Effect of catechol-O-methyl-transferase (COMT) inhibition on the vascular and metabolic responses to noradrenaline,isoprenaline and sympathetic nerve stimulation in canine subcutaneous adipose tissue

Summary The effect of COMT-inhibitors (U-0521, H 22/54) on lipolysis and on vascular responses induced by noradrenaline, isoprenaline and nerve stimulation in canine adipose tissue in situ has been studied.COMT inhibition, before or after -adrenoceptor blockade, did not influence the -adrenoceptor mediated vasoconstriction due to nerve stimulation (2–5 Hz). Vasoconstriction due to close i.a. noradrenaline was unaffected at a dose of 2×10^–10 moles but at a dose of 10^–9 moles the peak vasoconstriction was enhanced but the duration of the response was not changed. Prior -adrenoceptor blockade did not alter the responses to U-0521.After -adrenoceptor blockade with Hydergine (120–300 g i.a.) a vasodilatation was induced by noradrenaline 2×10^–10 mole. This response and that produced by isoprenaline (2×10^–11 or 10^–10 moles) were enhanced after U-0521-treatment, but the vasodilating response to nerve stimulation (2 Hz) after -adrenoceptor blockade was unaffected.Lipolysis induced by nerve stimulation or noradrenaline in vivo was increased 50% and that induced by isoprenaline was increased 100% by COMT inhibition. U-0521 did not alter basal lipolysis in vitro but enhanced lipolysis induced by 0.1 M noradrenaline significantly, both in the presence and in the absence of theophylline.The present results shows that COMT influences the effective concentration of isoprenaline more than that of noradrenaline. In the vasculature the effects of exogenous noradrenaline is influenced to a larger degree than those induced by nerve stimulation. However, COMT blockade increased lipolysis induced by equieffective doses of exogenous noradrenaline and nerve stimulation to an equal extent. These results are in agreement with the idea that COMT is of physiological importance mainly at receptor sites that are not in close contact with sympathetic nerve endings and supports the idea that vascular -adrenoceptors are innervated receptors. Vascular -adrenoceptors on the other hand seem to be humoral receptors unrelated to sympathetic nerve endings and preferentially stimulated by circulating noradrenaline. The -adrenoceptors on the fat cells may be of both types, i.e. innervated and humoral.     

Actions of dopamine in canine subcutaneous adipose tissue

Summary Subcutaneous adipose tissue in the inguinal region was isolated from surrounding tissues in fed female dogs and perfused with the dog's own blood.When dopamine (DA) was administered as a single i.a. injection in doses of 5 g, or higher, there was an increased vascular resistance and lipolysis. The vasoconstriction was abolished by phentolamine and phenoxybenzamine but was unaffected or potentiated by desipramine (5 mg i.a.). Desipramine abolished the lipolytic effect of DA as well as its capacity to increase the ³H-outflow from tissues prelabelled with L-³-H-noradrenaline.Infusion of DA (0.02 or 0.2 g/ml blood) had no effect on resting vascular resistance or on the glucose uptake. The higher DA concentration caused a significant increase in resting glycerol and free fatty acid release, while the lower was ineffective. DA did not significantly change the vasoconstrictor effect of sympathetic nerve stimulation (4 cps for 10 min) in either dose, but caused an increased lipolytic response.The results indicate that DA has many different actions in canine subcutaneous adipose tissue. Some of the possible mechanisms of action are discussed.     

Stimulation of cyclic adenosine 3′,5′-monophosphate accumulation and lipolysis in fat cells by adenosine deaminase

