A Dihydropyridine-Resistant Component in the Rat Adrenal Secretory Response to Splanchnic Nerve Stimulation

A study of the effects of dihydropyridine Ca2+ channel modulators on the release of catecholamines from perfused rat adrenal glands, evoked by electrical stimulation of their splanchnic nerves, is presented. Electrically mediated secretory responses were compared to chemically mediated responses (exogenous acetylcholine, nicotine, or high K+). Intensities of stimuli were selected to produce quantitatively similar secretory responses (between 100 and 200 ng per stimulus). The main finding of the study is that responses to transmural stimulation (300 pulses at 1 or 10 Hz) and to acetylcholine were inhibited only partially (about 50%) by isradipine, an L-type Ca2+ channel blocker. In contrast, responses to high K+ (17.5 mM for 2 min) were highly sensitive to isradipine (IC50 = 8.2 nM). Responses to nicotine were also fully inhibited by this drug. Bay K 8644 (an L-type Ca2+ channel activator) potentiated mildly the secretory responses to electrical stimulation at 10 Hz and to acetylcholine, but increased threefold the responses to K+ and nicotine. It is, therefore, likely that responses mediated by high K+ or nicotinic receptors are triggered by external Ca2+ gaining access to the internal secretory machinery through L-type, dihydropyridine-sensitive voltage-dependent Ca2+ channels. However, in addition to nicotinic receptors, the physiological stimulation of adrenal medulla chromaffin cells through splanchnic nerves has other components, i.e., muscarinic receptor stimulation or the release of cotransmitters such as vasoactive intestinal polypeptide. The poorer sensitivity to dihydropyridines of secretory responses triggered by electrical stimulation of splanchnic nerve terminals or exogenous acetylcholine speaks in favor of alternative Ca2+ pathways, probably some dihydropyridine-resistant Ca2+ channels, in modulating the physiological adrenal catecholamine secretory process.     

Stimulation of Calcium Uptake in PC12 Cells by the Dihydropyridine Agonist BAY K 8644

Abstract: Methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate (BAY K 8644), an analog of dihydropyridine calcium channel antagonists, stimulated ⁴⁵Ca uptake into PC12 pheochromocytoma cells. Half-maximal stimulation occurred at 80 n M BAY K 8644. Enhancement of uptake was inhibited by cationic and organic calcium channel blockers, but not by tetrodotoxin, which is consistent with an effect on voltage-dependent calcium channels. Stimulation of ⁴⁵Ca uptake by BAY K 8644 occurred only at elevated concentrations of extracellular K⁺, suggesting that BAY K 8644 may interact with calcium channels in the open (activated) state.     

Studies on the Inhibitory Action of Opiate Compounds in Isolated Bovine Adrenal Chromaffin Cells: Noninvolvement of Stereospecific Opiate Binding Sites

In isolated bovine adrenal chromaffin cells, beta-endorphin, dynorphin, and levorphanol caused a dose-dependent inhibition of catecholamine (CA) secretion elicited by acetylcholine (ACh), with an ID50 of 50, 1.3, and 4.3 microM, respectively. The inhibition by the opiate compounds was specific for the release evoked by ACh and nicotinic drugs and was noncompetitive with ACh. Stereospecific binding sites for the opiate agonist [3H]etorphine were found in homogenates of bovine adrenal medulla (KD = 0.59 nM). beta-Endorphin, dynorphin, levorphanol, and naloxone were potent inhibitors of the binding of [3H]etorphine with an ID50 of 12, 0.4, 5.2, and 6.2 nM, respectively. However, [3,5-I2Tyr1]-beta-endorphin, [3,5-I2Tyr1]-dynorphin, and dextrorphan, three opiate compounds with no or little activity in the guinea pig ileum assay, were relatively ineffective in inhibiting the binding of [3H]etorphine (ID50 700, 600, and 10,000 nM, respectively). On the other hand, these three compounds were equipotent with beta-endorphin, dynorphin, and levorphanol, respectively, in inhibiting the ACh-evoked release of CA from the adrenal chromaffin cells (ID50 of 10, 1.5, and 6 microM, respectively). Inhibition of CA release was also obtained with naloxone (ID50 = 14) microM) and naltrexone (ID50 greater than 10(-4) M), two classical antagonists of opiate receptors, and this effect was additive to that of beta-endorphin. These data indicate that the opiate modulation of CA release from adrenal chromaffin cells is not related to the stimulation of the high affinity stereospecific opiate binding sites of the adrenal medulla. The physiological function of these sites remains to be determined.     

