Karin Becker

Endothelin Receptors in the Failing and Nonfailing Human Heart

BACKGROUND In patients with chronic heart failure (CHF), plasma endothelin-1 (ET-1) levels are increased. We studied whether the cardiac ET-receptor system is altered in CHF patients. METHODS AND RESULTS We assessed ET-evoked inositol phosphate (IP) formation in slices from right atria and left ventricles from 6 potential heart transplant donors (NFH) and 15 patients with end-stage CHF; in membranes from the same tissues, we studied ET-induced inhibition of isoprenaline- and forskolin-stimulated adenylyl cyclase and ET-receptor density. ET (10[-9] to 10[-6] mol/L, ET-1 >>> ET-3) increased IP formation in right atria and left ventricles through ET(A)-receptor stimulation in a concentration-dependent manner; no difference in potency or efficacy between NFH and CHF hearts was observed. ET-1 (10[-10] to 10[-6] mol/L), via ET(A)-receptor stimulation, inhibited isoprenaline- and forskolin-stimulated adenylyl cyclase in right atria but not in left ventricles, whereas carbachol inhibited adenylyl cyclase in both tissues; again, the potency and efficacy of ET- or carbachol-induced adenylyl cyclase inhibition was not different between NFH and CHF hearts. [125I]ET-1 binding revealed the coexistence of ET(A) and ET(B) receptors in both tissues; however, the density of ET(A) receptors was not significantly different between NFH and CHF hearts. Finally, the immunodetectable amount of left ventricular Gq/11 protein did not differ between NFH and CHF hearts. CONCLUSIONS In the human heart, ET(A) and ET(B) receptors coexist; however, only ET(A) receptors are of functional importance. In right atria, ET(A) receptors couple to IP formation and inhibition of adenylyl cyclase; in left ventricles, they couple only to IP formation. In end-stage CHF, the functional responsiveness of the cardiac ET(A)-receptor system is not altered.

Cardiac muscarinic receptors decrease with age. In vitro and in vivo studies.

The M1 muscarinic receptor antagonist pirenzepine in low doses decreases resting heart rate; this effect declines with age (Poller, U., G. Nedelka, J. Radke, K. Pönicke, and O.-E. Brodde. 1997. J. Am. Coll. Cardiol. 29:187-193). To study possible mechanisms underlying this effect, we assessed (a) in six young (26 yr old) and six older volunteers (61 yr old), pirenzepine effects (0.32 and 0.64 mg intravenous [i.v.] bolus) on isoprenaline-induced heart rate increases; (b) in five heart transplant recipients, pirenzepine effects (0.05-10 mg i.v. bolus) on resting heart rate in the recipients native and transplanted sinus nodes; and (c) in right atria from 39 patients of different ages (5 d-76 yr) undergoing open heart surgery, M2 muscarinic receptor density (by [3H]N-methyl-scopolamine binding) and adenylyl cyclase activity. (a) Pirenzepine at both doses decreased heart rate in young volunteers significantly more than in older volunteers; (b) pirenzepine (< 1 mg) decreased resting heart rate in the recipients native but not transplanted sinus node; and (c) M2 receptor density and carbachol-induced inhibition of forskolin-stimulated adenylyl cyclase activity decreased significantly with the age of the patients. We conclude that pirenzepine decreases heart rate via inhibition of presynaptic M1 autoreceptors, thereby releasing endogenous acetylcholine, and that the heart rate-decreasing effect of acetylcholine declines with age because right atrial M2 receptor density and function decrease.

Trophic effect of angiotensin II in neonatal rat cardiomyocytes: role of endothelin-1 and non-myocyte cells

