Alteration of brown adipocyte Ca2+ responses in culture by adrenergic activation

Thermogenic capability of brown adipose tissue is controlled by norepinephrine. Interaction of norepinephrine with adipocyte at- and P3-adrenergic receptors results in the increase of Ca2+ and cAMP concentrations. The [Ca2+]i changes initiated by norepinephrine and selective agonists of alpha1- and beta-adrenergic receptors, cirazolin and isoproterenol, were recorded in single cells of primary culture on the 1st, 3rd and 6th days in vitro. On the first day, isoproterenol-induced [Ca2+]i changes as compared to cirazolin-induced ones were characterized by greater amplitude and lesser impulse duration over the entire range of physiological concentrations used. These differences were negligible after 3 days and kinetic differences were practically absent after 6 days of cultivation. The agonist-induced [Ca2+]i changes in proliferating and differentiated cells differed significantly: in the process of cell growth in culture, the amplitude of calcium response increased, the duration of impulse signal decreased and the sensitivity to adrenergic agonists increased. The Ca2+ store in endoplasmic reticulum increased during the cell growth and development in culture, according to thapsigargin-induced Ca2+ response amplitude increase in Ca2+ free medium. The rate of Ca2+ pumping out of cell characterizing PMCA-activity also increased.     

Blockade of tyramine-induced responses by short-acting 2-halogenoethylamines in the rat vas deferens.

A comparative study was made of the blockade of responses of the rat vas deferens to Norepinephrine (NE) and tyramine (TYR) by short-acting 2-halogenoethylamines (2-HEA). The recovery of NE-induced responses after blockade by 2-HEA was rapid and complete within 120 min. The recovery of responses to TYR was considerably slower and remained incomplete (<50%) at the end of 180 min. Pretreatment of tissues by cocaine (10⁻⁵M/5 min) or TYR (5×10⁻⁴M/5 min) prior to exposure to 2-HEA did not alter the magnitude of initial blockade or recovery of NE-induced responses but increased the magnitude of TYR-induced responses. Pretreatment by Desipramine (DMI) did not protect TYR-induced responses from blockade by 2-HEA. In control tissues, cocaine (10⁻⁵M/10 min) was devoid of any effect on TYR-induced responses while DMI produced concentration-dependent inhibitory effects lasting for 180 min.     

Hepatic potassium movements induced by sympathomimetic amines before and after adrenergic blockade.

The hepatic K+-mobilizing effects of phenylephrine and isoproterenol were studied in dogs equipped with chronic indwelling portal vein catheters. Animals anesthetized with sodium pentobarbital, received intraportal injections of these sympathomimetic amines, alone or in combination, before and after alpha, or beta, or combined adrenergic blockade. Hepatic K+ movements were assessed by measuring systemic arterial and hepatic venous K+ levels. It was concluded that adrenergic blockade exerted no significant influence on the ability of these agents to provoke the initial release and subsequent uptake of K+ by the liver.     

Platelet alpha-2 adrenergic receptor-mediated phosphoinositide responses in endogenous depression.

We have previously indicated that epinephrine stimulates phosphoinositide (PI) hydrolysis by activating alpha-2 adrenergic receptors in human platelets [H. Mori et. al. Life Sci., 741-747 44 (1989)]. This method involves the measurement of the accumulation of [3H]-inositol-1-phosphate (IP-1) as an index of PI hydrolysis; lithium is added to inhibit the metabolism of IP-1, thus giving an enhanced signal. In the present study, we assessed the platelet alpha-2 adrenergic receptor-mediated PI responses in samples from 15 unmedicated patients with endogenous depression and 15 age- and sex-matched control subjects. The responses to epinephrine (10 microM and 100 microM) in the depressed patients were significantly higher than those of the controls, whereas the basal values did not differ significantly. These results support the hypothesis that platelet alpha-2 adrenergic receptors may be supersensitive in patients with endogenous depression.     

Blockade of thromboxane responses in the airway of the cat by SQ 29,548

The effects of SQ 29,548, a thromboxane receptor antagonist, on airway responses were investigated in paralyzed, anesthetized, mechanically ventilated cats. Intravenous injections of the thromboxane and prostaglandin precursor, arachidonic acid (AA), and the thromboxane mimic, U 46619, produced dose-related increases in transpulmonary pressure and lung resistance and decreases in dynamic compliance. After administration of SQ 29,548 (0.5 mg/kg iv), bronchoconstrictor responses to AA were reduced by approximately 50%, whereas responses to U 46619 were reduced by approximately 90%. The cyclooxygenase inhibitor, sodium meclofenamate (2.5 mg/kg iv), blocked the component of the airway response to AA remaining after treatment with SQ 29,548. The thromboxane receptor antagonist had no significant effect on bronchoconstrictor responses to prostaglandins F2 alpha, and D2, methacholine, 5-hydroxytryptamine, histamine, or BAY K 8644, an agent that promotes calcium entry. Reductions in systemic arterial pressure in response to AA were enhanced by the thromboxane receptor antagonist and abolished by meclofenamate. SQ 29,548 had no effect on terminal enzyme activity in microsomal fractions from cat lung. These data support the hypothesis that AA-induced bronchoconstriction in the cat is mediated in large part by the actions of thromboxane A2. These data also suggest that U 46619 and U 44069 stimulate the same airway receptor as thromboxane A2 and mimic the bronchomotor effects of this hormone, which has not yet been isolated as a pure substance. These data demonstrate that SQ 29,548 is a selective thromboxane receptor antagonist in the airways of the closed-chest cat and may be a useful probe for studying responses to thromboxane A2 in physiological and pathophysiological processes in the lung.     

