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Differential Control of Adrenal and Sympathetic Catecholamine Release by ₂-Adrenoceptor Subtypes

Institut für Pharmakologie und Toxikologie (M.B., M.P., M.J.L., L.H.), Universität Würzburg, 97078 Würzburg, Germany; and Max-Planck-Institut für Biophysikalische Chemie (G.N., J.B.S.), 37077 Göttingen, Germany

Address all correspondence and requests for reprints to: Lutz Hein, Institut für Pharmakologie und Toxikologie, Universität Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany. E-mail: hein{at}toxi.uni-wuerzburg.de.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
In the adrenergic system, release of the neurotransmitter norepinephrine from sympathetic nerves is regulated by presynaptic inhibitory ₂-adrenoceptors, but it is unknown whether release of epinephrine from the adrenal gland is controlled by a similar short feedback loop. Using gene-targeted mice we demonstrate that two distinct subtypes of ₂-adrenoceptors control release of catecholamines from sympathetic nerves (_2A) and from the adrenal medulla (_2C). In isolated mouse chromaffin cells, ₂-receptor activation inhibited the electrically stimulated increase in cell capacitance (a correlate of exocytosis), voltage-activated Ca²⁺ current, as well as secretion of epinephrine and norepinephrine. The inhibitory effects of ₂-agonists on cell capacitance, voltage-activated Ca²⁺ currents, and on catecholamine secretion were completely abolished in chromaffin cells isolated from _2C-receptor-deficient mice. In vivo, deletion of sympathetic or adrenal feedback control led to increased plasma and urine norepinephrine (_2A-knockout) and epinephrine levels (_2C-knockout), respectively. Loss of feedback inhibition was compensated by increased tyrosine hydroxylase activity, as detected by elevated tissue dihydroxyphenylalanine levels. Thus, receptor subtype diversity in the adrenergic system has emerged to selectively control sympathetic and adrenal catecholamine secretion via distinct ₂-adrenoceptor subtypes. Short-loop feedback inhibition of epinephrine release from the adrenal gland may represent a novel therapeutic target for diseases that arise from enhanced adrenergic stimulation.

	INTRODUCTION

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ADRENOCEPTORS FORM THE interface between the endogenous catecholamines, epinephrine and norepinephrine, and a wide array of target cells in the body to mediate the biological effects of the sympathetic nervous system. To date, nine different adrenoceptor subtypes have been identified (three ₁, three ₂, three ß receptors) (1). Transgenic mouse models have recently become very valuable in the identification of the physiological significance of individual adrenergic receptor subtypes in vivo (2, 3, 4, 5, 6, 7).

The adrenergic system can be divided into three parts: adrenergic neurons in the central nervous system, sympathetic neurons that innervate many organs and tissues, and the adrenal medulla. Norepinephrine is the principal neurotransmitter of sympathetic neurons, whereas epinephrine, and to a smaller degree also its precursor norepinephrine, is secreted from the chromaffin cells of the adrenal medulla into the circulation (8). Thus, the actions of norepinephrine are mostly restricted to the sites of release from sympathetic nerves, whereas epinephrine acts as a hormone to activate cardiovascular and metabolic responses via the blood stream (9). As adrenal chromaffin cells and sympathetic neurons are both derived from a common neural crest precursor (10), it may be hypothesized that both cell types share similar patterns of regulation, including feedback control of neurotransmitter release.

Norepinephrine released from sympathetic nerves not only activates adrenoceptors on effector cells, but also stimulates presynaptic adrenoceptors to inhibit further transmitter secretion (11, 12). _2A- and _2C-adrenergic receptors were previously identified as presynaptic inhibitory receptors that control the release of norepinephrine from peripheral sympathetic nerves and central catecholaminergic neurons in vitro (13, 14, 15). Furthermore, the deletion of the _2A- and _2C-adrenergic receptor subtypes caused increased susceptibility to development of heart failure after chronic pressure overload in vivo (16). In contrast, it is unknown at present whether secretion of catecholamines from the adrenal medulla is under similar feedback control. To assess the existence and physiological relevance of feedback regulation in the two limbs of the sympathetic system, we used mice lacking individual ₂-adrenoceptor subtypes (14, 17). Here we demonstrate for the first time that two distinct ₂-receptor subtypes control sympathetic nerve (_2A) and adrenal (_2C) catecholamine release and that autocrine feedback regulation of epinephrine secretion in the adrenal gland is necessary to control circulating epinephrine levels.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Regulation of Plasma and Urine Catecholamine Levels by ₂-Adrenoceptors
Norepinephrine and epinephrine levels were determined in the plasma of anesthetized wild-type (WT) and ₂-receptor knockout (₂-KO) mice. Surprisingly, plasma catecholamines were differentially affected by deletion of the different ₂-receptor subtypes (Fig. 1). Deletion of the _2A-receptor led to increased plasma levels of norepinephrine whereas epinephrine concentrations were selectively elevated in _2C-receptor-deficient mice (Fig. 1B). These findings were confirmed in urine samples collected over a period of 24 h in awake, freely moving animals (Fig. 1, C and D). Excretion (24 h) of norepinephrine was increased by 155% in _2A-KO mice and epinephrine excretion was enhanced by 95% in _2C-KO animals. Urine volume did not differ between WT and _2B- or _2C-KO mice, but was significantly elevated in _2A-KO mice (+95 ± 15%, _2A-KO vs. WT; P < 0.05).

