Biomedical subjects

R Germack

Publications and source records attributed to R Germack.

10 recordsLinked to original sources

PubMed2005

Adenosine triggers preconditioning through MEK/ERK1/2 signalling pathway during hypoxia/reoxygenation in neonatal rat cardiomyocytes.

Three subtypes of adenosine receptors (A(1), A(2A) and A(3) ARs) are functionally expressed in cardiomyocytes. Adenosine released during ischemia and ischemia/reperfusion plays a major role in cardioprotection. Phosphatidylinositol 3-kinase (PI-3K)/protein kinase B (PKB) and MEK/ERK1/2 pathways are involved in cell survival. Since the role of these pathways in AR-mediated preconditioning is poorly understood, we have investigated whether PI-3K/PKB and/or MEK1/ERK1/2 pathways are involved in AR-induced cardioprotection in neonatal rat cardiomyocytes. Cells were pre-treated (15 min) with adenosine (non-selective), CPA (A(1)), CGS 21680 (A(2A)) or Cl-IB-MECA (A(3)) before 4 h hypoxia (0.5% O(2)) and 18 h reoxygenation (HX4/R). HX4/R-induced increase in LDH release was significantly reduced by adenosine (70%), CPA (59%) and Cl-IB-MECA (46%). The MEK1 inhibitor PD 98059 suppressed the effects of adenosine, CPA, and Cl-IB-MECA on LDH release, whereas the PI-3K inhibitor wortmannin did not reverse this cardioprotection. Western blotting of phosphorylated ERK1/2 and PKB during HX4/R supported the involvement of ERK1/2 and not PKB in A(1) and A(3) agonist-mediated cardioprotection. In addition, adenosine, CPA and Cl-IB-MECA inhibited HX4/R-induced caspase 3 activity by 75%, 70% and 59%, respectively, and this inhibition was abolished by PD 98059. Interestingly, wortmannin inhibited by 66% the anti-apoptotic response triggered by Cl-IB-MECA but had no effect on adenosine or CPA-induced inhibition of caspase 3. CGS 21680 did not modify cell survival or caspase 3 activity. In conclusion, these data show that the preconditioning effect of adenosine requires A(1) and A(3) but not A(2A) ARs and involves an anti-apoptotic effect via MEK1/ERK1/2 pathway in neonatal rat cardiomyocytes. In addition, A(3)AR-induced preconditioning also involves a PI-3K dependent pathway.

Adenosine↗

PubMed2002

Effect of intermittent hypoxia on cardiovascular function, adrenoceptors and muscarinic receptors in Wistar rats.

The usual model of intermittent hypoxia (sleep apnoea) corresponds to repeated episodes of hypoxia from a few seconds to a few hours interspersed with episodes of normoxia. The aim of this study was to evaluate in rats the effect of two periods of intermittent exposure for 2 months to hypoxia (IHX1, 24 h in hypoxia (428 Torr), 24 h in normoxia; IHX2, 48 h in hypoxia (428 Torr), 24 h in normoxia) as a new model of hypoxia simulating intermittent exposure to high altitude experienced by Andean miners. We assessed the haematological parameters, time course of resting heart rate and systolic blood pressure. We also evaluated the expression of adrenergic and muscarinic receptors. IHX1 and IHX2 produced an increase in haematocrit, haemoglobin concentration and mean corpuscular volume as previously seen in most hypoxic models. IHX1 and IHX2 induced a similar sustained elevation of systolic blood pressure (132 +/- 2 and 135 +/- 3 mmHg, respectively, vs. the control level of 121 +/- 16 mmHg) after 10 days of exposure without change in heart rate. Right ventricular (RV) hypertrophy (225 +/- 13 and 268 +/- 15 mg g(-1), vs. 178 +/- 7 mg g(-1) and downregulation of alpha1-adrenoceptor (RV: 127 +/- 21 and 94 +/- 16 fmol mg(-1) vs. 157 +/- 8 fmol mg(-1); left ventricle (LV): 141 +/- 5 and 126 +/- 9 fmol mg(-1) vs. 152 +/- 5 fmol mg(-1)) have been found in both groups, with right ventricular hypertrophy being greater and alpha1-adrenoceptor density being lower in IHX2 than in HX1 groups. These data indicate that both parameters are related to the time of exposure to hypoxia. IHX1 and IHX2 produced the same magnitude of upregulation of muscarinic receptors (LV, 60%; RV, 40%), and no change in beta-adrenoceptors. In conclusion, exposure to intermittent hypoxia led to polycythaemia and RV hypertrophy as observed in other types of hypoxia. A specific cardiovascular response was seen, that is an increase in blood pressure without change in heart rate, which was different from the one observed in episodic and chronic hypoxia. Furthermore, this model involved specific modifications of alpha1-adrenergic and muscarinic expression.

