HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (23) Citing Articles via Google Scholar Google Scholar Articles by García, A. G. Articles by García-Sancho, J. Search for Related Content PubMed PubMed Citation Articles by García, A. G. Articles by García-Sancho, J.

Calcium Signaling and Exocytosis in Adrenal Chromaffin Cells

Instituto Teófilo Hernando, Departamento de Farmacología y Terapéutica, and Servicio de Farmacología Clínica e Instituto Universitario de Investigación Gerontológica y Metabólica, Hospital Universitario de la Princesa, Facultad de Medicina, Universidad Autónoma de Madrid; Unidad de Farmacología, Facultad de Medicina, Universidad de la Laguna; and Instituto de Biología y Genética Molecular, Universidad de Valladolid y CSIC, Departamento de Fisiología, Facultad de Medicina, Valladolid, Spain

ABSTRACT

I. INTRODUCTION

II. CALCIUM ENTRY THROUGH VOLTAGE-ACTIVATED CHROMAFFIN CELL CALCIUM CHANNELS

A. T-Type Channels

B. L-Type Channels

C. N-Type Channels

D. P-Type Channels

E. Q-Type Channels

F. R-Type Channels

G. Differences Between Species

III. MODULATION OF CHROMAFFIN CELL CALCIUM CHANNELS

A. Calcium Channel Current Facilitation and Voltage Dependence of Their Modulation Is a G Protein-Linked Membrane-Limited Phenomenon

B. The Chromaffin Cell Is a Good Model to Study Autoreceptor Modulation of Calcium Channels

C. Flow-Stop Experiments Unmask the Modulation and Facilitation of Calcium Channel Currents

D. Direct Approaches Demonstrate That Endogenously Released Neurotransmitters Modulate Calcium Channels

E. Manipulation of the Rate of Secretion: Modulation of Calcium Channels in Cell Clusters

F. Modulation of L-Type Versus Non-L-Type Calcium Channels: Some Conflicting Points

G. Physiological Relevance of Calcium Channel Modulation

IV. CONTRIBUTION OF EACH CALCIUM CHANNEL SUBTYPE TO TRIGGERING EXOCYTOSIS IN CHROMAFFIN CELLS OF DIFFERENT ANIMAL SPECIES

A. Cat Chromaffin Cells

B. Bovine Chromaffin Cells

C. Rat Chromaffin Cells

D. Dog Chromaffin Cells

E. Mouse Chromaffin Cells

F. Specialization of Calcium Channel Subtypes

V. SPATIAL ORGANIZATION OF CALCIUM CHANNELS AND THEIR SECRETORY MACHINE

A. Action Potentials and Exocytosis

B. Use of Calcium Chelators Suggests the Lack of Colocalization Between Calcium Channels and Secretory Vesicles

C. Specialized Zones

D. Polarization of Chromaffin Cells

VI. CALCIUM MODULATION OF EXOCYTOSIS STEPS

A. Separation of Vesicle Pools by the Analysis of Exocytotic Kinetics

B. Relationship Between Ca2+ Entry and Exocytosis

C. Modulation by Ca2+ and Protein Kinase C

D. Modulation of the Final Steps of Exocytosis

VII. CALCIUM ENTRY AND REDISTRIBUTION INSIDE THE CHROMAFFIN CELL: ROLE OF ORGANELLES AND FUNCTIONAL IMPLICATIONS FOR EXOCYTOSIS

VIII. CONCLUSIONS AND PERSPECTIVES

GRANTS

ACKNOWLEDGMENTS

REFERENCES

	ABSTRACT

Top Next References

	I. INTRODUCTION

Top Previous Next References

 
Fear, stress, or conflicts trigger a surge of the catecholamines epinephrine and norepinephrine that mobilize the body for the "fight or flight" response; the heart rate, the strength of myocardial contraction, and blood pressure increase; the blood flow switches to skeletal muscle; glucose is mobilized from the liver and rises in the circulation; and the pupils and bronchioles dilate (200). The body is thus prepared to survive by combating an enemy or to flee from danger. This highly coordinated and precise response is regulated by the sympathetic nervous system and the adrenal gland in an attempt to maintain the equilibrium of the internal milieu (48, 71).

The "fight or flight" response is the end result of a secretory event that takes place in the adrenal medulla, the inner part of the two adrenal glands located just above the kidneys. The adrenal medulla is composed of chromaffin cells that secrete epinephrine and norepinephrine. These cells are of interest not only as the basis of the "fight or flight" response, but also because they have been excellent models to study the working of other secretory cells, in particular neurons.

Feldberg et al. (141) established that the main physiological neurotransmitter at the splanchnic nerve-chromaffin cell synapse was acetylcholine. Acetylcholine causes the release of catecholamines from the gland. The secretory response is suppressed in the absence of extracellular Ca²⁺ (126), and secretion is accompanied by an enhancement of ⁴⁵Ca²⁺ entry into chromaffin cells (125). On the basis of these observations and other experiments, William W. Douglas (123) coined the expression stimulus-secretion coupling as the source of neurotransmitter and hormone secretion; Ca²⁺ was the coupling agent between the stimulus and the exocytotic response.

Because of their unlimited availability, particularly from bovine species, their common origin with sympathetic neurons in the neural crest (154), and their ease of isolation and preparation in primary cultures (238), chromaffin cells have been widely used in biochemical, electrophysiological, and neuropharmacological studies. Their usefulness has been further enhanced by the development of techniques to separate norepinephrine- from epinephrine-containing cells (274, 275). Thus fundamental findings on catecholamine synthesis, storage, and release were extrapolated, with success, from these cells to basic neurotransmission mechanisms in the central and peripheral nervous systems.

From fertilization of cells at their origin to cell death by apoptosis, Ca²⁺ are essential to multiple physiological and pathological processes (78, 309). The physiological function of chromaffin cells consists in the exocytotic release of the catecholamines epinephrine and norepinephrine into the circulation in response to stress (71). Because this release is a Ca²⁺-dependent process (126), it is not surprising that chromaffin cells have been widely used as models to study the correlation between Ca²⁺ and exocytosis (286). They contain all the elements required for a strict control, both spatial and kinetic, of the Ca²⁺ transients required during the various steps of exocytosis (see Ref. 65 for a review).

