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α1-Adrenergic Stimulation Potentiates the Thermogenic Action of β3-Adrenoreceptor-generated cAMP in Brown Fat Cells

1997; Elsevier BV; Volume: 272; Issue: 52 Linguagem: Inglês

10.1074/jbc.272.52.32847

1083-351X

Jin Zhao, Barbara Cannon, Jan Nedergaard,

Neuropeptides and Animal Physiology

The relationship between cAMP levels and thermogenesis was investigated in brown fat cells from Syrian hamsters. Irrespective of whether the selective β3-, β2-, and β1-agonists BRL 37344, salbutamol, and dobutamine or the physiological agonist norepinephrine was used to stimulate the cells, increases in cAMP levels were mediated via the β3-receptor, as were the thermogenic effects. However, the relationship "thermogenesis per cAMP" was much lower for agents other than norepinephrine. Similarly, forskolin, although more potent than norepinephrine in elevating cAMP, was less potent in inducing thermogenesis. The selective α1-agonist cirazoline was in itself without effect on cAMP levels or thermogenesis, but when added to forskolin-stimulated cells, potentiated thermogenesis, up to the norepinephrine level, without affecting cAMP. This potentiation could not be inhibited by chelerythrine, but could be mimicked by Ca2+ ionophores. It was apparently not mediated via calmodulin-dependent protein kinase and was not an effect on mitochondrial respiratory control. The ability of all cAMP-elevating agents to induce thermogenesis in brown fat cells has earlier been interpreted to mean that it is only through the β-receptors and the resulting increase in cAMP levels that thermogenesis is induced. However, it is here concluded that the thermogenic response to norepinephrine involves two interacting parts, one mediated via β-receptors and cAMP and the other via α1-receptors and increases in cytosolic Ca2+ levels. The relationship between cAMP levels and thermogenesis was investigated in brown fat cells from Syrian hamsters. Irrespective of whether the selective β3-, β2-, and β1-agonists BRL 37344, salbutamol, and dobutamine or the physiological agonist norepinephrine was used to stimulate the cells, increases in cAMP levels were mediated via the β3-receptor, as were the thermogenic effects. However, the relationship "thermogenesis per cAMP" was much lower for agents other than norepinephrine. Similarly, forskolin, although more potent than norepinephrine in elevating cAMP, was less potent in inducing thermogenesis. The selective α1-agonist cirazoline was in itself without effect on cAMP levels or thermogenesis, but when added to forskolin-stimulated cells, potentiated thermogenesis, up to the norepinephrine level, without affecting cAMP. This potentiation could not be inhibited by chelerythrine, but could be mimicked by Ca2+ ionophores. It was apparently not mediated via calmodulin-dependent protein kinase and was not an effect on mitochondrial respiratory control. The ability of all cAMP-elevating agents to induce thermogenesis in brown fat cells has earlier been interpreted to mean that it is only through the β-receptors and the resulting increase in cAMP levels that thermogenesis is induced. However, it is here concluded that the thermogenic response to norepinephrine involves two interacting parts, one mediated via β-receptors and cAMP and the other via α1-receptors and increases in cytosolic Ca2+ levels. It is the generally accepted view (1Smith R.E. Horwitz B.A. Physiol. Rev. 1969; 49: 330-425Crossref PubMed Scopus (525) Google Scholar, 2Nedergaard J. Lindberg O. Int. Rev. Cytol. 1982; 74: 187-286Crossref PubMed Scopus (155) Google Scholar, 3Nicholls D.G. Locke R.M. Physiol. Rev. 1984; 64: 1-64Crossref PubMed Scopus (1353) Google Scholar, 4Cannon B. Nedergaard J. Essays Biochem. 