View larger version (31K): [in this window] [in a new window]   Fig. 1. Plasma and Urine Catecholamine Levels in 2-Adrenoceptor-Deficient Mice Norepinephrine (A and C) was found to be elevated in blood plasma (obtained under anesthesia) and in urine (collected in metabolic cages over 24 h) from mice lacking 2A-adrenoceptors as compared with WT mice. In contrast, epinephrine levels were significantly higher in 2C-adrenoceptor-deficient mice when compared with the other genotypes (B and D). These results suggest that 2A- and 2C-receptors differentially control catecholamine release from sympathetic nerves and from adrenal chromaffin cells, respectively (schematic drawing). *, P < 0.05, KO vs. WT, means ± SEM, n = 13–25.

Expression of _2C-Adrenoceptors in Adrenal Chromaffin Cells
Before investigating the function of isolated mouse chromaffin cells, we analyzed adrenal histology and expression patterns of ₂-adrenoceptor subtypes. Adrenal glands from male _2C-receptor-deficient mice were significantly larger than organs from WT or _2A- or _2B-KO mice [_2C-KO adrenal weight: +31 ± 8% vs. WT; P < 0.05; _2C-KO, n = 14; WT, n = 12]. Cross-sections of adrenal glands did not reveal any structural defects in specimens from ₂-receptor-deficient mice (Fig. 2), except for mild hypertrophy of chromaffin cells from _2C-KO mice (Fig. 2C; cross-sectional area +28 ± 5%, _2C-KO vs. WT). In isolated chromaffin cells from WT mice, only _2C-receptor mRNA expression could be readily detected (Fig. 2D). _2A- or _2B-receptor mRNA could not be amplified reproducibly from chromaffin cells that were either isolated from WT mice or from _2C-receptor-deficient animals (Fig. 2D). Thus, genetic deletion of the _2C-receptor gene was not accompanied by a compensatory up-regulation of _2A- or _2B-receptor expression. In the human adrenal gland, _2C-adrenoceptors are also the predominating ₂-receptor subtype (27, 28).

View larger version (51K): [in this window] [in a new window]   Fig. 2. Adrenal Gland Morphology and 2-Adrenoceptor Expression in WT and 2C-Adrenoceptor-Deficient Mice Cross-section through the adrenal gland of a male WT mouse (A) and high-magnifications of adrenal medulla chromaffin cells of WT (B) or 2C-KO (C) mice. Chromaffin cells in the adrenal cortex of 2C-KO mice showed normal morphology but appeared enlarged by 28% in their cross-sectional area as compared with WT cells (B vs. C). C, Adrenal cortex; M, adrenal medulla; arrow, border between adrenal cortex and medulla. Bars, 300 µm (A); 5 µm (B and C). D, Isolated chromaffin cells from WT mice expressed 2C-adrenoceptors as detected by RT-PCR but no 2A- or 2B-adrenoceptors. In 2C-deficient chromaffin cells, no compensatory expression of 2A- or 2B-receptor was detectable. As a possible control, RT-PCR signals for 2A-, 2B-, and 2C-receptors from WT mice at embryonic day 9.5 (E9.5) are shown (42 ). No signals were observed in the absence of reverse transcriptase (not shown). Lower panel, Ethidium bromide-stained gel of 18S and 28S RNA after extraction from cells.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Elevated Tissue L-dopa Levels in 2-Adrenoceptor-Deficient Mice Adrenal gland (A) and cardiac (B) concentrations of L-dopa were determined as an indicator of tyrosine hydroxylase activity. Adrenal L-dopa levels were selectively elevated in mice lacking 2C-adrenoceptors, whereas L-dopa in the heart was significantly increased only in 2A-KO mice. *, P < 0.05 KO vs. WT; n = 6–8 animals per genotype; means ± SEM.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Inhibition of Exocytosis and Voltage-Activated Calcium Currents by 2C-Adrenoceptors in Mouse Adrenal Chromaffin Cells Voltage protocol applied to stimulate secretion (A, upper panel) and a typical voltage clamp recording (A, lower panel). Average capacitance traces (B and C) and average Ca2+ currents evoked by ramp depolarization (D and E). UK, 2-Agonist UK14,304. In isolated WT mouse chromaffin cells, the 2-agonist UK14,304 inhibited the depolarization-evoked capacitance increase (B and F) as well as the peak Ca2+ current (D and G). The inhibitory effect of the 2-agonist on cell capacitance and calcium currents was completely abolished in chromaffin cells from 2C-receptor-deficient mice (C, E, F, and G). *, P < 0.01, means ± SEM (WT, n = 20; WT + UK, n = 19; KO, n = 22; KO + UK, n = 22).

