Role of membrane depolarization and T-type Ca2+ channels in angiotensin II and K+ stimulated aldosterone secretion.

The hypothesis that Ca2+ influx necessary for angiotensin II (AngII) and K+ stimulation of aldosterone secretion is primarily mediated by membrane depolarization and activation of T-type Ca2+ channels was examined in isolated rat adrenal glomerulosa cells. Perforated-patch clamp recordings of membrane potential (Vm) demonstrated that AngII and K+ induce concentration-dependent depolarizations capable of activating T channels and, at high K+ and AngII concentrations, activating L channels and inactivating T channels. K+-induced depolarizations were stable and readily reversible. Vm was proportional to K+ concentration, exhibiting a linear slope of 53.7 mV per 10-fold increase in K+. AngII-induced depolarizations were complex, consisting of a slow maintained component superimposed with small amplitude depolarizing fluctuations. Slow oscillations in Vm were occasionally observed in response to 10(-9) M AngII or greater. The slow, maintained component of depolarization coincided with inhibition of K+ conductance. Neither rapid fluctuations nor slow oscillations in Vm were blocked by mibefradil or other treatments that inhibit voltage-gated Ca2+ channels. Perforated-patch clamp experiments also demonstrated that AngII (10(-8) M) inhibited L channels by 45.6% without affecting T channels. Thus AngII activates T channels by depolarization rather than T channel modulation in rat cells. The concentration dependencies of mibefradil inhibition of T channels and AngII- and K+-induced aldosterone secretion were compared. Under whole-cell patch clamp mibefradil induced a concentration-dependent inhibition of T channels, exhibiting a K(app) of 0.62 microM. Mibefradil inhibition was use-dependent but mibefradil neither acted as an open channel blocker nor significantly affected T channel inactivation or activation. Mibefradil inhibited K+- and AngII-induced secretion at concentrations similar to that for T channel inhibition; at high concentrations (10 microM) mibefradil inhibited AngII-induced secretion by 88% and completely inhibited K+-induced secretion. The IC50 for K+-induced secretion was dependent on K+ concentration, increasing from 0.2 microM for 6 mM K+ to 2.5 microM for 10 mM K+ or greater. Mibefradil exhibited an IC50 of 1.1 microM for inhibition of secretion at all AngII concentrations examined (0.1, 1.0, and 10 nM). Mibefradil also exhibited multiple nonspecific effects, which complicated the assessment of T channel function, including; inhibition of leak and voltage-dependent K+ conductances, inhibition of Ca2+-independent aldosterone secretion, and inhibition of secretion under conditions expected to completely inactivate T channels (10 nM AngII or 20 mM K+). In summary, these results indicate that voltage-gated T channels represent the primary Ca2+ influx pathway activated by physiological concentrations of AngII and K+ but other Ca2+ influx pathways must mediate aldosterone secretion induced by high K+ or AngII concentrations.

Divalent cation permeability and blockade of Ca2+-permeant non-selective cation channels in rat adrenal zona glomerulosa cells.