Angiotensin II (AII) and the endothelins (ET) are known to be potent trophic stimuli in various cells including cardiomyocytes. In order to characterize further these effects we studied, in neonatal rat ventricular cardiomyocytes, the effects of several endothelin‐receptor antagonists and the AT1‐receptor antagonist losartan on AII‐ and endothelin‐induced inositol phosphate (IP)‐formation (assessed as accumulation of total [3H]‐IPs in myo‐[3H]‐inositol prelabelled cells) and increase in rate of protein synthesis (assessed as [3H]‐phenylalanine incorporation). Endothelin (10 pM–1 μM) concentration‐dependently increased IP‐formation (max. increase at 100 nM ET‐1: 130±14% above basal, n=25) and [3H]‐phenylalanine incorporation (max. increase at 1 μM: 52±4% above basal, n=16) with an order of potency: ET‐1>>ET‐3. Both effects were antagonized by the ETA/ETB‐receptor antagonist bosentan and the ETA‐receptor antagonist BQ‐123, but not affected by the ETB‐receptor antagonist IRL 1038 and the AT1‐receptor antagonist losartan. Pretreatment of the cells with 500 ng ml−1 pertussis toxin (PTX) overnight that completely inactivated PTX‐sensitive G‐proteins did not attenuate but rather enhance ET‐1‐induced IP‐formation. On the other hand, in PTX‐pretreated cardiomyocytes ET‐1‐induced [3H]‐phenylalanine incorporation was decreased by 39±5% (n=5). AII (1 nM–1 μM) concentration‐dependently increased IP‐formation (max. increase at 1 μM: 42±7% above basal, n=16) and [3H]‐phenylalanine incorporation (max. increase at 1 μM: 29±2%, n=9). These effects were antagonized by losartan, but they were also antagonized by bosentan and BQ‐123. In well‐defined cultures of cardiomyocytes (not contaminated with non‐myocyte cells) AII failed to increase [3H]‐phenylalanine incorporation; addition of non‐myocyte cells to the cardiomyocytes restored AII‐induced increase in [3H]‐phenylalanine incorporation. We conclude that, in rat neonatal ventricular cardiomyocytes, (a) the ET‐1‐induced increase in rate of protein synthesis (through ETA‐receptor stimulation) involves at least two signalling pathways: one via a PTX‐insensitive G‐protein coupled to IP‐formation, and the other one via a PTX‐sensitive G‐protein, and (b) the trophic effects of AII are brought about via local ET‐1 secretion upon AT1‐receptor stimulation in neonatal rat ventricular non‐myocyte cells.

FP‐receptor mediated trophic effects of prostanoids in rat ventricular cardiomyocytes

The aim of this study was to characterize the receptor subtype involved in cardiac effects of prostanoids. For this purpose we determined in neonatal and adult rat cardiomyocytes effects of prostanoids on inositol phosphate (InsP)‐formation (assessed as accumulation of total [3H]‐InsPs in myo‐[3H]‐inositol pre‐labelled cells) and on rate of protein synthesis (assessed as [3H]‐phenylalanine incorporation), and on contractile force in left ventricular strips of the rat heart. For comparison, effects of prostanoids on InsP‐formation and contractile force were determined in rat thoracic aorta, a classical TP‐receptor containing tissue. Prostanoid increased InsP‐formation and rate of protein synthesis in neonatal as well as adult rat cardiomyocytes; the order of potency was in neonatal (PGF2α>PGD2PGE2U 46619>PGE1) and adult (PGF2α>PGD2PGE2>U 46619) rat cardiomyocytes well comparable. Moreover, in electrically driven left ventricular strips PGF2α caused positive inotropic effects (pD2 7.5) whereas U 46619 (up to 1 μM) was uneffective. In contrast, in rat thoracic aorta U 46619 was about 100 times more potent than PGF2α in increasing InsP‐formation and contractile force. The TP‐receptor antagonist SQ 29548 only weakly antagonized prostanoid‐induced increases in rate of protein synthesis (pKB about 6) in rat cardiomyocytes but was very potent (pKB about 8–9) in antagonizing prostanoid‐induced increases in InsP‐formation and contractile force in rat aorta. We conclude that, in cardiomyocytes of neonatal and adult rats, the prostanoid‐receptor mediating increases in InsP‐formation and rate of protein synthesis is a FP‐receptor. Moreover, stimulation of these cardiac FP‐receptors can mediate increases in contractile force.

