The antilipolytic effect of endogenous and exogenous adenosine in canine adipose tissue in situ

The effects of adenosine, 2-Cl-adenosine, two adenosine uptake inhibitors (dipyridamole and dilazep) and the adenosine deaminase (ADA) inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) were studied on basal and stimulated lipolysis in subcutaneous adipose tissue. The basal lipolysis was unaffected by all agents. Lipolysis induced by nerve stimulation (4 Hz, 5 min) was dose-dependently antagonized (up to 100%) by close i.a. infusions of adenosine (1–40 μM in blood); if the nerve induced vasoconstriction was prevented by α-adrenoceptor-blockade. 2-Cl-adenosine was a more potent antilipolytic agent than adenosine. EHNA (3–10 μM in blood) did not inhibit stimulated lipolysis in vivo possibly because of the low ADA activity in fat cells. Dipyridamole (0.5-1.5 μM in blood) in combination with EHNA increased the venous plasma concentration of adenosine from 0.3±0.05 to 0.7±0.1 μM and enhanced the tissue concentration close to 3-fold. Lipolysis induced by nerve stimulation (4 Hz) was reduced by about 40% by dipyridamole + EHNA and that induced by close i.a. noradrenaline injection (20 nmol) by approximately 60%. It is concluded that adenosine is an antagonist of stimulated lipolysis in subcutaneous adipose tissue in situ in concentrations that are reached during prolonged sympathetic nerve stimulation.     

Presynaptic inhibitory actions of adenine nucleotides and adenosine on neurotransmission in the rat vas deferens.

Adenine nucleotides and adenosine inhibited the isometric contractions of the rat vas deferens in vitro in response to field stimulation but had no effect on the responses to exogenous noradrenaline. The inhibitions were potentiated by dipyridamole and compound 555, antagonized by theophylline and unchanged by indomethacin, 2-2′-pyridylistogen, phenoxybenzamine and atropine. Adenosine and adenosine 5′-triphosphate inhibited the release of [³H]noradrenaline produced by field stimulation.These results indicate that adenine nucleotides, probably acting via the common metabolite adenosine, inhibit adrenergic neurotransmission at a presynaptic site. Their antagonism by theophylline suggests that a presynaptic ‘purinergic’ receptor system could be involved.     

Theophylline interferes with the modulatory role of endogenous adenosine on cholinergic neurotransmission in guinea pig ileum

The ability of theophylline and other phosphodiesterase inhibitors to alter contractile responses to cholinergic nerve stimulation was investigated in isolated longitudinal muscle of the guinea pig ileum. Theophylline in low concentrations (10–100 μM), having no or little effect on measured phosphodiesterase activity, antagonized inhibitory effects of exogenous adenosine. In higher concentrations (0.1–10 mM), shown to be effective in inhibiting phosphodiesterase, theophylline as well as a “pure” cAMP phosphodiesterase inhibitor, ZK 62, 711, inhibited contractile responses. Dipyridamole and dilazep, inhibitors of adenosine inactivation, and also selective inhibitors of cAMP and cGMP phosphodiesterase, respectively, were found to enhance effects of exogenous adenosine and to cause a marked leftward shift of adenosine threshold dose. When dipyridamole and dilazep by themselves had inhibitory effects these could be antagonized by theophylline, suggesting an action through increased levels of endogenous adenosine. As a further indication of endogenous adenosine modulating neurotransmission low concentrations of theophylline enhanced responses to transmural stimulation. Endogenous purine concentrations in tissues and bath media were measured by HPLC. Because of tissue and microbial adenosine inactivation direct estimates of extracellular adenosine concentration could not be obtained. However, adenosine levels increased during transmural stimulation, and during inhibition of adenosine inactivation were sufficient, even in the bath medium, to interfere with the cholinergic neurotransmission.     

Vascular and Metabolic Effects of Theophylline,Dibuturyl Cyclic AMP and Dibuturyl Cyclic GMP in Canine Subcutaneous Adipose Tissue in situ1

In canine subcutaneous adipose tissue theophylline (2×10^--⁴ M), cAMP and ATP (10^--⁵ M), and DBcAMP (8×10^--⁴ M) increased blood flow by by approximately 100 per cent. These compounds also antagonized sympathetic vasoconstriction. Theophylline and DBcAMP increased glycerol release dose-dependently, while cAMP and ATP were ineffective up to I mM. Theophylline (0.4–0.8 mM) potentiated the lipolytic effect of nerve stimulation, while 2–8 mM apparently caused maximal stimulation Per se. DBcAMP did not affect FFA release following nerve stimulation, while DBcGMP potentiated. The apparent rate of re-esterification, glucose uptake and lactate release was decreased by theophylline. DBcAMP (0.1–0.4 mM) had no effect on these parameters, while DBcGMP at the same concentration decreased re-esterification and lactate release. Stimulated overflow of ³H from tissues prelabelled with L-³H-noradrenaline was reduced to 50 per cent by ATP (0.1–0.4 mM), but was unaffected by DBcAMP and DBcGMP at the same concentration. The results support the view that cAMP mediates the metabolic actions of sympathetic nerve stimulation in canine subcutaneous adipose tissue. The relationship between cAMP and vascular reactions may be more complex.     