1. The effects of the divalent cations Ca2+, Mg2+ and Ni2+ on unitary Na+ currents through receptor-regulated non-selective cation channels were studied in inside-out and cell-attached patches from rat adrenal zona glomerulosa cells. 2. External Ca2+ caused a concentration-dependent and voltage-independent inhibition of inward Na+ current, exhibiting an IC50 of 1.4 mM. The channel was also Ca2+ permeant and external Ca2+ shifted the reversal potential as expected for a channel exhibiting a constant Ca2+ : Na+ permeability ratio near to 4. 3. External and internal 2 mM Mg2+ caused voltage-dependent inhibition of inward and outward Na+ current, respectively. Modelling Mg2+ as an impermeant fast open channel blocker indicated that external Mg2+ blocked the pore at a single site exhibiting a zero voltage Kd of 5.1 mM for Mg2+ and located 19 % of the distance through the transmembrane electric field from the external surface. Internal Mg2+ blocked the pore at a second site exhibiting a Kd of 1.7 mM for Mg2+ and located 36% of the distance through the transmembrane electric field from the cytosolic surface. 4. External Ni2+ caused a voltage- and concentration-dependent slow blockade of inward Na+ current. Modelling Ni2+ as an impermeant slow open channel blocker indicated that Ni2+ blocked the pore at a single site exhibiting a Kd of 1.09 mM for Ni2+ and located 13.7% of the distance through the transmembrane electric field from the external surface. 5. External 2 mM Mg2+ increased the Kd for external Ni2+ binding to 1.27 mM, consistent with competition for a single binding site. Changing ionic strength did not substantially affect Ni2+ blockade indicating the absence of surface potential under physiological ionic conditions. 6. It is concluded that at least two divalent cation binding sites, separated by a high free energy barrier (the selectivity filter), are located in the pore and contribute to Ca2+ selectivity and permeability of the channel.

Effects of K+ channel blockers on K+ channels, membrane potential, and aldosterone secretion in rat adrenal zona glomerulosa cells.

The hypothesis that angiotensin II (ANG II)-induced aldosterone secretion is mediated through inhibition of plasma membrane K+ channels was examined by measuring the effects of K+ channel blockers on K+ currents, membrane potential, and aldosterone secretion in rat adrenal glomerulosa cells. Effective K+ channel blockers were identified and studied using patch clamp methods on isolated glomerulosa cells in cell culture. Extracellular Cs+ (2-20 mm) caused a voltage-dependent inhibition of macroscopic K+ currents, exhibiting an apparent Kd of 2 mM for blockade of K+ current at membrane potentials near the K+ equilibrium potential. Outward K+ current opposed the Cs+ block, imparting a steep voltage dependence to this block. In single channel studies Cs blocked inward, but not outward, unitary currents through ANG II-regulated weakly voltage-dependent K+ channels, which are thought to control resting membrane potential. Cs+ reversibly depolarized the resting membrane potential at concentrations greater than or equal to the apparent Kd for K+ conductance inhibition (> or =2 mM). Depolarization consisted of a slow, maintained phase proportional to Cs+ concentration superimposed with 2- to 5-mV transient depolarizing events. Cs+ induced a Ca2+-dependent stimulation of aldosterone secretion in acutely dissociated cells, exhibiting an EC50 of approximately 3 mM. Maximal Cs+-induced secretion was quantitatively similar to 1 nM ANG II- or 8 mM K+-induced secretion. Cs+-induced secretion was not additive with that of ANG II. K+ channel blockers that did not inhibit weakly voltage-dependent K+ channels at rest (quinidine, apamin, and charybdotoxin) did not cause depolarization or stimulate aldosterone secretion. Furthermore, charybdotoxin did not significantly affect ANG II-induced aldosterone secretion, indicating that Ca2+-dependent maxi-K+ channels did not contribute to the control of aldosterone secretion in acutely dissociated cells. These data strongly support involvement of weakly voltage-dependent K+ channels in ANG II-induced aldosterone secretion, but also implicate roles for other channel classes in controlling membrane potential during ANG II-induced aldosterone secretion.

Characterization of angiotensin II-regulated K+ conductance in rat adrenal glomerulosa cells.

