Fundam Clin Pharmacol (1992) 6 , Suppl 1, 23s-29s (9 Elsevier. Paris
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IMIDAZOLINE RECEPTORS IN THE NERVOUS SYSTEM Donald J. Reis, S. Regunathan, Hong Wang, Douglas L. Feinstein and Mary P. Meeley Div. of Neurobiol., Dept. of Neurol. & Neurosci., Cornell Univ. Med. Coll., New York, NY 10021. 1.
INTRODUCTION
A. lmldarollne receptors In brain. The original insights of Karppanen (1) and Bousquet (2) that the central antihypertensive actions of the imidazoline clonidine could not be attributed to interactions with q-adrenergic receptor (AAR) but related to the imidazoline structure of the drug gave birth to the concept of a novel receptor referred to here as the imidazoline receptor (IR). The original concept also indicated per force that IRs were localized in brain. More direct evidence for the existence of IRs in brain (3)and other kidney (4) was subsequently established by ligand binding studies which indicated that the binding of 3H-para-aminocbnidine(3H-PAC)to membranes of ventral medulla, the site of the antihypertensive actions of clonidine (5) and rilmenidine (6)was fully displaceable by clonidine yet only partially by adrenergic ligands. Non-adrenergic binding was displaceable by agents which were imidazoles, imidazolines (e.g. imidazole 4-acetic acid, cimetidine, cirazoline), some guanidines (e.g. guanabenz, amiloride) and oxazoles (rilmenidine).Thus, these agents bound to both AARs and another binding site considered to be the IR. That IRs in the CNS are functional has, however, largely been indirect. That IRs mediate the hypotensive action of clonidine and rilmenidine in the ventral medulla has been assumed based on evidence that the reduction in arterial pressure elicited by the microinjection of agents into the rostra1 ventrolateral medulla (RVL) correlates with binding at IRs and not AARs and that the microinjection of idazoxan but not of selective AAR antagonists into RVL will block the systemic fall of AP elicited by intravenous clonidine or rilmenidine. There is also indirect evidence that the neuroprotectiveactions of idazoxan and rilmenidine can be attributed to actions at nonadrenergic receptors presumably representing IRs (7). On the other hand several of the central actions of clonidine including analgesia and sedation appear to represent the actions of the agents at AARs.
B. Subtypes of imldazoline receptors. The earliest ligand-binding studies used 3H-PACas ligand and assumed that the IR in brain was a single entity with a distribution differing in part from AARs. Thus, IRs were detected in membranes of the ventral medulla but not in cerebral cortex (3).However, the discovery of a high-affinity binding of 3Hidazoxan, a radiolabeled imidazoline and o;-adrenergic antagonist, to non-adrenergic receptors in cerebral cortex suggested the existence of an "idazoxan receptor" also distinct from AARs ( 8 ) . In the light of more recent studies, it is now evident that IRs, like most other receptors, exit as multiple subtypes one of which is identical to the so-called "idazoxan receptor" (9). Two principal subtypes have been identified based on ligand binding. One, a clonidine-preferring subtype, is labeled by 3H-PAC(and also 3H-idazoxan)exhibits a rank order of potency for displacing agents (K,s) of: phentolamine > para-aminoclonidine > clonidine > idazoxan > cimetidine > imidazole 4-acetic acid >> cirazoline >> amiloride. A second, an idazoxan-preferring subtype labeled by 3H-idazoxan (but not 3H-PAC)has a rank order for displacement of 3H-idazoxanof: cirazoline > idazoxan > naphazoline > amiloride > clonidine >> imidazole 4-acetic acid. We have referredto the clonidine-preferringreceptor as the 1-1, and the idazoxanpreferring receptor as the 1-2 receptor subtypes with the latter subdivided on the basis of whether it shows high (I-2a) or low (I-2b) affinity for amiloride. C. Some issues relating to characterization of imidazoline binding sites as "receptors". The identification of the IR and its subtypes has demonstratedthat the IR in brain and other tissues represents a novel ligand binding site. However, remaining to be established is: (a) characterization of the topographic, cellular and subcellular distribution of IRs in neural tissue; (b) evidence that the binding site is functional receptor by demonstration of a signal transduction process and cellular event associated with its stimulation; (c) identification of an endogenous ligand for the receptor; and (d) structural evidence that IRs and AARs are distinct. Complicating the analysis of IR function in general and in CNS in particular are the facts that all agents that bind to IRs also bind to AARs (although the converse is not true) and all tissues expressing IRs also express AARs. We have recently, however, made two discoveries which have permitted a more detailed analysis of IRs in brain and related neuronal tissue. The first is that IRs but not AARs are expressed in chromaffin cells of adrenal medulla, a cell which is phenotypically and developmentally neuronal (10). This discovery has permitted these cells to serve as models in which to analyze signal transduction and functional responses to occupancy of IRs uncontaminated by responses of AARs and, moreover, are a source of genes, message and receptor-related proteins distinct from AARs. Second is the discovery that astrocytes express IRs providing a cell of CNS in which function and structure of IRs can be analyzed in vitro.