Inhibition of neuropeptide Y-induced augmentation of noradrenaline-induced vasoconstriction by D-myo-inositol 1,2,6-trisphosphate in the rat mesenteric arterial bed

The effect of the neuropeptide Y antagonist D-myo-inositol-l,2,6-trisphosphate (α-trinositol) was tested against modulatory actions mediated by neuropeptide Y in the isolated rat mesenteric arterial bed. Neuropeptide Y (1 and 10 nM) had no direct postjunctional effects, but augmented vasoconstrictor responses to noradrenaline and to sympathetic nerve stimulation to an extent which was greater with the higher concentration of neuropeptide Y. The augmenting effect of neuropeptide Y at 1 nM on vasoconstriction induced by lower doses of noradrenaline was antagonized by α-trinositol (1 μM), producing a shift to the right of the dose-response curve. A lower concentration of α-trinositol (0.1 μM) had no inhibitory effect on responses to noradrenaline. Augmentation by the higher concentration of neuropeptide Y (10 nM) of noradrenaline-induced vasoconstriction was not affected by α-trinositol at concentrations of up to 10μM. α-Trinositol did not significantly antagonize neuropeptide Y-induced augmentation of vasoconstrictor responses to sympathetic nerve stimulation, α-Trinositol alone did not affect vasoconstrictor responses to noradrenaline, potassium, or to sympathetic nerve stimulation. In the raised-tone preparation (tone raised with methoxamine) in the presence of guanethidine (5 μM) to block sympathetic neurotransmission, perivascular nerve stimulation caused vasodilatation due to activation of sensory-motor nerves. Neuropeptide Y inhibited sensory-motor nerve induced vasodilatation in a concentration-dependent manner but this was not affected by α-trinositol (1 μM). These results suggest that α-trinositol can be a useful functional antagonist of neuropeptide Y-induced augmentation of vasoconstrictor responses to noradrenaline in the rat mesenteric arterial bed. Antagonistic effects of α-trinositol on neuropeptide Y-mediated pre-junctional inhibition of sensory-motor neurotransmission were not evident.     

Activation of renal pelvic chemoreceptors in rats: role of calcitonin gene-related peptide receptors

Substance P and calcitonin gene-related peptide (CGRP) increase afferent renal nerve activity (ARNA). A substance P receptor antagonist but not a CGRP receptor antagonist, h-CGRP (8–37), blocks the ARNA response to renal mechanoreceptor (MR) stimulation. We have examined whether calcitonin gene-related peptide activates renal pelvic sensory receptors and whether such activation contributes to renal chemoreceptor stimulation. The calcitonin gene-related peptide receptor antagonist, h-CGRP (8–37) [0.01–10 μmol L^?1] dose-dependently decreased (29 ± 4–86 ± 13%, P < 0.01) the ipsilateral afferent renal nerve activity in response to the renal pelvic administration of calcitonin gene-related peptide (0.26 μmol L^?1). Renal pelvic perfusion with 900 m M NaCl also increased ipsilateral ARNA (23 ± 3% increase, P < 0.02) and contralateral urinary sodium excretion (13 ± 4% increase, P < 0.05). However, these responses to hypertonic NaCl were unaltered by h-CGRP (8–37). Renal pelvic perfusion with 1 or 10 μM h-CGRP (8–37) also failed to alter the ARNA responses to KCl (31.25, 62.5 and 125 m M ). These results indicate that there are sensory receptors in the renal pelvic area that are responsive to calcitonin gene-related peptide. The activation of these receptors elicits a contralateral natriuretic response. In contrast, the activation of renal calcitonin gene-related peptide receptors does not contribute to renal chemoreceptor activation.     

