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Mechanisms of ADRF release from rat aortic adventitial adipose tissue

Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Charité University Hospitals, Humboldt University of Berlin, HELIOS Klinikum Berlin, 13125 Berlin, Germany

Submitted 14 July 2003 ; accepted in final form 17 November 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Blood vessels are surrounded by variable amounts of adipose tissue. We showed earlier that adventitial adipose tissue inhibits rat aortic contraction by release of a transferable factor, adventitium-derived relaxing factor (ADRF), which activates smooth muscle K⁺ channels. However, little is known about the mechanisms of ADRF release. Using isolated rat aortic rings and isometric contraction measurements, we show that ADRF release depends on extracellular [Ca²⁺] (EC₅₀ 4.7 mM). ADRF effects do not involve neuronal presynaptic N-type Ca²⁺ and Na⁺ channels or vanilloid, cannabinoid, and CGRP receptors. ADRF release is strongly inhibited by the protein tyrosine kinase inhibitors genistein and tyrphostin A25. In contrast, daidzein, an inactive genistein analog, and the protein tyrosine kinase inhibitor ST638 had no effect. Protein kinase A inhibition by H89 also inhibited ADRF release, whereas the protein kinase G inhibitor KT-5823 had no effect. We propose that ADRF release is Ca²⁺ dependent and is regulated by intracellular signaling pathways involving tyrosine kinase and protein kinase A. Furthermore, ADRF release does not depend on perivascular nerve endings.

adipocyte-derived relaxing factor; smooth muscle; tyrosine kinase; protein kinase A; obesity; vascular dysfunction; arterial tone; adventitia-derived relaxing factor

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Local authorities approved the studies according to the Institutional Animal Care and Use Committee guidelines. Male Sprague-Dawley rats (200–300 g, 6–8 wk) were euthanized with ether. The thoracic aortas were removed, quickly transferred to cold (4°C) oxygenated (95% O₂-5% CO₂) physiological salt solution (PSS), and dissected into 5-mm-thick rings. Perivascular fat and connective tissue was either removed [(–)fat] or left intact [(+)fat]. The endothelium was not removed because we (19) previously observed that the anticontractile effects of perivascular adipose tissue and ADRF transferred between bath chambers were independent of the endothelium. After 1 h of equilibration, aortic ring contractile force, with or without perivascular fat, was measured isometrically using standard bath procedures, as described earlier (19). The basal tone was continuously monitored and adjusted to 1 g. The composition of PSS (in mM) was 119 NaCl, 4.7 KCl, 1.2 KH₂PO₄, 25 NaHCO₃, 1.2 Mg₂SO₄, 11.1 glucose, and 1.6 CaCl₂. The bath solution volume was 20 ml. Tension is expressed as a percentage of the steady-state tension (100%) obtained with isotonic external 60 mM KCl. Serotonin (5-HT; 1 or 2 µM) or phenylephrine (100 nM) was used to induce vasoconstriction of (–)fat or (+)fat aortic rings.

In the first series of bioassay experiments (protocol 1), we transferred aliquots (3–5 ml) of the bath solution from (+)fat preparations incubated in a donor bath chamber with different concentrations of external Ca²⁺ to (–)fat vessel preparations in an acceptor bath. The transfer interval of aliquots was 15–20 min; the aliquot volume was 3 or 5 ml. Transfer of bath solution aliquots from (–)fat aortic vessels or fresh PSS did not affect contraction of (–)fat vessel preparations in the acceptor bath chamber (19).

In the second series of experiments (protocol 2), (+)fat and (–)fat rings were first contracted with 1 to 2 µM 5-HT or 100 nM phenylephrine and then rinsed three times in PSS solution. The vessels were treated with different drugs. The second application of 5-HT or phenylephrine was repeated in the absence and presence of the drugs at desired concentrations. In some experiments, aortic contraction was induced by the cumulative addition of 5-HT to the bath solution (total concentrations, 0.7, 1, or 2 µM).

In the third series of bioassay experiments (protocol 3), we transferred aliquots (3–5 ml) of the bath solution from (+)fat preparations incubated in a donor bath chamber containing PSS plus different drugs. The aliquots were transferred to nontreated (–)fat vessel preparations in an acceptor bath (19). The transfer interval of aliquots was 15–20 min; the aliquot volume was 3 or 5 ml.