Animals↗

PubMed2001

Differential alterations in cardiac adrenergic signaling in chronic hypoxia or norepinephrine infusion.

Norepinephrine (NE)-induced desensitization of the adrenergic receptor pathway may mimic the effects of hypoxia on cardiac adrenoceptors. The mechanisms involved in this desensitization were evaluated in male Wistar rats kept in a hypobaric chamber (380 Torr) and in rats infused with NE (0.3 mg. kg(-1). h(-1)) for 21 days. Because NE treatment resulted in left ventricular (LV) hypertrophy, whereas hypoxia resulted in right (RV) hypertrophy, the selective hypertrophic response of hypoxia and NE was also evaluated. In hypoxia, alpha(1)-adrenergic receptors (AR) density increased by 35%, only in the LV. In NE, alpha(1)-AR density decreased by 43% in the RV. Both hypoxia and NE decreased beta-AR density. No difference was found in receptor apparent affinity. Stimulated maximal activity of adenylate cyclase decreased in both ventricles with hypoxia (LV, 41%; RV, 36%) but only in LV with NE infusion (42%). The functional activities of G(i) and G(s) proteins in cardiac membranes were assessed by incubation with pertussis toxin (PT) and cholera toxin (CT). PT had an important effect in abolishing the decrease in isoproterenol-induced stimulation of adenylate cyclase in hypoxia; however, pretreatment of the NE ventricle cells with PT failed to restore this stimulation. Although CT attenuates the basal activity of adenylate cyclase in the RV and the isoproterenol-stimulated activity in the LV, pretreatment of NE or hypoxic cardiac membranes with CT has a less clear effect on the adenylate cyclase pathway. The present study has demonstrated that 1) NE does not mimic the effects of hypoxia at the cellular level, i.e., hypoxia has specific effects on cardiac adrenergic signaling, and 2) changes in alpha- and beta-adrenergic pathways are chamber specific and may depend on the type of stimulation (hypoxia or adrenergic).

Adenylate Cyclase Toxin↗

PubMed2001

Myocardial adrenergic and cholinergic receptor function in hypoxia: correlation with O(2) transport in exercise.

The time course of changes in rat myocardial alpha(1)- and beta-adrenoceptors and of muscarinic cholinergic (M-Ach) receptor characteristics was studied parallel with the changes in exercise systemic O(2) transport during a 21-day period of hypoxia (barometric pressure 380 Torr) to assess the effects of receptor modification during acclimatization on maximal exercise capacity. Hypoxia resulted in polycythemia, pulmonary hypertension, right ventricular hypertrophy, and transient left ventricular weight loss. Maximal O(2) consumption at 30 min of hypoxia was reduced to 60% of the normoxic value and remained unchanged. This was partly due to a gradual decrease in maximal cardiac output and heart rate (HR(max)), which offset the increase in blood O(2) content. HR(max) correlated positively (r = 0.994) with beta-adrenoceptor density and negatively (r = -0.964) with M-Ach-receptor density, suggesting that HR(max) reduction results from intrinsic changes in myocardial receptor characteristics leading to reduced responses to adrenergic stimulation and elevated responses to cholinergic stimulation. alpha-Adrenoceptor density in both ventricles increased initially to eventually fall below normoxic values. The dissociation between the different patterns of right and left ventricular weight and the similar pattern of alpha-adrenoceptor change in both ventricles do not support a role for these receptors on right ventricular myocardial hypertrophy.