Vasodilatation and modulation of vasoconstriction in canine subcutaneous adipose tissue caused by activation of β-adrenoceptors

The present experiments were undertaken to study the balance between vascular α- and β-adrenoceptors in canine subcutaneous adipose tissue during sympathetic nerve stimulation and noradrenaline injections. Propranolol potentiated and prolonged the vasoconstrictor response to close i.a. injections of noradrenaline. The vasoconstriction induced by brief nerve stimulation (0.5 to 8 Hz) was, however, unaltered by the β-adrenoceptor blockade. During prolonged nerve stimulation the vasoconstrictor response was well maintained at 1.5 Hz but at 4 Hz there was a gradual escape. The escape phenomenon at 4 Hz was diminished by propranolol. The β₁-selective antagonist practolol, like propranolol, potentiated and prolonged the vasoconstriction induced by noradrenaline injections and reduced the vasoconstrictor escape during prolonged nerve stimulation at 4 Hz. Furthermore, the vasodilatation induced by noradrenaline injection or nerve stimulation during α-adrenoceptor blockade was diminished by practolol. Practolol also blocked the lipolytic response to noradrenaiine and nerve stimulation. The β₂-selective antagonist H35/25 blocked the effects of the β₂-selective agonist salbutamol but failed to alter noradrenaline as well as nerve stimulation induced vascular and lipolytic β-adrenoceptor responses. The present results provide further support for the hypothesis that vascular β-adrenoceptors in adipose tissue are humoral (noninnervated), preferentially activated by circulating noradrenaline. Moreover, both vascular and lipolytic β-adrenoceptors activated by noradrenaline in adipose tissue are best classified as β₁-adrenoceptors.     

Endogenous Adenosine Curtails Lipopolysaccharide-Stimulated Tumour Necrosis Factor Synthesis

Recent studies have demonstrated the inhibitory effect of exogenous adenosine on TNF production. During inflammation endogenous adenosine levels are elevated and may be one of several anti-inflammatory mediators that reduce TNF synthesis. In the present study the authors investigated this role of adenosine in freshly isolated human PBMC. The effect of endogenous adenosine on TNF formation was studied by four different approaches. First, adenosine deaminase was added to LPS-stimulated mononuclear cells. This enzyme specifically deaminates extracellular adenosine to the inactive metabolite inosine. TNF production was augmented from baseline stimulation (LPS alone) of 3.5 ± 0.4 ng ml^?1— 5.2 ± 0.9 ng ml^?1 in the presence of 10 U ml^?1 adenosine deaminase. Second, TNF production was determined after stimulation in the presence of dipyridamole, an inhibitor of cellular re-uptake of adenosine which increases extracellular concentrations. TNF synthesis was reduced dose-dependently from 3.1 ± 0.9 ng ml^?1— 1.1 ± 0.2 ng ml^?1 by 10 μm dipyridamole. Third, the adenosine A₂ receptor antagonist 8-(3-chlorostyryl)caffeine (100 nm ) enhanced TNF synthesis from a baseline of 3.7 ± 0.5 ng ml^?1— 5.5 ± 0.9 ng ml^?1. In contrast, no increase resulted from the addition of 100 nm of the specific A₁ receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. Finally, the authors were able to show that suppression of TNF formation by the specific type IV phosphodiesterase inhibitor rolipram can be completely reversed by adenosine deaminase or by the application of the A₂ receptor antagonist. The authors conclude that endogenous adenosine controls TNF production. This effect of adenosine may not only have a physiological role but also appears to contribute to the pharmacological inhibition of TNF synthesis by exogenous agents such as the specific type IV phosphodiesterase inhibitor rolipram.     

Mechanism of presynaptic actions of adenosine and acetylcholine on noradrenaline release in the guinea-pig heart

Isolated heart of the guinea-pig was stimulated transmurally (1 Hz), or by excess [K⁺] to evoke release of [³H]noradrenaline. The interactions of tetraethylammonium, acetylcholine and adenosine were investigated on [³H]noradrenaline overflow at different [Ca²⁺] concentrations. One mM tetraethylammonium doubled [³H]noradrenaline overflow, and at 30 mM the overflow was facilitated by about 50-fold. The facilitatory effect of 30 mM tetraethylammonium gradually decreased with reduction in [Ca²⁺] concentration. In 0.1 mM [Ca²⁺] overflow of [³H]noradrenaline was completely blocked. However, addition of 30 mM tetraethylammonium to 0.1 mM [Ca²⁺]-Krebs solution greatly facilitated the overflow. 35 mM K-induced [³H]noradrenaline overflow was facilitated by about 5-fold with 20 mM tetraethylammonium, and the facilitatory effect was blocked by 0.37 μM tetrodotoxin; 75 mM K-induced overflow was facilitated by only 1.5-fold by tetraethylammonium.Electrically induced overflow of [³H]noradrenaline was significantly reduced (30%) by 0.13 μM acetylcholine, and the effect increased up to about 75% with higher concentrations of acetylcholine (1.3 μM). 1.3 μM acetylcholine reduced 35 mM K-induced overflow by 30% and had no effect on 75 mM K-induced overflow. 0.37 μM tetrodotoxin had an almost similar effect on K-induced overflow. Ten-times higher concentrations of acetylcholine were needed to obtain over 70% inhibition of overflow produced by low or high K. The inhibitory effect of acetylcholine on [³H]noradrenaline overflow by 1 Hz was antagonized by increasing concentrations of tetraethylammonium (3 to 30 mM) in 2.5 mM Ca. The antagonism between acetylcholine and tetraethylammonium persisted even when Ca was lowered to 0.02 mM. However, if the overflow was enhanced by other means (high [Ca²⁺] plus phentolamine) to the same extent as with tetraethylammonium and low [Ca²⁺], then acetylcholine blocked overflow by 75%. 3.7 μM adenosine reduced [³H]noradrenaline overflow by about 54%, and this effect was completely prevented by 30 mM tetraethylammonium, either in 2.5 or 0.02 mM Ca.It is proposed that acetylcholine and adenosine interfere with nerve stimulation-evoked release of [³H]noradrenaline, presumably by altering electrical properties (i.e. conduction, resting membrane potential, duration of nerve action potential, etc.) of cardiac sympathetic nerve terminals. This primary action would subsequently lead to a reduction in the availability of [Ca²⁺] for the release process.     