In the fourth series of bioassay experiments (protocol 4), we transferred aliquots (3–5 ml) of the bath solution from nontreated (+)fat preparations incubated in a donor bath chamber containing PSS to (–)fat vessel preparations in an acceptor bath. The (–)fat vessel preparations were pretreated with different drugs. The transfer interval of aliquots was 15–20 min; the aliquot volume was 3 or 5 ml.

The following chemicals and drugs were used (in µM): 1 CGRP8–37, 1 capsazepine, 1 AM-251, 1 AM-630, 1 tetrodotoxin (TTX), 1 -conotoxin GVIA, 10 AM-404, and 0.5 KT-5823. The dosages of the drugs and chemicals used have been reported to be adequate to produce the necessary antagonism of CGRP (13), vanilloid (capsaicin) VR1 receptors (23), cannabinoid CB1 receptors (37), cannabinoid CB2 receptors (37), Na⁺ and N-type Ca²⁺ channels (7, 22), carrier-mediated anandamide transport into nerves in vivo (6), and protein kinase G (25), respectively.

Genistein, tyrphostin A25 (AG82), daidzein, and ST-638 were used at 10 µM. The dosages of genistein and AG82 used have been reported to inhibit tyrosine kinase (34), whereas 10 µM daidzein and 10 µM ST-638 have been reported to act as inactive genistein analog and inactive tyrosine kinase inhibitor, respectively (34). H-89 was used at 9 µM. This dosage has been reported to inhibit protein kinase A (30, 35), but in some other studies also Rho kinase (3, 18, 31) and protein kinase G (2).

AM-251, AM-630, and capsazepine were from Tocris (Biotrend; Köln, Germany), KT-5823, H-89, genistein, daidzein, and AG82 were from Calbiochem (Beeston; Nottingham, UK). CGRP8–37 and TTX were obtained from Sigma (Taufkirchen, Germany). -Conotoxin GVIA (-CTX) was from Alomone Labs (Jerusalem, Israel).

All values are given as means ± SE. Paired and unpaired Student's t-tests were used as appropriate. A value of P < 0.05 was considered statistically significant; n represents the number of arteries tested. The data points in Fig. 1B were fitted with the use of Origin 5.0 software.

View larger version (13K): [in this window] [in a new window]   Fig. 1. A: dose-response relationship for serotonin-induced contractions of aortic rings without periadventitial fat [(–)fat], aortic rings after longitudinal removal of 50% perivascular adipose tissue (1/2 fat), and intact [(+)fat] aortic ring preparations (n 12). Thus the presence of periadventitial fat reduced the contractile force. The anticontractile effect depends on the amount of fat. B: adventitium-derived relaxing factor (ADRF) release is Ca2+ dependent. Doseresponse curves for the aortic relaxation of rings without perivascular fat [(–)fat] by ADRF released from aortic rings with perivascular fat tissue [(+)fat] at different external [Ca2+] in the donor bath solution. Aliquots (5 ml) of the bath solution from intact (+)fat aortic preparations incubated in a closed donor bath chamber were transferred to (–)fat vessel preparations in a closed acceptor bath chamber. C: representative experiment with donor and acceptor vessels incubated in normal PSS. The aliquots induced vasorelaxation. In B, donor preparations were incubated in 2 µM serotonin-containing bath solutions with different external [Ca2+] for 20 min. The volume of the bath chambers was 20 ml. After transfer of the aliquots from the 10 mM Ca2+-containing donor bath solution to the acceptor bath chamber, the bath solution was simultaneously replaced by a Ca2+-free physiological salt solution (PSS containing 0 mM Ca2+ and 0 mM EGTA). In this experiment, the total external [Ca2+] in the acceptor bath solution increased to 2.5 mM. In all other transfer experiments, the total external [Ca2+] in the acceptor bath chamber was kept constant at 1.6 mM after transfer of the aliquots. At 0, 0.3, 1.0, 1.6, 2.5, 5.0, and 10 mM [Ca2+], n = 4, 3, 3, 8, 5, 6, and 4 rings, respectively, from different animals for each [Ca2+]. 5-HT is serotonin (protocol 1).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Anticontractile effects of perivascular adipose tissue. Figure 1A shows the dose-response relationships for 5-HT-induced contractions of aortic rings without periadventitial fat [(–)fat], aortic rings after longitudinal removal of 50% perivascular adipose tissue (1/2 fat), and intact [(+)fat] aortic ring preparations. The dose-response curves were shifted to the right with increasing amounts of fat. Thus the inhibition of the contractile response to 5-HT by fat depends on the amount of fat on each ring.