Nystatin perforated-patch clamp and single-channel recording methods were used to characterize macroscopic and single-channel K+ currents and the effects of angiotensin II (AngII) in cultured rat adrenal glomerulosa cells. Two basic patterns of macroscopic current-voltage relationships were observed: type 1 exhibited a rapidly activating, noninactivating, voltage-dependent outward current and type 2 exhibited an inactivating voltage-dependent outward current attributed to charybdotoxin sensitive Ca++-dependent K+ channels. Most cells exhibited the type 1 pattern and experiments focused on this cell type. Cell-attached and inside-out patches were dominated by a single K+ channel class which exhibited an outward conductance of 12 pS (20 mm K+ pipette in cell-attached and inside-out configurations, 145 mm K+in), a mean open time of 2 msec, and a weakly voltage-dependent low open probability that increased with depolarization. Channel open probability was reversibly inhibited by bath stimulation with AngII. At the macroscopic level, type 1 cell macroscopic K+ currents appeared comprised of two components: a weakly voltage-dependent current controlling the resting membrane potential (-85 mV) which appeared mediated by the 12 pS K+ channel and a rapidly activating, noninactivating voltage-dependent current activated above -50 mV. The presence of the second voltage-dependent K+ channel class was suggested by the effects of AngII, the blocking effects of quinidine and Cs+, and the properties of the weakly voltage-dependent K+ channel described. The K+ selectivity of the macroscopic current was demonstrated by the dependence of current reversal potentials on the K+ equilibrium potential and by the effects of K+ channel blockers, Cs+ and quinidine. AngII (10 pm to 1 nm) reversibly inhibited macroscopic K+ currents and this effect was blocked by the AT1 receptor antagonist losartin.

Angiotensin II activation of Ca(2+)-permeant nonselective cation channels in rat adrenal glomerulosa cells.

A Ca(2+)-permeant, nonselective cation channel was observed in cell-attached and inside-out membrane patches from rat adrenal glomerulosa cells maintained in primary cell culture. In cell-attached patches under near physiological ionic conditions, single-channel currents exhibited a reversal potential near -10 mV, inward rectification, a nearly linear slope conductance between 0 and -80 mV of 17.4 pS, and voltage-dependent block at potentials more negative than -80 mV. Channels exhibiting identical conductance and gating properties were observed in inside-out patches; however, channel gating ran down within minutes in this configuation. In the inside-out configuration, channel gating did not require cytosolic Ca2+ (Ca2+ < 10(-9) M), and inward rectification was relieved by removal of intracellular Mg2+. Relative ionic permeability was calculated using reversal potential measurements from inside-out patches under bi-ionic conditions. The channel discriminated poorly among monovalent cations (PLi > PK > PCs > PNa) and was not significantly permeable to anions. The channel was permeable to Ca2+, exhibiting a relative permeability ratio of 0.29 PCa/PNa) when measured with 110 mM Ca2+ on the intracellular face and a permeability ratio of 4.38 (PCa/PNa) with 110 mM Ca2+ on the extracellular face. Channel gating behavior was episodic with open times ranging from milliseconds to tens of seconds and closed times lasting up to several minutes or longer. Channel gating appeared to be relatively voltage independent except that mean channel open time and open probability were reduced by membrane hyperpolarization. In cell-attached patches, bath application of 1 nM angiotensin II (ANG II) increased the channel open probability, primarily affecting channels exhibiting a low open probability, primarily affecting channels exhibiting a low open probability before stimulation. With the use of nystatin perforated-patch current clamp to measure membrane potential, ANG II was observed to induce large transient membrane depolarizations, consistent with activation of an inward current. We hypothesize that this channel is an important component of ANG II-induced membrane depolarization and Ca2+ influx during stimulation of aldosterone secretion.

Atrial natriuretic peptide inhibition of calcium ionophore A23187-stimulated aldosterone secretion in rat adrenal glomerulosa cells.