Sympathetic vascular control of the pig nasal mucosa (III): co-release of noradrenaline and neuropeptide Y

The overflows of noradrenaline (NA) and neuropeptide Y like immunoreactivity (NPYLI) and vascular responses upon sympathetic nerve stimulation were analysed in the nasal mucosa of pentobarbital anaesthetized pigs. In controls, a frequency-dependent increase in NA overflow was observed whereas detectable release of NPY-LI occurred only at 6.9 Hz. Parallel decreases in blood flow in the sphenopalatine artery and vein and in nasal mucosa volume (reflecting blood volume in the venous sinusoids) were observed. The laser Doppler flowmeter signal (reflecting superficial blood flow) increased upon low and decreased upon high frequency stimulation. Twenty-four hours after reserpine pretreatment and preganglionic decentralization, the NA overflow was abolished while a frequency-dependent release of NPY-LI occurred. Forty, 60 and 80% of the vasoconstrictor responses then remained upon stimulation with a single impulse, 0.59 and 6.9 Hz, respectively. Both the vasoconstriction and NPY-LI overflow, however, were subjected to fatigue upon repeated stimulation. In reserpinized animals release of NPY-LI and vasoconstrictor responses were larger upon stimulation with irregular bursts at 0.59 Hz compared to effects seen at stimulation with continuous impulses. Pre-treatment with the a-adrenoceptor antagonist phenoxybenzamine or the monoamine reuptake inhibitor, desipramine, enhanced NA overflow by 2–3 and 1.5 times at 0.59 and 6.9 Hz, respectively. Phenoxybenzamine significantly reduced the nerve-evoked vascular responses while the release of NPY-LI at 6.9 Hz was increased. Desipramine increased the functional responses but reduced the NPY-LI overflow. During tachyphylaxis to the vasoconstrictor effects of the stable adenosine 5′-triphosphate (ATP) analogue α-β-methylene ATP (mATP) in controls, the vasoconstrictor responses as well as the NA and NPY-LI overflow to nerve stimulation were unmodified. In reserpinized animals, however, the vascular responses and the overflow of NPY-LI were reduced after mATP tachyphylaxis. These data show that both NA and NPY are released upon sympathetic nerve stimulation in the nasal mucosa in vivo and this release seems to be regulated via prejunctional a-adrenoceptors. The lack of effect of mATP tachyphylaxis under control conditions makes it less likely that ATP serves as a major mediator of the large nonadrenergic vasoconstrictor component.     

Evidence that prejunctional adenosine receptors regulating acetylcholine release from rat hippocampal slices are linked to an N-ethylmaleimide-sensitive G-protein,but not to adenylate cyclase or dihydropyridine-sensitive Ca2+-channeIs