Chromaffin cells are excitable cells and fire action potentials that open plasma membrane Ca²⁺ channels and produce Ca²⁺ entry; the resulting cytosolic Ca²⁺ signal triggers exocytosis (36, 90, 239, 308). Because cytoplasmic organelles can take up and release Ca²⁺ to the cytosol, understanding the cytosolic Ca²⁺ signal requires understanding Ca²⁺ redistribution between the cytosol and the different organelles. Being able to code one of the photoprotein aequorin genes (344, 345) has made it possible to introduce targeting sequences, and measure selective [Ca²⁺] changes in different organelles (62, 329, 331). The practical efficiency of measuring aequorin expression has been very much improved and simplified by the use of viral vectors (14, 354), to the point where it is possible to image a single cell (387). This methodology has been recently applied to gain insight into the role of organelles in shaping Ca²⁺ signaling and exocytosis in these cells. This review focuses on the pathways for Ca²⁺ influx into the chromaffin cell, on the intracellular organelles that contribute to the redistribution of the Ca²⁺ entering the cell, and on the mechanisms that terminate the Ca²⁺ signals and extrude the cation outside the cell. Finally, by obtaining a more unified picture of Ca²⁺ signaling and exocytosis in chromaffin cells, we try to correlate these Ca²⁺ signals with the exocytotic responses.

	II. CALCIUM ENTRY THROUGH VOLTAGE-ACTIVATED CHROMAFFIN CELL CALCIUM CHANNELS

Top Previous Next References

 
Four approaches helped with the discovery of the rich diversity of voltage-activated calcium channels in excitable cells (167, 299). The improvement of patch-clamp techniques has made it possible to characterize the biophysical properties of calcium channels (kinetics of activation, inactivation, and deactivation, voltage range for activation, conductance), both at the single-channel and at the whole cell levels (185). The invention of suitable fluorescent Ca²⁺ probes (327, 378) has allowed us to follow changes on cytosolic Ca²⁺ concentration ([Ca²⁺]_c) in living cells. Tracers other than Ca²⁺, particularly Mn²⁺ (184), pass through Ca²⁺ channels (151, 384, 386) and can be extremely useful in tracing activity, since their appearance in the cytosol can be accurately followed with fluorescent probes without interference from Ca²⁺ released from the intracellular Ca²⁺ stores (17, 172). On the other hand, the isolation, purification, and synthesis of different neurotoxins have provided ligands with remarkable discrimination for different subtypes of high-threshold calcium channels (299). And finally, molecular biology and genetic approaches clarified the molecular structure of calcium channels (42).

Calcium channels are formed by a multiple subunit protein complex consisting of a pore-forming ₁-subunit with several other auxiliary proteins, which include the intracellular -subunit and a disulfide-linked --subunit. In some tissues, a fifth subunit may exist, such as the transmembrane -subunit, which is a part of the channel complex in skeletal muscle or the neuronal p95 subunit. The functional diversity between different subtypes of calcium channels can be explained by 1) the existence of multiple genes encoding different classes of ₁- and -subunits as well as diverse variants from a single gene generated by alternative splicing, and 2) multiple possible combinations among the subunits which make up the channel complex.

Calcium channels can be classified according to their range of activation into two main groups: one has a low activation threshold (LVA channels; T channels) and the other has a high threshold for activation (HVA channels; L, N, P/Q, and R channels). Table 1 summarizes the properties of these channels and also presents their old and new nomenclatures.

View this table: [in this window] [in a new window]   TABLE 1. Calcium channel subtypes according to their 1-containing subunit

In addition to their low activation threshold, LVA calcium channels are characterized by a similar permeability to Ca²⁺ and Ba²⁺ (80, 152, 153). A single subtype of LVA calcium channel, termed T (for "transient" or "tiny"), has been identified. T channel’s main characteristics include fast inactivation, which generates a transient current, and inactivation at holding potentials between –60 and –50 mV. The single conductance is around 8 pS. Pharmacologically, T-type channels can be distinguished from other subtypes because they are more sensitive to blockade by the inorganic calcium channel blocker Ni²⁺ than by Cd²⁺ (152, 153). It has also been reported that T-type channels can be blocked by l-octanol, amiloride, and the antihypertensive drug mibefradil (265).

T-type currents are difficult to record in chromaffin cells. Although we have detected T-type channel mRNA in bovine chromaffin cells (169), we have been unable to record T-type currents. However, there are three studies reporting T-type Ca²⁺ currents in bovine (118) and rat chromaffin cells (59, 201). It has been suggested that T-type calcium channels are mainly expressed in immature developing chromaffin cells (59). Recently, T-type channels of the _1H class have been found to be expressed in rat chromaffin cells exposed to cAMP (290), and those channels were found to trigger a secretory response (173). This _1H T-type calcium channel has also been identified in rat adrenal glomerulosa zone (340).

B. L-Type Channels

L-type (for "long lasting") calcium channels are kinetically characterized by showing little inactivation during depolarizing steps (_inact >500 ms) and their lower sensitivity to depolarized holding potentials; however, when Ca²⁺ is used as a charge carrier, these channels are completely inactivated in chromaffin cells (193). Single-channel conductance is between 18 and 25 pS in 100 mM Ba²⁺. These L-type calcium channel subtypes seem to be present in almost all excitable cells. They are the main pathway for Ca²⁺ entry in heart and smooth muscle, as well as in the control of hormone and transmitter release from endocrine cells and some neurons. Four different ₁-subunits (_1C, _1D, _1F, and _1S) are responsible for L-type currents in different tissues (Table 1).

Pharmacologically, L-type calcium channels are characterized by their sensitivity to 1,4-dihydropyridines (DHP), whether agonists (i.e., BAY K 8644) or antagonists (i.e., nifedipine, nitrendipine, nisoldipine, nimodipine, furnidipine; Table 1). DHP agonists prolong the mean time duration of channel opening (291), typically observed in whole cell electrophysiological recordings as a prolongation of tail currents (315). Other organic compounds that effectively block L-type calcium channels (149, 355) include the arylalkylamines (i.e., verapamil) and benzothiazepines (i.e., diltiazem). They are particularly useful in cardiac and smooth muscle cells, where they exert negative inotropic and relaxing effects. Some piperazine derivatives (cinnarizine, flunarizine, dotarizine, R56865) also block L-type calcium channels, but they also block other subtypes of calcium channels and thus have been considered "wide-spectrum" calcium channel blockers (164, 234, 389). The same is true for imidazole antimycotics (386). Some toxins have also been shown to block L-type calcium channels, either in a selective (calciseptine and calcicludine) or a nonselective manner (-agatoxin IA, -agatoxin IIA, and -agatoxin IIIA).

