Catecholamines are the key for explaining the biological relevance of insulin-melatonin antagonisms in type 1 and type 2 diabetes.

In this paper, we analyze the biological relevance of melatonin in diabetogenesis. As has recently been demonstrated, melatonin decreases insulin secretion via specific melatonin receptor isoforms (MT1 and MT2) in the pancreatic ß-cells. In addition, type 2 diabetic rats, as well as patients, exhibit decreased melatonin levels, whereas the levels in type 1 diabetic rats are increased. The latter effects were normalized by insulin substitution, which signifies that a specific receptor-mediated insulin-melatonin antagonism exists. These results are in agreement with several recent genome-wide association studies, which have identified a number of single nucleotide polymorphisms in the MTNR1B gene, encoding the MT2 receptor, that were closely associated with a higher prognostic risk of developing type 2 diabetes. We hypothesize that catecholamines, which decrease insulin levels and stimulate melatonin synthesis, control insulin-melatonin interactions. The present results support this assertion as we show that catecholamines are increased in type 1 but are diminished in type 2 diabetes. Another important line of inquiry involves the fact that melatonin protects the ß-cells against functional overcharge and, consequently, hinders the development of type 2 diabetes. In this context, it is striking that at advanced ages, melatonin levels are reduced and the incidence of type 2 diabetes is increased. Thus, melatonin appears to have a protective biological role. Here, we strongly repudiate misconceptions, resulting from observations that melatonin reduces the plasma insulin level, that the blockage of melatonin receptors would be of benefit in the treatment of type 2 diabetes.

Noradrenaline-induced increase in protein synthesis in adult rat cardiomyocytes: involvement of only alpha1A-adrenoceptors.

Adult rat ventricular cardiomyocytes contain alpha1A- and alpha1B-adrenoceptors (ARs, 20%:80%, assessed by [3H]prazosin binding). We studied which alpha1-AR subtype mediates noradrenaline (NA)-induced increase in rate of protein synthesis, and which signalling pathway is involved. NA (10-9-10-4 M) concentration-dependently increased inositol phosphate (IP) formation (pEC50-value=6.1+/-0.1, n=5) and protein synthesis (assessed as [3H]phenylalanine incorporation; pEC50-value=6.6+/-0.1, n=6). NA-induced IP-formation was partly inhibited by the alpha1B-AR antagonist chloroethylclonidine (CEC, 30 microM; 33+/-9% inhibition, n=5); following CEC-treatment the alpha1A-AR-selective 5-methyl-urapidil (5-MU) inhibited NA-induced IP-formation with a pKi-value of 9.2+/-0.2 (n=6); the alpha1D-AR-selective BMY 7378 was only a weak antagonist (pKi-value <7). NA-induced increase in protein synthesis was insensitive to CEC whereas 5-MU inhibited it with a pKi-value of 9.1+/-0.2 (n=6). NA (1 microM)-induced increase in protein synthesis was inhibited by the protein kinase C (PKC) inhibitor bisindolylmaleimide (IC50-value: 206 nM), the PI 3-kinase inhibitors wortmannin (IC50=3.4 nM) and LY 294002 (IC50=10 microM), and p70s6-kinase inhibitor rapamycin (IC50=123 pM) but not by the p38 MAP-kinase inhibitor SB 203580 (10 microM) or the MEK-inhibitor PD 98059 (25 microM). Moreover, 5-MU (30 nM) but not CEC inhibited NA-induced activation of p70s6-kinase. We conclude that, in adult rat cardiomyocytes, alpha1A- and alpha1B-AR mediate NA-induced IP-formation but only alpha1A-ARs mediate increase in protein synthesis. Alpha1A-AR-mediated increase in protein synthesis involves activation of a PKC, PI 3-kinase and p70s6-kinase but not of ERK- or p38 MAP-kinase.

Impaired beta-adrenergic control of immune function in patients with chronic heart failure: reversal by beta1-blocker treatment.