Electrophysiological responses of crayfish oocytes to biogenic amines

Intracellular recordings were made from immature, growing oocytes of the crayfish Pacifastacus leniusciulus. Oocytes had a relatively negative resting potential of -74.7+/-2.2 mV (n=26; range -53 to -90) and a mean input resistance of 0.86+/-0.19 MOmega (n=22; range 0.17-3.3). Octopamine induced a long-lasting response involving biphasic changes in input resistance, together with bi- or multiphasic changes in membrane potential. The resistance-decreasing phase involved (in different oocytes) membrane hyperpolarization, depolarization or both. The resistance-increasing phase was usually a depolarization. The hyperpolarizing form of the resistance-decreasing response, and the depolarizing resistance-increasing response reversed in polarity at membrane potentials of (respectively) -90 and -92 mV, suggesting increases and decreases in K(+) conductance underly the biphasic changes in input resistance. The threshold concentration for the response was remarkably low (>10(-12) M) and showed little or no dose-dependence over the concentration range 10(-12)-10(-6) M. Similar responses were evoked by dopamine and serotonin (at 10(-9) M), although a higher proportion of oocytes responded to octopamine and/or dopamine than to serotonin.     

Pharmacological curve fitting to analyze cutaneous adrenergic responses

Although dose-response curves are commonly used to describe in vivo cutaneous α-adrenergic responses, modeling parameters and analyses methods are not consistent across studies. The goal of the present investigation was to compare three analysis methods for in vivo cutaneous vasoconstriction studies using one reference data set. Eight women (22 ± 1 yr, 24 ± 1 kg/m(2)) were instrumented with three cutaneous microdialysis probes for progressive norepinephrine (NE) infusions (1 × 10(-8), 1 × 10(-6), 1 × 10(-5), 1 × 10(-4), and 1 × 10(-3) logM). NE was infused alone, co-infused with NG-monomethyl-l-arginine (l-NMMA, 10 mM) or Ketorolac tromethamine (KETO, 10 mM). For each probe, dose-response curves were generated using three commonly reported analyses methods: 1) nonlinear modeling without data manipulation, 2) nonlinear modeling with data normalization and constraints, and 3) percent change from baseline without modeling. Not all data conformed to sigmoidal dose-response curves using analysis 1, whereas all subjects' curves were modeled using analysis 2. When analyzing only curves that fit the sigmoidal model, NE + KETO induced a leftward shift in ED(50) compared with NE alone with analyses 1 and 2 (F test, P < 0.05) but only tended to shift the response leftward with analysis 3 (repeated-measures ANOVA, P = 0.08). Neither maximal vasoconstrictor capacity (E(max)) in analysis 1 nor %change CVC change from baseline in analysis 3 were altered by blocking agents. In conclusion, although the overall detection of curve shifts and interpretation was similar between the two modeling methods of curve fitting, analysis 2 produced more sigmoidal curves.     

Structure--activity relations for the acceleration of efflux of noradrenaline from adrenergic nerves in rabbit atria by sympathomimetic amines.

Atria from reserpine-pretreated rabbits were exposed to pargyline to inhibit monoamine oxidase (amine oxidase (flavin-containing) EC 1.4.3.4) and subsequently incubated in (-)-[3H]noradrenaline to allow the cytoplasmic accumulation of amine in adrenergic nerves. The structure-activity relations for acceleration of efflux of cytoplasmic amine were examined. The most potent agents studied were (+)- and (-)-amphetamine, beta-phenethylamine, phentermine, and mephentermine. Ability to accelerate efflux was reduced by addition of phenolic hydroxyl groups, by phenolic methylation, by beta-hydroxylation, and by N-substitution. The structure-activity relations for acceleration of efflux differ notably from those for uptake, inhibition of uptake, or release of noradrenaline from adrenergic nerves, reported in previous studies. The ability and potency of a given phenethylamine derivative to accelerate the efflux of cytoplasmic noradrenaline is probably determined by such factors as the lipid solubility of the amine, the affinity of the amine for the uptake and efflux site(s) for noradrenaline, and competition for any reserpine-resistant intraneuronal binding sites.     