Reserpine Increases Chromaffin Cell Enkephalin Stores Without a Concomitant Decrease in Other Proenkephalin-Derived Peptides

Reserpine increases the levels of enkephalins in adrenal medullary chromaffin cells; however, the origin of the newly apparent pentapeptides has been the subject of debate, because no increase in the levels of proenkephalin mRNA has been observed. The present study was performed for determining if the reserpine-induced increase in context of enkephalins was derived from processing of preexisting fragments of proenkephalin. Bovine chromaffin cell enkephalins and larger enkephalin-containing peptides were separated by reversed-phase HPLC and identified by approximate molecular weight, elution with peptide standards, and enkephalin sequences contained. Treatment of the cells with reserpine increased the levels of enkephalins and of enkephalin-containing peptides of up to approximately 3 kilo-daltons without reducing the levels of larger enkephalin-containing peptides. Similar results were obtained with another catecholamine-depleting drug, tetrabenazine. In contrast, treatment of chromaffin cells with theophylline or forskolin increased the levels of both enkephalins and enkephalin-containing peptides of all sizes. The results suggest that new synthesis of proenkephalin is required for the effects of reserpine, although proenkephalin processing is also altered by this drug.     

Real-Time Monitoring of the Secretory Function of Cultured Adrenal Chromaffin Cells

A system to discriminate the real-time dynamics of the secretory function in cultured adrenal chromaffin cells, using a cell bed perfusion technique and an amperometric detector, was established. Examination of basal conditions revealed that the electrode potential and flow rate are crucial factors for monitoring precise dynamics of the secretory process. Stimulation of the cells either with acetylcholine (ACh) or with high K+ concentration caused a transient current response. The current responses showed concentration dependence for both stimuli, and also showed a high correlation with the amount of catecholamines (CA) in the respective peak fraction of perfusate. Either prolonged cholinergic stimulation or maintained depolarization produced a transient response, which is not attributable to a depletion of releasable storage of CA as indicated by double-stimulation experiments. Stimulation with high K+ concentration evoked an additional release of CA even after the cellular response to prolonged ACh was inactivated, whereas maintained depolarization with high K+ produced both facilitatory and inhibitory effects on the cell responsiveness to ACh. Most probably the transient natures of the secretory responses to ACh and to high K+ are mediated by different mechanisms. All the results suggest that the direct monitoring is profitable for studies on the regulatory mechanisms of the secretory function.     

Stimulation-Evoked Ca2+ Fluxes in Cultured Bovine Adrenal Chromaffin Cells Are Enhanced by Forskolin

Forskolin, 1 microM, increased acetylcholine (ACh)-stimulated 45Ca uptake by chromaffin cells. The stimulatory effects of forskolin decreased with increasing concentration of ACh. The attenuation of the effect of forskolin on 45Ca uptake as a function of ACh concentration correlated well with changes in the forskolin effect on ACh-evoked catecholamine (CA) release. Forskolin increased excess KCl- and veratrine-evoked CA release and 45Ca uptake. Forskolin by itself stimulated 45Ca efflux and enhanced ACh-, excess KCl-, and veratrine-stimulated 45Ca efflux. High doses of forskolin inhibited both ACh-evoked 45Ca uptake and CA release. The inhibitory action of forskolin was specific to receptor-mediated response because excess KCl- and veratrine-stimulated 45Ca uptake and CA release were not inhibited. Forskolin, 0.3-30 microM, dose-dependently increased caffeine-stimulated CA release and 45Ca efflux in the absence of Ca2+ in the medium, and the effects were mimicked by dibutyryl cyclic AMP. These results suggest that cyclic AMP increases stimulation-induced CA release by enhancing calcium uptake across the plasma membrane and/or altering calcium flux in an intracellular calcium store.     