BACKGROUND: In patients with chronic heart failure (CHF) and heightened sympathetic activity alterations in immune function have been described. OBJECTIVES: To find out whether, in CHF patients, beta-blocker treatment might beneficially affect immune function. METHODS: We studied activation of circulating lymphocytes (assessed as concanavalin A (CON A)-induced inositol phosphate (IP) formation and proliferation ([3H]-thymidine incorporation) from 8 CHF patients on standard medication (Group A, mean age 54 +/- 6 yrs, NYHA class II - IV, mean 3.1 +/- 0.3) and in 9 CHF patients on standard medication and additional treatment with the beta1-blocker metoprolol (Group B, mean age: 56 +/- 3 yrs, NYHA class II - IV, mean 2.9 +/- 0.2); 8 age-matched healthy volunteers (mean age 49 +/- 3 yrs) served as controls. RESULTS: Compared to controls, in group A isoprenaline-induced lymphocyte cyclic AMP-increase was reduced, CON A-evoked IP formation significantly enhanced and isoprenaline-induced inhibition of CON A-evoked IP formation and proliferation almost abolished. In group B, however, all these parameters were not significantly different from controls. CONCLUSION: In CHF patients lymphocyte cyclic AMP response to beta-adrenoceptor stimulation is blunted and the inhibitory effect of cyclic AMP on lymphocyte activation is almost abolished; this could result in a non-regulated increased production and release of proinflammatory cytokines that might contribute to the progression of the disease. Chronic treatment of CHF patients with the beta1-blocker metoprolol (at least partly) restores lymphocyte cyclic AMP responses to beta-adrenoceptor stimulation and the inhibitory effects of cyclic AMP on lymphocyte activation; the resulting "normalization" of the immune function might contribute to the beneficial effects of beta1-blockers in treatment of CHF.

ET-receptor function in relation to blood pressure in spontaneously hypertensive and aortic-banded rats.

Endothelin-1 (ET-1), a potent endogenous vasoconstrictor, has been proposed to play a pathophysiologic role in hypertension. The aim of this study was to find out whether changes in ET-receptor function are cause or consequence of blood pressure elevation in hypertension. For this purpose, we assessed ET-receptor function [as ET-1-induced [3H]inositol phosphate (IP) accumulation] in slices of left ventricle and renal cortex and in rings of thoracic and abdominal aorta from spontaneously hypertensive rats (SHR) at the age of 8 weeks (i.e. developing hypertension), 12 and 24 weeks (established hypertension) vs. normotensive age-matched Wistar-Kyoto (WKY) rats, and from supra-renal aortic-banded (AOB) rats at the age of 8, 12 and 24 weeks (i.e. 4, 8 and 20 weeks after AOB) vs. sham-operated (SOP) age-matched WKY rats. In the SHR with established hypertension ET-1-induced IP formation was altered in all tissues investigated: it was significantly increased vs. WKY rats in left ventricle, and significantly decreased in renal and aortic tissues. Similarly, in AOB rats at all ages ET-1-induced IP formation was changed in those tissues that were under pressure load [heart (increase) and thoracic aorta (decrease)] vs. SOP rats, whereas in those tissues not under pressure load (kidney and abdominal aorta) ET-1-induced IP formation was not different between AOB and SOP rats. Moreover, in 8-week-old SHR (where hypertension is not yet established) ET-1-induced IP formation was not significantly different compared to WKY rats (with the exception of thoracic aorta). We conclude that, at least in SHR and AOB rats, changes in ET-1 signalling are secondary to the elevation in blood pressure.

Inositolphosphate formation in thoracic and abdominal rat aorta following Gq/11-coupled receptor stimulation.

