Guinea-pig Adrenal Medullary Cells Research Articles

Mechanisms for pituitary adenylate cyclase-activating polypeptide-induced increase in excitability in guinea-pig and mouse adrenal medullary cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) acts on adrenal medullary (AM) cells as a neurotransmitter of the sympathetic preganglionic nerve. In guinea-pig AM cells, PACAP induces little catecholamine secretion, but enhances secretion evoked by stimulants, whereas in other animals, such as mouse, PACAP itself induces depolarization and/or catecholamine secretion. The present studies aim to explore the physiological implication of these species differences in PACAP actions, the ion channel mechanism for PACAP-induced depolarization, and the mechanism for facilitation of muscarinic receptor-mediated cation currents in mouse and guinea-pig AM cells. The perforated patch clamp technique was used to record the whole-cell current in isolated AM cells. The amplitudes of 3 nM PACAP-induced inward currents were significantly larger in mouse AM cells than guinea-pig, whereas 1 μM muscarine-induced currents were larger in guinea-pig AM cells than mouse. Exposure to PACAP consistently resulted in enhancement of muscarine-induced currents in guinea-pig AM cells and facilitation of cell membrane insertion of heteromeric TRPC1-TRPC4 channels in response to muscarine in PC12 cells. The PACAP-induced current was inhibited by 30 μM 9-phenanthrol, a specific TRPM4 channel inhibitor, and abolished by replacement of external Na+ with N-methyl D-glucamine. TRPM4-like immunoreactivity was located at the cell periphery in AM cells. The present results indicate that PACAP and muscarinic receptors are major metabotropic receptors mediating generation of depolarizing inward currents in mouse and guinea-pig AM cells, respectively. We conclude that PACAP activates TRPM4-like channels and enhance the muscarinic current through facilitating the membrane insertion of TRPC1-TRPC4 channels in AM cells.

STIM1-dependent membrane insertion of heteromeric TRPC1-TRPC4 channels in response to muscarinic receptor stimulation.

Muscarinic receptor stimulation results in activation of nonselective cation (NSC) channels in guinea pig adrenal medullary (AM) cells. The biophysical and pharmacological properties of the NSC channel suggest the involvement of heteromeric channels of TRPC1 with TRPC4 or TRPC5. This possibility was explored in PC12 cells and guinea pig AM cells. Proximity ligation assay (PLA) revealed that when exogenously expressed in PC12 cells, TRPC1 forms a heteromeric channel with TRPC4, but not with TRPC5, in a STIM1-dependent manner. The heteromeric TRPC1-TRPC4 channel was also observed in AM cells and trafficked to the cell periphery in response to muscarine stimulation. To explore whether heteromeric channels are inserted into the cell membrane, tags were attached to the extracellular domains of TRPC1 and TRPC4. PLA products developed between the tags in cells stimulated by muscarine, but not in resting cells, indicating that muscarinic stimulation results in the membrane insertion of channels. This membrane insertion required expression of full-length STIM1. We conclude that muscarinic receptor stimulation results in the insertion of heteromeric TRPC1-TRPC4 channels into the cell membrane in PC12 cells and guinea pig AM cells.

Differences among muscarinic agonists in M1 receptor-mediated nonselective cation channel activation and TASK1 channel inhibition in adrenal medullary cells

Muscarinic receptor stimulation induces depolarizing inward currents and catecholamine secretion in adrenal medullary (AM) cells from various mammals. In guinea-pig AM cells muscarine and oxotremorine at concentrations ≤ 1 μM produce activation of nonselective cation channels with a similar potency and efficacy, whereas muscarine at higher concentrations produces not only nonselective cation channel activation, but also TASK1 channel inhibition. In rat AM cells, the muscarinic M1 receptor is involved in TASK1 channel inhibition in response to muscarinic agonists, and the efficacy of oxotremorine is half that of muscarine. These pharmacological findings might indicate that different muscarinic receptor subtypes are responsible for the regulation of nonselective cation and TASK1 channel activities. The present study aimed to determine the muscarinic receptor subtypes involved in nonselective cation channel activation in guinea-pig and mouse AM cells. The inward current evoked by 1 μM muscarine was completely suppressed by 100 μM quinine, whereas 30 μM muscarine-induced inward currents were comprised of quinine-sensitive and -insensitive components. The electrophysiological and pharmacological properties of the muscarine-induced currents indicated that the quinine-sensitive and insensitive components are due to nonselective cation channel activation and TASK1 channel inhibition, respectively. Muscarine at 30 μM failed to induce any current in AM cells treated with muscarinic toxin 7 or genetically deleted of the M1 receptor. The KD value of VU0255035 against the muscarinic receptor mediating nonselective cation channel activation was 17.5 nM. These results indicate that the M1 receptor mediates nonselective cation channel activation as well as TASK1 channel inhibition.