Opiate Receptor-Mediated Inhibition of Catecholamine Release in Primary Cultures of Bovine Adrenal Chromaffin Cells

Abstract: Primary cultures of chromaffin cells from bovine adrenal medulla were used to evaluate the ability of several opiates to reduce the release of catecholamines induced by stimulation of nicotinic receptors. Etorphine, β-endorphin, Met-enkephalin[Arg⁶,Phe⁷], and the synthetic peptide [d -Ala²,Me-Phe⁴,Met(O)^s-ol]enkephalin inhibited the acetylcholine-induced release of catecholamines with an IC₃₀ varying from 10^?7 to 1 × 10^?6M. The effect was stereospecific because levorphanol (IC₃₀= 7.5 × 10^?7M) was approximately two orders of magnitude more potent than dextrorphan. Morphine (μ-receptor agonist), [d -Ala², d -Leu⁵]enkephalin (δ-receptor agonist), ethylketazocine (k -receptor agonist), and N-allylnormetazocine (σ-receptor agonist) were at least 100–1000 times less potent than etorphine. Diprenorphine (IC₅₀= 5 × 10^?7M) and naloxone (IC₅₀= 10^?6M) antagonized the effect of etorphine. High-affinity, saturable, and stereospecific binding sites for [³H]etorphine, [³H]dihydromorphine, [³H-d -Ala²,d -Leu⁵]enkephalin, [³H]ethylketazocine, and for [³H]N-allylnormetazocine, [³H]diprenorphine, and [³H]naloxone were detected in chromaffin cell membranes and in membranes obtained from adrenal medulla homogenates. However, the number of binding sites for [³H]etorphine and [³H]diprenorphine was 10–70 times higher than the number of sites measured with the other ³H ligands. The rank order of potency of these compounds for the displacement of [³H]etorphine binding correlates (r = 0.90) with the rank order of potency of the same compounds for the inhibition of acetylcholine-induced catecholamine release. These data suggest that a stereoselective opiate receptor (different from the classic μ-, δ-, k -, or σ-receptor) with high affinity for etorphine, diprenorphine, β-endorphin, and Met-enkephalin[Arg⁶,Phe⁷] modulates the function of the nicotinic receptor in adrenal chromaffin cells.     

Enhancement of Stimulation-Evoked Catecholamine Release from Cultured Bovine Adrenal Chromaffin Cells by Forskolin

Acetylcholine (ACh) increased cyclic AMP levels in cultured bovine chromaffin cells with a peak effect at 1 min after the addition. Pretreatment with forskolin (0.3 microM) enhanced the ACh-evoked cyclic AMP increase. The catecholamine (CA) release induced by ACh was enhanced by forskolin, but forskolin alone did not enhance the CA release. The effect of forskolin increased dose-dependently up to 1 microM, but decreased at higher concentrations. Dibutyryl cyclic AMP (DBcAMP) also enhanced ACh-evoked CA release, but the effect was less potent than that of forskolin. Forskolin enhanced both [3H]norepinephrine ([3H]NE) and endogenous CA release evoked by 30 mM K+ from cells that were preloaded with [3H]NE. The effects of forskolin were substantial when CA release was evoked with low concentrations of ACh or excess K+, but decreased with higher concentrations of the stimulants. Forskolin also enhanced the CA release induced by ionomycin and veratrine, or by caffeine in Ca2+-free medium. The potentiation by forskolin of the ACh-evoked CA release was manifest in low Ca2+ concentrations in the medium, but decreased when Ca2+ concentration was increased. These results suggest that cyclic AMP may play a role in the modulation of CA release from chromaffin cells.     