_2C-Adrenergic Receptors Inhibit Adrenal Secretion of Epinephrine and Norepinephrine
Acetylcholine-stimulated secretion of epinephrine could be inhibited by the ₂-agonist UK14,304 in chromaffin cells from WT, _2A-KO, and _2B-KO mice but not in cells from _2C-deficient mice (Fig. 5). Similarly, the secretion of norepinephrine from isolated chromaffin cells was inhibited by an ₂-agonist in WT cells but not in _2C-receptor-deficient cells (not shown). In WT chromaffin cells, epinephrine secretion induced by 80 mM K⁺-mediated depolarization was inhibited by the ₂-agonist UK14,304 by 72 ± 9%. Basal catecholamine release and the degree of acetylcholine-induced stimulation of release did not differ between WT and ₂-KO chromaffin cells (not shown). These findings demonstrate that in isolated chromaffin cells as in the adrenal, the _2C-adrenergic receptor subtype is the dominant regulator of catecholamine secretion. Due to their slower deactivation kinetics and higher affinity for catecholamines (31), _2C-receptors may be ideal receptors to sense and control the hormone epinephrine, whereas low-affinity _2A-receptors act as fast inhibitory regulators at the sympathetic nerve terminals (14, 32). Taken together, these results not only confirm that the _2A-subtype is the predominant receptor involved in the presynaptic regulation of norepinephrine from sympathetic nerves, but also definitely establish that the _2C-adrenergic receptor is an essential feedback regulator of epinephrine release from the adrenal gland.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Inhibition of Epinephrine Secretion by 2C-Adrenoceptors in Mouse Adrenal Chromaffin Cells Acetylcholine-induced epinephrine secretion from WT or 2A-KO and 2B-KO chromaffin cells was inhibited by the 2-agonist UK14,304. In chromaffin cells from 2C-KO mice, no effect of the 2-receptor agonist was observed (n = 4–6). *, P < 0.05, agonist vs. control; means ± SEM.

Conclusions
This is, to our knowledge, the first report demonstrating that _2C-adrenoceptors control secretion of the stress hormone epinephrine via an autocrine feedback loop in adrenal chromaffin cells and that genetic disruption of this feedback mechanism leads to enhanced blood levels of epinephrine.

The paradigm for endocrine feedback control is the hypothalamic-pituitary system, which releases tropic hormones to control peripheral endocrine glands, including the gonads, thyroid gland, and adrenal cortex. The conventional view is that hormones produced in the peripheral organs (e.g. glucocorticoids, thyroid hormone) feed back on the release of the hypothalamic-pituitary tropic hormones (e.g. CRH, TRH) via a long feedback circuit. However, recent experimental evidence suggests that, even in these systems, ultrashort feedback loops operate to control the tropic hormones, e.g. TRH inhibits its own synthesis (33). Ultrashort feedback control of GnRH may actually be required for pulsatile secretion of GnRH release from hypothalamic neurons (34). These examples illustrate that short and long feedback circuits operate together in the regulation of endocrine secretion. In this regulatory network, short feedback loops may have greater physiological and pathophysiological relevance than previously appreciated. Thus, _2C-adrenoceptors may represent novel therapeutic targets to attenuate or prevent the development of diseases that arise from overactivity of the adrenergic system.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
₂-Adrenoceptor-Deficient Mice
The generation of the mouse lines lacking ₂-adrenoceptor subtypes has been described previously (13, 17, 35). All animal procedures were approved by the responsible university and government authorities (Protocol No. 621-2531.01-28/01).