Although thoracic and abdominal rat aorta are often used as a classical pharmacological preparation for the assessment of vascular drug effects, little is known on regional differences among these two parts of the aorta with regard to their reaction to Gq/11-coupled receptor activation. Thus, we determined, in rings from thoracic and abdominal aorta from 12-week-old male Wistar rats, the effects of noradrenaline (NA; 10(-8)-10(-4) M), endothelin-1 (ET-1; 10(-10)-10(-6) M) and the thromboxane A2 mimetic U 46619 (10(-8)-10(-5) M) on inositolphoshate (IP) formation (assessed as accumulation of total [3H]IPs in [3H]myoinositol prelabelled rings). NA, ET-1 and U 46619 concentration-dependently increased IP formation; maximum increases were, however, significantly more pronounced in thoracic than in abdominal aorta. Similarly, NA, ET-1 and U 46619 evoked significantly larger maximum contractions in thoracic than in abdominal aorta. NA-induced [3H]IP formation could be inhibited with BMY 7378 (10(-9)-10(-4) M) and with 5-methyl-urapidil (5-MU; 10(-9)-10(-5) M) both exhibiting biphasic concentration-inhibition curves. The pKi-values for BMY 7378 at the high affinity site were in thoracic aorta 8.93+/-0.28 (n=5), and in abdominal aorta 8.76+/-0.35 (n=4) and at the low affinity site 6.45+/-0.2 (thoracic aorta) and 6.55+/-0.27 (abdominal aorta). pKi-Values for 5-MU in thoracic aorta at the high affinity site were 8.25+/-0.34 (n=4), and at the low affinity site 6.61+/-0.39 . In abdominal aorta reliable pKi-values could not be calculated for 5-MU due to a low signal-to-noise ratio. On the other hand, in both preparations the ETA-receptor antagonist BQ-123 (10(-9)-10(-5) M) and the TP-receptor antagonist SQ 29548 (10(-9)-10(-5) M) inhibited ET-1- and U 46619-induced IP formation, respectively, with monophasic concentration-inhibition curves: pKi-values for BQ-123 were: 8.16+/-0.24 (thoracic aorta) and 8.10+/-0.35 (abdominal aorta) and for SQ 29548: 8.2+/-0.3 (thoracic aorta) and 8.5+/-0.3 (abdominal aorta). The amount of immunodetectable Gq/11-protein was similar in both tissues. We conclude that responses to NA, ET-1 and U 46619 (IP formation and contractile force) are larger in thoracic than in abdominal aorta. ET-1 effects on IP formation are mediated by ETA-receptors and U 46619 effects by TP-receptors. NA effects are mediated by alpha1D- and alpha1A-adrenoceptors; alpha1B-adrenoceptors seem to play a minor role.

Beta-adrenoceptor stimulation attenuates the hypertrophic effect of alpha-adrenoceptor stimulation in adult rat ventricular cardiomyocytes.

OBJECTIVES: The study investigated whether beta-adrenoceptor antagonists augment the hypertrophic response of cardiomyocytes evoked by norepinephrine. BACKGROUND: In adult ventricular cardiomyocytes, stimulation of alpha- but not beta-adrenoceptors induces myocardial hypertrophy. Natural catecholamines, like norepinephrine, stimulate simultaneously alpha- and beta-adrenoceptors. We investigated whether beta-adrenoceptor stimulation interferes with the hypertrophic response caused by alpha-adrenoceptor stimulation. METHODS: Adult ventricular cardiomyocytes isolated from rats were used as an experimental model. Hypertrophic parameters under investigation were stimulation of phenylalanine incorporation and protein mass, stimulation of 14C-uridine incorporation and RNA mass, and increases in cell shape. RESULTS: Norepinephrine (0.01 to 10 micromol/liter) increased concentration-dependent phenylalanine incorporation; pEC50 value was 5.9 +/- 0.1 (n = 8). The alpha1-adrenoceptor antagonist prazosin (0.1 micromol/liter) suppressed norepinephrine-induced increase in rate of protein synthesis. Conversely, propranolol (1 micromol/liter) and the beta1-adrenoceptor selective antagonists CPG 20712A (300 nmol/liter) or atenolol (1 micromol/liter) augmented increases in phenylalanine incorporation caused by norepinephrine. Addition of the beta2-adrenoceptor antagonist ICI 118,551 (55 nmol/liter) did not influence the hypertrophic effect of norepinephrine. Atenolol augmented the norepinephrine-induced increases of all hypertrophic parameters investigated (i.e., protein mass, uridine incorporation, RNA mass, cell volume, and cross-sectional area). In the presence of norepinephrine, inhibition of beta1-adrenoceptors increased the amount of protein kinase C-alpha and -delta isoforms translocated into the particulate fraction. The effect of pharmacological inhibition of beta1-adrenoceptors could be mimicked by Rp-cAMPS (adenosine-3', 5'-cyclic phosphorothiolate-Rp). The inhibitory effect of beta1-adrenoceptor stimulation on the alpha-adrenoceptor-mediated effect persisted in cardiomyocytes isolated from hypertrophic hearts of rats submitted to aortic banding. CONCLUSIONS: In isolated ventricular cardiomyocytes from rats, beta1-adrenoceptor stimulation attenuates the hypertrophic response evoked by alpha1-adrenoceptor stimulation.