Calcium Dependence of Muscarinic Receptor-Mediated Catecholamine Secretion from the Perfused Rat Adrenal Medulla

It had previously been thought that muscarinic cholinergic receptors utilize an influx of extracellular calcium for activation of adrenomedullary catecholamine secretion. However, it has recently been demonstrated that muscarinic receptors on isolated adrenal chromaffin cells can elevate cytosolic free calcium levels in a manner independent of extracellular calcium, presumably by mobilizing intracellular calcium stores. We now demonstrate that muscarinic receptor-mediated catecholamine secretion from perfused rat adrenal glands can occur under conditions of extracellular calcium deprivation that are sufficient to block both nicotine- and electrically stimulated release. Three independent conditions of extracellular calcium deprivation were used: nominally calcium-free perfusion solution (no calcium added), EGTA-containing calcium-free perfusion solution, and perfusion solution containing the calcium channel blocker verapamil. Secretion was evoked from the perfused glands by either transmural electrical stimulation or injection of nicotine or muscarine into the perfusion stream. Each condition of calcium deprivation was able to block nicotine- and electrically stimulated catecholamine release in an interval that left muscarine-evoked release largely unaffected. The above results demonstrate that muscarine-evoked catecholamine secretion from perfused rat adrenal glands can occur in the absence of extracellular calcium, presumably by mobilization of intracellular calcium. The latter may be due to muscarinic receptor-mediated generation of inositol trisphosphate.     

Nicotine Tolerance in Chromaffin Cell Cultures: Acute and Chronic Exposure to Smoking-Related Nicotine Doses

Abstract: Nicotine tolerance and dependence are key aspects of tobacco addiction; however, the cellular mechanisms underlying these phenomena are poorly understood. Adrenal chromaffin cells release catecholamines upon exposure to nicotine and with repeated exposure this response exhibits nicotine tolerance. Using bovine adrenal chromaffin cells in culture, we have demonstrated acute and chronic nicotine tolerance at doses relevant to that in the blood and tissues of smokers (10–⁷M to 10–⁶M). Chromaffin cells are preexposed to low doses of nicotine for time periods ranging from 10 min to 7 days and then subsequently challenged with a maximally stimulating dose of nicotine (10–⁵M) for 10 min, all at 37°C. Preexposure to nicotine results in a depression of ⁴⁵Ca uptake and catecholamine release upon subsequent nicotine challenge. Acute tolerance or desensitization of nicotinestimulated catecholamine release begins to occur in minutes after preexposure to 10–⁶M nicotine at 37°C. The depression of catecholamine release upon preexposure to nicotine is both dose and temperature dependent and is not seen with potassium-evoked release. Chronic exposure to 10–⁷M nicotine for 3 days led to a depression of the secretory response to ～70% of control responses. There was a trend toward recovery of full response by days 5 and 7 of 10–⁷M nicotine preexposure. Nearly complete depression of the nicotine-evoked release occurs within the first day of exposure to 10–⁶M nicotine and persists for at least a week of nicotine exposure at 37°C. Nicotine tolerance in the catecholamine release response was reversible after nicotine washout. Cross-tolerance between nicotine, acetylcholine, and dimethylphenylpiperazinium was observed after 5 days’exposure to one agonist and subsequent challenge with a different agonist. Acute tolerance is likely to be related to nicotinic receptor desensitization. The mechanisms of chronic tolerance and potential adaptive cellular changes remain to be determined.     

Influence of the CNS Environment on Chromaffin Cell Survival and Catecholamine Secretion Patterns