DJ Reis ef a/
24s
We review here some of these studies which have indicated that both cell types express IRs of the 1-2 subclass which are concentrated on mitochondria1membranes, that stimulationof 1-2 receptors of chromaffin cells stimulated Ca'* fluxes and release of catecholamines, replicated in part by the purported endogenous ligand for IRs, clonidine displacing substance (CDS), that the absence of AARs in chromaffin cells has permitted the isolation of a selective imidazoline binding protein and that stimulation of astrocytic IRs results in increased expressionof mRNA for a glial selective protein, glial fibrillary acidic protein (GFAP). IRS OF THE ADRENAL CHROMAFFIN CELL
11.
A. Blndlng of llgands and CDS. Membranes of chromaffin cells of bovine adrenal medulla exhibit saturable, high affinity binding for 3H-idazoxanbut not 3H-PAC. Binding is to a single site with a K, of 5 nM. 3H-ldazoxanbinding is displaceable by drazoline and idazoxan (with respective Kis of 1 and 5 nM) but not by epinephrine nor rauwolscine (Fig. 1). Thus, chromaffin cells express IRs but not AARs and the IR is of the 1-2 subclass (10).
5
100
U
80
C
3
0
rn
$
60
Flg. 1. The displacement of binding of 3 H - i d a ~ ~ ~ a n to chromaffin cell membranes by various agents. Membranes (100-150 pg protein) were incubated in Tris-HCI buffer at 25°C for 30 min with 5 nM 3Hidazoxan. The total counts ranged between 2500 to 3000 dpm and the non-specific binding, defined using 10 pM idazoxan. was about 30% of total binding.
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p
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m V ._
._
v
20
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a
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-8
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Log (M)
When we examined the subcellular distribution of idazoxan binding sites in nuclear, mitochondrial, and plasma membrane fractions of chromaffin cells and compared them with membranes of the entire cell (crude fraction) we observed that the receptor was heavily concentrated in mitochondria, poorly represented in the plasma membrane and absent in the cell nucleus. Thus, while the K, of 3H-idazoxanbinding to crude, mitochondrial and plasma membranes were comparable (from 5-7 nM), mitochondriahad 7-fold more binding sites (750 fmoVmg protein) than crude membrane (Fig. 2).
a
7501
F/g. 2. Binding of 3H-idazoxan and activities of marker enzymes in subcellular fractions of adrenal medulla. The number of binding sites (6,) was calculated from Scatchard analysis of specific binding to each fraction. Enzyme activities in crude membrane fractions were: cytochrome oxidase, 0.601 2 0.051 unitslmg protein and 5' nucleotidase, 2.8 2 0.24 unitslmg protein and enrichment was calculated as percent over crude membrane fraction.
Session I
-
Central imidazoline preferring receptors : comparison with a,-adrenoceptors
25s
The potency profile for other ligands, assessed by displacement of 3H-idazoxanfrom mitochondria1membranes was: cirazoline > idazoxan > naphazoline > amiloride > detomedine > clonidine >> phentolamine = cimetidine = piodoclonidine = imidazole 4-acetic acid >> epinephrine = norepinephrine = rauwolscine. This order indicates that the IR of adrenal chromaffin cells is of the 1-2 subclass. Since binding is sensitive to arniloride it is similar to kidney and liver receptors in rabbit (11,12) but distinct from brain receptors which are insensitive to amiloride (8). We term the amiloridesensitive receptor as I-2a subtype in contrast to the amiloride-insensitive I-2b subtype. The binding of 3H-idazoxanto mitochondrial membranes of chromaffin cells was inhibited by K+ (K,: 75 mM) and the K' channel antagonists 4-aminopyridine and tetraethylammoniumbromide (K,s: 20 pM, 100 pM, respectively) but not by Na'. Since adrenergic receptors are sensitive to Na+the finding further supports the independence of IRs and AARs and indicates the close relationship between IRs and AARs. We also investigated the binding to mitochondrialmembranes of chromaffin cells of clonidine displacing substance (CDS) the putative endogenous ligand for IRs (13). CDS potently displaces %I-idazoxan binding to chromaffin cell membranes with an IC, of 5 Units (Fig. 3). The displacement of ?i-idazoxan binding by CDS is not modified by Gpp(NH)p, suggesting that IRs of the 1-2 type are not G-protein coupled receptors (see below). Since CDS also displaces 3H-PACfrom membranes of bovine ventral medulla (14) the results indicate that CDS binds to both 1-1 and 1-2 receptors, a pattern to be expected of an endogenous ligand.