Ca²⁺ dependence of ADRF release. To demonstrate that release of ADRF is a Ca²⁺-dependent process, we performed bioassay experiments (protocol 1), where we transferred aliquots of the bath solution from an intact donor preparation with perivascular fat incubated in 2 µM 5-HT-containing solution, to acceptor vessel preparations without periadventitial fat precontracted with 5-HT. This maneuver transferred ADRF released by either intact preparations or isolated perivascular adipose tissue to arteries without perivascular fat (19). Transfer of the donor bath solution aliquots containing the proposed ADRF to 5-HT-precontracted (–)fat aortic rings without periadventitial fat resulted in a dose-dependent relaxation (Fig. 1C). The effects of ADRF were reversed after washout of ADRF (Fig. 1C). There was no change in the biological activity when transfer intervals were increased up to 45 min. The bath solution aliquots containing ADRF were kept at 37°C in closed tubes. However, the relaxation did not occur when donor aortic rings with periadventitial fat were incubated in Ca²⁺-free external solution (PSS containing 0 mM Ca²⁺ and 0.5 mM EGTA) (Fig. 1B). Relaxation increased with increasing concentrations of [Ca²⁺] in the bath solution, in which (+)fat donor aortic rings with periadventitial fat were incubated. Dose-response curves for the aortic relaxation by ADRF released at different [Ca²⁺] are shown in Fig. 1B. The data points were well fitted (nonlinear least-squares fitting, ² = 93.7) by the following equation

where R is percent vasorelaxation, EC₅₀ is the [Ca²⁺] with half-maximal relaxation, N is the Hill coefficient, and [Ca²⁺] is the external Ca²⁺ concentration in which (+)fat donor aortic rings with periadventitial fat were incubated. Relaxation was half-maximal at 4.7 ± 1.2 mM [Ca²⁺] and N was 0.9 ± 0.2.

Role of Ca²⁺ and Na⁺ channels, vanilloid, cannabinoid, and CGRP receptors. We next studied whether or not synthesis or action of ADRF is dependent on vanilloid, cannabinoid, and CGRP receptors. Vanalloid and cannabinoid receptor agonists and CGRP can be released by perivascular nerve endings involving neuronal N-type Ca²⁺ and Na⁺ channels (5, 11, 15, 17, 20, 21) and induce vasorelaxation (9, 23, 28, 29, 36), at least in part by opening smooth muscle ATP-sensitive K⁺ (K_ATP) channels (12, 26, 35). The endocannabinoid anandamide causes vasorelaxation of sheep coronary arteries by cellular uptake and conversion of anandamide to a vasodilatory prostanoid (8). To test the hypothesis, we challenged (+)fat intact aortic rings and (–)fat aortic rings without periadventitial fat with 1 to 2 µM 5-HT in the absence or presence of pharmacological drugs. At 1–2 µM 5-HT, the contractile response of (+)fat rings was significantly lower than that of (–)fat vessels in all groups of pharmacological drugs. The CGRP antagonist CGRP8–37 (1 µM, n = 4, paired test) (13), the vanilloid (capsaicin) VR1 receptor antagonist capsazepine (1 µM, n = 4, paired test) (23), the cannabinoid CB1 receptor antagonist AM-251 (1 µM, n = 6, paired test) (37), the cannabinoid CB2 receptor antagonist AM-630 (1 µM, n = 8, paired test) (37), Na⁺ channel blocker TTX (1 µM, n = 4, paired test), and the presynaptic N-type Ca²⁺ channel blocker -conotoxin GVIA (-CTX, 1 µM, n = 4, paired test) (7, 22) did not affect vasocontraction of (+)fat and (–)fat rings (data not shown). AM-404 (10 µM), an inhibitor of carrier-mediated anandamide transport into nerves inhibiting anandamide inactivation in vivo (6), did not affect vasocontraction of (+)fat and (–)fat rings (n = 4 each; data not shown). These results are in line with our previous data indicating that ADRF is released from perivascular adipocytes rather than from nerve endings (19) and that ADRF is distinct from CGRP and cannabinoids.