Abstract: Chromaffin cells implanted into the CNS have been used as a potential source of sustained catecholamine delivery, although their survival and continued catecholamine secretion are controversial. In addition, chromaffin cells exhibit a high degree of neurochemical plasticity in response to environmental factors. The present aims were to determine whether the CNS provides a supportive environment for sustained catecholamine production in transplanted chromaffin cells and to assess whether this novel environment alters patterns of catecholamine secretion. Catecholamine release from bovine chromaffin cells implanted into the rat midbrain was determined in brain slices. In addition, alterations in catecholamine secretion patterns, particularly adrenaline/noradrenaline ratios, were compared in vitro versus in transplants. Results indicated that brain slices containing chromaffin cell implants released high basal and nicotine-stimulated levels of adrenaline and noradrenaline. It is surprising that although adrenaline/noradrenaline ratios steadily declined in culture, this did not occur when cells were transplanted to the CNS in the early postharvesting phases. However, if cells were transplanted following longer periods in culture, adrenaline/noradrenaline ratios remained low. Together, these results suggest that the CNS can provide a supportive environment for chromaffin cell survival and that the pattern of catecholamine secretion can be optimized by prior in vitro manipulation.     

Modulation of catecholamine release by endogenous adenosine in the rat adrenal medulla

Adenosine was shown to inhibit norepinephrine (NE) release from sympathetic nerve endings. The purpose of this study was to examine whether endogenous adenosine restrains NE and epinephrine release from the adrenal medulla. The effects of an adenosine receptor antagonist, 1,3-dipropyl-8-(p-sulfophenyl) xanthine (DPSPX), on epinephrine and NE release induced by intravenous administration of insulin in conscious rats were examined. Plasma catecholamines were measured by HPLC with an electrochemical detector. DPSPX significantly increased plasma catecholamine in both control rats and rats treated with insulin. The effect of DPSPX on plasma catecholamine was significantly greater in rats treated with insulin. Additional experiments were performed in adrenalectomized rats to investigate the contribution of the adrenal medulla to the effect of DPSPX on plasma catecholamine. The effect of DPSPX and insulin on epinephrine in adrenalectomized rats was significantly reduced compared with that of the controls. Finally, we tested whether endogenous adenosine restrains catecholamine secretion partially through inhibiting the renin-angiotensin system. The effect of DPSPX on plasma catecholamine in rats pretreated with captopril (an angiotensin-converting enzyme inhibitor) was reduced. These results demonstrate that under basal physiological conditions, endogenous adenosine tonically inhibits catecholamine secretion from the adrenal medulla, and this effect is augmented when the sympathetic system is stimulated. The effect of endogenous adenosine on catecholamine secretion from the adrenal medulla is achieved partially through the inhibitory effect of adenosine on the renin-angiotensin system.     

Parallel but Separate Release of Catecholamines and Acetylcholinesterase from Stimulated Adrenal Chromaffin Cells in Culture

Abstract: A pharmacological study was made of the effects of veratridine and lasalocid on the release of catecholamines, acetylcholinesterase (AChE) and dopamine-β-hydroxylase (DBH) from cultures of isolated bovine adrenal chromaffin cells. Exposure of the cultures to veratridine resulted in concomitant release of catecholamines and AChE into the external medium in a dose-dependent and Ca^{2 +}-dependent manner. A Ca²⁺ iono-phore, lasalocid, also produced a dose-dependent and parallel release of both catecholamines and AChE. The release of the two components was accompanied by release of DBH. The present results provide pharmacological evidence for a parallel release of catecholamines, AChE, and DBH from cultured adrenal chromaffin cells, and the stoichiometry of the release evoked by different secretagogues suggests that AChE and catecholamines are released from different cellular compartments.     

BAY K 8644, a 1,4-Dihydropyridine Ca2+ Channel Activator: Dissociation of Binding and Functional Effects in Brain Synaptosomes

K+-stimulated 45Ca2+ uptake into rat brain and guinea pig cerebral cortex synaptosomes was measured at 10 s and 90 s at K+ concentrations of 5-75 mM. Net increases in 45Ca2+ uptake were observed in rat and guinea pig brain synaptosomes. 45Ca2+ uptake under resting or depolarizing conditions was not increased by the 1,4-dihydropyridine BAY K 8644, which has been shown to activate Ca2+ channels in smooth and cardiac muscle. High-affinity [3H]nitrendipine binding in guinea pig synaptosomes (KD = 1.2 X 10(-10) M, Bmax = 0.56 pmol mg-1 protein) was competitively displaced with high affinity (IC50 2.3 X 10(-9) M) by BAY K 8644. Thus high-affinity Ca2+ channel antagonist and activator binding sites exist in synaptosome preparations, but their relationship to functional Ca2+ channels is not clear.     