I
Fig. 3. Inhibition of 3H-idazoxanbinding to adrenal chromaffin cell membranes by CDS. Membranes were incubated with 5 nM 3H-idazoxanat 25°C for 30 min.
B. Signal transduction and secretion. The fact that chromaffin cells express IR without AARs provides a cell system in which the biological actions of IR occupancy can be examined without contamination by actions upon AARs. We therefore investigated classical signal transduction pathways following the activation of IR. We observed that treatment of chromaffin cells with a series of agents acting at IRs had no effect on PI turnover and did not stimulate the accumulation nor inhibit the forskolin-activatedaccumulation of CAMP (15). Although clonidine elicited a slow, modest and PDE- inhibitable accumulation of cGMP the response was idiosyncratic and limited to clonidine. On the other hand, imidazolines like clonidine, oxymetazoline, rilmenidine and naphazoline increased the flux of "Ca" into chromaffin cells. Incubation of chromaffin cells with agents binding to IRs including CDS also released catecholamines. The response to drugs was modest (1.3-2.0-fold),and dose and Ca'+ dependent. However, CDS produced a very substantial dose-dependentrelease of catecholamines, preponderantly epinephrine (Fig. 4). The release of catecholamines by CDS was at lower doses Ca" dependent and blocked by Co". At higher doses of CDS, a Ca" independent release became evident. The calcium-dependent release reached a plateau above 5 Units of CDS, with a maximal response at 15 min. In addition to releasing catecholamines,the exposure of chromaffin cells to clonidine also increases the expression of the adrenaline synthesizing enzyme phenylethanolamineN-methyltransferase(PNMT) (16). This observation indicates that activation of IRs may have long-term as well as short-term effects within target cells. The findings, therefore, indicate that the binding sites in adrenal chromaffin cell probably represent a true receptor in so far that binding of a ligand elicits functional responses. However, the signal transduction mechanism coupled to IR is not known. That it is not coupled to classical G-protein pathways provides additional evidence that AAR and IRs are distinct receptor systems. However, while the study excludes the common G-protein coupled second messenger systems, it leaves unanswered the primary mechanism facilitating Ca" entry. Is it due to direct activation of Ca'+ channels and, if so, what type? The fact that the gastric contraction elicited by CDS is blocked by verapamil (Ernsberger
26s
DJ Reis ef a/
Meeley and Reis, unpublished) suggest that an L-rypechannel may be engaged albeit secondarily. Ifnot, is it attributable to inhibition of K' channels, since K' and K+-channelblockers bind to IRs (17,18) and imidazoline agents inhibit K'currents (19), which may in turn secondarily augment Ca" influx? (c) If not, is it the result of interaction with Na'/H+ exchanger since, in other tissues, amiloride, an inhibitor of the exchanger, also binds to IRs? (d) Does it increase intracellular mobilization of Ca", possibly from the mitochondria that may harbor the receptor (11,12)? (e) Since 1-2 receptors are localized to mitochondria, how does mitochondria1signaling influence ion shifts within the whole cell? (1) Are comparable signal transduction mechanisms utilized in other tissues besides adrenal medulla, notably brain? These are many of the questions yet to be addressed in future understanding of the cellular actions mediated by IRs.