L-type currents have been characterized in bovine (9, 30, 31, 57, 58, 157), rat (158, 322), mouse (191), pig (226), cat (5), and human chromaffin cells (162, 322). Recent studies have presented molecular evidence that L-type currents in chromaffin cells are carried out by two different calcium channels: _1C and _1D (42).

C. N-Type Channels

N-type calcium channels inactivate faster than L-type channels and do not persist at less negative holding potentials. In some preparations N-type calcium channels can contain a noninactivating component, for instance, in bovine chromaffin cells, in which N-type channels have been described as "nonclassical N-type" (31). Pharmacologically, N-type calcium channels are characterized by irreversible blockade by the Conus geographus toxin -conotoxin GVIA (219, 291, 299) and reversible blockade by the Conus magus toxin -conotoxin MVIIA (Table 1) (383, 385). Other wide-spectrum toxins such as -conotoxin MVIIC and -conotoxin MVIID (267, 385) can also block N-type calcium channels in a nonselective manner. This is also the case for -agatoxin IIA, -agatoxin IIIA, and -grammotoxin SIA (isolated from the venom of the tarantula Grammostola spatulata).

N-channel currents have been characterized in chromaffin cells of various species including bovine (31, 249), pig (226), cat (5), rat (158), mouse (191), and human (162). This current suffers voltage-dependent inactivation (152, 390, but see Ref. 31) and is irreversibly blocked by -conotoxin GVIA (298) and -conotoxin MVIIC (160, 198) or reversibly blocked by -conotoxin MVIID (160, 267).

D. P-Type Channels

P-type calcium channels were first described by Llinás et al. (242) in cerebellar Purkinje cells, in which Ca²⁺ currents were resistant to blockade by DHP and -conotoxin GVIA. The toxin fraction from the venom of the funnel web spider Agelenopsis aperta (FTX) effectively blocked this current. These results led to the suggestion of the existence of a new subtype of HVA calcium channel, which was named P (for "Purkinje"). P-type calcium channels are characterized by their relative insensitivity to changes in the holding potential and because they do not inactivate during depolarizing steps (262–264, 324). Pharmacologically, P-type calcium channels can be blocked by FTX, its synthetic analog sFTX, and by -agatoxin IVA at nanomolar concentrations (Table 1). P-type calcium channels can also be blocked in a nonselective manner by -conotoxin MVIIC (198, 267), -conotoxin MVIID, and by -grammotoxin SVIA (231, 312, 313, 380).

Nanomolar concentrations of -agatoxin IVA, known to fully and selectively block P-type channels (262, 264), cause only a 5–10% blockade of calcium channel current in bovine chromaffin cells (10). Previous studies reported larger contributions of P-type channels to the whole chromaffin cell Ca²⁺ currents; however, this blockade is now attributable to inhibition of Q-type channels (407) by sFTX (157) or large concentrations of -agatoxin IVA (9, 26). In cat chromaffin cells, combined -conotoxin GVIA plus nisoldipine blocked 90% of the current, leaving little room for P-type channels (5). In rat (158) and mouse chromaffin cells (191), the -agatoxin IVA-sensitive current fraction was only 10–15%. Thus, in all species studied, P-type channels are barely expressed in chromaffin cells. This, together with the difficulty of separating the _1A-subunit into P- and Q-type channels (339), suggests the convenience of speaking of P/Q-type channels rather than of two separate calcium channel subtypes.

E. Q-Type Channels

The isolation, purification, and synthesis of the toxin from the marine snail Conus magus -conotoxin MVIIC (198, 267) led to the identification and characterization of a new subtype of HVA channel termed Q (323, 407). The characterization of Q-type calcium channels rests mainly on pharmacological criteria. Q-type channels are resistant to blockade by DHPs, -conotoxin GVIA, and low concentrations (<100 nM) of -agatoxin IVA, but are sensitive to -conotoxin MVIIC (1–3 µM). Higher concentrations of -agatoxin IVA (up to 2 µM) can also block Q-type calcium channels (407). It should be noted that these toxins are not selective for this subtype of channel since they also nonselectively block N and P channels. Other toxins that can block Q channels as well include the Conus magus snail toxin -conotoxin MVIID (267) and the Grammostola spatulata tarantula toxin, -grammotoxin SIA (231, 312, 313, 380).

The P/Q component of the whole cell calcium channel current has been widely studied in chromaffin cells. This component is voltage inactivated (390), and it is pharmacologically isolated by 2 µM -conotoxin MVIIC, -conotoxin MVIID, or -agatoxin IVA. In bovine chromaffin cells, -conotoxin MVIID reversibly blocks the N current, but blockade by -conotoxin MVIIC is irreversible (160). Thus the use of -conotoxin MVIID followed by its washout can be a convenient tool to isolate the P/Q channel. The blocking effects of -conotoxin MVIIC are extraordinarily slowed down and decreased in the presence of high concentrations (i.e., more than 2 mM) of Ba²⁺ (10, 258) or Ca²⁺ (385).

F. R-Type Channels

In neuronal tissues, a residual Ca²⁺ current, characterized by its insensitivity to blockade by DHPs, -conotoxin GVIA, -agatoxin IVA, and -conotoxin MVIIC, has also been described and named "R type" for "resistant" (323). This subtype of calcium channel belongs to the HVA group, inactivates rapidly, and is more sensitive to blockade by Ni²⁺ than by Cd²⁺. Newcomb et al. (289) described the first selective R channel blocker SNX-482, a peptide from the African tarantula Hysterocrates gigas. We found, however, that this toxin also blocks P/Q channels in the bovine chromaffin cell (23). Thus caution should be exerted when using this toxin to target R-type currents.

Differences have been reported in various laboratories concerning the expression of R-type calcium channels in chromaffin cells, and they may be due to the configuration of the patch-clamp technique (whole cell vs. perforated-patch recordings). In some initial studies, an R-type component of the calcium channel current could not be detected in bovine (9, 10, 26, 28, 30, 157, 251, 382), cat (5), human (162), pig (226), or mouse chromaffin cells (11, 191). In contrast, using the perforated-patch configuration instead of whole cell patch configuration, an R-type component was found in slices of mouse adrenal medulla and mouse chromaffin cells (11, 12). The most obvious explanation for this finding is that some soluble cytosolic factor, which is necessary for chromaffin cell R-channel activity, is dialyzed with the whole cell but not with the perforated-patch configuration. R-type currents have also been reported in rat chromaffin cells (75, 85, 201).