Characterization and Ca2+ Requirement of Histamine-Induced Catecholamine Secretion in Cultured Bovine Chromaffin Cells

The stimulation of cultured bovine chromaffin cells with histamine induced a continuous catecholamine secretion (EC50 = 3 x 10(-7) M) via the H1 receptor, in addition to an initial catecholamine burst due to a nonspecific stimulatory effect at higher doses (greater than or equal to 10(-4) M). The continuous secretion showed little desensitization and lasted for more than 1 h. In fura-2-loaded cells, the stimulation with histamine evoked a transient rise of intracellular free Ca2+ concentration ([Ca2+]i) which lasted only for a few minutes and was followed by a sustained [Ca2+]i rise which continued for more than 20 min. The addition of an activator for the L-type voltage-sensitive Ca2+ channel, i.e., Bay K 8644 (1 microM), facilitated the sustained [Ca2+]i rise, as well as the secretion, whereas the addition of relatively high concentrations of Ca(2+)-channel blockers (10 microM) suppressed the sustained [Ca2+]i rise and part of the secretion. Removal of extracellular Ca2+ completely abolished continuous secretion and sustained [Ca2+]i rise. When the external Ca2+ level was elevated, both sustained [Ca2+]i rise and continuous secretion were enhanced in a similar Ca(2+)-dependent manner, showing saturation with around 1-3 mM Ca2+. This Ca2+ dependence was clearly different from that observed with high K+ and nicotine, which is mediated by the L-type Ca2+ channel, in which the responses showed little or no saturation when the Ca2+ level was increased. The results indicate that stimulation with histamine induces a continuous secretion via the H1 receptor, in addition to a transient and nonspecific secretion at higher doses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+/Calmodulin-Dependent Protein Kinase II Inhibitor KN-62 Inhibits Adrenal Medullary Chromaffin Cell Functions Independent of Its Action on the Kinase

Abstract: KN-62, an inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II), inhibited significantly catecholamine secretion and tyrosine hydroxylase activity stimulated by acetylcholine in cultured bovine adrenal medullary cells. KN-62, however, showed an additional inhibitory effect on acetylcholine-induced ⁴⁵Ca²⁺ influx, which is essential for functional responses. Carbachol-stimulated ²²Na⁺ influx, veratridine-induced ²²Na⁺ influx, and 56 m M K⁺-evoked ⁴⁵Ca²⁺ influx were also attenuated by KN-62. Inhibitions by KN-62 of these ion influxes were correlated closely with those of catecholamine secretion. KN-04, which is a structural analogue of KN-62 but does not inhibit CaM kinase II activity, elicited inhibitory effects on the three kinds of stimulant-evoked ion influxes with an inhibitory potency similar to KN-62. These results suggest that KN-62 inhibits catecholamine secretion and tyrosine hydroxylase activation due to mainly its ion channel blockade on the plasma membrane rather than the inhibition of CaM kinase II activity in the cells.     

Hypoxia-Induced Catecholamine Release and Intracellular Ca2+ Increase via Suppression of K+ Channels in Cultured Rat Adrenal Chromaffin Cells