The cardiac beta-adrenoceptor-G-protein(s)-adenylyl cyclase system in monocrotaline-treated rats.

In rats, injection of the alkaloid monocrotaline (MCT) causes right ventricular hypertrophy and cardiac failure. In order to study whether, in MCT-treated rats, changes in the cardiac beta -adrenoceptor-G-protein(s)-adenylyl cyclase system might be comparable to those found in human primary pulmonary hypertension, we assessed in right and left ventricles from MCT-treated rats the components of the beta -adrenoceptor system: the receptor number and subtype distribution (by (-)-[(125)I]iodocyanopindolol binding), the G-proteins (by quantitative Western blotting), and the activity of adenylyl cyclase. A single injection of 60 mg/kg i.p. MCT caused in rats right ventricular hypertrophy (RVH); part of the rats developed cardiac failure (RVF). In these rats the cardiac beta -adrenoceptor-G-protein(s)-adenylyl cyclase system was markedly changed beta -adrenoceptors were desensitized due to a decrease in receptor number, an uncoupling of the receptor from the G(s)-adenylyl cyclase system, a decrease in G(s)and a decrease in the activity of the catalytic unit of adenylyl cyclase. In general, these changes were more pronounced in right ventricles v left ventricles, and in rats with RVF v rats with RVH. On the other hand, cardiac muscarinic receptors and G(i)appeared not to be altered. We conclude that in MCT-treated rats changes in the cardiac beta -adrenoceptor-G-protein(s)-adenylyl cyclase system occur that resemble those observed in human primary pulmonary hypertension. Thus, MCT-treated rat appears to be a suitable animal model to study in more detail the pathophysiology of the development of right heart failure, and to identify new therapeutic possibilities.

Heterogeneity of cardiac rat and human elongation factor 2.

Elongation factor 2 (EF-2) catalyses the last step of the elongation cycle, translocation, in the course of protein biosynthesis. A system for analyzing post-translational modifications of EF-2, which is a single polypeptide of 857 amino acids, is reported and its application to cytosolic extracts of cultured neonatal rat heart myocytes, neonatal and adult rat cardiac tissue, and extracts of human left ventricular myocardium is described. Comparing different pH ranges in immobilized pH gradient-isoelectric focusing (IPG-IEF), a range of pH 3 - 10 and 4 - 9 resulted in a highly defined and reproducible resolution of six different EF-2 variants of all extracts in the first dimension. These six variants were detected by the "imaging plate" (phosphor radiation image sensor) after specific labeling with Pseudomonas exotoxin A catalyzed [32P]ADP-ribosylation. This finding could be confirmed in Western blot analysis with a specific polyclonal rabbit antibody. Using two-dimensional polyacrylamide gel electrophoresis (2-D-PAGE), five to six EF-2 variants could be demonstrated in all extracts. By application of a second IPG indicator strip to the 2-D gel, they could be aligned with corresponding spots in a silver-stained 2-D separation of human myocardial tissue, revealing that the EF-2 variants belong to the group of low-abundance proteins.

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 [(3)H]-InsP's in myo-[(3)H]-inositol pre-labelled cells) and on rate of protein synthesis (assessed as [(3)H]-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 (PGF(2alpha)>PGD(2)> or =PGE(2)> or =U 46619>PGE(1)) and adult (PGF(2alpha)>PGD(2)> or =PGE(2)>U 46619) rat cardiomyocytes well comparable. Moreover, in electrically driven left ventricular strips PGF(2alpha) caused positive inotropic effects (pD(2) 7.5) whereas U 46619 (up to 1 microM) was uneffective. In contrast, in rat thoracic aorta U 46619 was about 100 times more potent than PGF(2alpha) in increasing InsP-formation and contractile force. The TP-receptor antagonist SQ 29548 only weakly antagonized prostanoid-induced increases in rate of protein synthesis (pK(B) about 6) in rat cardiomyocytes but was very potent (pK(B) 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.