The effect of atrial natriuretic peptide (ANP) on calcium ionophore A23187-stimulated aldosterone secretion was investigated using collagenase-dispersed rat adrenal glomerulosa cell suspensions. A23187 treatment induced a dose-dependent stimulation of aldosterone secretion, exhibiting an EC50 of approximately 75 nM. In agreement with the presumed action of A23187 as a Ca2+ ionophore, stimulation was dependent on the extracellular Ca2+ concentration, being completely inhibited in nominally Ca(2+)-free medium. In such Ca(2+)-free medium, stimulation of aldosterone secretion by bath applied 25-hydroxycholesterol was not inhibited, indicating that cells and biosynthetic pathway enzymes were not inhibited by low extracellular Ca2+ levels. A23187-induced aldosterone secretion was also inhibited by more than 90% when cells were simultaneously treated with ANP. Maximal ANP inhibition of A23187-stimulated aldosterone secretion was not overcome by concentrations of A23187 up to 10 microM or by increasing the extracellular Ca2+ concentration from 1.25 to 5 mM in the presence of A23187 and ANP. Addition of A23187 to ACTH-, angiotensin II-, or K(+)-stimulated glomerulosa cells did not overcome ANP-induced inhibition of aldosterone secretion stimulated by these secretagogues. In contrast to ANP inhibition of Ca(2+)-dependent A23187 stimulation of aldosterone secretion, ANP inhibition of dBcAMP-stimulated aldosterone secretion was readily overcome by increasing the dBcAMP concentration. These results indicated that ANP selectively and noncompetitively inhibited an intracellular step necessary for Ca(2+)-dependent stimulation of the early pathway of aldosterone biosynthesis in rat adrenal glomerulosa cells.

Local generation of angiotensin II as a mechanism of aldosterone secretion in rat adrenal capsules.

Angiotensin-converting enzyme (ACE) is found in the adrenal gland, but the role of adrenal ACE in the formation of angiotensin II (AII) and subsequent stimulation of aldosterone is unclear. We examined the effect of adrenal ACE activity on aldosterone secretion by superfusing rat adrenal capsules with angiotensin I (AI) in the presence and absence of the ACE inhibitor, lisinopril. Angiotensin I (10 microM) stimulated aldosterone secretion from 914 +/- 41 to 1465 +/- 118 pg/min/capsule (P less than 0.05). Simultaneous superfusion of AI plus lisinopril (100 microM) inhibited the stimulation of aldosterone by 73% (P less than 0.05). Perfusion of the capsules with angiotensin II (1 microM) stimulated aldosterone from 893 +/- 180 to 1466 +/- 181 pg/min/capsule (P less than 0.01). In contrast, simultaneous superfusion of AII plus lisinopril (100 microM) did not inhibit the AII stimulation of aldosterone. The failure of lisinopril to inhibit AII stimulation of aldosterone argues against a toxic or nonspecific action of lisinopril. The inhibition of AI stimulation of aldosterone release by lisinopril is mostly due to lisinopril inhibition of ACE and resulting decreased conversion of AI to AII. These results demonstrate that adrenal ACE may generate AII from AI in the adrenal gland, and this locally produce AII stimulates aldosterone.

Guanabenz-induced inhibition of aldosterone secretion from isolated rat adrenal glomerulosa cells.

The authors examined the effects of the alpha 2-adrenergic agonist guanabenz and other alpha-adrenergic ligands on aldosterone secretion and cyclic nucleotide content in isolated rat adrenal glomerulosa cells. Guanabenz inhibited aldosterone secretion stimulated by potassium, angiotensin II (AII), and adrenocorticotropic hormone (ACTH), exhibiting IC50 values of 35 microM, 43 microM, and 58 microM for stimulation by 10 mM K+, 1 nM AII, and 10 pM ACTH, respectively. Guanabenz did not affect the cGMP content of purified adrenal glomerulosa cells but inhibited ACTH stimulation of cAMP accumulation. Guanabenz inhibition of ACTH-induced cAMP may represent a mechanism for inhibition of aldosterone secretion, however, guanabenz also inhibited aldosterone secretion stimulated by the cAMP analog dibutyryl cAMP. The effect of guanabenz on the early and late pathways of steroidogenesis was tested in the isolated rat glomerulosa cells using 25-OH cholesterol and steroid precursors to aldosterone. Guanabenz inhibited the steroidogenic response to 25-OH cholesterol stimulation of aldosterone secretion but induced a much smaller inhibition of the steroidogenic response to exogenous pregnenolone, progesterone, and 11-deoxycorticosterone. These results suggested that guanabenz inhibited aldosterone secretion primarily through inhibition of the early component of the steroidogenic pathway prior to pregnenolone formation. The effects of guanabenz were not mimicked by other alpha-adrenergic ligands suggesting that these effects of guanabenz were not mediated through activation of alpha-adrenergic receptors.