1985; 20: 110-164PubMed Google Scholar, 5Bukowiecki L.J. Trayhurn P. Nicholls D.G. Brown Adipose Tissue. Edward Arnold, London1986: 105-121Google Scholar, 6Himms-Hagen J. Prog. Lipid Res. 1989; 28: 67-115Crossref PubMed Scopus (279) Google Scholar, 7Lafontan M. Berlan M. J. Lipid Res. 1993; 34: 1057-1091Abstract Full Text PDF PubMed Google Scholar) that the initial steps in the pathway leading from norepinephrine stimulation of brown fat cells to the final thermogenic reaction are quite well understood: through interaction with β-adrenergic receptors and transduction via Gs proteins, norepinephrine activates adenylyl cyclase. The sympathetic stimulation is thus intracellularly mediated by an increase in cAMP levels. cAMP, through its activation of protein kinase A and a subsequent activation of hormone-sensitive lipase, leads to release of free fatty acids from the triglycerides in the cells. The free fatty acids are combusted in the mitochondria; they are thus the substrate for thermogenesis, and they are also, in a less well understood way, involved in the activation of the uncoupling protein UCP1 (see for example, Ref. 8Nedergaard J. Cannon B. New Compr. Biochem. 1992; 23: 385-420Crossref Scopus (106) Google Scholar for review). It is an inherent implication of this model that it is the cAMP level in the brown fat cells that, during acute thermogenesis, solely determines the final outcome of the acute sympathetic stimulation. Thus, the means by which the cAMP is elevated is implicitly not of interest: any agent that has the ability to increase cAMP levels is also expected to elicit a proportionate increase in thermogenesis. However, this view has been so generally accepted that no detailed analysis has been presented. We present here data challenging this simple relationship and indicating that both a β3-receptor-induced increase in cAMP and an increase in Ca2+ caused by α1-adrenergic stimulation may in reality be of significance for the norepinephrine-induced thermogenic process. The presence of α1-adrenergic receptors in brown adipose tissue has long been demonstrated (9Mohell N. Svartengren J. Cannon B. Eur. J. Pharmacol. 1983; 92: 15-25Crossref PubMed Scopus (47) Google Scholar, 10Raasmaja A. Mohell N. Nedergaard J. Eur. J. Pharmacol. 1985; 106: 489-498Crossref Scopus (32) Google Scholar, 11Costain W.J. Mainra R. Desautels M. Sulakhe P.V. Can. J. Physiol. Pharmacol. 1996; 74: 234-240PubMed Google Scholar), but until now, the role of α1-receptors in the acute thermogenic function of the tissue has been considered to be small (12Mohell N. Nedergaard J. Cannon B. Eur. J. Pharmacol. 1983; 93: 183-193Crossref PubMed Scopus (57) Google Scholar, 13Schimmel R.J. McCarthy L. McMahon K.K. Am. J. Physiol. 1983; 244: C362-C368Crossref PubMed Google Scholar) or nonexistent (14Bukowiecki L. Follea N. Paradis A. Collet A. Am. J. Physiol. 1980; 238: E552-E563PubMed Google Scholar). We have thus here unveiled a hitherto overlooked role of α1-adrenergic stimulation and confer to the α1-receptors on brown fat cells a significant role also in the acute thermogenic process. Brown adipocytes were isolated with the collagenase method described earlier (15Zhao J. Unelius L. Bengtsson T. Cannon B. Nedergaard J. Am. J. Physiol. 1994; 267: C969-C979Crossref PubMed Google Scholar). Each preparation was from two adult (10–30-week old) Syrian hamsters (Mesocricetus auratus) of either sex. The hamsters had been kept at 20 ± 1 °C, one per cage, with food and water ad libitum. For parallel measurements of cAMP levels and thermogenesis (rate of oxygen consumption), 50,000–80,000 brown fat cells were added to a magnetically stirred oxygen electrode chamber (thermostated at 37 °C) containing 1.1 ml of Krebs-Ringer bicarbonate buffer (see "Buffers") and fitted with a Yellow Springs Model 4004 Clark-type oxygen probe. The suspension was covered with a lid, and the oxygen tension and oxygen consumption rate were continuously monitored. After 4 min, the agent was added with a syringe through a hole in the lid, and the oxygen consumption was followed. After 10 min, the incubation was terminated by taking a 0.5-ml aliquot of the suspension and transferring it to 1 ml of 99.5% ethanol. This suspension was dried in a Speedvac centrifuge for 12 h at room temperature. The dried samples were dissolved in 200 μl of buffer 1 provided with the [3H]cAMP assay system from Amersham Corp. and centrifuged in an Eppendorf centrifuge at 12,000 rpm for 12 min. Two 50-μl aliquots of the supernatants were analyzed according to the description provided with the assay system. In certain experiments, only oxygen consumption was followed in the same experimental system. In certain experiments, only cAMP levels were followed in the same experimental system, but without the lid. For the time course experiments, a similar setup was used, but a chamber containing 4 ml of buffer was used, and successive sampling was performed. Krebs-Ringer phosphate buffer (used only for cell preparation and storage) had the following composition: 148 mm Na+, 6.9 mm K+, 1.5 mm Ca2+, 1.4 mm Mg2+, 119 mm Cl−, 1.4 mmSO42−, 5.6 mmH2PO4−, 16.7 mmHPO42−, 10 mm glucose, and 10 mm fructose. 