Gq/11-coupled receptors and protein synthesis in rat cardiomyocytes: role of Gi-proteins and protein kinase C-isozymes.

Our recent findings indicate that, in rat neonatal ventricular cardiomyocytes, endothelin-1 (ET-1) induces increases in the rate of protein synthesis in a partly pertussis toxin (PTX)-sensitive manner, and that angiotensin II-evoked increases in the rate of protein synthesis are brought about via local secretion of ET-1. The aim of this study was to find out: (1) whether noradrenaline (NA) and the thromboxane A2 (TXA2)-mimetic U 46619-induced increases in the rate of protein synthesis may be also partly PTX-sensitive and/or mediated by ET-1, and (2) whether the growth-promoting effects of NA and U 46619 as well as ET-1 might involve activation of the same set of protein kinase C (PKC) isozymes. For this purpose we first studied the effects of NA and U 46619 on inositol phosphate (IP)-formation (assessed as accumulation of total [3H]IPs in myo-[3H]inositol prelabelled cells) and on the rate of protein synthesis (assessed as [3H]phenylalanine incorporation) (1) in the presence and absence of the ET(A)-receptor antagonist BQ-123, and (2) in nontreated and PTX-pretreated cells. Second, we assessed the effects of the PKC-inhibitors bisindolylmaleimide I and Gö 6976 and of phorbol-12-myristate-13-acetate (PMA; 1 microM overnight)-pretreatment on U 46619-, NA- and ET-1-induced increases in the rate of protein synthesis. NA (0.01-10 microM) concentration-dependently increased IP-formation (maximum increase: 115-/+23% above basal, n=4) and [3H]phenylalanine incorporation (maximum increase: 40+/-3% above basal, n=20). Both responses were antagonized by the alpha1-adrenoceptor antagonist prazosin (1 microM), but were not significantly affected by BQ-123 (1 microM). U 46619 (0.01-100 microM) concentration-dependently increased IP-formation (maximum increase: 89+/-12% above basal, n=8) and [3H]phenylalanine incorporation (maximum increase: 33+/-4% above basal, n=16). Both responses were slightly but significantly antagonized by the TP-receptor antagonist SQ 29548 (1 microM), but were not affected by BQ-123 (1 microM). Pretreatment of the cardiomyocytes with 250 ng ml(-1) PTX overnight did not significantly affect NA- and U 46619-evoked increases in IP-formation and [3H]phenylalanine incorporation. The PKC-inhibitor bisindolylmaleimide I (5 microM) as well as pretreatment of the cells with PMA (1 microM) significantly reduced the effects of NA, U 46619 and ET- I on the rate of protein synthesis; in contrast, the PKC-inhibitor Gö 6976 (5 microM) was without any effects. We conclude that, in rat neonatal ventricular cardiomyocytes, stimulation of Gq/11-coupled receptors increases the rate of protein synthesis; this involves activation of the same PKC-isozymes (very likely PKC-delta and/or -epsilon). NA and U 46619 cause their growth-promoting effects in a PTX-insensitive manner; ET-1 is not involved in their effects.

Muscarinic receptors in the failing human heart.

In the human heart, as in the heart of several other species, muscarinic receptors are predominantly of the M2-subtype that couple via a pertussis toxin-sensitive Gi-protein to inhibit adenylyl cyclase. However, it is not clear whether an additional muscarinic receptor subtype exists in the human heart. In human right atrium, stimulation of muscarinic M2 receptors causes direct negative inotropic and chronotropic effects; in human ventricular myocardium, however, the negative inotropic effect can be only achieved when basal force of contraction has been pre-stimulated by cyclic AMP-elevating agents such as beta-adrenoceptor agonists, forskolin or phosphodiesterase inhibitors (indirect effect); this has been shown in various in vitro and in vivo studies. Evidence has accumulated that in chronic heart failure vagal activity is decreased. Cardiac muscarinic M2 receptor density and functional responsiveness (inhibition of adenylyl cyclase activity and negative inotropic effects), however, are not considerably changed when compared with non-failing hearts although cardiac Gi-activity is increased.

Cardiac effects of beta-adrenoceptor antagonists with intrinsic sympathomimetic activity in humans: beta1- and/or beta2-adrenoceptor mediated?

