 +/- 22.1% vs 142.3 +/- 28.7%) or KD (5.0 nmol l-1 vs 3.5 nmol l-1) compared to the controls. 9. In conclusion, pretreatment of human bronchi with beta-adrenoceptor agonists leads to functional desensitization and, in lung tissue, to down-regulation of beta-adrenoceptors. This effect can be counteracted by additional administration of dexamethasone. Our model of desensitization has proved useful for the identification of mechanisms of beta-adrenoceptor desensitization and could be relevant for the evaluation of therapeutic strategies to counteract undesirable effects of long-term beta-adrenoceptor stimulation.     

Na/H exchange and H-K ATPase increase distal tubule acidification in chronic alkalosis

We examined whether H(+)-ATPase, H(+)-K(+)-ATPase, and or Na+/H+ exchange mediates increased distal tubule acidification in animals with chronic metabolic alkalosis using pharmacological inhibitors of these H+ transporters in in vivo-perfused tubules of anesthetized rats. Chronic metabolic alkalosis was induced with furosemide followed by minimum electrolyte diet and HCO3 drinking water. The reduction in net HCO3 reabsorption was greater in distal tubules of alkalotic compared to control animals perfused with Schering 28080 to inhibit H(+)-K(+)-ATPase (-6.4 +/- 0.9 vs. -1.4 +/- 0.5 pmol/mm.min-1, P < 0.02) and with EIPA to inhibit Na+/H+ exchange (-11.1 +/- 1.7 vs. -6.6 +/- 0.9 pmol/mm.min-1, P < 0.01) but was similar in distal tubules of alkalotic and control animals perfused with bafilomycin to inhibit H(+)-ATPase. The greater reduction of distal tubule net HCO3 reabsorption in alkalotic compared to control animals induced by EIPA was eliminated by systemic infusion of the endothelin receptor antagonist bosentan (-4.6 +/- 0.7 vs. -4.4 +/- 0.7 pmol/mm.min-1, P = NS) but the greater reduction induced by Schering 28080 persisted. Urine endothelin-1 (ET-1) excretion was higher in animals with maintained alkalosis (164.5 +/- 23.7 vs. 76.6 +/- 10.8 fmol/day, P < 0.03), but decreased following KCl repletion to a value (86.7 +/- 10.0 fmol/day, P < 0.02 vs. respective before-KCl value) that was not different from that for KCl-repleted control animals (79.9 +/- 8.7 fmol/day, P = NS vs. KCl-repleted alkalotic animals). The data support that augmented distal tubule acidification in alkalotic animals is due to increased H(+)-K(+)-ATPase and Na+/H+ exchange activity, the latter stimulated by endogenous endothelins.     

On the high affinity binding site for [3H]-1,3-dipropyl-8-cyclopentylxanthine in frog brain membranes

1. Radioligand binding properties of the adenosine receptor ligands, [3H]-1,3-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), and [3H]-R-phenylisopropyladenosine ([3H]-R-PIA) were investigated in frog brain membranes. 2. The specific binding of the adenosine antagonist, [3H]-DPCPX to frog brain membranes showed one binding site with Kd and Bmax values of 43.8 nM and 0.238 +/- 0.016 pmol mg-1 protein, respectively. Guanosine 5'-triphosphate (GTP, 100 microM) decreased to 72 +/- 7% and Mg2+ (8 mM) increased to 121 +/- 3% [3H]-DPCPX (40 nM) binding to frog brain membranes. 3. [3H]-DPCPX saturation binding experiments performed in the presence of Mg2+ (8 mM), or in the presence of GTP showed that Mg2+ ions decreased the Kd value of [3H]-DPCPX to 14 nM, and GTP increased this value to 65.6 nM. Bmax values were not significantly (P > 0.05) modified (0.261 +/- 0.018 pmol mg-1 protein, with Mg2+, and 0.266 +/- 0.026 pmol mg-1 protein, in presence of GTP) by the presence of Mg2+ or GTP. 4. The specific binding of [3H]-R-PIA (15 nM) was decreased to 37 +/- 6% by GTP (100 microM) and increased to 123 +/- 4% by Mg2+ (8 mM). [3H]-R-PIA saturation binding experiments performed in the presence of Mg2+ (8 mM) showed one binding site with Kd and Bmax values of 0.9 nM and 0.229 +/- 0.008 pmol mg-1 of protein, respectively. 5. The concentration-inhibition curves of adenosine agonists and antagonists versus [3H]-DPCPX binding showed the following order of potencies: CPA> R-PIA~ NECA> S-PIA> > CGS 21680, for the agonists, and XAC ~-DPCPX> > XCC> PACPX, for the antagonists.6. The present results suggest that the adenosine binding site in the frog brain membranes is G-protein coupled, but that the antagonist affinities and the pharmacological profile is different from the Al or A2 adenosine receptors.     

Sudden unexpected deaths in epilepsy--where are we now?