Free and Total Carnitine Concentrations in Pig Plasma after Oral Ingestion of Various L-Carnitine Compounds

This study was undertaken to investigate the bioavailability of various L-carnitine esters (acetyl-L-carnitine and lauroyl-L-carnitine) and salts (L-carnitine L-tartrate, L-carnitine fumarate, L-carnitine magnesium citrate) relative to base of free L-carnitine. Six groups of five or six piglets each were administered orally a single dose of 40 mg L-carnitine equivalents/kg body weight of each of those L-carnitine compounds. A seventh group served as a control. Free and total plasma carnitine concentrations were determined 1, 2, 3.5, 7, 24, and 32 hours after administration of the single dose. Area-under-the-curve (AUC) values were calculated to assess the bioavailability of the L-carnitine compounds. AUC values, calculated for the time interval between 0 and 32 hours, for both free and total carnitine were similar for base of free L-carnitine and the three L-carnitine salts (L-carnitine L-tartrate, L-carnitine fumarate, L-carnitine magnesium citrate) while those of the two esters (acetyl-L-carnitine, lauroyl-L-carnitine) were lower. Administration of L-carnitine L-tartrate yielded a higher plasma free carnitine AUC value for the time interval between 0 and 3.5 hours than administration of the other compounds. The data of this study suggest that L-carnitine salts have a similar bioavailability to that of free L-carnitine while L-carnitine esters have a lower one. The study also suggests that L-carnitine L-tartrate is absorbed faster than the other L-carnitine compounds.

Endothelin-induced inositol phosphate formation in rat kidney. Studies on receptor subtypes, G-proteins and regulation during ontogenesis

The aim of this study was to characterize the properties of endothelin (ET)-receptor subtypes mediating inositol phosphate (IP)-formation in rat kidney and their regulation during ontogenesis. In renal cortical slices of adult rats (12–16 weeks old) ETs concentration-dependently increased IP-formation with an order of potency ET -1 ≫ ET 3. While the non-selective ET receptor antagonist bosentan (10 μM) completely suppressed ET-induced IP-formation, the ETA-receptor antagonist BQ-123 (10 μM) inhibited it only by 70%, the ETB-receptor antagonist IRL 1038 (1 μM) by 25%; combined application of BQ-123 + IRL 1038 caused complete inhibition of ET-1-induced IP-formation. Pretreatment of isolated renal cells with pertussis toxin (PTX, 500 ng/ml) overnight did not attenuate but significantly increased ET-1-induced IP-formation. Ontogenetic studies in renal slices from neonatal, 1, 2, 3, 6, 12 and 24 weeks old rats revealed that ET-1-induced IP-formation maturation-dependently declined being highest in neonatal rats (increase: 169% over basal) and lowest in 24 weeks old rats (increase: 47% over basal). This decline in ET-induced IP-formation was accompanied by a decrease in renal ET receptor number and the amount of immunodetectable Gq/11 (assessed by Western-blotting using the QL-antiserum). Moreover, ET receptor subtypes changed during the maturation process: from neonates to 12 weeks old rats number and functional responsiveness of ETA-receptors declined, while that of ETB-receptors increased. We conclude that in adult rat renal cortex ET-induced IP-formation is mediated by activation of both ETA- and ETB-receptors and does not involve a PTX-sensitive G-protein. ET-induced IP-formation declines during the maturation process; this is associated with a decrease in ET-receptor number and the immunodetectable amount of Gq/11.

The effects of cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acids on delta4-desaturation in HepG2 cells.

The objective of this study was to determine the effects of cis-9, trans-11 and trans-10, cis-12 CLA on the delta4-desaturation process in HepG2 cells. Experiments were conducted in which HepG2 cells were incubated with 25 micromol/L of those fatty acids and the concentrations of (n-6) and (n-3) polyunsaturated fatty acids in phosphatidyl choline and phosphatidyl ethanolamine were determined. In the presence of eicosapentaenoic acid, cells treated with trans-10, cis-12 CLA had a lower ratio of docosahexaenoic acid [22:6 (n-3)], the product of delta4-desaturation, to docosapentaenoic acid [22:5 (n-3)], the substrate of delta4-desaturation in both phospholipids, than control cells or cells treated with cis-9, trans-11 CLA (p < 0.05). This suggests that trans-10, cis-12 CLA suppresses the delta4-desaturation process in HepG2 cells.