Regulation of α3-containing GABAA receptors in guinea-pig adrenal medullary cells by adrenal steroids

GABA is thought to function as a paracrine factor in adrenal medullary (AM) cells. Thus, we electrophysiologically and immunologically examined the properties of GABAA receptors (GABAARs) in guinea-pig AM cells. Bath application of GABA produced an inward current at −60mV in a dose-dependent manner with an EC50 of 32.3μM. This GABA-induced current was enhanced by allopregnanolone at concentrations of 0.01μM and more. A prior exposure to allopregnanolone resulted in a decrease in an EC50 for GABA in activating GABAARs. The GABA-induced current was suppressed by Zn2+ in a dose-dependent manner with an IC50 of 18μM, whereas it was enhanced by 100μM La3+. The benzodiazepine analog diazepam was three times more potent than zolpidem in enhancing the GABA current, and it was also augmented by L-838,417, which has no action on α1-containing GABAARs. The GABAAR α3, but not α1, and γ2 subunits were immunologically detected at the cell periphery. The expression of α3 subunits in PC12 cells was enhanced by glucocorticoid activity. The results indicated that GABAARs in guinea-pig AM cells mainly comprise α3, β, and γ2 subunits and are enhanced by allopreganalone and glucocorticoids may play a major role in the expression of α3 subunits.

Mechanisms and roles of muscarinic activation in guinea-pig adrenal medullary cells

Muscarinic receptors are expressed in the adrenal medullary (AM) cells of various mammals, but their physiological roles are controversial. Therefore, the ionic mechanism for muscarinic receptor-mediated depolarization and the role of muscarinic receptors in neuronal transmission were investigated in dissociated guinea-pig AM cells and in the perfused guinea-pig adrenal gland. Bath application of muscarine induced an inward current at -60 mV. This inward current was partially suppressed by quinine with an IC(50) of 6.1 μM. The quinine-insensitive component of muscarine-induced currents changed the polarity at -78 mV and was inhibited by bupivacaine, a TWIK-related acid-sensitive K(+) (TASK) channel inhibitor. Conversely, the current-voltage relationship for the bupivacaine-insensitive component of muscarine currents showed a reversal potential of -5 mV and a negative slope below -40 mV. External application of La(3+) had a double action on muscarine currents of both enhancement and suppression. Immunoblotting and immunocytochemistry revealed expression of TASK1 channels and cononical transient receptor potential channels 1, 4, 5, and 7 in guinea-pig AM cells. Retrograde application of atropine reversibly suppressed transsynaptically evoked catecholamine secretion from the adrenal gland. The results indicate that muscarinic receptor stimulation in guinea-pig AM cells induces depolarization through inhibition of TASK channels and activation of nonselective cation channels and that muscarinic receptors are involved in neuronal transmission from the splanchnic nerve.

Two distinct inhibitory actions of steroids on cholinoceptor-mediated secretion of catecholamine from guinea-pig adrenal medullary cells

We investigated the effects of dexamethasone, cortisol and aldosterone on responses to nicotine and muscarine in guinea-pig isolated adrenal medullary cells. Nicotine-induced inward currents were reversibly inhibited by these steroids in a dose-dependent and non-competitive manner. These steroids inhibited an increase in [Ca 2+] i in response to nicotine but not muscarine. Muscarine-induced catecholamine secretion was inhibited by cortisol and aldosterone but not dexamethasone. Phorbol 12-myristate 13-acetate, a protein kinase C activator, caused catecholamine secretion which was inhibited by cortisol and aldosterone but not dexamethasone. These results suggest that catecholamine secretion induced by cholinoceptor stimulation is inhibited by steroids via two distinct mechanisms; one is the inhibition of nicotinic receptors, another is the inhibition of protein kinase C activation in guinea-pig adrenal medullary cells.

Cytochrome b558 (p22phox) in the guinea‐pig adrenal medulla

Paraganglionic cells are sensitive to hypoxia, and the involvement of a plasmalemmal cytochrome b558-like protein in oxygen sensing by these cells has been suggested, but neither the identity of the immunoreactive protein detected by immunohistochemistry nor its anticipated subcellular (i.e., plasmalemmal) localization were directly proven. Thus, we extended these studies to the largest paraganglion, i.e., the adrenal medulla, in the guinea-pig, which, due to its size and accessibility, allowed us to address both of these issues utilizing antisera raised against synthetic peptides of the small (22 kD) subunit of cytochrome b558, p22phox. Cytochrome b558 was originally identified in granulocytes and macrophages, and antisera against this phagocyte p22phox were utilized. Immunoreactivity to p22phox was observed in all adrenal medullary endocrine cells, and the identity of the immunoreactive protein to the small cytochrome b558-subunit was confirmed by Western blotting. Immuno-electron microscopy of ultrathin cryosections and of resin-embedded tissue demonstrated its subcellular localization in the dense core vesicles of endocrine A-cells but not in the plasma membrane. In conclusion, the present study documents the presence of the small subunit of cytochrome b558 in guinea-pig adrenal medullary cells, but its subcellular vesicular localization does not support the initial interpretation of cytochrome b558 serving as a plasmalemmal oxygen sensor. Microsc. Res. Tech. 47:215–220, 1999. © 1999 Wiley-Liss, Inc.