/
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F/g. 4. CDS-stimulated catecholamine release from adrenal chromaffin cells after 10 min in the presence and absence ( 0 ) of 1 mM cobalt. Values are mean 2 SE of two experiments done in triplicate. 'p
L3
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23s
IMIDAZOLINE RECEPTORS IN THE NERVOUS SYSTEM Donald J. Reis, S. Regunathan, Hong Wang, Douglas L. Feinstein and Mary P. Meeley Div. of Neurobiol., Dept. of Neurol. & Neurosci., Cornell Univ. Med. Coll., New York, NY 10021. 1.
INTRODUCTION
A. lmldarollne receptors In brain. The original insights of Karppanen (1) and Bousquet (2) that the central antihypertensive actions of the imidazoline clonidine could not be attributed to interactions with q-adrenergic receptor (AAR) but related to the imidazoline structure of the drug gave birth to the concept of a novel receptor referred to here as the imidazoline receptor (IR). The original concept also indicated per force that IRs were localized in brain. More direct evidence for the existence of IRs in brain (3)and other kidney (4) was subsequently established by ligand binding studies which indicated that the binding of 3H-para-aminocbnidine(3H-PAC)to membranes of ventral medulla, the site of the antihypertensive actions of clonidine (5) and rilmenidine (6)was fully displaceable by clonidine yet only partially by adrenergic ligands. Non-adrenergic binding was displaceable by agents which were imidazoles, imidazolines (e.g. imidazole 4-acetic acid, cimetidine, cirazoline), some guanidines (e.g. guanabenz, amiloride) and oxazoles (rilmenidine).Thus, these agents bound to both AARs and another binding site considered to be the IR. That IRs in the CNS are functional has, however, largely been indirect. That IRs mediate the hypotensive action of clonidine and rilmenidine in the ventral medulla has been assumed based on evidence that the reduction in arterial pressure elicited by the microinjection of agents into the rostra1 ventrolateral medulla (RVL) correlates with binding at IRs and not AARs and that the microinjection of idazoxan but not of selective AAR antagonists into RVL will block the systemic fall of AP elicited by intravenous clonidine or rilmenidine. There is also indirect evidence that the neuroprotectiveactions of idazoxan and rilmenidine can be attributed to actions at nonadrenergic receptors presumably representing IRs (7). On the other hand several of the central actions of clonidine including analgesia and sedation appear to represent the actions of the agents at AARs.
B. Subtypes of imldazoline receptors. The earliest ligand-binding studies used 3H-PACas ligand and assumed that the IR in brain was a single entity with a distribution differing in part from AARs. Thus, IRs were detected in membranes of the ventral medulla but not in cerebral cortex (3).However, the discovery of a high-affinity binding of 3Hidazoxan, a radiolabeled imidazoline and o;-adrenergic antagonist, to non-adrenergic receptors in cerebral cortex suggested the existence of an "idazoxan receptor" also distinct from AARs ( 8 ) . In the light of more recent studies, it is now evident that IRs, like most other receptors, exit as multiple subtypes one of which is identical to the so-called "idazoxan receptor" (9). Two principal subtypes have been identified based on ligand binding. One, a clonidine-preferring subtype, is labeled by 3H-PAC(and also 3H-idazoxan)exhibits a rank order of potency for displacing agents (K,s) of: phentolamine > para-aminoclonidine > clonidine > idazoxan > cimetidine > imidazole 4-acetic acid >> cirazoline >> amiloride. A second, an idazoxan-preferring subtype labeled by 3H-idazoxan (but not 3H-PAC)has a rank order for displacement of 3H-idazoxanof: cirazoline > idazoxan > naphazoline > amiloride > clonidine >> imidazole 4-acetic acid. We have referredto the clonidine-preferringreceptor as the 1-1, and the idazoxanpreferring receptor as the 1-2 receptor subtypes with the latter subdivided on the basis of whether it shows high (I-2a) or low (I-2b) affinity for amiloride. C. Some issues relating to characterization of imidazoline binding sites as "receptors". The identification of the IR and its subtypes has demonstratedthat the IR in brain and other tissues represents a novel ligand binding site. However, remaining to be established is: (a) characterization of the topographic, cellular and subcellular distribution of IRs in neural tissue; (b) evidence that the binding site is functional receptor by demonstration of a signal transduction process and cellular event associated with its stimulation; (c) identification of an endogenous ligand for the receptor; and (d) structural evidence that IRs and AARs are distinct. Complicating the analysis of IR function in general and in CNS in particular are the facts that all agents that bind to IRs also bind to AARs (although the converse is not true) and all tissues expressing IRs also express AARs. We have recently, however, made two discoveries which have permitted a more detailed analysis of IRs in brain and related neuronal tissue. The first is that IRs but not AARs are expressed in chromaffin cells of adrenal medulla, a cell which is phenotypically and developmentally neuronal (10). This discovery has permitted these cells to serve as models in which to analyze signal transduction and functional responses to occupancy of IRs uncontaminated by responses of AARs and, moreover, are a source of genes, message and receptor-related proteins distinct from AARs. Second is the discovery that astrocytes express IRs providing a cell of CNS in which function and structure of IRs can be analyzed in vitro.