Electrically evoked [³H]acetylcholine ([³H]ACh) release from slices of the rat hippocampus was reduced in a dose-dependent manner by the adenosine A,-receptor agonist R-phenylisopropyladenosine (R-PIA) in the concentration range 0.1–10 μM. The maximal effect was observed with I μM R-PIA. Treatment with N-ethylmaleimide (NEM, 100 μ M, 10 min), which inactivates nucleotide-binding proteins (G-proteins), caused a slight increase in the basal overflow (0.17 ± 0.01%v. 0.10 ± 0.003% in the control slices), but did not affect stimulated release (0.73 ± 0.05%vs. 0.74 ± 0.03% in the control slices). N-ethylmaleimide pretreatment significantly reduced the prejunctional inhibitory effect of R-PIA on [³H]ACh release in a non-competitive manner. The S₂/S₁ ratio was 0.92 ± 0.03 in controls and was reduced to 0.32 ± 0.02 by I μ Mm R-PIA in the control slices and to 0.57 ± 0.03 after NEM pretreatment. Stimulation of cyclic AMP-accumulation by forskolin (I μ M) and rolipram (30 μ M) before the second stimulation (S₂) enhanced the S₂/S₁ ratio by about 30% to 1.26 ± 0.12, but did not reduce the inhibitory effect of R-PIA (I μ MM). The Ca²⁺-channel agonist Bay K 8644 (I μ MM), a concentration that increases K⁺-evoked noradrenaline release, did not affect the basal or electrically evoked [³H]ACh overflow, or the prejunctional effects of R-PIA (0.1 and I μ MM) on [³H]ACh release. Our results suggest that the presynaptic inhibitory effects of A₁-receptor agonists on [³H]ACh release are exerted via a nucleotide-binding protein that can be inhibited by NEM. However, the inhibitory effect is apparently not caused by a change in adenylate cyclase activity or by affecting dihydropyridine-sensitive Ca²⁺-channels.     

Pancreatic polypeptide family (APP,BPP, NPY and PYY) in relation to sympathetic vasoconstriction resistant to α-adrenoceptor blockade

Electrical stimulation of the cat cervical sympathetic trunk caused submandibular salivary secretion and vasoconstriction simultaneously with a contraction of the nictitating membrane. Following α- and β-adrenoceptor blockade by phentolamine or phenoxybenzamine combined with propranolol, the salivary response and the nictitating membrane contraction upon sympathetic stimulation were almost abolished. A considerable vasoconstrictor response (up to 40% of control) however still remained in the submandibular gland. This yasoconstriction, which persisted after α-adrenoceptor blockade, was rather slow in onset and had a long duration without any poststimulatory hyperemia. Local intra-arterial infusions of noradrenaline caused submandibular vasoconstriction, salivary secretion and nictitating membrane contraction. The blood flow response to exogenous noradrenaline did, however, not mimic the effects of sympathetic nerve stimulation with regard to vascular escape. Whereas the vascular escape after nerve stimulation was followed by a prolonged vasoconstriction with a gradual decline, the escape after noradrenaline infusions was accompanied by a normalization of blood flow. Local intra-arterial infusions of pancreatic polypeptide (PP)-related peptides caused a slowly developing vasoconstriction with a long duration in the submandibular gland, but no salivary secretion or contraction of the nictitating membrane. The relative molar potencies as vasoconstrictory agents were about PYY: 1, neuropeptide Y (NPY): 5, avian and bovine pancreatic polypeptid 100. The vasoconstrictor effects of PP-related peptides were resistant to α-adrenoceptor blockade and present also in sympathectomized animals, suggesting a direct action on vascular smooth muscle. Combined local infusions of noradrenaline and NPY caused a vascular response in the submandibular salivary gland which was similar to that seen upon sympathetic nerve stimulation. PYY and NPY caused increase in systemic arterial blood pressure upon systemic administration which indicates general vasoconstrictor actions. This effect was accompanied by a transient bradycardia which was due to inhibition of sympathetic tone, since it was absent in animals treated with propranolol. In conclusion, the present findings illustrate the differential sensitivity to α-adrenoceptor antagonists of the submandibular vasoconstriction and salivation as well as smooth muscle contraction of the nictitating membrane induced by sympathetic nerve stimulation. This remaining vasoconstriction may be explained by release of a nonadrenergic, PP-related transmitter such as NPY which may be present together with noradrenaline in the vascular nerves. Release of an additional vasoconstrictory factor may also account for the finding that infusions of noradrenaline do not mimic the vascular effects of sympathetic nerve stimulation in vivo.     

Adenosine as a modulator of sympathetic nerve-stimulation-induced release of noradrenaline from the isolated rabbit heart

The ability of adenosine to inhibit sympathetic nerve-stimulation-induced overflow of noradrenaline was studied in isolated rabbit hearts with intact sympathetic innervation. Noradrenaline in the heart effluents was measured by HPLC with electrochemical detection. The drugs used, adenosine, theophylline, and 8-parasulphophenyltheophylline, were administered via the perfusion fluid. Adenosine (1-100 microM) dose-dependently inhibited stimulation-evoked outflow of noradrenaline from the heart, by up to 47%: the inhibition was completely antagonized by theophylline (200 microM), and by 8-parasulphophenyltheophylline (100 microM). Neither theophylline nor 8-parasulphophenyltheophylline did per se affect basal or stimulation-evoked outflow of noradrenaline. Simultaneous infusion of adenosine (100 microM) and theophylline (200 microM) significantly increased the outflow of noradrenaline during nerve stimulation, by almost 40%. No such effect was observed by adenosine (100 microM) and 8-parasulphophenyltheophylline (100 microM), indicating that theophylline may facilitate transmitter release by an action dissociated from purinoceptor antagonism. It is concluded that (a) adenosine inhibits depolarization-induced liberation of sympathetic transmitter in the rabbit heart, (b) this inhibition is mediated by activation or purinoceptors, probably located on the presynaptic nerve terminals, and (c) brief periods of sympathetic stimulation in the normoxic heart does not release sufficient amounts of adenosine to cause significant inhibition of transmitter release.     