Summary The basal lipolytic activity of isolated fat cells of the rat was greatly enhanced in the presence of 0.01 to 30 g adenosine deaminase (ADA) per ml. This effect was more pronounced in dilute (20000 cells/ml) than in concentrated cell suspensions (100000 cells/ml); this is possibly due to the presence, in the incubation medium, of a high concentration of inosine which is formed by the deamination of the large amounts of adenosine released from high concentrations of fat cells. Inosine, although less potent than adenosine as an antilipolytic agent, markedly inhibited ADA-induced lipolysis at concentrations between 10 to 100 M. The lipolytic effect of ADA was identical with the stimulation of lipolysis by 1 M noradrenaline or 1 mM theophylline, while 1 mM dibutyryl cyclic AMP yielded two-fold higher values. The effects of ADA and lipolytic agents at maximally stimulating concentrations were not additive.After 5 min of incubation maximally effective concentrations of ADA which were also maximal with respect to lipolysis caused a 3- to 6-fold elevation of cyclic AMP levels in fat cells. A similar increase was observed with maximally effective concentrations of theophylline, whereas noradrenaline produced a 100- to 200-fold elevation. This indicates that a small accumulation of cyclic AMP may be sufficient to trigger the full lipolytic response. Furthermore, ADA, like theophylline, acted synergistically with noradrenaline and prevented the fall of cyclic AMP levels during 30 min of incubation.Insulin (100 U/ml) and nicotinic acid (0.1 M) decreased cyclic AMP accumulation and glycerol production induced by ADA.The results support the hypothesis that adenosine is released from isolated fat cells and that this nucleoside may serve as an inhibitor of adenyl cyclase activity, thus regulating cyclic AMP-dependent processes in adipose tissue.     

Role of pre- and postjunctional inhibition by prostaglandin E2 of lipolysis induced by sympathetic nerve stimulation in dog subcutaneous adipose tissue in situ

1. Canine subcutaneous adipose tissue was isolated and autoperfused in situ after labelling of the noradrenaline stores by (3)H-(-)noradrenaline.2. Prostaglandin E(2) (10-200 ng/ml) increased blood flow and glucose uptake, and caused a dose-dependent inhibition of lipolysis induced by sympathetic nerve stimulation (4 Hz). The actions of exogenous prostaglandin E(2) are therefore similar to those of prostaglandin E(1) in this tissue. There were no consistent effects of prostaglandin E(2) on the vasoconstriction or on the (3)H-noradrenaline overflow induced by nerve stimulation.3. Phenoxybenzamine (1.5-2 mg i.a.) caused a 5-fold increase in (3)H-noradrenaline overflow and a 95% reduction of the vasoconstrictor response to nerve stimulation. The lipolytic response was similar to that of the control. Prostaglandin E(2) (100-200 ng/ml) administered after phenoxybenzamine caused a 90% inhibition of lipolysis, while the vasoconstrictor response was enhanced to about 50% of control. Prostaglandin E(2) inhibited (3)H-noradrenaline overflow by about 50% but it was still larger than that of the control.4. It is suggested that exogenous prostaglandin E(2) inhibits lipolysis induced by sympathetic nerve stimulation mainly by a postjunctional action in canine subcutaneous adipose tissue.     

Modulation by prostaglandins of the release of [3H]noradrenaline evoked by potassium and nerve stimulation in the isolated rat heart

In the isolated rat heart perfused with Krebs solution and prelabeled with [³H]noradrenaline, we examined the effect of prostaglandins (PG) I₂, E₂, 6-keto-PGF_1α and their precursor, arachidonic acid, on the overflow of tritium elicited by potassium (K⁺) and by stimulation of cardiac sympathetic nerve plexus. Prostaglandins E₂, I₂ and arachidonic acid but not 6-keto-PGF_1α reduced K⁺ and nerve stimulation-induced overflow of tritium. Administration of indomethacin, an inhibitor of cyclooxygenase, increased tritium overflow elicited by either K⁺ or by nerve stimulation. During infusion of indomethacin, the inhibitory effect of both PGE₂ and PGI₂ on the K⁺ or nerve stimulation-induced overflow of tritium remained unaltered. In contrast, the effect of arachidonic acid to reduce K⁺ or nerve stimulation-induced overflow of tritium was abolished by indomethacin, indicating that the fatty acid inhibits release of tritium by its conversion to a product(s) of cyclooxygenase, presumably PGI₂ and PGE₂. These data suggest that prostaglandins, particularly PGI₂ and PGE₂ sythesized in the isolated rat heart act on prejunctional sites to modulate release of the adrenergic transmitter.     