G. Differences Between Species

Notable species differences have been found among the subtypes of calcium channels to be expressed by different cell types. For instance, the K⁺-evoked Ca²⁺ entry in brain cortex synaptosomes is controlled by N channels in chicks and by P channels in rats (60). On the other hand, neurotransmitter release at the muscle end-plate is controlled by N channels in fish (3, 140, 346) and amphibians (214) and by P channels in mammals (402).

Detailed comparative electrophysiological studies from six different mammalian species have been performed in adrenal medullary chromaffin cells (Fig. 1). L-type calcium channels account for nearly half of the whole cell calcium channel current in the cat (5), rat (158), and mouse chromaffin cells (191). But in pigs (226), bovine (9, 157), and humans (162), L channels carry only 15–20% of the whole cell Ca²⁺ current.

View larger version (38K): [in this window] [in a new window]   FIG. 1. Relative proportions of different neuronal calcium channel subtypes in primary cultures of chromaffin cells isolated from bovine, rat, mouse, cat, pig, and human adrenal medullary tissues. In fresh mouse adrenal slices, the R-type calcium channels carry as much as 22% of the whole cell current; no data are available in other animal species.

The fraction of current carried by P/Q channels in bovine chromaffin cells amounts to 50% (10). This fraction is even higher (60%) in human chromaffin cells (162). The opposite occurs in pig (226) and cat chromaffin cells (5) where P/Q channels carry only 5% of the current. Finally, in rat chromaffin cells, P/Q channels contribute 20% to the current (158) and 30% in mouse chromaffin cells in the earlier study (191), but more recent studies report that P/Q channels make only 15% of the current in the mouse cells (12).

We do not yet know the physiological relevance of these extreme interspecies differences. But, surely they have clear consequences for the fine control of the differential exocytotic release of epinephrine and norepinephrine in response to different stressors. Differing autocrine/paracrine regulation by catecholamines and other coexocytotic vesicular components of the L- and non-L-types of calcium channels might be a reason. Other regulatory mechanisms (i.e., voltage- or Ca²⁺-dependent inactivation of calcium channels) (261, 390), could also explain the preferential expression of one or another channel type in a given species. On the other hand, the selective segregation of a particular channel type to exocytotic microdomains, and the uneven geographic distribution of other channel types, might also result in a neurosecretory cell preferentially expressing one or another type of channel. An alternative explanation for this channel diversity rests on the assumption that chromaffin cells probably have the same function in the six animal species studied to date, i.e., the sudden release of catecholamines in response to stressors. This might explain the lack of an evolutionary pressure to conserve a particular pattern of expression of calcium channel subtypes. In any case, the differences of channel type expression provide different models of chromaffin cells to study the dominant role of a calcium channel subtype in controlling exocytosis. Research in the next few years will probably develop interesting techniques with isolated adrenal slices from various species that should allow the study of the expression of a particular pattern of calcium channels, [Ca²⁺]_c signals, and exocytosis in more physiological conditions.

	III. MODULATION OF CHROMAFFIN CELL CALCIUM CHANNELS

Top Previous Next References

 
Dunlap and Fischbach (133) were the first to report that the exogenous application of norepinephrine, GABA, or serotonin onto the surface of chick sensory neurons inhibited their Ca²⁺ conductance. This observation was soon corroborated in rat sympathetic neurons (132, 133, 156) and was demonstrated to affect HVA, but not LVA, calcium channels (115). The inhibition of the current was associated with many neurotransmitters, receptors, and neurons (79, 120, 165, 197) and was shown to be a membrane-delimited mechanism that was directly coupled to G proteins (122, 195, 203, 211, 235, 399).

A. Calcium Channel Current Facilitation and Voltage Dependence of Their Modulation Is a G Protein-Linked Membrane-Limited Phenomenon

Marchetti et al. (257) first observed that the neurotransmitter-mediated inhibition of calcium channel currents was modulated by voltage. Dopamine slows down HVA channel activation, and the effect is stronger at more negative membrane potentials. Another intriguing observation has been that strong depolarizing prepulses augment, instead of produce, the expected voltage-dependent channel inactivation of the calcium channel current induced by milder depolarizing test pulses, an effect that is called "facilitation" (143). We believe that both observations have the same underlying mechanism, a membrane-delimited G protein-mediated inhibition of calcium channels that is relieved by voltage. Figure 2 schematically shows the G protein-mediated effects of exogenous neurotransmitters on calcium channel currents and the voltage-dependent facilitation by prepulses of the currents. The typical HVA current shows fast activation and meager inactivation, if any (Fig. 2A). When a neurotransmitter binds to its receptor, i.e., ATP in chromaffin cells, it activates a G protein that couples negatively to the calcium channel, promoting a process of slow activation, and the peak current decreases by 50% (Fig. 2B). When a prepulse protocol is applied in the absence of a neurotransmitter, the normal "test pulse" current is little affected; however, in the presence of a neurotransmitter, i.e., ATP in chromaffin cells, which can slow the activation and inhibition of the current, the prepulse changes the structural conformation of the G protein coupled to the calcium channels and converts any current into another with fast activation and higher peak (facilitation; Fig. 2C). Thus the greater the inhibition of the current produced by a given neurotransmitter, the greater the facilitation by the prepulses. Thus the facilitation depends then on the degree of inhibition of the current, and this inhibition may depend on the secretory activity of the studied cell (autocrine modulation) or neighboring cells (paracrine modulation). These mechanisms will be described in the next sections.

View larger version (40K): [in this window] [in a new window]   FIG. 2. The mechanism of calcium channel inhibition by G proteins. In resting conditions, G proteins are not coupled to calcium channels, and when channels open, a large amount of Ca2+ flows through them (A). When ATP binds to its purinergic receptor, G protein couples to the channel, slowing down the current activation and decreasing its peak amplitude (B). Application of a strong positive depolarizing prepulse uncouples the G protein from the channel, and the current recovers its control profile (facilitation; C).