Vasodilatation and modulation of vasoconstriction in canine subcutaneous adipose tissue caused by activation of beta-adrenoceptors.

The present experiments were undertaken to study the balance between vascular alpha- and beta-adrenoceptors in canine subcutaneous adipose tissue during sympathetic nerve stimulation and noradrenaline injections. Propranolol potentiated and prolonged the vasoconstrictor response to close i.a. injections of noradrenaline. The vasoconstriction induced by brief nerve stimulation (0.5 to 8 Hz) was, however, unaltered by the beta-adrenoceptor blockade. During prolonged nerve stimulation the vasoconstrictor response was well maintained at 1.5 Hz but at 4 Hz there was a gradual escape. The escape phenomenon at 4 Hz was diminished by propranolol. The beta1-selective antagonist practolol, like propranolol, potentiated and prolonged the vasoconstriction induced by noradrenaline injections and reduced the vasoconstrictor escape during prolonged nerve stimulation at 4 Hz. Furthermore, the vasodilatation induced by noradrenaline injection or nerve stimulation during alpha-adrenoceptor blockade was diminished by practolol. Practolol also blocked the lipolytic response to noradrenaline and nerve stimulation. The beta2-selective antagonist H35/25 blocked the effects of the beta2-selective agonist salbutamol but failed to alter noradrenaline as well as nerve stimulation induced vascular and lipolytic beta-adrenoceptor responses. The present results provide further support for the hypothesis that vascular beta-adrenoceptors in adipose tissue are humoral (noninnervated), preferentially activated by circulating noradrenaline. Moreover, both vascular and lipolytic beta-adrenoceptors activated by noradrenaline in adipose tissue are best classified as beta1-adrenoceptors.     

Effects of adenosine on ex vivo filtragometry platelet aggregation in relation to plasma levels

The aim of the present study was to investigate the concentration effect of adenosine on unstimulated platelet aggregation in humans. Adenosine infusion was given intravenously to 12 volunteers in the antecubital vein with infusion rates increasing from 20 to 100 μg kg^?1 min^?1. Filtragometry measurements were obtained from the contralateral antecubital vein before and during 100 μg kg^?1 min^?1 or during maximal tolerable infusion rate. In another set of experiments with 10 volunteers, basal filtragometry measurements were obtained before and after infusion of various concentrations of adenosine into the filtragometer test unit. With intravenous infusion aggregation time tended to increase from 333±42 to 418±8 s (mean±SEM) and increased the venous plasma adenosine concentration from 0.42±0.09 μM to 1.52±0.38 μM . Adenosine infusion into the filtragometer tubing system dose-dependently inhibited aggregation (P<0.05). Adenosine was rapidly eliminated with a half-life of adenosine in the filtragometry tubing system calculated to be about 6 s. These data extend our knowledge from an in vitroto an ex vivo situation that adenosine dose-dependently has a platelet antiaggregatory effect.     

Nervous regulation of the tone of the retractor penis muscle in the goat

The nervous control of the retractor penis muscle (rp) was investigated in the anaesthetized goat. Also, isolated field stimulated strips of the muscle were studied. The noradrenaline (NA) and acetylcholine (ACh) content of the rp was determined, and histochemistry for adrenergic and acetylcholinesterase (AChE) positive nerves was performed. The muscle exhibited spontaneous activity that persisted after section of all nerves. There was, however, also a tendency of the activity to follow the general vasomotor tone, which disappeared after section of the sympathetic chains. The excitatory adrenergic nerves which innervate the muscle come from the sympathetic chains and run along the pudendal, the hypogastric and the pelvic nerves. The rp has a dense network of adrenergic fibres and is very sensitive to excitatory adrenergic stimulation. It has a fairly large NA content, which is higher in old goats (5.95 ± 0.42 μg g^-1) than in young goats (2.87 ± 0.78 μg g-¹). Inhibitory non-adrenergic non-cholinergic (NANC) innervation reaches it via the pelvic and the hypogastric nerves. The maximum inhibitory response is reached at low frequencies (2–4 Hz). Cholinergic prejunctional inhibition of the excitatory response to sympathetic chain stimulation was effected by simultaneous stimulation of the hypogastric nerves. In vitro experiments confirmed the presence of endogenous cholinergic muscarinic suppression of the excitatory adrenergic neurotransmission. Significant amounts of ACh (0.81 7 plusmn; 0.18 μg g^-1) are present in the muscle, and it contains strongly AChE positive nerve fibres and nerve cell bodies. It is concluded that the goat rp is innervated by sympathetic adrenergic excitatory nerves and parasympathetic NANC inhibitory nerves. It further has a direct sympathetic inhibitory NANC innervation, and an indirect inhibitory cholinergic innervation which at least in part is sympathetic.     