The aim of this study was to find out whether cardiac responses to the beta-adrenoceptor antagonists with intrinsic sympathomimetic activity (ISA) xamoterol and celiprolol are mediated by cardiac beta1- or beta2-adrenoceptors or both. For this purpose we assessed, in six healthy male volunteers, the effects of xamoterol (100 and 200 mg, p.o.) and celiprolol (200, 600, and 1,200 mg, p.o.) on blood pressure, heart rate, and heart rate-corrected duration of the electromechanical systole (QS2c, as a measure of inotropism). Xamoterol, in both doses, increased systolic blood pressure and heart rate, transiently decreased diastolic blood pressure, and shortened QS2c; all these effects were attenuated after pretreatment of the volunteers with the beta1-adrenoceptor antagonist bisoprolol. Celiprolol, in all three doses, increased heart rate, decreased diastolic blood pressure, and shortened QS2c but only marginally increased systolic blood pressure. Bisoprolol did not attenuate these celiprolol effects but rather enhanced celiprolol effects on systolic blood pressure and heart rate. In a further set of experiments, we studied cardiovascular effects of celiprolol in six healthy volunteers whose beta2-adrenoceptors had been desensitized by a 2-week treatment with 3x5 mg/day terbutaline. Under these conditions, celiprolol failed to increase heart rate or to shorten QS2c. We conclude that, under resting conditions, in healthy volunteers, beta-adrenoceptor antagonists with ISA can exert increases in heart rate and contractility that are mediated by either cardiac beta1-adrenoceptor (xamoterol) or cardiac beta2-adrenoceptor (celiprolol) stimulation. Thus in the human heart, the ISA of beta-adrenoceptor antagonists can be a beta1- or beta2-adrenoceptor agonistic component.

Mechanism of ET(A)-receptor stimulation-induced increases in intracellular Ca2+ in SK-N-MC cells.

The mechanism underlying endothelin-1 (ET-1)-induced increases in intracellular Ca2+ concentrations in the human neuroblastoma cell-line SK-N-MC was investigated. ET-receptor agonists increased inositol phosphate (IP)-formation (assessed as accumulation of total [3H]-IPs in [3H]-myo-inositol prelabelled cells) and intracellular Ca2+ (assessed by the FURA-2 method) with an order of potency: ET-1 > sarafotoxin 6b (S6b)> ET-3 = S6c; the ETA-receptor antagonist BQ-123 inhibited both responses with apparent pKi-values of 8.3 and 8.6, respectively, while the ETB-receptor antagonist BQ-788 did not. Pretreatment of the cells with pertussis toxin (PTX, 500 ng ml(-1) overnight) reduced ET-1-induced Ca2+ increases by 46+/-5%, but rather enhanced ET-1-induced IP-formation. Chelation of extracellular Ca2+ by 5 mM EGTA did not affect ET-1-induced IP-formation. However, in the presence of 5 mM EGTA or SKF 96365, an inhibitor of receptor mediated Ca2+ influx (1.0-3.0 x 10(-5) M) ET-1-induced Ca2+ increases were inhibited in normal, but not in PTX-treated cells. [125I]-ET-1 binding studies as well as mRNA expression studies (by RT-PCR) detected only ETA-receptors whereas expression of ETB-receptor mRNA was marginal. ET-1 (10(-8) M) inhibited isoprenaline-evoked cyclic AMP increases; this was antagonized by BQ-123, not affected by BQ-788 and abolished by PTX-treatment. We conclude that SK-N-MC cells contain a homogeneous population of ETA-receptors that couple to IP-formation and inhibition of cyclic AMP formation. Stimulation of these ETA-receptors increases intracellular Ca2+ by at least two mechanisms: a PTX-insensitive IP-mediated Ca2+ mobilization from intracellular stores and a PTX-sensitive influx of extracellular Ca2+.

Effects of uremic plasma on alpha- and beta-adrenoceptor subtypes.