B. The Chromaffin Cell Is a Good Model to Study Autoreceptor Modulation of Calcium Channels

Pioneering experiments using ATP showed that bovine chromaffin cells were susceptible to neurotransmitter modulation of their calcium channel currents (118, 159). This nucleotide delayed the whole cell calcium channel current activation and inhibited the amplitude of the current. These effects were mediated by P_2y purinergic receptors through a G protein pathway, as in neurons; they disappear by dialyzing the cells with guanosine 5'-O-(2-thiodiphosphate) (GDPS), or by pretreating the cell with Pertussis toxin, and are mimicked by guanosine 5'-O-(3-thiotriphosphate) (GTPS). Similar effects by ATP were corroborated 3 years later also in bovine chromaffin cells (111). Opiates (methionine-enkephalin, leucine-enkephalin), through the activation of µ- and -receptors, also exert a modulatory action on bovine chromaffin cell calcium channels, i.e., they slow down channel activation, and decrease the amplitude of the current through a G protein membrane-limited pathway (6).

Various properties make the chromaffin cell a suitable model to study autoreceptor modulation of calcium channels and neurosecretion. 1) Like sympathetic neurons, the chromaffin cell is derived from the neural crest and has all the machinery to manufacture, store, and release catecholamines. 2) The chromaffin cell can be easily isolated from the adrenal glands of various animal species, even in large quantities as in the case of the bovine, and maintained in primary cultures that can survive 1–2 wk. 3) The chromaffin cell expresses various subtypes of voltage-dependent calcium channels that vary considerably with each animal species. 4) The participation of each channel subtype in the control of exocytosis varies markedly with species, stimulation pattern, or cell type (noradrenergic, adrenergic). 5) Chromaffin cells that store epinephrine or norepinephrine also contain a rich cocktail of other chemicals (i.e., ATP, opiates, chromogranins) that are coreleased with the catecholamines. 6) Manipulation of the superfusion system can enhance or decrease the release of these materials, or to direct them to the same patch-clamped cell, or wash materials quickly out from the cell surface. 7) Cells can be cultured in isolation or in clusters to study the influence on the voltage-clamped cells by the materials being released from neighboring cells on the voltage-clamped cells. 8) Simultaneous measurements of calcium channel activity, changes in [Ca²⁺]_c, and catecholamine release can be monitored, and correlations between these three parameters can be established in the same cell. Although properties 6–8 can be valid for any cell and culture, chromaffin cells offer the unique advantage that the rich chemical cocktail of their chromaffin vesicles is well known (410), and thus the effects of endogenous chemicals (i.e., ATP and opiates) on calcium channel currents can be studied and characterized.

C. Flow-Stop Experiments Unmask the Modulation and Facilitation of Calcium Channel Currents

In chromaffin cells (and probably also in several neuronal cell types), the experimental conditions used to record whole cell calcium channel currents under voltage-clamp conditions trigger the exocytotic release of endogenous neurotransmitters; this release is activated by Ca²⁺ (or Ba²⁺) entering during the depolarizing test pulses applied to elicit the currents. So, the released neurotransmitter could act backwards onto autoreceptors on the surface of the cell being recorded. If these receptors are coupled to calcium channels via G proteins, they will obviously inhibit the current flowing through them. An experiment to enhance the probability of the released transmitter combining with its autoreceptors, that increases its concentration near these autoreceptors, consists of stopping the flow of the extracellular solution that bathes the cell being recorded. This manipulation was first used in perfused cat spleen in a pioneering study that was basic to the hypothesis of the modulation of norepinephrine release by presynaptic -adrenergic receptors (225). Blockade of these receptors enhances, while their stimulation inhibits, the norepinephrine release induced by low-frequency stimulation of sympathetic nerves. This -mediated modulation could not be shown in chromaffin cells (301, 321), even though they are very close relatives of sympathetic neurons. However, we now know that this modulation does exist, but it is associated with purinergic P_2y and opiate µ- and -receptors that act at sympathetic nerve terminals.

In other studies, Doupnik and Pun (128), Albillos et al. (8), and Currie and Fox (111) observed that the rate of activation and the amplitude of Ba²⁺ currents in bovine chromaffin cells critically depend on the experimental superfusion conditions of the patch-clamped cell. Cell activity under stop-flow conditions (unperfused cell) favors the local rise of secreted products close to the plasmalemma, the subsequent activation of membrane autoreceptors, and the rapid inhibition of spatially localized calcium channels. This tonic inhibition, induced by low-molecular-weight compounds of the vesicle content (i.e., ATP and opiates), coreleased with the catecholamines during application of depolarizing pulses under flow-stop conditions, can be markedly reversed ("facilitated") by strong depolarizing prepulses. The tonic inhibition of the current is also reversed upon resuming the rapid flow over the surface of the cell, which quickly washes the released materials out (Fig. 3).

View larger version (13K): [in this window] [in a new window]   FIG. 3. Drawing illustrating the technical approaches followed to study the regulation of calcium channel currents by endogenously released materials. A: fast superfusion conditions. B: flow-stop conditions.

If ATP and enkephalins modulate calcium channels, and ATP and opiates are co-stored with catecholamines at high concentrations in chromaffin vesicles (410), it would be interesting to test whether the vesicular contents could produce the same effect as the exogenous application of these compounds. Chromaffin vesicles were purified from a bovine adrenal medulla homogenate to prepare a soluble vesicle lysate (SVL). When applied with the extracellular solution onto the surface of a voltage-clamped bovine chromaffin cell, SVL inhibited HVA currents in a concentration- and voltage-dependent manner (8). The modulated current exhibited the same slow activation kinetics as those produced by exogenously applied ATP or methionine-enkephalin; in fact, a mixture of purinergic and opiate receptor blockers antagonized the effects of SVL. Also, depolarizing prepulses evoked a strong current facilitation in the presence of SVL, indicating that the facilitated current was originated in the suppression of the tonic inhibition of calcium channels by ATP and opiates secreted during cell stimulation.

E. Manipulation of the Rate of Secretion: Modulation of Calcium Channels in Cell Clusters

If a procedure could be found to block exocytosis without suppressing calcium channel currents, the currents would not be inhibited and then facilitated as they switch from superfusion to flow-stop. This was true in voltage-clamped bovine chromaffin cells dialyzed with tetanus toxin, which does not affect calcium channel current, but does hydrolyze synaptobrevin and block exocytosis (A. Albillos and A. G. García, unpublished results).

However, there are conditions that can enhance exocytosis and the concentration of materials available to the cell in which the calcium channel currents are being recorded. For instance, the use of Ca²⁺ instead of Ba²⁺ as charge carrier can drastically change the secretion rate, since Ba²⁺ is a more powerful secretagogue than Ca²⁺ (192) and is poorly chelated by the EGTA that is present in the intracellular solution. Thus, in the presence of 10 mM Ca²⁺, stopping the flow had effect on modulation of the current; however, when the same cell was bathed with 10 mM Ba²⁺, flow-stop caused a drastic slowing down of current activation and a large decrease in peak amplitude (192).