Abstract: Hypoxia (5% O₂) enhanced catecholamine release in cultured rat adrenal chromaffin cells. Also, the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increased within 3 min in ∼50% of the chromaffin cells under hypoxic stimulation. The increase depended on the presence of extracellular Ca²⁺. Nifedipine and ω-conotoxin decreased the population of the cells that showed the hypoxia-induced [Ca²⁺]_i increase, showing that the Ca²⁺ influx was attributable to L- and N-type voltage-dependent Ca²⁺ channels. The membrane potential was depolarized during the perfusion with the hypoxic solution and returned to the basal level following the change to the normoxic solution (20% O₂). Membrane resistance increased twofold under the hypoxic condition. The current-voltage relationship showed a hypoxia-induced decrease in the outward K⁺ current. Among the K⁺ channel openers tested, cromakalim and levcromakalim, both of which interact with ATP-sensitive K⁺ channels, inhibited the hypoxia-induced [Ca²⁺]_i increase and catecholamine release. The inhibitory effects of cromakalim and levcromakalim were reversed by glibenclamide and tolbutamide, potent blockers of ATP-sensitive K⁺ channels. These results suggest that some fractions of adrenal chromaffin cells are reactive to hypoxia and that K⁺ channels sensitive to cromakalim and glibenclamide might have a crucial role in hypoxia-induced responses. Adrenal chromaffin cells could thus be a useful model for the study of oxygen-sensing mechanisms.     

Phosphoproteins of the Adrenal Chromaffin Granule Membrane

A fraction of chromaffin granule membranes contained a number of substrates for endogenous protein kinase activity as well as endogenous phosphatase activity. The major 32P-labelled polypeptide of molecular weight 43,000 appeared to be the alpha-subunit of pyruvate dehydrogenase of residual mitochondria. Several polypeptides showed cyclic AMP stimulation of phosphorylation of which the major polypeptide of molecular weight 59,000 shows half-maximal phosphorylation with 0.49 microM cyclic AMP. The phosphorylation of several other polypeptides is inhibited at high cyclic AMP concentrations. From studies with immunoprecipitation and two-dimensional electrophoresis it was found that alpha- and beta-tubulin and actin were absent from the granule membranes. However 32P labelling of a proportion of the copies of dopamine-beta-hydroxylase was demonstrated. The majority of the substrates for endogenous protein kinase activity are probably on the cytoplasmic side of the granule membrane.     

Chromogranin C: A Third Component of the Acidic Proteins in Chromaffin Granules

We characterized a group of acidic proteins of bovine chromaffin granules with an antiserum raised against a protein described by Rosa and Zanini [Eur. J. Cell Biol. 31, 94-98 (1983)] in pituitary gland. In adrenal medulla the proteins reacting with this antiserum are confined to chromaffin granules. Their largest component has a Mr of 86,000 and a pI of 5.0. In addition six proteins of lower molecular weight are recognized by this antiserum. In a cell-free system only one protein is synthesized that can be precipitated with this antiserum. The properties of these proteins are very similar to those of the previously described chromogranins A and B; however, there is no immunological cross-reaction between these protein groups. We suggest this third group of acidic proteins of chromaffin granules be named chromogranins C.     

Internal Ca2+ Mobilization by Muscarinic Stimulation Increases Secretion from Adrenal Chromaffin Cells Only in the Presence of Ca2+ Influx

The cytosolic free Ca2+ concentration ([Ca2+]in) in single cat and bovine adrenal chromaffin cells was measured to determine whether or not there was any correlation between the [Ca2+]in and the catecholamine (CA) secretion caused by muscarinic receptor stimulation. In cat chromaffin cells, methacholine (MCh), a muscarinic agonist, raised [Ca2+]in by activating both Ca2+ influx and intracellular Ca2+ mobilization with an accompanying CA secretion. In bovine cells, MCh elevated [Ca2+]in by mobilizing intracellular Ca2+ but did not cause CA secretion. The MCh-induced rise in [Ca2+]in in cat cells was much higher than that in bovine cells, but when Ca2+ influx was blocked, the rise was reduced, with a concomitant loss of secretion, to a level comparable to that in bovine cells. Intracellular Ca2+ mobilization due to muscarinic stimulation substantially increased secretion from depolarized bovine and cat cells, where a [Ca2+]in elevated above basal values was maintained by a continuous Ca2+ influx. These results show that Ca2+ released from internal stores is not effective in triggering secretion unless Ca2+ continues to enter across the plasma membrane, a conclusion suggesting that secretion depends on [Ca2+]in in a particular region of the cell.