Role of adenosine in exercise‐induced human skeletal muscle vasodilatation

The role of adenosine in exercise‐induced human skeletal muscle vasodilatation remains unknown. We therefore evaluated the effect of theophylline‐induced adenosine receptor blockade in six subjects and the vasodilator potency of adenosine infused in the femoral artery of seven subjects. During one‐legged, knee‐extensor exercise at ～48% of peak power output, intravenous (i.v.) theophylline decreased (P < 0.003) femoral artery blood flow (F_aBF) by ～20%, i.e. from 3.6 ± 0.5 to 2.9 ± 0.5 L min^?1, and leg vascular conductance (VC) from 33.4 ± 9.1 to 27.7 ± 8.5 mL min^?1 mmHg^?1, whereas heart rate (HR), mean arterial pressure (MAP), leg oxygen uptake and lactate release remained unaltered (P = n.s.). Bolus injections of adenosine (2.5 mg) at rest rapidly increased (P < 0.05) F_aBF from 0.3 ± 0.03 L min^?1 to a 15‐fold peak elevation (P < 0.05) at 4.1 ± 0.5 L min^?1. Continuous infusion of adenosine at rest and during one‐legged exercise at ～62% of peak power output increased (P < 0.05) F_aBF dose‐dependently to level off (P = ns) at 8.3 ± 1.0 and 8.2 ± 1.4 L min^?1, respectively. One‐legged exercise alone increased (P < 0.05) F_aBF to 4.7 ± 1.7 L min^?1. Leg oxygen uptake was unaltered (P = n.s.) with adenosine infusion during both rest and exercise. The present findings demonstrate that endogenous adenosine controls at least ～20% of the hyperaemic response to submaximal exercise in skeletal muscle of humans. The results also clearly show that arterial infusion of exogenous adenosine has the potential to evoke a vasodilator response that mimics the increase in blood flow observed in response to exercise.     

Transmitter characteristics of cutaneous,renal and skeletal muscle small arteries in the rat

Aim: We studied transmitter characteristics of proximal and distal arteries supplying skin (saphenous artery and its medial tarsal branch), kidneys (terminal branches of renal artery and interlobar arteries) and skeletal muscle (proximal and distal sections of external sural artery). Methods: Artery segments were mounted in an isometric myograph and intramural nerves were activated by electrical field stimulation. Adrenergic and purinergic components of the neurogenic response were blocked using phenoxybenzamine and α,β‐methylene adenosine triphosphate (mATP), respectively. Results: Arteries from skin or kidney developed rapid and prominent neurogenic contractile responses, with half‐maximal amplitude reached within 5–15 s; responses in proximal vessels were greater than in distal vessels. Arteries from skeletal muscle responded to sympathetic stimulation with a moderate contraction developing over 1 min or more, the response of distal segments was greater than that of proximal segments. In skeletal muscle vessels the sympathetically evoked contraction was completely blocked by phenoxybenzamine, whereas in skin and renal vessels it was the combined effect of noradrenaline and adenosine triphosphate (ATP). Given alone, mATP did not change the magnitude of the response to nerve stimulation, but increased its latency and also potentiated the response to exogenous noradrenaline. In all vascular beds, distal vessels were more sensitive to noradrenaline and mATP. Conclusion: It thus appears that the noradrenaline/ATP ratio of the sympathetic vasoconstrictor response differs between vascular beds in a way that is consistent with known differences in the selective regulation of regional vascular resistance by the sympathetic nervous system.     