DJ Reis ef a/
24s
We review here some of these studies which have indicated that both cell types express IRs of the 1-2 subclass which are concentrated on mitochondria1membranes, that stimulationof 1-2 receptors of chromaffin cells stimulated Ca'* fluxes and release of catecholamines, replicated in part by the purported endogenous ligand for IRs, clonidine displacing substance (CDS), that the absence of AARs in chromaffin cells has permitted the isolation of a selective imidazoline binding protein and that stimulation of astrocytic IRs results in increased expressionof mRNA for a glial selective protein, glial fibrillary acidic protein (GFAP). IRS OF THE ADRENAL CHROMAFFIN CELL
11.
A. Blndlng of llgands and CDS. Membranes of chromaffin cells of bovine adrenal medulla exhibit saturable, high affinity binding for 3H-idazoxanbut not 3H-PAC. Binding is to a single site with a K, of 5 nM. 3H-ldazoxanbinding is displaceable by drazoline and idazoxan (with respective Kis of 1 and 5 nM) but not by epinephrine nor rauwolscine (Fig. 1). Thus, chromaffin cells express IRs but not AARs and the IR is of the 1-2 subclass (10).
5
100
U
80
C
3
0
rn
$
60
Flg. 1. The displacement of binding of 3 H - i d a ~ ~ ~ a n to chromaffin cell membranes by various agents. Membranes (100-150 pg protein) were incubated in Tris-HCI buffer at 25°C for 30 min with 5 nM 3Hidazoxan. The total counts ranged between 2500 to 3000 dpm and the non-specific binding, defined using 10 pM idazoxan. was about 30% of total binding.
-
X
0 N
m
p
40
I
m V ._
._
v
20
0,
a
Rauwolscine
cn
0
-10
-9
-8
-7
-6
-5
Log (M)
When we examined the subcellular distribution of idazoxan binding sites in nuclear, mitochondrial, and plasma membrane fractions of chromaffin cells and compared them with membranes of the entire cell (crude fraction) we observed that the receptor was heavily concentrated in mitochondria, poorly represented in the plasma membrane and absent in the cell nucleus. Thus, while the K, of 3H-idazoxanbinding to crude, mitochondrial and plasma membranes were comparable (from 5-7 nM), mitochondriahad 7-fold more binding sites (750 fmoVmg protein) than crude membrane (Fig. 2).
a
7501
F/g. 2. Binding of 3H-idazoxan and activities of marker enzymes in subcellular fractions of adrenal medulla. The number of binding sites (6,) was calculated from Scatchard analysis of specific binding to each fraction. Enzyme activities in crude membrane fractions were: cytochrome oxidase, 0.601 2 0.051 unitslmg protein and 5' nucleotidase, 2.8 2 0.24 unitslmg protein and enrichment was calculated as percent over crude membrane fraction.
Session I
-
Central imidazoline preferring receptors : comparison with a,-adrenoceptors
25s
The potency profile for other ligands, assessed by displacement of 3H-idazoxanfrom mitochondria1membranes was: cirazoline > idazoxan > naphazoline > amiloride > detomedine > clonidine >> phentolamine = cimetidine = piodoclonidine = imidazole 4-acetic acid >> epinephrine = norepinephrine = rauwolscine. This order indicates that the IR of adrenal chromaffin cells is of the 1-2 subclass. Since binding is sensitive to arniloride it is similar to kidney and liver receptors in rabbit (11,12) but distinct from brain receptors which are insensitive to amiloride (8). We term the amiloridesensitive receptor as I-2a subtype in contrast to the amiloride-insensitive I-2b subtype. The binding of 3H-idazoxanto mitochondrial membranes of chromaffin cells was inhibited by K+ (K,: 75 mM) and the K' channel antagonists 4-aminopyridine and tetraethylammoniumbromide (K,s: 20 pM, 100 pM, respectively) but not by Na'. Since adrenergic receptors are sensitive to Na+the finding further supports the independence of IRs and AARs and indicates the close relationship between IRs and AARs. We also investigated the binding to mitochondrialmembranes of chromaffin cells of clonidine displacing substance (CDS) the putative endogenous ligand for IRs (13). CDS potently displaces %I-idazoxan binding to chromaffin cell membranes with an IC, of 5 Units (Fig. 3). The displacement of ?i-idazoxan binding by CDS is not modified by Gpp(NH)p, suggesting that IRs of the 1-2 type are not G-protein coupled receptors (see below). Since CDS also displaces 3H-PACfrom membranes of bovine ventral medulla (14) the results indicate that CDS binds to both 1-1 and 1-2 receptors, a pattern to be expected of an endogenous ligand.