AIM: To study the effects of menidipine (Men) on the affinity and density of dihydropyridines (DHP) binding sites in the cell membranes of left ventricle (LV) and brain in elderly renovascular hypertensive rats (RVHR) with LV hypertrophy. METHODS: Renovascular hypertension was produced by clipping the left renal artery in 20-month-old rats. The affinity and density of DHP binding sites in the cell membranes of LV and brain were assessed by radioligand assay. RESULTS: Men (20 mg.kg-1.d-1 ig for 9 wk) decreased markedly the systolic blood pressure and the LV weight (P < 0.01). Though not affecting the density of DHP receptor (Bmax), Men markedly decreased the total number of DHP binding sites in hypertrophied LV (from 5.95 +/- 0.62 to 4.0 +/- 1.1 pmol.LV). Men also reduced Bmax of DHP binding sites in the thalamus (from 522 +/- 27 to 371 +/- 24 pmol/g protein) and hippocampus (from 498 +/- 26 to 332 +/- 32 pmol/g protein). CONCLUSION: Men reversed the LV hypertrophy from renovascular hypertension accompanied with reduced total number of DHP binding sites in the cell membranes of LV and Bmax of the cell membranes of thalamus and hippocampus from elderly LV hypertrophied rats.     

The effect of oxygen free radicals on calcium current and dihydropyridine binding sites in guinea-pig ventricular myocytes

1. We used electrophysiological and binding techniques to determine the effects of oxygen free radicals (OFRs) generated by dihydroxyfumaric acid (DHF, 5 mM) on calcium current and dihydropyridine binding sites in guinea-pig isolated ventricular myocytes. 2. Binding of [3H]-PN200-110 to isolated ventricular myocytes revealed one population of binding sites with a KD of 0.11 +/- 0.01 nM and Bmax of 139.1 +/- 6.9 fmol mg-1 protein (n = 24). After 15 min of exposure to DHF, the density, but not the affinity of [3H]-PN200-110 binding sites was significantly (P < 0.01) reduced to 35% of the control value (Bmax = 49.4 +/- 3.7 fmol mg-1 protein, KD = 0.11 +/- 0.01 nM, n = 15). In the presence of superoxide dismutase (SOD) and catalase (CAT) the reduction in [3H]-PN200-110 binding sites was almost completely prevented (Bmax = 120.5 +/- 7.4 in control, n = 4 and 98.8 +/- 7.4 fmol mg-1 protein in DHF plus SOD and CAT, n = 4). KD values were not modified (0.08 +/- 0.01 in control and 0.09 +/- 0.01 nM in DHF plus SOD and CAT). 3. The time-course of the reduction of [3H]-PN200-110 binding sites by OFRs was paralleled by the decrease in L-type calcium current (Ica,L) measured in patch-clamped guinea-pig ventricular myocytes either in the absence or in the presence of EGTA in the patch pipette. In the former conditions OFRs induced the appearance of calcium-dependent alterations, i.e. the transient inward current, within 10 min. After 30 min of incubation with DHF, [3H]-PN200-110 binding sites were reduced to 25% of the control value. 4. In myocytes incubated with the antilipoperoxidant agent, butylated hydroxytoluene (BHT, 50 microM), the decrease in [3H]-PN200-110 binding sites caused by DHF was partially prevented (Bmax values after 30 min exposure to DHF were 55.5 +/- 1.9 and 23.7 +/- 5.9 fmol mg-1 protein in the presence and in the absence of BHT respectively, P < 0.05). BHT did not affect the decrease in [3H]-PN200-110 binding sites during the first 15 min of exposure to DHF, but was able to prevent completely the further decrease occurring during the following 15 min of incubation with OFRs. 5. Our results demonstrate that the OFR-induced decrease in calcium current is associated with a reduction in DHP binding sites. The decrease in calcium current and in calcium channels may be implicated in the mechanical dysfunction associated with oxidative stress.     

Effect of cyclic GMP reduction on regional myocardial mechanics and metabolism in experimental left ventricular hypertrophy

We tested the hypotheses that decreased myocardial cyclic GMP levels produced by intracoronary injection of methylene blue would increase local myocardial work and O2 consumption while decreasing intracellular cyclic GMP and that the relation between work, O2 consumption, and cyclic GMP may be altered in left ventricular hypertrophy (LVH) produced by aortic valve plication. In 8 control and 8 LVH open-chest anesthetized dogs, 1 mg/kg/min methylene blue was infused into the left anterior descending coronary artery (LAD); the circumflex region (CFX) served as control area. Regional work was calculated as the integrated product of force (miniature transducer) and segment shortening (sonomicrometry). Regional myocardial O2 consumption was calculated from flow measurements (radioactive microspheres), and regional O2 saturations (microspectrophotometry). A radioimmunoassay was used to determine intracellular level of cyclic GMP in the myocardium. Global hemodynamics and blood gases were unchanged by methylene blue in both control and LVH animals. Intracoronary methylene blue increased regional work from 762 +/- 129 to 1,451 +/- 307 g center dot mm/min in controls and from 912 +/- 173 to 1581 +/- 253 g center dot mm/min in the LVH groups. No significant changes in CFX regional work were observed. Regional blood flow, O2 extraction, and O2 consumption remained unchanged after injection of methylene blue in both control and LVH animals. The basal levels of cyclic GMP in the LVH group were fivefold higher than that in controls. In both groups, cyclic GMP levels were significantly decreased by methylene blue and to a greater extent in the LVH animals (from 6.16 +/- 1.2 to 3.34 +/- 0.44 pmol/g) than in the control animals (from 1.32 +/- 0.20 to 1.09 +/- 0.19 pmol/g). Therefore, intracoronary methylene blue increased regional myocardial work equally in control and LVH hearts without affecting regional metabolism (i.e., increased efficiency). For the same increased mechanical function, the hypertrophic myocardium exhibited a greater reduction in cyclic GMP pool size.