Regulation of ADRF release by intracellular kinases. The protein kinase A/Rho kinase inhibitor H-89 (9 µM) (30, 35) reduced the anti-contractile response to perivascular fat (Fig. 2A), whereas H-89 (9 µM) showed no effect on aortic rings without perivascular fat (Fig. 2B). Pretreatment of (–)fat and (+)fat aortic rings with the Rho kinase inhibitor Y-27632 (3 µM) abolished completely aortic contraction by 5-HT (0.7, 1, and 2 µM) (n = 8, not shown). These results suggest that the difference in response to 5-HT between intact vessels and vessels without periadventitial fat is dependent on activation of protein kinase A.

View larger version (29K): [in this window] [in a new window]   Fig. 2. The anticontractile effect of periadventitial adipose tissue [(+)fat] was inhibited by the protein kinase A antagonist H-89 (n = 6, paired test, protocol 2) (A), whereas H-89 had no effect on aortic rings without perivascular adipose tissue [(–)fat] (n = 6, paired test, protocol 2) (B). Aliquots (3–5 ml) of the bath solution from (+)fat aortic preparations incubated in a closed donor bath chamber were transferred to (–)fat aortic preparations in a closed acceptor bath chamber. The aliquots induced relaxation of H-89-treated (–)fat acceptor aortic rings (n = 4, protocol 4) C: in contrast, aliquots of the bath solution of H-89 (9 µM) treated (+)fat aortic rings did not induce relaxation of (–)fat aortic rings (n = 4, protocol 3). D: aortic contraction was induced by cumulative addition of serotonin to the bath solution (total concentrations, 0.7, 1, or 2 µM). The bath chamber (20 ml) was replaced by equal volumes of the aliquots. The total volume of added aliquots is indicated in ml (3, 8, or 13 ml). The protein kinase G inhibitor KT-5823 (0.5 µM) had no effect on tension induced by serotonin (0.7 µM, 1 µM, 2 µM) in both (+)fat aortic rings (n = 6, paired test, protocol 2) (E) and (–)fat aortic rings (n = 6, paired test, protocol 2) (F). *P < 0.05; n.s., not significant.

We next performed bioassay experiments where we transferred aliquots of the bath solution from an intact donor preparation incubated in 2 µM 5-HT-containing solution, to 5-HT precontracted vessel preparations without periadventitial fat. This maneuver transferred ADRF released by perivascular adipose tissue to arteries without perivascular fat. Transfer of the donor bath solution aliquots containing the proposed ADRF to 5-HT-precontracted H-89 (9 µM) pretreated (–)fat aortic rings without periadventitial fat resulted in a dose-dependent relaxation (Fig. 2C). In contrast, transfer of the donor bath solution aliquots of the bath solution from an (+)fat intact donor preparation incubated in 2 µM 5-HT and 9 µM H-89-containing solution to 5-HT-precontracted (–)fat aortic rings without periadventitial fat did not induce relaxation (Fig. 2D). These results suggest that H-89 reduced the anticontractile effect of perivascular fat tissue by an inhibition of ADRF release. The protein kinase G inhibitor KT-5823 (0.5 µM) (25) did not affect the anticontractile response to perivascular fat (Fig. 2E), whereas KT-5823 (0.5 µM) showed no significant effect on aortic rings without perivascular fat (Fig. 2F).

We next tested the hypothesis that tyrosine kinase is involved in ADRF release. Figures 3A and 4A show that the tyrosine kinase inhibitors genistein (10 µM) and AG82 (10 µM) (34) reduced the anti-contractile response to perivascular fat (Fig. 3A; for other genistein data, see also Ref. 19 and Fig. 5A), whereas the inactive genistein analog daidzein (10 µM) and the inactive tyrosin kinase inhibitor ST-638 (10 µM) (34) showed no effect (Fig. 3A). Genistein (10 µM) and AG82 (10 µM) showed no effect on aortic rings without perivascular fat (Figs. 3B and 4B). Daidzein (10 µM) and ST-638 (10 µM) showed only small inhibitory effects on aortic rings without perivascular fat (Fig. 3B). At 100 nM phenylephrine, the contractile response of (+)fat rings was significantly lower than that of (–)fat vessels. These data suggest involvement of tyrosine kinase in action or synthesis of ADRF.