4% crude bovine serum albumin was also included. The pH was adjusted with Tris-OH or Tris-HCl to 7.4. Krebs-Ringer bicarbonate buffer (used for all experiments) had the following composition: 145 mm Na+, 6.0 mm K+, 2.5 mm Ca2+, 1.2 mm Mg2+, 128 mm Cl−, 1.2 mm SO42−, 25.3 mm HCO3−, 1.2 mm H2PO4−, 10 mm glucose, 10 mm fructose, and 2% fatty acid-free bovine serum albumin. This buffer was purchased as a sterile solution from Statens Veterinärmedicinska Anstalt (Uppsala, Sweden). The buffer was bubbled with 5% CO2 in air, and the pH was adjusted with Tris-OH or Tris-HCl to 7.4; the buffer was continuously bubbled with a small stream of 5% CO2 in air until use. Crude bovine serum albumin and fatty acid-free bovine serum albumin (albumin fraction V) were from Boehringer Mannheim. l-Norepinephrine bitartrate (Arterenol), forskolin, dl-propranolol, prazosin, yohimbine, A23187, ionomycin, chelerythrine, EGTA, and crude collagenase (type II; clostridiopeptidase A, EC 3.4.24.3) were obtained from Sigma (as thedl-form of propranolol was used, p A 2values for l-propranolol are probably 0.3 units higher than the values given below). BRL 37344 was a gift from SmithKline Beecham Pharmaceuticals. Dobutamine (Dobutrex) was obtained as a solution for infusion from Lilly (Fegersheim, France). Salbutamol (Ventoline) was obtained as a solution for inhalation from Glaxo (Middlesex, UK). Cirazoline, KN-92, and KN-93 were obtained from Research Biochemicals International (Natick, MA). All adrenergic agents were freshly dissolved in water, except prazosin, which was dissolved in ethanol and diluted 1:10 in water for use. Forskolin and chelerythrine were dissolved and diluted in Me2SO. A23187, KN-92, and KN-93 were dissolved in Me2SO and diluted in water for use. EGTA was initially dissolved in dilute HCl. Dose-response curve data were, if not otherwise indicated, analyzed with the general fit option of the KaleidaGraph application for Macintosh for adherence to simple Michaelis-Menten kinetics, i.e. V(x) = basal +V max·(x/(EC50 +x)). The indicated uncertainties are those obtained from the fitting procedures. Where indicated, Michaelis-Menten kinetics with a free Hill coefficient were used for fitting, i.e. V(x) =V max·(x H/(EC50H+ x H)), where H indicates the Hill coefficient. When estimated from single-dose antagonist curve shifts, p A 2 values were calculated as p A 2 = log(C R − 1) − log[antagonist], where C R is the ratio between the EC50 values in the presence and absence of antagonist. For multiple-dose antagonist curve shifts, Schild plots were used as illustrated. To investigate the relationship between cAMP accumulation and thermogenesis in hamster brown fat cells, it was first necessary to establish the time course for adrenergically induced increases in cAMP levels. In Fig. 1 A, a time curve is shown for the norepinephrine-induced increase in cAMP content in suspensions of isolated brown fat cells. This experiment, as all following, was performed in the absence of any phosphodiesterase inhibitor; thus, it is the endogenously attained level of cAMP that is followed, rather than adenylyl cyclase activation as such, and the response includes the resultant hormonal effects, irrespective of whether they affect cAMP production or degradation. As seen, the initial increase was rapid, and the elevated cAMP levels were maintained for at least 20 min. There was thus no tendency to the transient "overshoot" behavior earlier observed in brown fat cell preparations incubated under somewhat different conditions (16Pettersson B. Vallin I. Eur. J. Biochem. 1976; 62: 383-390Crossref PubMed Scopus (61) Google Scholar, 17Pettersson B. Eur. J. Biochem. 1977; 72: 235-240Crossref PubMed Scopus (26) Google Scholar), and the cAMP levels were at least as well maintained as the norepinephrine-induced thermogenesis (18Nedergaard J. Lindberg O. Eur. J. Biochem. 