When the cell whose calcium channel currents are being explored belongs to a cell cluster (Fig. 4B), strong inhibition and prepulses facilitation of the whole cell current are observed, if 10 mM Ba²⁺ (but not Ca²⁺) is used (Fig. 4, C and D). This result was clearly obtained in human (162) as well as in bovine chromaffin cell clusters (192). Ba²⁺ (but not Ca²⁺) induced a powerful secretory response (395) in the unpatched, surrounding cells; the secreted materials would be reaching the patch-clamped cell at high concentrations, thereby causing visible modulatory effects on the calcium channel current (Fig. 4D). The experiments shown in Figure 4, E and F, were performed in cells superfused with 10 mM Ca²⁺ (instead of Ba²⁺). Ca²⁺ requires K⁺ depolarization to activate exocytosis in chromaffin cells. So, in Figure 4F, secretion was stimulated in a cell cluster using a brief application of a K⁺-rich solution (70K⁺, 1 s); under these conditions, the Ca²⁺ current that was being recorded from the patch-clamped cell within the cluster activated with a slow kinetics and halved its amplitude; this indicates that the voltage-clamped cell is under the modulatory influence of materials released from neighboring cells by the K⁺ pulse. Modulation of the Ca²⁺ current (after the K⁺ pulse) is not seen in an isolated cell (Fig. 4E). Experiments on cell clusters were performed by Callewaert et al. (69) in bovine chromaffin cells; they concluded that calcium channel currents were regulated through the release of protons, rather than ATP or opiates. Cell clusters are more similar than isolated cells to intact adrenal medullary tissue, in which secretory materials from neighboring cells can impinge on a given cell and strongly modulate its electrical properties and secretory activity. Hence, studies in adrenal medulla slices should provide further insight into these autocrine-paracrine modulatory mechanisms.

View larger version (17K): [in this window] [in a new window]   FIG. 4. Modulation of calcium channel currents in a single chromaffin cell (A, C, E) or in a cell immersed in a cell cluster (B, D, F). Cells C and D were superfused with an extracellular solution containing 10 mM Ba2+; cells E and F were superfused with 10 mM Ca2+. Before the test pulse to 0 mV (with or without a prepulse to +100 mV), a 70 mM K+ solution was applied during 1 s (70 K+). See text for further details. [Adapted from Gandia et al. (162) and Hernandez-Guijo et al. (192).]

Because chromaffin cells express four types of HVA calcium channels (L, N, P/Q, and R), the question is whether all of them are equally modulated by endogenously released ATP and opiates. It is interesting that L- and non-L-type calcium channels in bovine chromaffin cells seem to be modulated by opioids through different mechanisms (6). L channels are modulated mostly through a voltage-independent mechanism (no facilitation was observed associated to L channels); non-L channels appear to be inhibited through a voltage-dependent pathway. Thus nifedipine blocks the voltage-independent component with high selectivity (60% block of the voltage-independent component, 5% block of the voltage-dependent component). While -conotoxin MVIIC (a blocker of N and P/Q channels) completely blocks the voltage-dependent component. The voltage-dependent component was distributed 58% from N channels and 42% from P/Q channels. These data are similar to those obtained by Currie and Fox (111) also in bovine chromaffin cells when they use ATP to modulate the calcium channel currents: N-type channels contributed 69%, and P/Q channels 47%, to the voltage-dependent modulation.

Contrary to our results, a series of papers from the laboratory of Aaron Fox and co-workers (25–28, 30–32) found that prepulse facilitation of calcium channel currents was entirely blocked by nisoldipine, a blocker of L-type calcium channels, and G proteins were not necessary for voltage-dependent facilitation recruitment (32). They argued that "the expression of the facilitation L channel is strongly dependent on the age of the animals from which chromaffin cells are prepared" (25), suggesting that L-type channel facilitation is a property of chromaffin cells from 10- to 12-wk-old young calves. Thus facilitation would be associated with an L-type channel that does not usually contribute to normal Ca²⁺ currents.

We believe that the reasons behind these conflicting results may have different origins living on the complexity of the chromaffin cell system, the autocrine/paracrine nature of calcium channel modulation (8, 128, 159), and a possible coupling between channel subunits and second messenger pathways affecting the up- or downregulation of calcium channel subtypes (197). We would like to stress that the membrane-delimited pathway of calcium channel modulation by neurotransmitters in neurons (197) is also a well-established phenomenon in chromaffin cells; it is highly reproducible and has been observed by six independent laboratories at the macroscopic (5, 6, 8, 111, 128, 159, 226) and single-channel level (77). A study in rat chromaffin cells (201) describes a voltage-dependent facilitation of L-type channels that deviates significantly from that observed in Fox’s laboratory with bovine cells. In the rat, facilitation associated with L-type channels contributes to 6% of the total current at most, and it is insensitive to D₁ dopamine agonists and to protein kinase A (PKA) activation, and is short lasting rather than persisting for seconds.

Modulation of chromaffin cell L-type calcium channels has been extensively studied in the laboratory of Emilio Carbone during the last years (see Ref. 42 for a review). Among the many modulatory pathways, two are of interest because of their autocrine nature: a G protein-dependent inhibition and cAMP/PKA-mediated potentiation (120, 121). In chromaffin cells both pathways are activated by autoreleased neurotransmitter molecules and produce opposite effects of similar magnitude (85). The inhibition is complete within seconds and is mediated by PTX-sensitive G proteins coupled to P_2y-purinergic, µ--opioid, ₂- and ₂-adrenoceptors (6, 8, 73, 76, 159, 190, 227) while the potentiation is triggered by ₁-adrenergic receptors and occurs slowly over a few minutes through the activation of a cAMP/PKA pathway, which may act at distant sites from receptors (218, 333). The increase of Ca²⁺ entry elicited by PKA activation also contributes to the increase of secreted vesicle’s quantal size (74, 253).