Peptidergic and serotonergic facilitation of a neuromuscular synapse in Aplysia.

(1) A buccal muscle motor neuron which synthesizes the neuromodulatory small cardioactive peptides (SCPs) was identified in the buccal ganglion of Aplysia by using a combination of electrophysiological and single cell biochemical experiments. This neuron was designated B38. (2) Exogenous SCPb enhanced B38-induced contractions when perfused over the target muscle, the rostral portion of the buccal I3 muscle. SCPB potentiation of muscle contraction was associated with an increase in the excitatory junction potential (EJP) amplitude recorded from the muscle fibers, increased muscle cyclic AMP (cAMP) content, hyperpolarization of the muscle fibers, and an increase in the muscle fiber membrane conductance. Exogenous SCPB also depolarized the cell body of B38 and increased electrical coupling between the symmetrically paired B38 neurons. (3) These results suggest that the SCPs may be co-released from B38 along with an unidentified conventional neurotransmitter to homosynaptically facilitate B38 synaptic transmission by modulating presynaptic and postsynaptic components. (4) Stimulation of the identified serotonergic metacerebral neuron or perfusion of exogenous serotonin (5-HT) over the 13 muscle also potentiated B38-induced muscle contractions and EJP amplitude. Thus the B38 neuromuscular synapse represents a peripheral site of serotonergic heterosynaptic facilitation in Aplysia. (5) Presynaptic and postsynaptic serotonergic effects were qualitatively similar to those of SCPB. Serotonergic effects on muscle fiber hyperpolarization and increase in muscle fiber membrane conductance were similar in magnitude to those of SCPB but 5-HT induced a much larger increase in the EJP amplitude which was additive with that of SCPB. (6) The effect of 5-HT on the EJP amplitude was associated with inhibition of a slowly decaying component of synaptic facilitation. Concentrations of SCPB that increased the EJP were much less effective at inhibiting the slow component of facilitation. These observations indicate that 5-HT also exerted a presynaptic effect on B38 transmitter release. (7) Both 5-HT and SCPB increased muscle cAMP levels and application of forskolin mimicked many of their effects. suggesting that at least some of the postsynaptic effects were mediated by increased cAMP levels in the 13 muscle.

Reciprocal modulation of calcium current by serotonin and dopamine in the identified Aplysia neuron R15.

Voltage-clamp methods were employed to study the effects of serotonin (5-HT) and dopamine on the pharmacologically isolated calcium current in the identified Aplysia neuron R15 grown in cell culture. Neurons were obtained from juvenile animals and had not yet developed the bursting pacemaker pattern of activity characteristic of R15 in mature animals. In R15 5-HT elicits a biphasic response consisting of excitatory depolarization followed by an inhibitory hyperpolarization and dopamine elicits an inhibitory hyperpolarization. 5-HT increased the Ca2+ current without affecting its voltage dependence. The 5-HT effect persisted when Ba2+ was employed to carry current through Ca2+ channels. 5-HT did not affect the rate of Ca2+-dependent Ca2+ current inactivation other than through its effect on the magnitude of the Ca2+ current. The adenylate cyclase activator forskolin, in the presence of a phosphodiesterase inhibitor, also increased the magnitude of the Ca2+ or Ba2+ current. This result suggested that the 5-HT-induced enhancement of Ca2+ current was mediated by cAMP. Dopamine inhibited Ca2+ current when either Ca2+ or Ba2+ was employed as the current carrier. Dopamine did not affect the rate of Ca2+-dependent inactivation of Ca2+ current other than through its effect on the magnitude of the Ca2+ current. Intracellular injection of the Ca2+ chelator EGTA inhibited serotonergic modulation of the Ca2+ current but not dopaminergic modulation. These results indicated that the putative neurotransmitters 5-HT and dopamine may regulate bursting activity in mature R15 neurons through modulation of Ca2+ current.