Influence of adrenergic β-receptor activity on blood flow and free fatty acid release in canine subcutaneous adipose tissue during hemorrhagic shock

Summary Irreversible hemorrhagic shock was produced in dogs by means of a standardized bleeding procedure. Blood flow and net release of free fatty acids were measured in subcutaneous adipose tissue. Infusion of isoprenaline (0.5 g/kg/ min i.v.) prevented the severe vasoconstriction during bleeding and promoted the net release of free fatty acids. Futhermore, isoprenaline infusion made it possible to restore a normal blood flow after reinfusion of the shed blood in contrast to the case in control animals. Propranolol (1 mg/kg i.v.) decreased the arterial free fatty acid concentration in the resting animal and also increased the mortality rate.It is concluded that-receptor stimulation by isoprenaline prevents the vascular damage in canine subcutaneous adipose tissue during development of hemorrhagic shock.This investigation was supported by the Hungarian Medical Research Council and the Swedish Medical Research Council 14X-731-05C, K70-14R-3040.     

Deletion of adipose triglyceride lipase abolishes blood flow increase after β3-adrenergic stimulation in visceral adipose tissue of mice

Dynamic changes in adipose tissue blood flow (ATBF) with nutritional status play a role in the regulation of metabolic and endocrine functions. Activation of the sympathetic nervous system via β-adrenergic receptors (β-AR) contributes to the control of postprandial enhancement of ATBF. Herein, we sought to identify the role of each β-AR subtype in the regulation of ATBF in mice. We monitored the changes in visceral epididymal ATBF (VAT BF), induced by local infusion of dobutamine, salbutamol, and CL316,243 (a selective β1-, β2-, and β3-AR agonist, respectively) into VAT of lean CD-1 mice and global adipose triglyceride lipase (ATGL) knockout (KO) mice, using laser Doppler flowmetry. Administration of CL316,243, known to promote lipolysis in adipocytes, significantly increased VAT BF of CD-1 mice to a greater extent compared to that of the vehicle, whereas administration of dobutamine or salbutamol did not produce significant differences in VAT BF. The increase in VAT BF induced by β3-AR stimulation disappeared in ATGL KO mice as opposed to their wild-type (WT) littermates, implying a role of ATGL-mediated lipolysis in the regulation of VAT BF. Different vascular reactivities occurred despite no significant differences in vessel density and adiposity between the groups. Additionally, the expression levels of the angiogenesis-related genes were significantly higher in VAT of ATGL KO mice than in that of WT, implicating an association of ATBF responsiveness with angiogenic activity in VAT. Our findings suggest a potential role of β3-AR signaling in the regulation of VAT BF via ATGL-mediated lipolysis in mice.     

Effects of GABA on noradrenaline release and vasoconstriction induced by renal nerve stimulation in isolated perfused rat kidney.

We examined effects of gamma-aminobutyric acid (GABA) on vasoconstriction and noradrenaline (NA) release induced by electrical renal nerve stimulation (RNS) in the isolated pump-perfused rat kidney. RNS (1 and 2 Hz for 2.5 min each, 0.5-ms duration, supramaximal voltage) increased renal perfusion pressure (PP) and renal NA efflux. GABA (3, 10 and 100 microM) attenuated the RNS-induced increases in PP by 10-40% (P<0.01) and NA efflux by 10-30% (P<0.01). GABA did not affect exogenous NA (40 and 60 nM)-induced increases in PP. The selective GABA(B) agonist baclofen (3, 10 and 100 microM) also attenuated the RNS-induced increases in PP and NA efflux, whereas the RNS-induced responses were relatively resistant to the selective GABA(A) agonist muscimol (3, 10 and 100 microM). The selective GABA(B) antagonist 2-hydroxysaclofen (50 microM), but not the selective GABA(A) antagonist bicuculline (50 microM), abolished the inhibitory effects of GABA (10 microM) on the RNS-induced responses. The selective alpha2-adrenoceptor antagonist rauwolscine (10 nM) enhanced the RNS-induced responses. GABA (3, 10 and 100 microM) potently attenuated the RNS-induced increases in PP by 40-60% (P<0.01) and NA efflux by 20-50% (P<0.01) in the presence of rauwolscine. Prazosin (10 and 30 nM) suppressed the RNS-induced increases in PP by about 70-80%. Neither rauwolscine (10 nM) nor GABA (10 microM) suppressed the residual prazosin-resistant PP response. These results suggest that GABA suppresses sympathetic neurotransmitter release via presynaptic GABA(B) receptors, and thereby attenuates adrenergically induced vasoconstriction in the rat kidney.     