1979; 95: 139-145Crossref PubMed Scopus (49) Google Scholar). Thermogenesis in these cells has been demonstrated to be mediated via β3-receptors (15Zhao J. Unelius L. Bengtsson T. Cannon B. Nedergaard J. Am. J. Physiol. 1994; 267: C969-C979Crossref PubMed Google Scholar), and we therefore also investigated the cAMP response to a β3-agonist, BRL 37344. This response had somewhat different kinetics than that to norepinephrine, and it did not reach the maximum before ≈10 min. At any time point, the level induced was slightly higher than that observed after norepinephrine. In all subsequent experiments, cAMP was determined 10 min after addition of agent. Dose-response curves for the action of norepinephrine and BRL 37344 on cAMP levels are shown in Fig. 1 B. High concentrations of norepinephrine were necessary to obtain a full effect on cAMP levels; the EC50 for norepinephrine for elevation of cAMP was ≈840 nm. This value should be contrasted with that associated with other effects of norepinephrine in these cells, such as thermogenesis (≤200 nm (e.g. Ref. 15Zhao J. Unelius L. Bengtsson T. Cannon B. Nedergaard J. Am. J. Physiol. 1994; 267: C969-C979Crossref PubMed Google Scholar and see below). In agreement with the results in the time course experiment, 1 μm BRL 37344 induced a higher level of cAMP than did 1 μm norepinephrine, although the maximal level attained by BRL 37344 was not different from that of norepinephrine. The EC50 was much lower than that for norepinephrine: 26 nm. Thus, as an elevator of cAMP levels in brown fat cells, BRL 37344 was 30-fold more potent than norepinephrine. That the elevation of cAMP levels was more efficiently induced by BRL 37344 than by norepinephrine may in itself be seen as an indication that it is via stimulation of the β3-adrenergic receptors that cAMP levels are increased. However, this does not unequivocally demonstrate that the stimulation occurs through β3-receptors. Therefore, to establish through which β-receptor norepinephrine (and BRL 37344) elevates cAMP levels in brown fat cells, we utilized the characteristic of β3-adrenergic receptors that they are relatively insensitive to conventional β-adrenergic antagonists, such as propranolol. For interaction of propranolol with β1- or β2-receptors, the p A 2 values should be 8–9, but with β3-receptors, only ≈6 (19Arch J.R.S. Proc. Nutr. Soc. 1989; 48: 215-223Crossref PubMed Google Scholar,20Arch J.R.S. Kaumann A.J. Med. Res. Rev. 1993; 13: 663-729Crossref PubMed Scopus (345) Google Scholar). Therefore, dose-response curves for elevation of cAMP levels were constructed in the absence and presence of a fixed dose of propranolol. In Fig. 2 A, it is shown that 8 μm propranolol was sufficient to induce a significant shift of the dose-response curve for BRL 37344 to the right. The p A 2 for propranolol on BRL 37344-induced cAMP elevation was only 5.9, indicating that, as expected, BRL 37344 increased cAMP by interaction with β3-receptors. When the effect of propranolol on norepinephrine was investigated, the p A 2 was also 5.9, implying that the physiological agonist norepinephrine also elevated cAMP levels through interaction with β3-receptors. The above interpretation of the response to norepinephrine being mediated through β3-receptors is based on the global response of the cells to norepinephrine (as if only one β-receptor type should be involved). It could not be excluded that a minor elevation was induced through other β-receptors. To increase the possibility of observing the activity of other β-adrenergic receptors, adrenergic agents that are selective for β1- or β2-receptors (here dobutamine and salbutamol) were tested. In the dose-response curve for dobutamine (Fig. 2 B), there was no measurable increase in cAMP at the low concentrations expected if dobutamine interacted with β1-receptors. No significant elevation of cAMP content was observed at agonist concentrations below 10 μm, and the EC50 was as high as 16 μm. In the presence of 8 μmpropranolol, only a marginal increase was observed at the highest dobutamine concentration, but this was still sufficient to estimate a p A 2 of 6.6. Thus, this selective β1-agonist did not display a high potency for increasing cAMP levels (or even an observable component with a high potency), and the observable action was probably through interaction with the β3-receptors. Thus, although β1-receptors are found on these cells (21Svoboda P. Svartengren J. Snochowski M. Houstek J. Cannon B. Eur. J. Biochem. 