The priming effect of halogenated phospholipids on the functional responses of human neutrophils

Halogenated lipids formed in the reactions with myeloperoxidase (MPO)-derived species may contribute to the regulation of the functional activity of cells. In the present study we have investigated the effects of chloro- and bromohydrins formed in the HOCl and HOBr reactions, respectively, with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) on three different functional responses of human neutrophils: H₂O₂ generation, degranulation (MPO exocytosis), and cell aggregation. It was shown that POPC chloro- and bromohydrins (POPC-Cl and POPC-Br) induced the priming of neutrophils, resulting in significant upregulation of cell responses to neutrophil stimulators such as N-formyl-Met-Leu-Phe and lectin from Solanum tuberosum. The stimulating effects of POPC-Cl and POPC-Br were observed at low micromolar concentrations (liposomal concentration of POPC, 0.5–5 μM; the content of POPC-Cl or POPC-Br, 38 ± 3% of total lipids) after a short exposure (about 5 min) of the neutrophils to POPC-Cl or POPC-Br. These results suggest that halogenated lipids formed in vivo via MPO-dependent reactions may be considered as a new class of biologically active substances that are potentially able to contribute to the priming of myeloid cells in the sites of inflammation and serve as inflammatory response modulators.     

Modulation of the responses of fetal sheep to adrenergic stimulation by adrenal demedullation and chemical sympathectomy

The responses to sympathetic stimulation of fetal sheep adrenal-demedullated or sympathectomised by infusion of guanethidine sulphate have been studied. Sympathetic responses in such denervated or sympathectomised fetuses was studied by intravenous infusion of adrenaline or noradrenaline at about 0.4 micrograms/min per kg. This infusion increased plasma concentration 100-200 fold and there was no significant difference between the control fetuses and those in the vasrious treatment groups. Catecholamine infusions at these rates normally have little effect upon fetal blood gas and pH values, but in adrenal-demedullated fetuses adrenaline infusion drepressed fetal arterial PO2 by 4-6 mmHg (P less than 0.05). The heart rate and blood pressure responses to catecholamine infusion in sympathectomised fetuses was, as expected, much increased. Similar observations were made on adrenal-demedullated fetuses, an unexpected finding, and this is taken to illustrate loss of the adrenal medulla is associated with enhanced responsiveness to adrenergic stimulation in peripheral tissues. The majority of the endocrine and metabolic responses, as reflected in fetal plasma concentrations of ACTH, cortisol, insulin, glucose, lactate and fatty acids, to catecholamine infusion were similarly much enhanced by adrenal-demedullation and chemical sympathectomy. Of particular note was a substantial increase in the responsiveness of the fetal adrenal, as reflected in plasma cortisol, to stimulation by ACTH, a change that usually induces labour, but not so in the present sheep. The results on increased sensitivity in adrenal-demedullated fetuses are discussed in relation to likely tissue mechanisms mediating the changes.     

Blockade of the RhoA-JNK-c-Jun-MMP2 cascade by atorvastatin reduces osteosarcoma cell invasion

Osteosarcoma is characterized by a high malignant and metastatic potential, which points to the need for new therapeutic strategies to prevent cell metastasis. In this study, we show that statin-induced HMG-CoA reductase inhibition reduces cell migration and invasion in human and murine osteosarcoma cells, independently of the genotype. The statin-induced reduction of cell migration and invasion was independent of induction of apoptosis and was geranylgeranylpyrophosphate-dependent. The statin reduced matrix metalloproteinase (MMP) 2, 9, and 14 and TIMP2 expression or activity in invading cells. Forced expression of MMP2 and MMP14 overcame the inhibitory effect of the statin on cell invasion, suggesting a role for these MMPs in invasive potential. We also investigated the mechanisms involved in the reduced MMP2 activity and cell invasion. Inhibition of JNK, but not ERK1/2 signaling, reduced MMP2 activity. Pharmacological or constitutive activation of JNK overcame the reduced MMP2 activity and cell invasion induced by the statin. The statin decreased JNK phosphorylation and c-Jun nuclear translocation, suggesting that HMG-CoA reductase inhibition targets the JNK-c-Jun signaling pathway. We showed that mevalonate or geranylgeranylpyrophosphate treatment prevented the statin-induced reduction in JNK phosphorylation, MMP2 activity, and cell invasion. Forced expression of a constitutively active form of RhoA increased JNK phosphorylation and overcame the inhibitory effect of atorvastatin on MMP2 activity and cell invasion. The data establish a link between RhoA, JNK, c-Jun, and MMP2 activity that is functionally involved in the reduction in osteosarcoma cell invasion by the statin. This suggests a novel strategy targeting RhoA-JNK-c-Jun signaling to reduce osteosarcoma cell tumorigenesis.