Nonexocytotic noradrenaline release induced by pharmacological agents or anoxia in human cardiac tissue

In acute myocardial ischemia, noradrenaline is released locally from sympathetic varicosities by a Ca²⁺-independent nonexocytotic release mechanism that is effectively suppressed by inhibitors of the neuronal noradrenaline carrier (uptake₁). The purpose of the present study was to elucidate the significance of free axoplasmic amine concentration and disturbed neuronal sodium homeostasis for nonexocytotic noradrenaline release in the human heart by comparing the release induced by anoxia with that induced by reserpine, tyramine, or veratridine. The overflow of endogenous noradrenaline and dihydroxyphenylethyleneglycol was assessed in human atrial tissue incubated in calcium-free Krebs-Henseleit-solution to prevent interferences by exocytotic release. The overflow of dihydroxy-phenylethyleneglycol served as indicator of the free axoplasmic noradrenaline concentration.When vesicular uptake was blocked by the reserpine-like agent Ro 4-1284, high dihydroxyphenylethyleneglycol overflow was observed without concomitant noradrenaline overflow. If, however, Ro 4-1284 was combined with sodium pump inhibition (by omission of extracellular potassium) or with alteration of the transmembrane sodium gradient (by lowering the extracellular sodium concentration), both dihydroxyphenylethyleneglycol and noradrenaline were released. The indirectly acting sympathomimetic tyramine induced a marked increase in noradrenaline overflow which was accompanied by overflow of high amounts of dihydroxyphenylethyleneglycol, indicating interference of the drug with both vesicular catecholamine transport and amine transport via uptake₁. Likewise, veratridine induced an overflow of noradrenaline (which was prevented by blockade of uptake₁) and dihydroxyphenylethyleneglycol indicating a reserpine-like action of the drug. A disturbed energy status of the sympathetic neuron induced by cyanide intoxication or anoxia caused noradrenaline overflow which was suppressed by uptake, blockade. Blockade of sodium channels by tetrodotoxin effectively reduced noradrenaline overflow during cyanide intoxication but not during anoxia. Anoxia-induced noradrenaline release, however, was markedly suppressed by inhibition of Na⁺/H⁺ exchange with ethylisopropylamiloride, indicating the Na⁺/H⁺ exchange as the predominant pathway for sodium entry into the sympathetic neuron during anoxia.The results demonstrate that disturbed neuronal sodium homoeostasis and impaired vesicular storage function are critical conditions, causing nonexocytotic noradrenaline release in anoxic human cardiac tissue.     

Increased noradrenaline release in rat portal vein during sympathetic nerve stimulation due to activation of presynaptic beta-adrenoceptors by noradrenaline and adrenaline.

With the aim of investigating whether exogenous noradrenaline (NA) and adrenaline (A) can modulate transmitter release via the stimulation of presynaptic beta-adrenoceptors, 3H-release from isolated portal veins was studied after pretreatment with 3H-1-NA, phenoxybenzamine, desipramine and normetanephrine. NA (10 muM) and A (0.05 muM) increased the fractional 3H-release elicited by sympathetic nerve stimulation by 30%. This effect could be blocked by d, 1-propranolol which per se reduced the release by 10%. It is concluded that NA can facilitate its own release via a presynaptic beta-adrenoceptor-mediated positive feed-back mechanism and that adrenaline can stimulate this beta-adrenoceptor-mediated mechanism.     