1979; 102: 203-210Crossref PubMed Scopus (35) Google Scholar), we found no evidence for them being coupled to increased cAMP levels. Very similar results were obtained with the selective β2-agonist salbutamol (Fig. 2 B). However, the absence of specific β2-receptor interaction was less unexpected, as no evidence has been presented for the presence of β2-receptors on these cells. It would therefore appear that in these isolated mature brown adipocytes, cAMP elevation is mediated only through β3-adrenergic receptors. All agents tested above increase cAMP and are therefore predicted to stimulate thermogenesis. This was naturally the case for norepinephrine and BRL 37344 (Fig.3 A), as amply observed earlier. In the present investigation, the EC50 for norepinephrine was 195 nm, and that for BRL 37344 was 24 nm. Thus, for thermogenesis, there was the same qualitative difference between the agents as for cAMP elevation: BRL 37344 was more potent than norepinephrine. There was, however, a remarkable quantitative difference: although BRL 37344 was 30-fold more potent than norepinephrine as an elevator of cAMP levels, it was only 8-fold more potent as a stimulator of thermogenesis. This in itself implied that the relationship between cAMP elevation and stimulation of thermogenesis was not simple. The stimulation of thermogenesis by norepinephrine and BRL 37344 has earlier been shown to have a p A 2 for propranolol of ≈6 (15Zhao J. Unelius L. Bengtsson T. Cannon B. Nedergaard J. Am. J. Physiol. 1994; 267: C969-C979Crossref PubMed Google Scholar), indicating that both these agents are coupled through β3-receptors (see also below). It is to be expected that the increase in cAMP brought about by the selective β1-agonist dobutamine or the selective β2-agonist salbutamol would also lead to thermogenesis, but this has not earlier been demonstrated. In Fig. 3 B, it is shown that these agents could also induce a full thermogenic response. To examine whether this response was mediated via a selective interaction of these agents with their intended receptors (β1 or β2) or unspecifically through β3-receptors, dose-response curves for thermogenesis were obtained for these agents (and, for comparison, for norepinephrine) in the presence of three different propranolol concentrations. From these curves (Fig. 4, A–C), Schild plots were constructed, and apparent p A 2 values for propranolol were calculated (Fig. 4 D). The p A 2 values were all between 5 and 6. Therefore, although the brown fat cells respond thermogenically to selective β1- and β2-agonists, it is clear that these agents mediated their effects through interaction with the β3-receptors. Since cAMP production and oxygen consumption were thus demonstrated (above) to be β3-receptor mediated processes, irrespective of which adrenergic agent was used, the prediction would be that, irrespective of which adrenergic agent is used, cAMP should be equally effective as a thermogenic stimulator. In Fig. 5 A, the rate of oxygen consumption (thermogenesis) stimulated by norepinephrine is plotted against the cAMP increase induced by norepinephrine; this is thus the dose-response curve for cAMP as a stimulator of thermogenesis. As expected, increases in cAMP levels correlated with increases in oxygen consumption until oxygen consumption peaked at the maximum capacity of the cells for thermogenesis. As can be understood from the graphs above and shown in Fig. 5 A, higher norepinephrine concentrations could further increase the cAMP levels, but this was not associated with a further increase in thermogenesis. Principally, a similar correlation was seen for the BRL 37344-stimulated increases in oxygen consumption and cAMP (Fig.5 A). However, there was an unexpected and important distinction: the relationships between cAMP levels and thermogenesis rates were not superimposable for the data sets for norepinephrine and BRL 37344. For any nonsaturating level of cAMP generated by BRL 37344 stimulation, the resulting thermogenesis was lower than that obtained with cAMP generated by norepinephrine stimulation. Only when cAMP levels were markedly increased by BRL 