Acclimatization↗

PubMed2000

Regulation of beta 1- and beta 3-adrenergic agonist-stimulated lipolytic response in hyperthyroid and hypothyroid rat white adipocytes.

1. This study examined the effects of thyroid status on the lipolytic responses of rat white adipocytes to beta-adrenoceptor (beta-AR) stimulation. The beta 1- and beta 3-AR mRNAs and proteins were measured by Northern and saturation analyses, respectively. Glycerol production and adenyl cyclase (AC) activity induced by various non-selective and selective beta 1/beta 3-AR agonists and drugs which act distal to the receptor in the signalling cascade were measured in cells from untreated, triiodothyronine (T3)-treated and thyroidectomized rats. 2. The beta 3-AR density was enhanced (72%) by T3-treatment and reduced (50%) by introduction of a hypothyroid state while beta 1-AR number remained unaffected. The beta 1- and beta 3-AR density was correlated with the specific mRNA level in all thyroid status. 3. The lipolytic responses to isoprenaline, noradrenaline (beta 1/beta 3/beta 3-AR agonists) and BRL 37344 (beta 3-AR agonist) were potentiated by 48, 58 and 48%, respectively in hyperthyroidism and reduced by about 80% in hypothyroidism. 4. T3-treatment increased the maximal lipolytic response to the partial beta 3-AR (CGP 12177) and beta 1-AR (xamoterol) agonists by 234 and 260%, respectively, increasing their efficacy (intrinsic activity: 0.95 versus 0.43 and 1.02 versus 0.42). The maximal AC response to these agonists was increased by 84 and 58%, respectively, without changing their efficacy. 5. In the hypothyroid state, the maximal lipolytic and AC responses were decreased with CGP (0.17 +/- 0.03 versus 0.41 +/- 0.08 mumol glycerol/10(6) adipocytes; 0.048 +/- 0.005 versus 0.114 +/- 0.006 pmol cyclic AMP min-1 mg-1) but not changed with xamoterol. 6. The changes in lipolytic responses to postreceptor-acting agents (forskolin, enprofylline and dibutenyl cyclic AMP, (Bu)2cAMP) suggest the modifications on receptor coupling and phosphodiesterase levels in both thyroid states. 7. Thyroid status affects lipolysis by modifying beta 3-AR density and postreceptor events without changes in the beta 1-AR functionality.

Adenylyl Cyclases↗

PubMed2000

Activation of protein kinase B by the A(1)-adenosine receptor in DDT(1)MF-2 cells.