View larger version (16K): [in this window] [in a new window]   Fig. 3. The anticontractile effect of periadventitial adipose tissue [(+)fat] was inhibited by the tyrosine kinase inhibitor genistein (10 µM, n = 4, paired test) but not by inactive analog of genistein daidzein (10 µM, n = 4, paired test) and protein tyrosine kinase inhibitor ST-638 (10 µM, n = 4, paired test) in (+)fat aortic rings (A), whereas genistein (10 µM, n = 4, paired test) had no effect on (–)fat aortic rings and daidzein and ST-638 only slightly reduced contraction of (–)fat aortic rings (n = 4, each, paired tests) (B). Aortic rings were contracted by 100 nM phenylephrine (PE). Protocol 2 was used. Paired data were analyzed in all experiments. For graphic illustration, the left bars in A and B summarize the effects of 100 nM PE in the absence of genistein, daidzein, and ST-638 (n = 12). *P < 0.05.

View larger version (25K): [in this window] [in a new window]   Fig. 4. The anticontractile effect of periadventitial adipose tissue was inhibited by the tyrphostin A25 (AG82, 10 µM, n = 5, paired test, protocol 2) in (+)fat aortic rings (A), whereas AG82 (10 µM) had no effect on aortic rings without perivascular adipose tissue [(–)fat] (n = 6, paired test, protocol 2) (B). At concentrations of 0.7, 1, and 2 µM serotonin, the contractile response of nontreated (+)fat rings was significantly lower than that of nontreated (–)fat vessels (nonpaired test). Aliquots (3–5 ml) of the bath solution from (+)fat aortic preparations induced relaxation of AG82 10 µM treated (–)fat acceptor aortic rings (n = 4, protocol 4) (C). In contrast, aliquots of the bath solution of AG82 10 µM treated (+)fat aortic rings did not induce relaxation of (–)fat aortic rings (n = 4, protocol 3) (D). *P < 0.05.

View larger version (16K): [in this window] [in a new window]   Fig. 5. The anticontractile effect of periadventitial adipose tissue was inhibited by H-89 (9 µM) plus tyrphostin A25 (AG82, 10 µM, n = 9, paired test, protocol 2) in (+)fat aortic rings (A), whereas H-89 (9 µM) plus AG82 (10 µM) had no effect on (–)fat aortic rings (n = 5, paired test, protocol 2) (B). At concentrations of 0.7, 1, and 2 µM serotonin, the contractile response of nontreated (+)fat rings was significantly lower than that of nontreated (–)fat vessels (nonpaired test). *P < 0.05.

To study whether or not these effects are mediated by protein tyrosine kinase-mediated activation of K_ATP channels in aortic smooth muscle cells (27), we next performed bioassay experiments where we transferred aliquots of the bath solution from an intact donor preparation incubated in 2 µM 5-HT-containing solution to vessel preparations without periadventitial fat, precontracted with 5-HT. Transfer of the donor bath solution aliquots containing the proposed ADRF to 5-HT-precontracted AG82 (10 µM) pretreated (–)fat aortic rings without periadventitial fat resulted in a dose-dependent relaxation (Fig. 4C). In contrast, transfer of the donor bath solution aliquots of the bath solution from an intact (+)fat donor preparation incubated in 2 µM 5-HT and 10 µM tyrphostin A25-containing solution to 5-HT-precontracted (–)fat aortic rings without periadventitial fat did not induce relaxation (Fig. 4D). These results suggest that tyrosine kinase is involved in the regulation of ADRF release.

Figure 5 shows that the administration of H-89 (9 µM) and AG82 (10 µM) together reduced the anti-contractile response to perivascular fat, whereas H-89 (9 µM) and AG82 (10 µM) showed no effect on aortic rings without perivascular fat. The effects of H-89 (9 µM) plus AG82 (10 µM) were not additive, compared with the effects of H-89 (9 µM) or AG82 (10 µM) alone.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We performed bioassay experiments to characterize cellular mechanisms of ADRF release by rat aortic perivascular adipose tissue. Our data suggest that the release of ADRF from adventitial adipose tissue is a Ca²⁺-dependent process and that intracellular signaling pathways involving tyrosine kinase and protein kinase A regulate the process. Our data support the view that ADRF is released from perivascular adipocytes rather than from perivascular nerve endings. In agreement with this suggestion, we found that ADRF release and action is neither affected by inhibitors of neuronal presynaptic N-type Ca²⁺ channels and voltage-gated Na⁺ channels, nor by inhibitors of vanilloid/cannabinoid and CGRP receptors.