Responsiveness of adrenergic receptors is decreased in patients on maintenance hemodialysis. In this study we investigated whether uremic plasma might affect adrenergic receptors. For this purpose we determined the effects of uremic plasma obtained from 10 patients on hemodialysis treatment (mean age 61 +/- 3 years, dialysis frequency 3 x 4 h/week, duration of treatment 3 +/- 1 years) before and at the end of the 4-hour dialysis treatment on binding of radioligands to beta1- and beta2- as well as alpha1- and alpha2-adrenoceptors. Plasma from 6 healthy volunteers served as control; the plasmas were studied in three dilutions: undiluted, 1:1 (v/v) and 1:4 (v/v) with saline diluted. Plasma from healthy control did not significantly affect the number of beta1- and beta2- or alpha1- and alpha2-adrenoceptors. On the other hand, uremic plasma significantly decreased the number of beta1- and beta2-adrenoceptors; this inhibitory effect was also observed when plasma obtained at the end of the 4-hour dialysis treatment was investigated. On the other hand, uremic plasma did not significantly decrease the number of alpha1- and alpha2-adrenoceptors. We conclude that in patients on maintenance hemodialysis, the presence of inhibitory substance(s) in uremic plasma could be - at least partly - responsible for the beta-adrenoceptor hyporesponsiveness; the mechanism leading ot alpha-adrenoceptor hyporesponsiveness, however, remains to be elucidated.

[Histamine in tears in allergic rhinoconjunctivitis]. / Histamin in der Tränenflüssigkeit bei allergischer Rhinokonjunktivitis.

UNLABELLED: The classic clinical symptoms of allergic conjunctivitis (type I allergy)--itching and lacrimation--are the effect of histamine. Determination of histamine levels in tears may be useful in evaluating the dynamics of local histamine release in connection with the clinical findings. PATIENTS AND METHODS: Between 1.7.1994 and 31.6.1995 we analyzed the histamine levels in tears and investigated the clinical symptoms (score of 0-3) of 32 hyposensitized pollen-sensitive patients (14 males and 18 females, aged 18-45 years, group I) and of 32 controls (group II) without any allergic disease, performed in each case once in season and once out of season. Tear production and composition were measured by Schirmer's test and tear break-up time at the same time. The histamine levels of the tear samples (obtained by microcapillary method) were analyzed by electrochemical determination. RESULTS: In group I there was a highly significant increase of the mean histamine level from 0.89 +/- 2.22 ng/ml (out of season) to 7.71 +/- 7.51 ng/ml (in season) for the right eye and from 0.73 +/- 2.36 ng/ml (out of season) to 9.51 +/- 9.07 ng/ml (in season) for the left eye (P = 0.0000). The histamine level in tears of the controls (group II) was below the detection limit in all samples. The seasonal histamine level were higher with the severity of atopy (Erlangen atopy score). There was no significant influence of age and gender. The reduction of allergic symptoms during hyposensitization was not comparable to the degree of seasonal histamine level. Compared with the clinical findings, the histamine level in tears did not correlate with the symptoms of lacrimation, itching and conjunctival hyperemia. CONCLUSION: The histamine level in tears alone is not useful as a marker for the clinical severity of this atopyassociated disorder and for the efficacy of the anti-allergic therapy. After standardization of the determination method and the identification of other soluble mediators simultaneously, the histamine level in tears can be used as one part of a profile of mediators to evaluate the clinical symptoms.

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.

Terbutaline-induced desensitization of human cardiac beta 2-adrenoceptor-mediated positive inotropic effects: attenuation by ketotifen.