In this study the effect of insulin and A(1)-adenosine receptor stimulation on protein kinase B (PKB) activation has been investigated in the hamster vas deferens smooth muscle cell line DDT(1)MF-2. Increases in PKB phosphorylation were determined by Western blotting using an antibody that detects PKB phosphorylation at Ser(473). Insulin, a recognized activator of PKB, stimulated a concentration-dependent increase in PKB phosphorylation in DDT(1)MF-2 cells (EC(50) 5+/-1 pM). The selective A(1)-adenosine receptor agonist N(6)-cyclopentyladenosine (CPA) stimulated time and concentration-dependent increases in PKB phosphorylation in DDT(1)MF-2 cells (EC(50) 1.3+/-0.5 nM). CPA-mediated increases in PKB phosphorylation were antagonized by the A(1)-adenosine receptor selective antagonist 1,3-dipropylcyclopentylxanthine (DPCPX) yielding an apparent K(D) value of 2.3 nM. Pre-treatment of DDT(1)MF-2 cells with pertussis toxin (PTX, 100 ng ml(-1) for 16 h), to block G(i)/G(o)-dependent pathways, abolished CPA (1 microM) induced phosphorylation of PKB. In contrast, responses to insulin (100 nM) were resistant to PTX pre-treatment. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin (IC(50) 10.3+/-0.6 nM) and LY 294002 (IC(50) 10.3+/-1.2 microM) attenuated the phosphorylation of PKB elicited by CPA (1 microM) in a concentration-dependent manner. Wortmannin (30 nM) and LY 294002 (30 microM) also blocked responses to insulin (100 nM). Removal of extracellular Ca(2+) and chelation of intracellular Ca(2+) with BAPTA had no significant effect on CPA-induced PKB phosphorylation. Similarly, pretreatment (30 min) with inhibitors of protein kinase C (Ro 31-8220; 10 microM), tyrosine kinase (genistein; 100 microM), mitogen-activated protein (MAP) kinase kinase (PD 98059; 50 microM) and p38 MAPK (SB 203580; 20 microM) had no significant effect on CPA-induced PKB phosphorylation. In conclusion, these data demonstrate that A(1)-adenosine receptor stimulation in DDT(1)MF-2 cells increases PKB phosphorylation through a PTX and PI-3K-sensitive pathway.

Adenosine↗

PubMed2000

[Hemodynamic consequences of endogenous hyperinsulinism in obese rats with lesions of the ventromedial hypothalamus].

The rat with ventromedian hypothalamus lesions (VMH) is characterized by massive obesity, hyperinsulinemia, increase in parasympathetic tonus and sympathetic depression. The aim of this study was to examine in this model the hemodynamic changes and the baroreflex response and to compare the data with the evaluation of beta adrenergic sensitivity. In VMH rats and Sham operated rats hemodynamic parameters were followed until 8 weeks after operation. Heart rate (HR) and blood pressure (BP) were monitored each week during 24 hours by a telemetric system, a catheter being implanted in aorta. In VMH, HR was significantly lower by the first week (p = 0.02) and until the last measurement. Systolic BP increased progressively in the two groups but was higher in VMH only at 8 weeks (p = 0.03). Compared with Sham rats, 5 days after operation, the percentage of HR acceleration in response to atropine and isoprenaline was significantly higher in VMH, whereas HR response to sodium nitroprussiate was similar in the two groups. Plasma epinephrine and norepinephrine levels were significantly higher in VMH rats. The density of cardiac beta receptors decreased from 15 days to 3 months after operation, similarly in VMH and Sham rats. The affinity of cardiac beta receptors remained stable during the same period and very similar in VMH and Sham rats. This study suggests that in VMH rats 1. bradycardia results mainly from an increase in parasympathetic tone; 2. the increase in reflex tachycardia described in normal rats after insulin infusion needs a normal activity of the sympathetic nervous system; 3. catecholamine levels may be increased despite sympathetic depression, probably as a result of an increase in adrenomedullary secretion possibly due to endogenous hyperinsulinemia; 4. the lack of hypertension in this model including a massive obesity is likely to result from the proper vasodilatory effect of insulin.

Adrenergic Agonists↗

PubMed1997

Beta-adrenoceptor subtype expression and function in rat white adipocytes.