An interesting recent finding relates to the expression of two L-channels subtypes in chromaffin cells, _1C and _1D (42, 169, 171, 409). These two channel types are not easily distinguishable on the basis of their affinities for dihydropyridines (Table 1) or their biophysical properties (i.e., single-channel conductance, mean open and mean closed times). The only parameter that appears to be significantly different between the two channel types is the ranges of their activation voltage, which shifts towards more negative potentials by 20–25 mV for the _1D-subunit (230, 415). Biophysical data at the single-channel recording level suggest that most of the functional L channels in chromaffin cells are probably associated with the _1C-subunit, in both bovine (74, 76) and rat chromaffin cells (42). According to the available data, it is likely that the G protein-mediated inhibition and cAMP/PKA-mediated potentiation converge on the same channel type, again probably the _1C. A voltage-independent mechanism for autocrine inhibition of P/Q-type calcium channel currents that requires Src family kinase activity has also been reported (401).

G. Physiological Relevance of Calcium Channel Modulation

The effects of neurotransmitters on Ca²⁺ currents and exocytosis have been studied in bovine chromaffin cells by combining membrane capacitance measurements and whole cell current recordings. Extracellular ATP markedly inhibits L-, N-, and P/Q-type currents and exocytosis in a parallel manner; the ATP does not alter the Ca²⁺-dependent fusion of vesicles to the plasmalemma or the vesicle supply to the release sites, suggesting that the inhibitory effects of ATP on exocytosis are primarily associated with calcium channels (320, 382). In rat chromaffin cells, ATP inhibits exocytosis either by depressing Ca²⁺ currents (L, N, P/Q) or by directly acting on the secretory machine through a Ca²⁺-independent pathway (236). In rat chromaffin cells, where L-type calcium channels dominate, the cAMP-permeant analog pCPT-cAMP potentiates both the L current and depolarization-evoked secretion; however, the current increase accounted for only 20% of the total secretory response. cAMP doubled the size of the readily-releasable pool of vesicles in these rat cells (75). It is also interesting that chronic stimulation of -adrenergic receptors and cAMP recruit T-type calcium channels that functionally control secretion in rat chromaffin cells (290).

Slowing down current activation, ATP plus opiate inhibition of peak calcium channel currents should cause profound alterations in Ca²⁺ entry, in local [Ca²⁺]_c transients at exocytotic subplasmalemmal sites, and in the rates of norepinephrine and epinephrine release. These effects may constitute the basis for the fine tuning of the quantity of adrenal medullary catecholamines delivered to the circulation during stressful incidents. This fine regulation is absolutely necesary to prevent massive uncontrolled release of catecholamines that could lead to a hypertensive crisis, arrhythmias, and myocardial damage. Catecholamines are required quickly and at the appropriate concentrations, by target organs needing to adapt to mental or physical stress, or in emergencies requiring a fast "fight or flight" response by the entire body. But the catecholamines stored in both adrenal glands can kill the animal, if they are suddenly released into the circulation; a precise and efficient control of their rate of secretion is needed. The data reported here would explain the regulation of catecholamine release to the circulation as follows (Fig. 5).

View larger version (66K): [in this window] [in a new window]   FIG. 5. Scheme showing our present view on how adrenal medullary calcium channels and catecholamine release are modulated by an autocrine/paracrine feedback mechanism activated by the ATP and opiates coreleased with norepinephrine and epinephrine.

It is noteworthy that although the modulation of the different calcium channel subtypes has been thoroughly studied in chromaffin cells, the functional consequences of this modulation on Ca²⁺ signals and exocytosis is very poorly documented. Simultaneously measuring the "modulated" Ca²⁺ currents through a given channel subtype, the ensuing [Ca²⁺]_c increase, and the secretory activity in the same cell using both capacitance and amperometric techniques should provide very interesting data and should provide physiological significance if done in cell clusters or adrenal slices as well.

	IV. CONTRIBUTION OF EACH CALCIUM CHANNEL SUBTYPE TO TRIGGERING EXOCYTOSIS IN CHROMAFFIN CELLS OF DIFFERENT ANIMAL SPECIES

Top Previous Next References

 
As described above, different calcium channel subtypes are found on the plasmalemmal membrane of chromaffin cells. This coexistence raises the question as to whether all of the channel types participate in the control of exocytosis and how their density and properties would condition their participation if any. Furthermore, the presence and proportion of the various calcium channels subtypes varies widely between the animal species (Fig. 1). Therefore, catecholamine secretion from these cells will presumably be controlled differently, in accordance with the calcium channels expressed by the cells. Here, we will review how catecholamine secretion is controlled in different animal species and how some subtypes of calcium channels are more directly implicated in the control of exocytosis. It is important to emphasize that, depending on the type of stimulus used (i.e., K⁺ depolarization, acetylcholine, step depolarizations, action potentials), one type of channel may be more favored over another in secretion. For this reason, the type of stimulus used is indicated in each of the following subsections.

A. Cat Chromaffin Cells

The K⁺-evoked secretion of catecholamines is effectively blocked in a concentration-dependent manner by DHPs and other drugs acting on L-type calcium channels like verapamil and diltiazem (161). Measuring differential secretion of epinephrine and norepinephrine, Cárdenas et al. (81) demonstrated that secretion of both amines is completely blocked when it is induced by either high K⁺ or the nicotinic agonist dimethylphenylpiperazinium (DMPP). Initially, these data indicated that an L-type channel controlled secretion in these cells. But, Albillos et al. (5) showed that cat chromaffin cells also contained -conotoxin GVIA-sensitive channels in addition to the L-type channels. It was then demonstrated that HVA L and N calcium channels in cat chromaffin cells were present in an approximate proportion of 50–50% and that the increase in [Ca²⁺]_c induced by short (10 s) depolarizing pulses (70 mM K⁺) could also be reduced 44% by furnidipine and 43% by -conotoxin GVIA. In a perfused adrenal gland or isolated cat chromaffin cells, catecholamine release induced by 10-s pulses of 70 mM K⁺ was blocked by more than 95% with furnidipine and only 25% with -conotoxin GVIA. These results show that although Ca²⁺ entry through both channels (N and L type) leads to similar increments of the average [Ca²⁺]_c, the control of the K⁺-evoked catecholamine release response in cat chromaffin cells is dominated by the Ca²⁺ entering through L-type calcium channels (247). However, more recent data suggest that when exocytosis is measured using capacitance techniques, and the membrane potential is held at –80 mV, the N-type channels also contribute to exocytosis (G. Arroyo, M. Aldea, A. Albillos, and A. G. García, unpublished data). It may be that previous experiments using cell populations or intact cat adrenal glands (81, 161) and long-duration (seconds) depolarizing stimuli inactivated the N-type calcium channels.