Facilitation of noradrenaline release by adenosine A(2A) receptors in the epididymal portion and adenosine A(2B) receptors in the prostatic portion of the rat vas deferens

The adenosine-receptor modulation of noradrenaline release was compared in prostatic and epididymal portions of rat vas deferens. In both portions, tritium overflow elicited by electrical stimulation (100 pulses/8 Hz) was reduced by the adenosine A(1) receptor agonist, N(6)-cyclopentyladenosine, and enhanced by the nonselective receptor agonist, 5'-N-ethylcarboxamidoadenosine, in the presence of the adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 20 and 100 nM). The adenosine A(2A) receptor agonist, 2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine, increased tritium overflow, but only in the epididymal portion. The enhancement caused by NECA was prevented by the adenosine A(2A) receptor antagonist, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 20 nM), in the epididymal and by the adenosine A(2B) receptor antagonist, alloxazine (1 microM), in the prostatic portion. Inhibition of adenosine uptake enhanced tritium overflow in both portions, an effect blocked by ZM 241385 in the epididymal and by alloxazine in the prostatic portion. The results indicate that adenosine exerts an adenosine A(1) receptor-mediated inhibition, in both portions, and facilitation mediated by adenosine A(2A) receptors in the epididymal and by A(2B) receptors in the prostatic portion.     

The outward transport of axoplasmic noradrenaline induced by a rise of the sodium concentration in the adrenergic nerve endings of the rat vas deferens

Summary The adrenergic nerve endings of the rat vas deferens were loaded with ³H-(–)-noradrenaline; COMT was inhibited by the presence of 10 mol/l U-0521, and all experiments were carried out with calcium-free solution. After 100 min of wash-out a neuronal efflux of tritium was obtained which remained constant with time (when expressed as fractional rate of loss; FRL); it contained more DOPEG than noradrenaline.The in vitro administration of reserpine-like drugs (reserpine and Ro 4-1284) increased the FRL of tritium, presumably because of an increase in the leakage of noradrenaline from storage vesicles; the efflux of DOPEG increased more than that of noradrenaline, and the ratio NA/DOPEG declined.Inhibition of the membrane ATPase (by omission of potassium from the medium or by the presence of 3 mmol/l ouabain) increased the FRL of tritium, presumably because of an increase in the net leakage of noradrenaline from the storage vesicles (as a consequence of the fall in the concentration of free axoplasmic noradrenaline; see below).Veratridine also increased the FRL of tritium, partly because of its known reserpine-like effect (Bönisch et al. 1983); in the presence of 1 mol/l veratridine, the efflux of DOPEG increased.Irrespective of the presence or absence of reserpine or Ro 4-1284, inhibition of the membrane ATPase or the presence of veratridine (agents or procedures which increase the axoplasmic sodium concentration) always resulted in a brisk increase of the efflux of noradrenaline that was accompanied by a simultaneous decrease in the efflux of DOPEG (see above for one exception). In all experiments the rise in internal sodium caused the ratio NA/DOPEG to increase.These results indicate that—as long as the sodium gradient is normal—the axonal membrane functions as a barrier that largely prevents any outward movement of axoplasmic noradrenaline. Consequently, the axoplasmic amine is largely deaminated, and the ratio NA/DOPEG is low. However, when the axoplasmic sodium concentration rises, axoplasmic noradrenaline is transported out of the nerve ending at such high rates that the axoplasmic noradrenaline concentration falls; the fall in the efflux of DOPEG is indicative of a fall in the intraneuronal formation of DOPEG. The results show that changes in the efflux of DOPEG (i.e., of a highly lipophilic metabolite that easily leaves adrenergic nerve endings) can serve as an index of changes in axoplasmic noradrenaline levels.Supported by the Deutsche Forschungsgemeinschaft (Tr.96)     

Sensitivity to selective adenosine A1 and A2A receptor antagonists of the release of glutamate induced by ischemia in rat cerebrocortical slices

