Muscarinic Receptor Stimulation Does Not Inhibit Voltage-dependent Ca2+ Channels in Rat Adrenal Medullary Chromaffin Cells

Abstract

Adrenal medullary chromaffin (AMC) and sympathetic ganglion cells are derived from the neural crest and show a similar developmental path. Thus, these two cell types have many common properties in membrane excitability and signaling. However, AMC cells function as endocrine cells while sympathetic ganglion cells are neurons. In rat sympathetic ganglion cells, muscarinic M₁ and M₄ receptors mediate excitation and inhibition via suppression of M-type K⁺ channels and suppression of voltage-dependent Ca²⁺ channels, respectively. On the other hand, M₁ receptor stimulation in rat AMC cells also produces excitation by suppressing TWIK-related acid sensitive K⁺ (TASK) channels. However, whether M₄ receptors are coupled with voltage-dependent Ca²⁺ channel suppression is unclear. We explore this issue electrophysiologically and biochemically. Electrical stimulation of nerve fibers in rat adrenal glands trans-synaptically increased the Ca²⁺ signal in AMC cells. This electrically evoked increased Ca²⁺ signal was not altered during muscarine-induced increase in Ca²⁺ signal, whereas it decreased significantly during a GABA-induced increase, due to a shunt effect of increased Cl^- conductance. The whole-cell current recordings revealed that voltage-dependent Ca²⁺ currents in AMC cells were suppressed by adenosine triphosphate, but not by muscarinic agonists. The fractionation analysis and immunocytochemistry indicated that Ca_V1.2 Ca²⁺ channels and M₄ receptors are located in the raft and non-raft membrane domains, respectively. We concluded that muscarinic stimulation in rat AMC cells does not produce voltage-dependent Ca²⁺ channel inhibition. This lack of muscarinic inhibition is at least partly due to physical separation of voltage-dependent Ca²⁺ channels and M₄ receptors in the plasma membrane.

Keywords: ATP; chromaffin cell; muscarinic receptor; raft membrane domain; voltage-dependent Ca2+ channel.

Fig. 1.

Effects of muscarine and GABA on trans-synaptically evoked increase in the intracellular Ca²⁺ concentration in rat adrenal medullary chromaffin (AMC) cells. (A and B) Relative changes in Ca²⁺ signal in Fluo-4-loaded AMC cells are plotted against time. The adrenal gland was retrogradely perfused through the adrenal vein with saline. Nerve fibers remaining in the gland were electrically stimulated with 50 V pulses of 1.5 msec in duration at 0.5, 1, and 5 Hz during the indicated periods (bars). Muscarine (MUS) and GABA, each at 30 μM, were added to the perfusion solution during the indicated period (interrupted lines). Fluorescence intensities in up to eight areas in each frame were measured. After correction for the decline due to photobleaching, the increase in fluorescence intensity in response to electrical stimulation and muscarine or GABA is expressed as a fraction of the resting level (see the Materials and Methods). (C) Summary of the relative changes in Ca²⁺ signal in response to electrical stimulation at 0.5, 1, and 5 Hz. The data represent the mean ± standard error of the mean (SEM) of eight adrenal glands. (D and E) Relative amplitudes of the increase in the Ca²⁺ signal electrically evoked at 1 and 5 Hz during application of MUS and GABA, respectively. The relative amplitudes were obtained by dividing the relative changes in Ca²⁺ signal during MUS or GABA application with an average of these before and after its application. Statistical significance was evaluated by a paired Student’s t test. The trans-synaptically evoked increase in Ca²⁺ signal significantly decreased during application of GABA, but not MUS. The data represent the mean ± SEM of six and eight adrenal glands for MUS and GABA, respectively.

Fig. 2.

Lack of muscarinic receptor-mediated inhibition of voltage-dependent Ca²⁺ channels in rat AMC cells. (A and B) Muscarine (MUS) and adenosine triphosphate (ATP) did not and did produce a rapid inhibition of voltage-dependent Ca²⁺ currents in dissociated rat AMC cells, respectively. The amplitudes of voltage-dependent Ca²⁺ currents are plotted against the time. The whole cell current was recorded with the perforated path clamp method where the pipette was filled with CsCl (see the Materials and Methods). The holding potential was −70 mV, and 70 mV test pulses of 50 msec in duration were applied every 10 sec. The amplitudes of the current at the end of test pulses were measured with reference to the current level just before the test pulse. The insets (a, b, and c) show the current responses to the 70 mV test pulses. a, b, and c in the insets correspond to a, b, and c in the plots. (C) Summary of the amplitudes of voltage-dependent Ca²⁺ currents (I_Ca) in the presence of ATP, MUS, or oxotremorine (OXO) at concentrations of 30 to 100 μM. The I_Ca amplitudes during application of chemicals are expressed relative to the averaged values of I_Ca before and after it. The data represent the mean ± SEM (ATP, n = 7; MUS, n = 6: OXO, n = 4). Statistical significance was evaluated with the Mann–Whitney rank sum test.

Fig. 3.

Different distribution of Ca_V1.2 channel and muscarinic M₄ receptor in the cell membrane in rat AMC cells. (A) Immunostaining of Ca_V1.2-YFP fusion protein expressed in HEK293T cells with a rabbit anti-Ca_V1.2 antibody (Ab). HEK293T transfected with a Ca_V1.2-YFP construct were labeled with the rabbit anti-Ca_V1.2 Ab. a and b represent confocal images of Ca_V1.2-like immunofluorescence and YFP fluorescence, respectively; c represents a differential interference contrast (DIC) image. The immunoreaction and YFP fluorescence were visualized with excitation at 514 nm and emission of 530–600 nm and with excitation at 633 and emission above 650 nm, respectively. (B) Fractionation analysis of rat adrenal medullae for integral membrane proteins. The cell membrane was divided into the raft and non-raft membrane domains by using discontinuous sucrose density gradient centrifugation (see the Materials and Methods). The same volume of each fraction with 5%–40% sucrose was immunoblotted for caveolin-1, transferrin receptor (R), muscarinic M₄ receptor, and TASK1 channel. Note that caveolin-1, a raft membrane marker, was enriched in the 20% fraction, whereas transferrin R, a non-raft membrane marker, was present in the 40% fraction. (C) Double staining for caveolin-1 and Ca_V1.2 and for M₄ receptor and Ca_V1.2 in rat AMC cells. The first column indicates confocal images of caveolin-1 and M₄ receptor-like immunofluorescence. The second column shows confocal images of Ca_V1.2-like immunofluorescence. The third column is a merge of immunofluorescence images. The fourth column shows DIC images. The calibration applies to all the images. Dissociated rat AMC cells were treated overnight with rabbit anti-Ca_V1.2 Ab (dilution, 1:50) and mouse anti-caveolin-1 Ab (1:20) or mouse anti-M₄ Ab (1:50). Ca_V1.2 and caveolin-1 or M₄ receptor-like immunoreactive material were visible as rhodamine and FITC-like fluorescence, respectively. (D) Summary of the coincidence rates of caveolin-1 (Cav1) and M₄ with Ca_V1.2. The data represent the mean ± SEM (Cav1/Ca_V1.2, n = 10; M₄/Ca_V1.2, n = 5). Statistical significance was evaluated with an unpaired Student’s t test.

Fig. 4.

Diagram showing localization of caveolin-1, Ca_V1.2, muscarinic M₄ receptor subtype, and TASK1 in the raft and non-raft membrane domains.