Phosphate and cyclic AMP excretion decreases during less than 12 hours of hypoxia in conscious rats

Hypocapnia is known to have an antiphosphaturic effect that overcomes the phosphaturic effect of hypoxia. The objective of this study was to examine whether conscious rats exposed to acute hypoxia show a decrease in phosphate excretion due to the concomitant hypocapnia. Wistar rats weighing 200 g were exposed to hypoxia (inspired oxygen fraction=0.10) or normoxia (inspired oxygen fraction=0.21) for 6 h; and rats were alternately exposed to hypoxia or normoxia every 12 h for a total 36 h. Renal clearance and hormone studies were performed. Rats exposed to 6 h of hypoxia (n = 11) showed significant hypophosphaturia and decreases in absolute and fractional excretion of phosphate (0.38±0.10 μg min^-1, mean ±SE, P<0.0001 and 0.59±0.15%, P<0.0001) as compared with normoxic rats (n = 11, 3.91±0.68 μg min-¹ and 5.62±0.85%). In addition, nephrogenous adenosine 3′,5′-cyclic monophosphate level per glomerular filtrate was significantly decreased (-0.87±0.64 nmol dL GF^-1, P<0.05) and plasma parathyroid hormone level was unchanged (45.2±9.5 pg mL-¹) after 6 h of hypoxia as compared with normoxic rats (4.03±1.83 nmol dL GF-¹ and 54.3±10.4 pg mL-¹). A parallel increase in urinary noradrenaline and a decrease in dopamine excretion was observed in rats after 6 h of hypoxia. The decreased phosphate and adenosine 3′,5′-cyclic monophosphate excretion during acute hypoxia were restored to normoxic levels by reoxygenation with 21% oxygen in the study of 12-h intermittent hypoxia. In summary, (1) hypoxia produced by inhalation of 10% oxygen for 12 h or less causes reduced phosphate and adenosine 3′,5′-cyclic monophosphate (cAMP) excretion by spontaneously breathing rats; (2) these effects are reversed by reoxygenation and (3) hypoxia elicits a parallel increase in noradrenaline excretion and a decrease in dopamine excretion. These data suggest that renal adrenergic and dopaminergic systems play important roles in hypophosphaturia during acute hypoxia in conscious rats.     

Calcium fluxes in rat thyroid FRTL-5 cells. Evidence for a functional Na+/Ca2+ exchange mechanism

Törnquist , K. 1992. Calcium fluxes in rat thyroid FRTL-5 cells. Evidence for a functional Na⁺/Ca²⁺ exchange mechanism. Acta Physzol Scand 144 , 341–348. Received 28 April 1 991 , accepted 30 October 1991. ISSN 0001–6772. Endocrine Research Laboratory, University of Helsinki, Minerva Foundation Institute for Medical Research, Helsinki, Finland. The effect of extracellular Na⁺ on cytosolic free Ca²⁺ and on influx and efflux of Ca²⁺ was investigated in FRTL-5 thyroid cells. Stimulating the cells with the purinergic agonist ATP induced a rapid efflux of ⁴⁵Ca²⁺ from cells loaded with ^4aCa²⁺. Replacement of extracellular Na⁺ with choline⁺, significantly decreased the adenosine triphosphate-induced efflux of ⁴⁵Ca²⁺. Furthermore, adenosine triphosphate-induced uptake of ⁴⁵Ca²⁺ was increased when extracellular Na⁺ was replaced with choline⁺, compared with the uptake seen in Na⁺ buffer. Replacing extracellular Na⁺ with choline⁺, increased resting levels of cytosolic free Ca²⁺ from 50 ± 2 nM (mean ± SE) to 81 ± 3 nM (P < 0.05) in Fura 2 loaded cells. In cells preincubated with 1 mM ouabain for 30 min, resting cytosolic free Ca²⁺ increased to 73 ± 3 nM (P < 0.05). In a Na⁺ buffer, the adenosine triphosphate-induced transient increase in cytosolic free Ca²⁺ was 872 ± 59 nM, compared with 1070 ±63 nM in choline' buffer (P < 0.05). The plateau level of cytosolic free Ca²⁺ in response to adenosine triphosphate was 130±16 nM in Na⁺ buffer, compared with 209±9 nM in choline⁺ buffer (P < 0.05). Readdition of Na⁺ to the plateau phase decreased cytosolic free Ca⁺² to 152 ± 5 nM. Stimulating the cells with 10 μM of the Na^+-selective monovalent ionophore monensin increased cytosolic free Ca²⁺ from 53 ± 9 nM to 12416 nM (P < 0.05). This increase in cytosolic free Ca²⁺ was dependent on both extracellular Na⁺ and extracellular Ca²⁺ If cells were first stimulated with monensin, and then with adenosine triphosphate, the transient increase in cytosolic free Ca²⁺ was 1027 ± 24 nM (P < 0. 05 , compared with control cells). The results thus indicate, that FRTL-5 cells have a functional Na⁺/Ca²⁺ exchange mechanism and that this mechanism is of importance in restoring adenosine triphosphate-induced transient increase in cytosolic free Ca²⁺ to resting cytosolic free Ca²⁺ levels.