37344 was a full thermogenic response reached. Therefore, the view that there is a simple relationship between cAMP and thermogenesis is clearly not an adequate description of the situation. A similar set of curves (Fig. 5 B) was generated for dobutamine and salbutamol and compared with the norepinephrine response from the same series. It is again evident that the curves were not superimposable: cAMP generated by stimulation with the selective pharmacological ligands was apparently less thermogenically potent than that derived from norepinephrine stimulation. These differences in apparent cAMP potency are even more unanticipated when it is remembered that the increase in cAMP levels in all cases (both with norepinephrine and with the three selective adrenergic agents) is mediated through the same receptor, the β3-receptor (as demonstrated above). The difference between the potency of cAMP to stimulate thermogenesis when it originates from norepinephrine stimulation of the cells and when it originates from stimulation with the selective adrenergic agents could be due to a negative effect on thermogenesis of the selective agents or to a positive effect of norepinephrine as compared with the selective agents. To distinguish between these possibilities, it was necessary to increase cAMP levels in a non-adrenergic receptor-mediated way. Therefore, we investigated the thermogenic potency of cAMP generated by stimulation with forskolin, which directly stimulates adenylyl cyclase. In Fig. 6 A, dose-response curves for the action of forskolin on the level of cAMP, compared with norepinephrine, are presented. It is evident (and in agreement with Ref. 22Thonberg H. Zhang S.-J. Tvrdik P. Jacobsson A. Nedergaard J. J. Biol. Chem. 1994; 269: 33179-33186Abstract Full Text PDF PubMed Google Scholar) that forskolin was able to massively stimulate cAMP production, far in excess of the levels generated by norepinephrine. However, when the effect of forskolin as a stimulator of thermogenesis was investigated (Fig. 6 B), a remarkable reversal was observed: although forskolin was able to stimulate thermogenesis, norepinephrine was more potent. Forskolin was not even able to fully reach the level of norepinephrine-induced thermogenesis. Curves were therefore again constructed to analyze the relationship between cAMP levels and oxygen consumption. As is clear from Fig.6 C, a similar, but even more dramatic difference was seen between norepinephrine and forskolin as between norepinephrine and the selective agents (Fig. 5). The thermogenic potency of forskolin-generated cAMP was apparently markedly lower than that generated by norepinephrine. Thus, the higher thermogenic potency of cAMP generated by norepinephrine stimulation compared with that generated by the selective adrenergic agents (Fig. 5) was likely due to an additional positive effect of norepinephrine. There is, of course, an important difference between norepinephrine and the other agents studied here: norepinephrine interacts with adrenergic receptor subtypes other than β, i.e. α2- and α1-adrenoreceptors. Concerning possible α2-adrenergic effects, it has earlier been demonstrated that hamster brown fat cells apparently lack α2-adrenergic receptors (23McMahon K.K. Schimmel R.J. Life Sci. 1982; 30: 1185-1192Crossref PubMed Scopus (17) Google Scholar), and this pathway is therefore probably not relevant. However, isolated brown fat cells have a high density of α1-adrenergic receptors (9Mohell N. Svartengren J. Cannon B. Eur. J. Pharmacol. 1983; 92: 15-25Crossref PubMed Scopus (47) Google Scholar) coupled to activation of phosphatidylinositol bisphosphate turnover (24Mohell N. Wallace M. Fain J.N. Mol. Pharmacol. 1984; 25: 64-69PubMed Google Scholar), protein kinase C activation (25Barge R.M. Mills I. Silva E. Larsen P.R. Am. J. Physiol. 1988; 254: E323-E327Crossref PubMed Google Scholar), inositol 1,4,5-trisphosphate production (26Nånberg E. Nedergaard J. Biochim. Biophys. Acta. 1987; 930: 438-445Crossref PubMed Scopus (9) Google Scholar), and an increase in cytosolic Ca2+ (27Wilcke M. Nedergaard J. Biochem. Biophys. Res. Commun. 