Characterization of adenosine receptors in bone. Studies on the effect of adenosine analogues on cyclic AMP formation and bone resorption in cultured mouse calvaria

The effect of different adenosine analogues on cyclic AMP (cAMP) formation and bone resorption in cultured mouse calvarial bones was investigated. 5'-N-ethylcarboxamidoadenosine (NECA), R-N6-phenylisopropyl-adenosine (PIA), N6-cyclohexyl-adenosine (CHA) and 2-chloroadenosine all stimulated cyclic AMP formation with a threshold close to 1 mumol l-1); NECA was the most potent agonist. Theophylline (10, 100 mumol l-1) inhibited the cAMP accumulation induced by NECA and 2-chloroadenosine (30 and 300 mumol l-1), dose dependently. There was no inhibition of cAMP formation by PIA and CHA in forskolin-treated bone tissue. SQ 22, 536 and 2',5'-dideoxyadenosine (100 mumol l-1) both inhibited rolipram-stimulated cAMP formation. Cyclic AMP accumulation in isolated osteoblast-like cells from neonatal mouse calvarial bones was stimulated by NECA (10 and 100 mumol l-1) and 2-chloroadenosine (100 mumol l-1). 2-chloroadenosine (10 and 30 mumol l-1), but not NECA, PIA nor CHA, caused a dose-dependent stimulation of 45Ca release in both 48- and 120-h culture. The effect of 2-chloroadenosine on 45Ca release could not be antagonized by theophylline. Neither NECA, PIA, CHA nor 2-chloroadenosine could affect PTH-stimulated 45Ca release in short term cultures (6, 24 h). By contrast, stimulation of cAMP formation by forskolin or dibutyryl cAMP caused a rapid (6 h) inhibition of PTH-stimulated bone resorption. The results demonstrate functional A2 and P-site receptors in mouse calvaria and osteoblast-like cells, but no A1-receptor was detected. These adenosine receptors regulate cAMP, but are not intimately linked to bone resorption. The calcium mobilization induced by 2-chloroadenosine appears to be unrelated to adenosine receptors.     

Direct Antilipolytic Effect of Acidosis in Isolated Rat Adipocytes

The possibility that acidosis inhibits lipolysis indirectly by causing ionic shifts or by favouring the accumulation of an inhibitor has been tested in isolated fat cells. Lipolysis induced by 3 μM noradrenaline (NA) was inhibited by 40–60% and that induced by 1 mM theophylline (THEO) by about 75% when the pH was reduced to 6.6. Lipolysis induced by NA+THEO was inhibited by 20–30%. Changing the concentration of Ca⁺⁺or Mg⁺⁺did not alter the degree of inhibition. Reducing the K⁺-ion concentration enhanced the inhibitory effect of low pH on lipolysis induced by NA or NA + THEO, whereas cyclic AMP accumulation was uninfluenced. Omitting glucose from the incubation medium caused a slight enhancement of pH-induced inhibition of lipolysis (from 60 to 70%, p<0.01). Reducing the concentration of albumin, which binds inhibitory substances such as FFA, reduced lipolysis more at normal than at reduced pH. At high FFA/albumin ratios (5 or above) lipolysis was similar at normal and reduced pH. The antilipolytic effect of decreased pH was equally pronounced in perifused fat cells, where inhibitory substances are not allowed to accumulate. Our results suggest that the antilipolytic effect of acidosis is mainly a direct effect of the increase in H⁺ion concentration. The inhibitory effect of acidosis on various responses to β-adrenoceptor stimulation may be caused by a decreased formation of cyclic AMP in turn caused directly by the decrease in pH.     

Adenosine A1-receptor-mediated inhibition of evoked acetylcholine release in the rat hippocampus does not depend on protein kinase C

The possible involvement of protein kinase C and/or a lipoxygenase product in the mechanism by which adenosine inhibits release of [3H]acetylcholine evoked by electrical pulses from [3H]choline-labelled hippocampal slices was examined. For comparison, the muscarinic autoreceptors were examined using carbachol. The order of potency of adenosine analogues (CHA = R-PIA greater than NECA much greater than CGS 21680, CV 1808) indicates that the adenosine receptor responsible is of the A1 subtype. Adenosine (10 microM) and R-PIA (0.1 microM) were virtually equiactive as inhibitors and were antagonized to an equal extent by 8-CPT with a potency (IC50 approximately 25 nM) which is also compatible with A1-receptor mediation. The effects of carbachol and of R-PIA were not antagonized by the lipoxygenase inhibitor NDGA (10 or 50 microM). Stimulation of protein kinase C by the phorbol ester 4 beta-phorbol 12,13-dibutyrate caused a concentration-dependent increase in stimulation-evoked 3H overflow, but did not antagonize the presynaptic inhibitory effect of R-PIA or carbachol (0.01-1 microM). Staurosporine (0.1 microM), which inhibited the stimulating effect of phorbol dibutyrate, did not alter the effects of carbachol or R-PIA. The presynaptic effects of phorbol dibutyrate, R-PIA and adenosine were reduced by pretreatment with N-ethylmaleimide (100 microM for 10 min), which inactivates G-proteins. The evoked transmitter release was unaffected by nifedipine (1 microM) in the presence and in the absence of phorbol dibutyrate. These results indicate that adenosine, by acting at presynaptic A1-receptors, reduces transmitter release by a mechanism that involves neither an NDGA-sensitive lipoxygenase nor protein kinase C. The results also indicate that the enhancement of transmitter release by phorbol esters is due to protein kinase C activation and that a G-protein may be involved in the effect but a dihydropyridine-sensitive L-type Ca2+ channel probably is not.