Serotonin and forskolin increase an inwardly rectifying potassium conductance in cultured identified Aplysia neurons.

1. The effect of serotonin (5-HT) and forskolin on an inwardly rectifying K+ conductance (IKR) was studied using voltage-clamp techniques in several identified Aplysia neurons isolated and maintained in primary cell culture. 2. Inward rectification was observed in the current-voltage relationship of the identified neurons R15, R2, B1, and B2 and was predominately due to IKR, as demonstrated by the dependence of inward rectification on the extracellular K+ concentration, instantaneous kinetics of the membrane current response to hyperpolarizing voltage clamp pulses, and voltage-dependent Ba2+ block of the inwardly rectifying current. 3. 5-HT increased IKR conductance between 100 and 400% in the identified neuron R15 in culture and increased IKR conductance approximately 50% in the identified neurons B1, B2, and R2 in culture. The adenylate cyclase activator, forskolin, plus a phosphodiesterase inhibitor, Ro 20-1724, also increased IKR conductance in these neurons. 4. 5-HT and forskolin modulated other ion conductances as well in all of these cultured neurons.

Serotonin and forskolin modulation of a chloride conductance in cultured identified Aplysia neurons.

1. The effect of serotonin (5-HT) and forskolin on a hyperpolarization activated Cl- conductance (gCl-) was studied using voltage-clamp techniques in identified Aplysia neurons maintained in primary cell culture. 2. The hyperpolarization-activated conductance induced by intracellular Cl- loading was carried by Cl- as determined by the following criteria: the extrapolated reversal potential of the current closely approximated the reversal potential of a cholinergic Cl- conductance, the current was not affected by extracellular ion substitutions other than Cl-, extracellular thiocyanate ions reversibly inhibited the current and the current exhibited slow voltage-dependent exponential kinetics similar to those described for the hyperpolarization-activated Cl- current in Aplysia neurons in situ. 3. In the identified neurons B1, B2, R15, and R2, 5-HT or forskolin reversibly inhibited gCl-, suggesting that 5-HT acted via an adenosine 3',5'-cyclic monophosphate-dependent mechanism. 4. Serotonergic inhibition resulted from a change in the voltage dependence of Cl- channel gating.

Serotonin induced protein phosphorylation in the Aplysia eye.

Serotonin (5-HT) increases the phosphorylation of two low molecular weight phosphoproteins of 23,000 and 15,000 daltons molecular weight and decreases the phosphorylation of a 20,000 dalton phosphoprotein in the isolated Aplysia eye. The cAMP analog 8-benzylthio cAMP increases and decreases the phosphorylation of the 23,000 and 20,000 dalton 5-HT sensitive phosphoproteins, respectively. The effect of 5-HT on protein phosphorylation is not affected by the phase of the circadian rhythm of spontaneous compound action potentials generated in the eye.

Fine tuning of neuronal electrical activity: modulation of several ion channels by intracellular messengers in a single identified nerve cell.