1989; 163: 292-300Crossref PubMed Scopus (50) Google Scholar). In the present circumstances, the possibility therefore existed that it could be through interaction with α1-receptors that norepinephrine potentiated the thermogenic effect of the β-adrenergically generated cAMP. Studies were therefore performed to investigate whether selective α1-adrenergic stimulation could potentiate the thermogenic response to a given cAMP level. To avoid possible problems with receptor specificity for selective adrenergic agents, we performed these studies in cells in which the cAMP level was elevated with forskolin. A dose of forskolin was chosen that (according to the data in Fig. 6 A) gave an increase in cAMP approximately similar to that induced by the dose of norepinephrine that gave maximal oxygen consumption. First, we investigated the effect of α1-adrenergic stimulation on thermogenesis. Results from a typical experiment are seen in Fig. 7. Forskolin, at the dose utilized, stimulated oxygen consumption to some 60% of the level stimulated maximally by norepinephrine. Cirazoline, a selective α1-adrenergic receptor agonist, was in itself unable to stimulate oxygen consumption at the concentration utilized here (and indeed at any concentration between 1 nm and 10 μm) (data not shown here, but compare with Fig.9 A). However, when the cells were stimulated with cirazoline in combination with forskolin, the forskolin-stimulated respiration markedly increased, irrespective of whether cirazoline was added together with or (as shown here) after forskolin.Figure 9Effect of cirazoline and calcium on the correlation between induced cAMP levels and stimulation of oxygen consumption. A, effect of cirazoline. Experiments were performed as described for Fig. 6. In each experimental series, the response to 1 μm norepinephrine (NE) was determined and used to normalize the results. The results of addition of different concentrations of cirazoline (1, 10, 100 nm, 1 and 10 μm) are plotted as open symbols. The effect of 3 μm forskolin (F) alone is indicated, and the results of adding forskolin plus the different cirazoline concentrations are shown (closed diamonds; from left to right, the diamonds represent the following cirazoline concentrations: 1 nm, 100 nm, 10 nm and 10 μm). The points are means ± S.E. from four independent cell preparations in which oxygen consumption and cAMP levels were determined in parallel.B, effect of Ca2+ ionophores. Experiments were performed as described for A, except that either ionomycin at different concentrations (closed triangles; from the lowest point: 0.1, 1, and 10 μm) was used instead of cirazoline or 10 μm A23187 (F + A23;inverted closed triangle) was used. The points are means ± S.E. from four independent cell preparations in which oxygen consumption and cAMP levels were determined in parallel.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In Fig. 8 A, a dose-response curve is shown for this cirazoline-mediated potentiation of forskolin-induced thermogenesis. The response was dose-dependent, with an EC50 of 23 nm, i.e. within the order of affinity expected for a cirazoline effect on α1-receptors. To confirm that the cirazoline-induced potentiation was indeed mediated via α1-adrenergic receptors, the effects of antagonists to α1- and α2-receptors on the potentiation were studied (Fig. 8 B). The selective α2-antagonist yohimbine was without significant effect, whereas the selective α1-antagonist prazosin completely blocked the cirazoline-mediated effect. Thus, provided that an increase in cAMP had been induced by forskolin, α1-adrenergic stimulation was able to nearly double the thermogenic effect of this amount of cAMP. To eliminate the possibility that the increase in thermogenesis caused by α1-adrenergic stimulation was due to an unexpected effect of cirazoline on cAMP accumulation, cAMP levels and thermogenesis were determined in parallel in the presence of forskolin and with varying cirazoline concentrations. The relationship between cAMP levels and oxygen consumption is plotted in Fig.9 A. It is shown (Fig. 9 A, open symbols, lower left corner) that cirazoline in itself increased neither thermogenesis nor cAMP levels. The concentration of forskolin used here (point F) gave a level of cAMP very close to that obtained with 1 μm norepinephrine (point NE), but only half the thermogenesis. The further presence of cirazoline (closed diamonds) had no dose-dependent effect on cA