B. Bovine Chromaffin Cells

K⁺-evoked catecholamine secretion from bovine chromaffin cells is greatly potentiated in the presence of the DHP L-type channels agonist BAY K 8644; the rise in secretion parallels the increase in ⁴⁵Ca uptake (168). Ceña et al. (84) showed that nitrendipine completely blocked catecholamine release ([³H]norepinephrine) in bovine chromaffin cells stimulated with high K⁺. These results do not agree with those obtained by other authors who found that in bovine chromaffin cells DHP did not block more than 40–50% of the secretion (163, 217, 303). The differences may be based on different stimulation patterns and the use of cultured chromaffin cells, fast-superfused cell populations, or the intact perfused adrenal gland.

When it was possible to selectively block specific subtypes of calcium channels with toxins, it was demonstrated that these cells contain other calcium channel subtypes besides L, i.e., N and the P/Q type (9, 43, 57, 58, 157). -Conotoxin GVIA was ineffective or just barely effective in blocking K⁺-evoked catecholamine secretion (26, 28, 129, 144, 217, 249, 303) in bovine chromaffin cells.

Reports on the contribution of P-type calcium channels to catecholamine secretion vary in the literature. Granja et al. (180) showed that catecholamine secretion induced by high K⁺ is not affected by -agatoxin IVA (100 nM); nevertheless, when secretion was activated by nicotine, the -agatoxin significantly decreased catecholamine release by 50%. Thus Granja et al. (180) conclude that -agatoxin IVA could also affect the nicotinic receptor. Duarte et al. (129) show that FTX decreases K⁺-evoked norepinephrine release to 25% and epinephrine release to 39% of the control levels; the combination of FTX plus nitrendipine further decreases norepinephrine and epinephrine release to 12 and 24% of the control levels. Baltazar et al. (44) showed that bovine chromaffin cells contain two types of -agatoxin IVA-sensitive calcium channels and that the contribution of the P-type channels to secretion is higher at low depolarization levels.

The L-N-P-insensitive portion of catecholamine release in bovine chromaffin cells seems to be -conotoxin MVIIC sensitive. López et al. (249) observed that catecholamine release from superfused bovine chromaffin cells (stimuli: 70 mM K⁺ for 10 s) was inhibited 50% by DHP furnidipine (3 µM). -Conotoxin MVIIC (3 µM) also reduced the secretory response by 50%. The combination of furnidipine with -conotoxin MVIIC completely abolished secretion. On the other hand, these authors also demonstrated that -conotoxin GVIA and -agatoxin IVA have no effect on secretion. These results strongly suggest that secretion in these cells is predominantly controlled by Ca²⁺ entering through the L- and Q-type calcium channels.

Further studies performed by Lara et al. (233) suggest that Q-type channels are coupled more tightly to active exocytotic sites than are the L-type channels. This hypothesis was suggested by the observation that the external Ca²⁺ that enters the cell through a calcium channel, located near chromaffin vesicles, will saturate the K⁺ secretory response at both extracellular Ca²⁺ concentrations ([Ca²⁺]_e), i.e., 0.5 and 5 mM. In contrast, Ca²⁺ entering through more distant channels will be sequestered by intracellular buffers and will therefore not saturate the secretory machinery at a lower [Ca²⁺]_e.

C. Rat Chromaffin Cells

DHPs block secretion in perfused rat adrenal glands in a concentration-dependent manner. The magnitude of this blockade is related to the type of stimuli employed to induce secretion. The DHP isradipine can fully block secretion when the stimuli used are K⁺ or nicotine. In contrast, when electrical field stimulation is used, the DHPs can only obtain a partial blockade and the inhibition is frequency dependent (248).

Measuring Ca²⁺ currents and capacitance, Kim et al. (223) have shown that -conotoxin GVIA (1 µM) blocks 40%, and nicardipine around 60% of the total capacitance increase in rat chromaffin cells. Therefore, secretion in these cells would be controlled by L- as well as by N-type calcium channels. Other recent studies suggest that secretion in rat chromaffin cells is supported by all the available calcium channels (74, 173).

The role of each calcium channel subtype in secretion has also been studied in intact whole adrenal glands from rats. Secretion evoked by depolarizing stimuli like high K⁺ was strongly inhibited (80%) by L-type calcium channel blockers, whereas acetylcholine-evoked responses were inhibited equally by either furnidipine or -conotoxin MVIIC (337). Electrical field stimulation of intact glands releases acetylcholine and other cotransmitters from the splanchnic nerves (397). Under these conditions, N-type calcium channels seem to contribute to the maintenance of the secretory responses, probably through acting on presynaptic channels at the splanchnic nerve terminals (337). In rat chromaffin cells treated with cAMP, a "low-threshold" exocytotic response was triggered at very low depolarizations; this unusual secretory response is associated with the _1H-subtype of calcium channels (173).

D. Dog Chromaffin Cells

Kimura et al. (224) have studied the effects of -conotoxin GVIA and L-type calcium channel blockers (nifedipine and verapamil) on catecholamine release in anesthetized dogs. Catecholamine release into the bloodstream was induced either by electrical stimulation of the splanchnic nerve or by intra-arterial injection of acetylcholine. Administration of 0.4 µg/ml of -conotoxin GVIA reduced catecholamine secretion by 30% in response to the electrical stimulation used, and nifedipine or verapamil had no effect under these experimental conditions. However, when catecholamine release was induced by acetylcholine, -conotoxin GVIA blocked secretion by 50% and nifedipine also reduced it by 50%. These results suggest that N- and L-type calcium channels contribute to the release of catecholamines in the dog adrenal gland. To our knowledge, there is no available patch-clamp study that determines the subtypes of calcium channels expressed by dog chromaffin cells.

E. Mouse Chromaffin Cells

Simultaneous recordings of Ca²⁺ current (I_Ca) and change in membrane capacitance (C_m) in isolated mouse chromaffin cells indicate that exocytosis is proportional to the relative density of each calcium channel subtype: 40% L, 34% N, 14% P/Q, and 11% R (12). This is in line with some of the observations reported above on bovine (139, 251) and rat chromaffin cells (74). In _1A-deficient mice, the L component of I_Ca rose to 53% while the P/Q channel contribution disappeared; the N and R contributions were similar (12). This indicates that under the perforated-patch configuration, the secretory response elicited by 200-ms depolarizing pulses is a strict function of the amount of Ca²⁺ entering the cell, by whatever calcium channel subtype, L, N, P/Q, or R. In addition, it seems that any calcium channel type colocalizes with the secretory machinery in a similarly random manner and shows the same relative efficacy in activating exocy