The identified neurone R15 in the abdominal ganglion of the marine mollusc, Aplysia californica, exhibits a rhythmic bursting pattern of electrical activity. This pattern, which is generated endogenously by the interaction of several voltage- and time-dependent ion currents in R15's membrane, is subject to long-term modulation by synaptic stimulation and application of several neurotransmitters. At micromolar concentrations the transmitter serotonin causes neurone R15 to hyperpolarize, as a result of the activation of an anomalously rectifying potassium conductance. Furthermore under some conditions serotonin can excite R15, as a result of the activation of a voltage-dependent calcium current. Both of these effects of serotonin are mediated by the intracellular second messenger cyclic AMP. In addition, serotonin can modulate a chloride current by a cyclic-AMP-dependent mechanism. In contrast to the activation of the voltage-dependent calcium current by serotonin/cyclic AMP, a cyclic GMP analogue alters the bursting pattern by inhibiting this current. The results indicate that a single neurotransmitter, acting via a single intracellular messenger, can modulate several classes of ion channels in a single nerve cell. Furthermore a single class of ion channel, that is responsible for a voltage-dependent calcium current, may be the target for modulation by at least two different intracellular messengers. These findings emphasize the intricacy of the regulatory pathways which contribute to fine tuning of neuronal electrical activity.

Involvement of protein synthesis in circadian clock of Aplysia eye.

The effects of the protein synthesis inhibitors anisomycin and puromycin were measured on protein synthesis and phase shifting of the circadian rhythm in the isolated Aplysia eye. Anisomycin pulses induce phase delays proportional in magnitude to the duration and percentage of protein synthesis inhibition. The phase-response curve to anisomycin pulses consisted of delays induced throughout the subjective night. Delays were maximal between circadian times (CT) 18 and CT 2; pulses initiated between CT 2 and CT 12 did not phase shift. Puromycin induced phase delays and advances. Delays were proportional to the duration and percentage of protein synthesis inhibition, occurring with increasing magnitude throughout the subjective night (CT 12-2). Peptidyl-puromycin formation may contribute to the magnitude of the delay. Advances, occurring between CT 2 and CT 8, required a greater drug concentration and pulse duration than delays and appeared to result from an effect other than protein synthesis inhibition. Our results support the hypothesis of a phase-dependent requirement for protein synthesis during the subjective night in this circadian clock.

Endocytosis of surface bound dopamine ?-hydroxylase and plasma membrane following catecholamine secretion by bovine adrenal chromaffin cells.

Cultured chromaffin cells were stimulated with either Ba(2+) or nicotine to secrete catecholamines. This resulted in the appearance of the chromaffin granule membrane protein, dopamine ?-hydroxylase (DBH), on the cell surface. The DBH exposed on the cell surface was labeled using fluorescently tagged anti-DBH Fab fragments and the cell surface was simultaneously labeled with fluorescently tagged concanavalin A. Immediately after labeling, both fluorescent markers were localized on or near the cell surface; anti-DBH fluorescence was distributed as patches, but Con A fluorescence was uniformly distributed. Approximately 30 min after labeling, anti-DBH fluorescence appeared to be almost completely internalized without apparent redistribution on the surface whereas much of the Con A fluorescence remained on the cell surface. The rate of DBH endocytosis was quantified using (125)I labeled anti-IgG to measure surface bound anti-DBH. Following stimulation of catecholamine secretion, DBH and DBH/anti-DBH complexes both disappeared from the cell surface at similar rates. The half-life on the cell surface was approximately 7 min. These results demonstrate that DBH was rapidly and selectively retrieved from the cell surface, probably from the site of exocytosis.

Light microscopic observations on the release of vesicles by isolated chromaffin cells.

Cultured bovine adrenal medullary chromaffin cells were stimulated with the secretogogues Ba2+ or carbamyl choline plus Ca2+. With video-enhanced contrast, differential interference contrast microscopy, small vesicles were found to appear on the cell surface during stimulation. The structures were of lower refractive index than the cytoplasm, and their appearance required several tenths of a second. The vesicles are thought to correspond to omega figures seen with electron microscopy due to exocytosis. Many of the structures disappeared within a few seconds, but some appeared to coalesce into larger structures. The large structures may lead to the vacuoles that have been demonstrated to be present following stimulation. The nature of the cellular elements responsible for the vesicle which appeared on the surface was not found with either differential interference or interference reflection microscopy. The simplest explanation is that the refractive index of the elements is similar to that of the cell, and therefore the elements cannot be seen.