We have previously demonstrated that periadventitial adipose tissue markedly attenuates the contractile response to 5-HT, phenylephrine, and angiotensin II in aortic ring preparations (19). In bioassay experiments, we showed that periadventitial fat appears to release a substance (ADRF) into the organ bath that induces vasorelaxation. This substance(s) is relatively stable and can be transferred to smooth muscle tissue, where it exerts a similar anticontractile effect (19). Synthesis or action of ADRF is not dependent on the endothelium, cyclooxygenase or P-450 pathway, the formation of NO, activation of adenosine receptors, or presence of functional leptin receptors. ADRF is inactivated by heating and not adsorbed by essentially fatty acid-free serum albumin, suggesting that ADRF is more likely a protein than a lipid (19). Bioassay data with cultured adipocytes and fibroblasts (19) are in line with our hypothesis that adipocytes represent the source for production of ADRF.

In the present study, we determined the [Ca²⁺] dependency of the ADRF release process. We found that the relationship of ADRF release and [Ca²⁺] is relatively steep at physiological external [Ca²⁺]. Release of ADRF release was half-maximal at 4.7 mM (EC₅₀). We suggest that perivascular adipose tissue exhibits Ca²⁺-sensing properties, providing positive feedback for ADRF release and control of vascular tone. We studied the possible involvement of vanilloid, cannabinoid CB1/CB2, and CGRP. These substances are possible candidates for ADRF because they 1) can be released by perivascular nerve endings, involving N-type Ca²⁺ and Na⁺ channels (5, 10, 15, 17, 20, 21, 39), and 2) can induce vasorelaxation by opening smooth K_ATP channels (26, 35). We observed that CGRP8–37, the vanilloid (capsaicin) receptor antagonist capsazepine, the cannabinoid CB1 receptor antagonist AM-251, and the cannabinoid CB2 receptor antagonist AM-630 did not affect the anticontractile effects of perivascular adipose tissue. We also found that the Na⁺ channel blocker TTX, and the presynaptic N-type Ca²⁺ channel blocker -conotoxin GVIA, had no effect. Therefore, we suggest that ADRF is distinct from CGRP, vanilloid, and cannaboid receptor agonists releasable by perivascular nerve endings. Although our data with -conotoxin GVIA and tetrodotoxin are in line with the hypothesis that perivascular adipocytes produce ADRF, we cannot rule out the possibility that ADRF is released by components of the adventitium other than adipocytes. Further studies are needed to clarify this point.

Tyrosine kinase, protein kinase A, and Rho kinase are important intracellular signaling molecules in adipocytes (1, 4, 16, 33) and vascular smooth muscle cells (14, 24). We found that the release of ADRF is strongly inhibited by the tyrosine kinase inhibitors genistein and AG82. In contrast, the inactive analogues of genistein daidzein and ST-638 had no effect. Inhibition of protein kinase A by H-89 also inhibited the release of ADRF, whereas the protein kinase G inhibitor KT-5823 had no effect. AG82 and H-89 did not exhibit additive effects. We therefore propose that the release of ADRF from adventitial adipose tissue is a Ca²⁺-dependent process that is regulated by intracellular signaling pathways involving tyrosine kinase and protein kinase A. The release mechanism does not involve protein kinase G.

Tyrosine kinase has been reported to be involved in ₁-adrenergic vasoconstriction (38). However, we believe that this signaling pathway plays only a minor role in our preparation because daidzein and ST-638 exerted rather small inhibitory effects on phenylephrine-induced contraction in (–)fat aortic rings. It is possible that daidzein and ST638 exhibited nonspecific relaxant effects.

In conclusion, these results demonstrate an important functional role of external [Ca²⁺] in ADRF release by perivascular adipose tissue, which was until now not clear. Furthermore, this study provides indirect support that intracellular signaling pathways involving tyrosine kinase and protein kinase A regulate the process. Our data support the hypothesis that ADRF is released from perivascular adipocytes. The data show that presynaptic neuronal N-type channels, voltage-gated Na⁺ channels, and neurotransmitters activating canabinoid/vanilloid and CGRP receptors are not involved, which may play a major role in vasorelaxation by perivascular nerve endings. Release of ADRF is of interest in various pathophysiological conditions, including obesity and obesity-associated hypertension. The role and nature of ADRF in health and cardiovascular disease remains to be identified and further studies are needed.

	ACKNOWLEDGMENTS

 
We thank Kirill Essin for help in data analysis.

GRANTS

The Deutsche Forschungsgemeinschaft supported this work. We thank Dr. Cecilia J. Hillard for helpful discussions.

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. Gollasch, Franz Volhard Clinic, Wiltbergstrasse 50, 13125 Berlin, Germany (E-mail: gollasch{at}fvk-berlin.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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