1. The pharmacological features of rat white adipocyte beta-adrenoceptor subtypes were investigated by saturation and beta-agonist competition studies with [3H]-CGP 12177 and by lipolysis induced by beta-agonists as well as their inhibition by CGP 20712A (selective beta 1-antagonist) and ICI 118551 (selective beta 2-antagonist) in an attempt to establish a relationship between the functionality and binding capacity of beta-adrenoceptor subtypes. 2. Two populations of binding sites were identified on adipocyte membranes, one with high affinity (0.22 +/- 0.07 nM) and the other with low affinity (23 +/- 7 nM). The low affinity binding sites constituted 90% of the total binding sites. 3. The competition curves, with 15 nM [3H]-CGP 12177, for the beta-agonists, isoprenaline (Iso), noradrenaline (NA) and adrenaline (Ad), and the selective beta 3-agonist, BRL 37344 (BRL), were clearly biphasic (P < 0.001). The rank orders of agonist potency (pKi) in competing for [3H]-CGP 12177 high affinity and low affinity binding sites, respectively, were Iso (9.28 +/- 0.24) > NA (8.90 +/- 0.12) > Ad (8.65 +/- 0.12) > > BRL (4.53 +/- 0.17) and BRL (7.38 +/- 0.19) > > Iso (2.96 +/- 0.26) > or = NA (2.80 +/- 0.17) > Ad (2.10 +/- 0.11) indicating the expression of beta 1- and beta 3-adrenoceptor subtypes on rat white adipocytes, respectively. Inversely, competition studies with the selective beta 1-agonist, xamoterol (Xam), provided evidence for a single homogeneous population of binding sites with low density (81 +/- 9 fmol mg-1) and high pKi value (7.23 +/- 0.26) confirming the presence of beta 1-adrenoceptors. 4. To assess a possible contribution of the beta 2-subtype, procaterol (Proc), a selective beta 2-agonist, was used to compete with 2 nM [3H]-CGP 12177. A single low affinity (4.61 +/- 0.07) population of binding sites was identified. The density of these sites (71 +/- 12 fmol mg-1) was similar to the one obtained with Xam, suggesting that Proc displaced [3H]-CGP 12177 from the beta 1-subtype. 5. The functional potency (pD2) order with BRL (9.07 +/- 0.20) and catecholamines (Iso: 7.26 +/- 0.06, NA: 6.89 +/- 0.02 and Ad: 6.32 +/- 0.07) was the same as that found for the low affinity binding sites in competition studies. Xam induced lipolysis with greater potency than dobutamine (Dob), 6.31 +/- 0.06 and 5.66 +/- 0.10, respectively. Proc stimulated lipolysis with a low potency (5.59 +/- 0.21). 6. The lipolytic response to 0.001 microM BRL was inhibited by both, selective beta 1- and beta 2-antagonist, in a monophasic manner with low potencies (CGP 20712A pKi: < 4.5 and ICI 118551 pKi: 5.57 +/- 0.13). Similar monophasic profiles were obtained for inhibition of Xam- and Dob-induced lipolysis. In this case, CGP 20712A was more potent (> 10 times) than ICI 118551. The monophasic inhibition was also observed with ICI 118551 in the presence of 0.05 microM Iso or 0.13 microM NA. In contrast, two populations of sites were identified with CGP 20712A in the presence of Iso as well as NA. The pKi values for the first sites were 8.41 +/- 0.09 and 8.58 +/- 0.17, respectively, and for the second population of sites 4.73 +/- 0.22 and 4.27 +/- 0.27, respectively. The proportion of the first sites was low: 19 +/- 4 and 22 +/- 5%, respectively. Biphasic curves were obtained with both antagonists using 2.5 microM Proc (CGP 20712A: pKi1: 8.17 +/- 0.08, site1: 23 +/- 6%, pKi2: 4.77 +/- 0.14; ICI 118551: pKi1: 7.78 +/- 0.03, site1: 37 +/- 2%, pKi2: 5.35 +/- 0.25). 7. Our results show that the radioligand [3H]-CGP 12177 allows the characterization of beta 1- and beta 3-adrenoceptor subtypes on rat white adipocytes. Lipolysis is highly dependent on beta 1- and beta 3-adrenoceptors. Finally, binding and functional studies confirm that lipolysis is mainly driven by the beta 3-subtype.

Adipocytes↗

PubMed1996

Triiodothyronine and amiodarone effects on beta 3-adrenoceptor density and lipolytic response to the beta 3-adrenergic agonist BRL 37344 in rat white adipocytes.