Catecholamine Determination
Catecholamines were measured in plasma obtained from tribromoethanol-anesthetized mice, from urine samples that were collected over a 24-h period in metabolic cages, or from the supernatant of chromaffin cells cultured in vitro. Adrenal and cardiac catecholamine levels were determined after extraction as described previously (13). Epinephrine, norepinephrine, and L-dopa were quantified by HPLC combined with electrochemical detection (14).

Chromaffin Cells
To obtain chromaffin cells, adrenal glands from 8- to 12-wk-old mice were incubated in Locke’s solution containing 1 mg/ml collagenase (>200 U/ml; Biochrom KG, Berlin, Germany) (36). Cells were resuspended in HEPES-buffered Tyrode’s solution (for determination of catecholamine release) or in enriched DMEM and plated on glass coverslips (for electrophysiological experiments). Enriched DMEM (Linaris, Wertheim, Germany) contained: 2.2 g/liter NaHCO₃, 4.5 g/liter D-glucose, 1.028 g/liter L-glutamine, 10 ml/liter insulin-transferrin-selenium-X (Life Technologies, Inc., Gaithersburg, MD), penicillin and streptomycin Life Technologies, Inc.). For detection of ₂-adrenoceptor expression, RNA was isolated from 100–150 chromaffin cells using the RNAqueous 4PCR kit (Ambion, Austin, TX). RT-PCR was performed with 0.2 µg of RNA as described previously (35, 37, 38).

Electrophysiological Measurements and Determination of [Ca²⁺]_I
The bath solution contained (in mM): 145 NaCl, 2.8 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, 1 mg/ml D-glucose (pH 7.2) (osmolarity, 310 mosmol/liter). After the whole-cell patch was established, the cell was perfused locally with a solution containing: 25 tetraethylammonium chloride, 48 Na-gluconate, 60 Na-acetate, 2.8 K-gluconate, 10 CaCl₂, 1 MgCl₂, 10 HEPES, 1 mg/ml D-glucose, 600 nM tetrodotoxin (pH 7.2) (osmolarity was adjusted to 310 mosmol/liter). The pipette solution contained: 70 Cs-glutamate, 30 CsCl, 8 NaCl, 0.125 CaCl₂, 32 HEPES, 2 MgATP, 0.3 GTP, 0.5 fura-2 (Molecular Probes, Eugene, OR) (pH 7.2) (osmolarity was adjusted to 300 mosmol/liter). The calculated free [Ca²⁺]_i was approximately 80 nM.

Conventional whole-cell recordings were performed at 30 C with sylgard-coated 3–5 M pipettes (Kimax-51; Kimble Products, Vineland, NY) 1–2 d after cell preparation. Access resistance ranged from 5–20 M. An EPC-9 patch clamp amplifier was used together with the Pulse software package (HEKA Electronics, Lambrecht, Germany). Capacitance measurements were performed using the Lindau-Neher technique as described previously (39). A 1000-Hz, 70-mV peak-to-peak sinusoid voltage stimulus was superimposed onto a direct current holding potential of minus 70 mV. Currents were filtered at 3 kHz and sampled at 12 kHz. A triple depolarization voltage protocol was applied 2 min after the whole-cell configuration was established and after 2-min local perfusion. Peak current was estimated from the first (ramp) depolarization after leak substraction as indicated in Fig. 2A, right panel. Cell size, leak current, and access resistance were not different between the groups (data not shown). Fluorescence measurements were conducted as described previously (40).

	ACKNOWLEDGMENTS

 
The authors are indebted to Professor E. Neher (Max Planck Institute, Göttingen, Germany) for critical discussions, to Kerstin Hadamek for technical assistance, and to Dr. Moritz Bünemann for assistance with the electrophysiology and critical discussion throughout this project.

	FOOTNOTES

 
This study was supported by the Deutsche Forschungsgemeinschaft (DFG) (in support of L.H. and M.J.L.). G.N. is a Ph.D. student of the International M.D./Ph.D. Program in the Neurosciences at the International Max Planck Research School and holds a fellowship from the DFG (DFG Graduiertenkolleg 521).

M.B. and G.N. have contributed equally to this manuscript.

Abbreviations: [Ca²⁺]_i, Intracellular calcium concentration; L-dopa, 3-(3,4-dihydroxyphenyl)-L-alanine; ₂-KO, ₂-receptor knockout; WT, wild-type.

Received for publication January 31, 2003. Accepted for publication May 12, 2003.

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