Cytochrome b558 (p22phox) in the guinea-pig adrenal medulla.

Paraganglionic cells are sensitive to hypoxia, and the involvement of a plasmalemmal cytochrome b558-like protein in oxygen sensing by these cells has been suggested, but neither the identity of the immunoreactive protein detected by immunohistochemistry nor its anticipated subcellular (i.e., plasmalemmal) localization were directly proven. Thus, we extended these studies to the largest paraganglion, i.e., the adrenal medulla, in the guinea-pig, which, due to its size and accessibility, allowed us to address both of these issues utilizing antisera raised against synthetic peptides of the small (22 kD) subunit of cytochrome b558, p22phox. Cytochrome b558 was originally identified in granulocytes and macrophages, and antisera against this phagocyte p22phox were utilized. Immunoreactivity to p22phox was observed in all adrenal medullary endocrine cells, and the identity of the immunoreactive protein to the small cytochrome b558-subunit was confirmed by Western blotting. Immuno-electron microscopy of ultrathin cryosections and of resin-embedded tissue demonstrated its subcellular localization in the dense core vesicles of endocrine A-cells but not in the plasma membrane. In conclusion, the present study documents the presence of the small subunit of cytochrome b558 in guinea-pig adrenal medullary cells, but its subcellular vesicular localization does not support the initial interpretation of cytochrome b558 serving as a plasmalemmal oxygen sensor.

Comparative study of different species of oxygen free radicals on the ATP-sensitive potassium channels of the heart

The effects of vasoactive intestinal polypeptide (VIP) on catecholamine (CA) secretion by isolated guinea pig adrenal medullary cells were studied. VIP (1 μM) alone induced only a slight secretion of CA, but it stimulated ACh-induced CA secretion. At concentrations of 0.01–1 μM, it stimulated muscarine-induced CA secretion, but not nicotine-induced CA secretion. It did not affect high K+ or Ca2+ ionophore-induced CA secretion. The stimulatory effect of VIP on muscarine-induced CA secretion was observed at muscarine concentrations of 2–200 μM and was detectable after 2 min incubation.

Glucocorticoids inhibit acetylcholine-induced current in chromaffin cells.

The effects of dexamethasone, a synthetic glucocorticoid, on membrane excitability and on the acetylcholine (ACh)-induced inward current (IACh) were studied in dispersed guinea pig adrenal medullary cells by using the whole cell version of the patch-clamp technique. Bath application of 10 microM dexamethasone had no effect on the resting membrane conductance or voltage-gated Na+, Ca2+, or K+ channels, whereas it reversibly inhibited the IACh at a holding potential of -70 mV. Intracellular application of dexamethasone through the patch electrode did not modify the IACh. Application of 10 microM dexamethasone neither shifted the dose-response curve of the peak IACh to the right [dissociation constant (Kd) = 50 microM] nor affected its Hill coefficient (1.2) but inhibited the current amplitudes by approximately 41% in the cells sufficiently pretreated with dexamethasone. Furthermore, fractional inhibition of the IACh at the end of approximately 50-s application was the same for any concentration of ACh (3-100 microM). The dose-response curve of the inhibition by dexamethasone showed a good fit to the theoretical line assuming an inhibition constant (Ki) of 10 microM and a Hill coefficient of 1. Hydrocortisone 21-hemisuccinate sodium salt (25 microM) and prednisolone 21-hemisuccinate sodium salt (25 microM) inhibited the IACh to a lesser extent than 25 microM dexamethasone. These results suggest that dexamethasone binds to the specific site on the outer cell membrane, probably on the ACh receptor-coupled channel, and inhibits the IACh in a noncompetitive manner, thus controlling the immediate catecholamine release from the adrenal medulla.

O-248 - Voltage clamp study of the ACh-induced excitation of dispersed guinea-pig adrenal medullary cells

O-248 - Voltage clamp study of the ACh-induced excitation of dispersed guinea-pig adrenal medullary cells

P-179 - Stimulation by vasoactive intestinal polypeptide of muscarinic receptor-mediated catecholamine secretion from isolated guinea pig adrenal medullary cells

P-179 - Stimulation by vasoactive intestinal polypeptide of muscarinic receptor-mediated catecholamine secretion from isolated guinea pig adrenal medullary cells

Stimulation by vasoactive intestinal polypeptide of muscarinic receptor-mediated catecholamine secretion from isolated guinea pig adrenal medullary cells

The effects of vasoactive intestinal polypeptide (VIP) on catecholamine (CA) secretion by isolated guinea pig adrenal medullary cells were studied. VIP (1 μM) alone induced only a slight secretion of CA, but it stimulated ACh-induced CA secretion. At concentrations of 0.01–1 μM, it stimulated muscarine-induced CA secretion, but not nicotine-induced CA secretion. It did not affect high K + or Ca 2+ ionophore-induced CA secretion. The stimulatory effect of VIP on muscarine-induced CA secretion was observed at muscarine concentrations of 2–200 μM and was detectable after 2 min incubation.