The beta-adrenergic effects of catecholamines are potentiated by thyroid hormones in adipose tissue. Amiodarone (AM) is structurally similar to thyroid hormones and was used to explore the mechanism of the triiodothyronine (T3) effect on beta-adrenergic receptors (beta-ARs) in adipose tissue. AM decreases the expression of some T3 sensitive genes in various tissues and antagonizes the effect of T3 on its nuclear receptors. In this study, the T3, AM and AM + T3 effects on the beta 1- and beta 3-AR density were assessed on rat white adipocytes by radioligand binding using [3H]CGP 12177 after characterization of these subtypes by displacement of [3H]CGP 12177 binding by isoproterenol, BRL 37344 and noradrenaline. BRL 37344 was used to study beta 3-AR lipolysis. White adipocytes from hyperthyroid rats had increased responsiveness (Emax x 2) and sensitivity (+ 38%) to BRL 37344, while those given AM alone had decreased values. Moreover, AM antagonized the T3 effect on lipolysis. The beta 1-binding characteristics (receptor density [Bmax]: 45 +/- 4 fmol/mg of proteins; dissociation factor [Kd]: 0.96 +/- 0.10 nM) were not modified by either compound. Finally, T3 significantly increased beta 3-AR density (587 +/- 69 versus 363 +/- 25 fmol/mg of proteins) and Kd (38 +/- 2 versus 23 +/- 3 nM), while AM alone had no effect and did not antagonize the T3 effect on beta 3-AR number. In conclusion, the hyperthyroid state in the rat potentiated the lipolytic response of white adipocytes to a specific beta 3-agonist and increased the beta 3-AR density without changing in beta 1-AR number and affinity. Furthermore, the lack of antagonism between AM and T3 on beta 3-AR expression suggests that T3 does not work directly on the beta 3-AR gene. Moreover, AM induced a functional tissular hypothyroid-like effect and its antilipolytic effect probably occurred at a postreceptor level.

Adipocytes↗

PubMed1994

Kinetics of a new iodolabeled MAO-B inhibitor in the rat brain and in cultured astrocytes.

3-Bromobenzyloxy phenyloxy hydroxymethyl propanol was labelled with iodine-125. Labeling yield was approximately 92%. Using HPLC and an RP18 column, Iodo*MD (MW = 412) was obtained at no-carrier-added conditions (specific activity 125 Ci/mmole). Biochemical experiments were carried out in vitro and showed a Ki for MAO-B of 5.4 nM and of 5000 for MAO-A (RA/B = 926). Using ex vivo kinetic inhibition in rat (dose: 5 mg/kg p.o.), the results demonstrated a strong similarity of action with BromoMD and IodoMD, with an inhibition percentage that decreased with time (91% at 1 hour, 48% at 8 hours, 2% at 24 hours). The rat brain Iodo*MD concentration was maximal after the first pass and inhibition decreased slowly with time (T1/2 = 1.8 hours). Uptake and wash-out of Iodo*MD was studied on two-day-old rat astrocytes in culture. Half-times of uptake and efflux were respectively 2.5 minutes and 7.5 minutes. The use of metabolic inhibitors (KCN and Digoxin) suggested the absence of any active transport. Binding studies with various concentration of cold MD 360194 showed that at 10(-8) M the uptake decreased significantly. Rats were dissected at different times post i.v. injection (0-2 hours), and the principal organs and brain were obtained (the brain was separated into 7 pieces). Radioactivity was concentrated mainly in the liver (24.6 +/- 4%), fat (12.4 +/- 3.4%) and muscles (18.4 +/- 3%). In the brain the concentration was approximately 1.2 +/- 0.3% within 30 minutes post i.v. injection and 0.84 +/- 0.15% thereafter. The hypothalamus and striatum were two-fold more active than the cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Propanol↗