BACKGROUND: In patients with chronic heart failure cardiac beta 1-adrenoceptors are desensitized whereas beta 2-adrenoceptors are only marginally affected. The mechanism underlying this differential regulation is not known. OBJECTIVES: To find out whether or not human cardiac beta 2-adrenoceptors might be 'resistant' to agonist-induced desensitization and whether or not the antiallergic drug ketotifen might attenuate possible desensitization. METHODS: We investigated, in a single blinded, randomised, placebo-controlled, cross-over study of ten healthy male volunteers (mean age, 25.3 +/- 0.7 years), the effects of two weeks treatment with the beta 2-adrenoceptor agonist terbutaline (3x5 mg/day p.o.) with and without simultaneous treatment with ketotifen (2x1 mg/day p.o. for three weeks) or placebo on beta-adrenoceptor-mediated cardiovascular effects. Cardiovascular effects were assessed as isoprenaline (3.5-35 ng/kg/min)- and terbutaline (25-150 ng/kg/min)-infusion-induced increases in heart rate and systolic blood pressure, decreases in diastolic blood pressure and shortening of the systolic time intervals (STIs), heart rate corrected duration of electromechanical systole (QS2c) and pre-ejection period (PEP; as a measure of inotropism). RESULTS: Ketotifen did not significantly affect basal haemodynamics in the volunteers. Isoprenaline- and terbutaline-infusion caused dose-dependent increases in systolic blood pressure and heart rate, decreases in diastolic blood pressure and shortening of QS2c and PEP, whereby isoprenaline effects were more pronounced. After two weeks of treatment with terbutaline p.o., isoprenaline- and terbutaline-infusion-induced increases in heart rate, shortening of QS2c and PEP were significantly reduced whereby terbutaline-infusion effects were markedly more attenuated than isoprenaline-infusion effects. Ketotifen significantly reduced terbutaline p.o. treatment-induced attenuation of all terbutaline-infusion effects (largely beta 2-adrenoceptor-mediated) and the isoprenaline-infusion-induced increase in heart rate (beta 1- and beta 2-adrenoceptor-mediated), but did not (or only marginally) affect reduction in isoprenaline-induced shortening of QS2c and PEP (largely beta 1-adrenoceptor-mediated). CONCLUSION: Human cardiac beta 2-adrenoceptors are not 'resistant' to agonist-induced desensitization: Ketotifen might prevent such beta 2-adrenoceptor-agonist-evoked desensitization.

Influence of atropine on the cardiovascular effects of noradrenaline and tyramine in elder volunteers.

The aim of this study, carried out in six elder healthy volunteers (mean age: 61 years), was to determine the influence of muscarinic receptor blockade with atropine (15 microg/kg i.v. loading dose followed by 0.15 microg/kg/min by i.v. infusion) on the effects of i.v. infusions of noradrenaline (5 incremental doses of 10-120 ng/kg/min) or tyramine, that releases endogenous noradrenaline (4 incremental doses of 5-20 microg/kg/min), on blood pressure, heart rate and systolic time intervals (STI's, as a measure of positive inotropism). These results were compared with those recently published for young healthy volunteers (mean age: 26 years; Schäfers et al. 1997). Noradrenaline caused increases in systolic and diastolic blood pressure, decreases in heart rate and a shortening of STI's that were not different from those in young volunteers. Atropine did not significantly affect these hemodynamic responses to noradrenaline, while in young volunteers it significantly enhanced noradrenaline-induced blood pressure increases and converted the heart rate decrease into an increase. In the present study in elder volunteers, tyramine caused a smaller increase in systolic blood pressure than in the previous study in young volunteers; in addition, it slightly increased diastolic blood pressure while it decreased diastolic blood pressure in young volunteers. Atropine did not significantly affect the hemodynamic effects of tyramine in the elder volunteers, while in the young volunteers it enhanced the increase in systolic blood pressure and converted the decreases in diastolic blood pressure and heart rate into increases. These results indicate a) that ageing is accompanied by a blunted baroreflex-mediated parasympathetic activation resulting in reduced cholinergic vasodilation and decreases in heart rate, and b) that ageing is associated with a decreased responsiveness of (cardiac) beta-adrenoceptors and (vascular) alpha1-adrenoceptors which is only unmasked when the counterregulatory action of parasympathetic activation is removed.

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

1. 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). 2. Endothelin (10 pM-1 microM) 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 microM: 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. 3. 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). 4. All (1 nM-1 microM) concentration-dependently increased IP-formation (max. increase at 1 microM: 42 +/- 7% above basal, n = 16) and [3H]-phenylalanine incorporation (max. increase at 1 microM: 29 +/- 2%, n = 9). These effects were antagonized by losartan, but they were also antagonized by bosentan and BQ-123. 5. In well-defined cultures of cardiomyocytes (not contaminated with non-myocyte cells) All failed to increase [3H]-phenylalanine incorporation: addition of non-myocyte cells to the cardiomyocytes restored All-induced increase in [3H]-phenylalanine incorporation. 6. 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 All are brought about via local ET-1 secretion upon AT1-receptor stimulation in neonatal rat ventricular non-myocyte cells.