I
Fig. 3. Inhibition of 3H-idazoxanbinding to adrenal chromaffin cell membranes by CDS. Membranes were incubated with 5 nM 3H-idazoxanat 25°C for 30 min.
B. Signal transduction and secretion. The fact that chromaffin cells express IR without AARs provides a cell system in which the biological actions of IR occupancy can be examined without contamination by actions upon AARs. We therefore investigated classical signal transduction pathways following the activation of IR. We observed that treatment of chromaffin cells with a series of agents acting at IRs had no effect on PI turnover and did not stimulate the accumulation nor inhibit the forskolin-activatedaccumulation of CAMP (15). Although clonidine elicited a slow, modest and PDE- inhibitable accumulation of cGMP the response was idiosyncratic and limited to clonidine. On the other hand, imidazolines like clonidine, oxymetazoline, rilmenidine and naphazoline increased the flux of "Ca" into chromaffin cells. Incubation of chromaffin cells with agents binding to IRs including CDS also released catecholamines. The response to drugs was modest (1.3-2.0-fold),and dose and Ca'+ dependent. However, CDS produced a very substantial dose-dependentrelease of catecholamines, preponderantly epinephrine (Fig. 4). The release of catecholamines by CDS was at lower doses Ca" dependent and blocked by Co". At higher doses of CDS, a Ca" independent release became evident. The calcium-dependent release reached a plateau above 5 Units of CDS, with a maximal response at 15 min. In addition to releasing catecholamines,the exposure of chromaffin cells to clonidine also increases the expression of the adrenaline synthesizing enzyme phenylethanolamineN-methyltransferase(PNMT) (16). This observation indicates that activation of IRs may have long-term as well as short-term effects within target cells. The findings, therefore, indicate that the binding sites in adrenal chromaffin cell probably represent a true receptor in so far that binding of a ligand elicits functional responses. However, the signal transduction mechanism coupled to IR is not known. That it is not coupled to classical G-protein pathways provides additional evidence that AAR and IRs are distinct receptor systems. However, while the study excludes the common G-protein coupled second messenger systems, it leaves unanswered the primary mechanism facilitating Ca" entry. Is it due to direct activation of Ca'+ channels and, if so, what type? The fact that the gastric contraction elicited by CDS is blocked by verapamil (Ernsberger
26s
DJ Reis ef a/
Meeley and Reis, unpublished) suggest that an L-rypechannel may be engaged albeit secondarily. Ifnot, is it attributable to inhibition of K' channels, since K' and K+-channelblockers bind to IRs (17,18) and imidazoline agents inhibit K'currents (19), which may in turn secondarily augment Ca" influx? (c) If not, is it the result of interaction with Na'/H+ exchanger since, in other tissues, amiloride, an inhibitor of the exchanger, also binds to IRs? (d) Does it increase intracellular mobilization of Ca", possibly from the mitochondria that may harbor the receptor (11,12)? (e) Since 1-2 receptors are localized to mitochondria, how does mitochondria1signaling influence ion shifts within the whole cell? (1) Are comparable signal transduction mechanisms utilized in other tissues besides adrenal medulla, notably brain? These are many of the questions yet to be addressed in future understanding of the cellular actions mediated by IRs.
/
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'
/
F/g. 4. CDS-stimulated catecholamine release from adrenal chromaffin cells after 10 min in the presence and absence ( 0 ) of 1 mM cobalt. Values are mean 2 SE of two experiments done in triplicate. 'p