Adenosine released during cerebral ischemia is considered to act as a neuroprotectant, possibly through the inhibition of glutamate release. The involvement of A(1) and A(2A) receptors in the control of the rise of extracellular glutamate during ischemia was investigated by monitoring the effects of selective A(1) and A(2A) receptor antagonists on ischemia-evoked glutamate release in rat cerebrocortical slices.Slices were superfused with oxygen- and glucose-deprived medium and [(3)H]D-aspartate or endogenous glutamate was measured in the superfusate fractions. Withdrawal of Ca(2+) ions or addition of tetrodotoxin more than halved the ischemia-evoked efflux of [(3)H]D-aspartate or glutamate, compatible with a vesicular-like release. The glutamate transporter inhibitor DL-TBOA prevented the ischemia-evoked efflux of [(3)H]D-aspartate by about 40%, indicating a carrier-mediated efflux. The ischemia-evoked efflux of [(3)H]D-aspartate or glutamate was increased by the A(1) receptor antagonist DPCPX. The A(2A) antagonist SCH 58261 decreased [(3)H]D-aspartate or endogenous glutamate efflux (50 and 55% maximal inhibitions; EC(50): 14.9 and 7.6 nM, respectively); the drug was effective also if added during ischemia. No effect of either the A(1) or the A(2A) receptor antagonist was found on the ischemia-evoked efflux of [(3)H]D-aspartate in Ca(2+)-free medium. Our data suggest that adenosine released during cerebral ischemia can activate inhibitory A(1) and stimulatory A(2A) receptors that down- or up-regulate the vesicular-like component of glutamate release.     

Sustained subcutaneous infusion of nicotine enhances cholinergic vasodilation in the cerebral cortex induced by stimulation of the nucleus basalis of Meynert in rats

The present study examined the effects of sustained nicotine exposure on the cholinergic vasodilative system originating in the nucleus basalis of Meynert (NBM) and projecting to the cerebral cortex in rats. Rats received sustained subcutaneous nicotine (100 μg/kg/h) for 14 days. Under urethane anesthesia, the vasodilation response and acetylcholine release in the parietal cortex induced by electrical stimulation of the NBM (10–200 μA) were measured. The basal level of acetylcholine release was significantly higher in nicotine-treated rats than in saline-treated control rats. In the control rats, both the acetylcholine release and blood flow were increased by NBM stimulation in a stimulus intensity-dependent manner, and a threshold of 50 μA. In nicotine-treated rats, the threshold intensity of NBM stimulation producing increases in acetylcholine release and blood flow was reduced to 20 μA. The stimulus intensity-dependent acetylcholine release and vasodilation by NBM stimulation were significantly larger in nicotine-treated rats than in control rats. We conclude that sustained subcutaneous infusion of nicotine enhances cholinergic vasodilative system in the cerebral cortex originating in the NBM.     

Effects of indomethacin on changes in renal blood flow induced by adenosine and its analogues in conscious dogs

Summary The effects of indomethacin on changes in renal blood flow induced by adenosine, NECA (adenosine-5-N-ethyl-carboxamide) and 2,3-dinitro-NECA were investigated in 6 chronically instrumented conscious dogs. Adenosine (187.5, 375 and 750 nmol/kg, i.v.) induced a dose-dependent initial decrease, followed by a reactive increase in renal blood flow. NECA (1.5 nmol/kg, i.v.) also induced an initial decrease, which was, however, followed by a prolonged reactive increase in renal blood fow. 2,3-dinitro-NECA (50 nmol/kg, orally) induced only an increase in renal blood flow. Indomethacin (27.9 mol/kg, i.v.) caused no relevant change of the initial decrease and a significant attenuation of the reactive increase in renal blood flow induced by adenosine. NECA-induced changes in blood flow were affected by indomethacin in the same direction but to a greater extent than were adenosine-induced changes in blood flow. Indomethacin reversed the increase to a decrease in renal blood flow induced by 2,3-dinitro-NECA. Thus, prostaglandins seem to be involved in mediating the response of renal blood flow to adenosine, NECA and 2,3-dinitro-NECA.Part of this study was presented at the fall meeting of the German Pharmacological Society, September 1982 in Vienna, Austria