Regulation by batrachotoxin, veratridine, and monensin of basal and carbachol-induced phosphoinositide hydrolysis in neurohybrid NCB-20 cells.

Batrachotoxin (BTX), veratridine and monensin induced a time- and dose-dependent increase of [3H]-inositol monophosphate (3H-IP1) accumulation in the presence of lithium in prelabeled neurohybrid NCB-20 cells. A decrease of NaCl concentration to less than 30 mM markedly increased basal 3H-IP1 accumulation; however, the percentage of stimulation induced by these three agents remained unchanged even in the complete absence of sodium. The stimulation of phosphoinositide hydrolysis induced by these agents was detected in the absence of lithium but was largely prevented in the calcium-free medium. Tetradotoxin (TTX) blocked effects of BTX and veratridine (IC50 approximately 20nM), but not that stimulated by monensin. Thus, calcium-dependent activation of phospholipase C by these agents did not involve the entry of sodium or lithium. BTX and monensin also induced greater than additive effects on carbachol-induced 3H-IP1 accumulation. These effects were also TTX-sensitive and involved an increase in the Vmax and a decrease in the EC50 for carbachol. Veratridine provoked strikingly different effects on carbachol-dependent phosphoinositide turnover, depending on the passage number of the cells.

Veratridine and other depolarizing agents counteract the inhibitory effect of Mg2+ ions on N-methyl-D-aspartate (NMDA)-induced noradrenaline release in vitro.

Rat brain cortex slices preincubated with 3H-noradrenaline were superfused with Krebs-Henseleit solution with or without Mg2+. In the absence of Mg2+ ions, NMDA evoked 3H-noradrenaline overflow above basal efflux; this effect was concentration-dependently inhibited by Mg2+ (IC50: 19 mumol/l). Despite the presence of 1.2 mmol/l Mg2+, which is known to block cation influx through the ion channel coupled to the NMDA receptor, NMDA evoked 3H-noradrenaline release if the membrane was permanently kept depolarized by 20 or 25 mmol/l K+, 1 mumol/l veratridine or 200 mumol/1 3,4-diaminopyridine; the stimulant effect of NMDA was counteracted by 2-amino-5-phosphonovaleric acid (2-APV), a competitive antagonist at the NMDA receptor and by (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohept-5,10-imine hydrogen maleate (MK 801), an antagonist acting at the cation channel associated with the NMDA receptor. In contrast, no stimulatory effect of NMDA in the presence of 1.2 mmol/l Mg2+ was observed when the membrane of the nerve terminals was intermittently depolarized by electrical impulses of 2 ms duration at a frequency of 1-3 Hz. It is concluded that continuous depolarization of the nerve membrane counteracts the blocking effect of Mg2+ on cation influx through the NMDA receptor-associated ion channel. Under this condition, noradrenaline release can be stimulated by NMDA receptor activation even in the presence of physiological Mg2+ concentrations.

17-beta-estradiol potentiates luteinizing hormone glycosylation and release induced by veratridine, diacylglycerol, and phospholipase C in rat anterior pituitary cells.

We determined the effect of 17 beta-estradiol (E2) on synthesis and release of luteinizing hormone (LH) induced by drugs which activate intracellular signal transduction mechanisms in rat anterior pituitary cells. Cells were pretreated with E2 (6 x 10(-10) M) or diluent for 24 h, then washed and incubated for 4 h with E2 or diluent, respectively, in the presence or absence of drugs. LH translation and glycosylation were monitored by measuring incorporation of [14C]alanine and [3H]glucosamine, respectively, into total (medium plus cells) immunoprecipitable LH. Immunoreactive LH (IRLH) was measured by radioimmunoassay. Gonadotropin-releasing hormone (GnRH, 1 nM), veratridine (5 microM), L-alpha-1,2-dioctanoyl glycerol (C8, 200 microM), and phospholipase C (PLC, 0.24 U/ml) all increased (p less than 0.01) medium IRLH, [3H]glucosamine-LH, and [14C]alanine-LH, and total [3H]glucosamine-LH in both E2- and diluent-treated cells. Total IRLH or [14C]alanine-LH were not increased by any treatment. E2 alone slightly increased (p less than 0.05) basal medium IRLH and [3H]glucosamine-LH. The stimulatory effects of E2 on basal medium [14C]alanine-LH and total [3H]glucosamine-LH were inconsistent. E2 potentiated (p less than 0.01) the effects of veratridine, C8, PLC, and GnRH on medium IRLH, and medium and total [3H]glucosamine-LH. E2 also potentiated (p less than 0.01) the effects of veratridine, PLC, and GnRH, but not of C8, on medium [14C]alanine-LH. In contrast, E2 did not increase either precursor uptake or incorporation of precursor into total protein in the presence of any secretagogue.(ABSTRACT TRUNCATED AT 250 WORDS)

Veratridine-induced oscillations in membrane potential of cultured rat skeletal muscle: role of the Na-K pump.

1. The acute effects of veratridine on membrane potential (Em) and Na-K pump activity in cultured skeletal muscle were examined. 2. At a concentration of 10(-4) M, veratridine caused depolarization of Em and a decrease in Na-K pump activity. At concentrations of 10(-5) and 10(-6) M, veratridine caused oscillations of Em and an increase in Na-K pump activity compared to untreated, control cells. The oscillations consisted of depolarization to about -40 mV followed by hyperpolarization to about -90 mV; the level of hyperpolarization was higher at 37 than at 23 degrees C. 3. Veratridine-induced oscillations could be prevented by pretreatment with tetrodotoxin (10(-6) M) and blocked or prevented by ouabain, which depolarizes Em of cultured myotubes. In contrast, depolarization of Em to -60 mV by excess K+ did not alter the amplitude or frequency of the oscillations. 4. The results demonstrate that veratridine-induced increase in Na influx both depolarizes cultured myotubes and increases the activity of the Na-K pump, which repolarizes Em to levels higher than control. This sequence accounts for veratridine-induced oscillations in Em. High concentrations of veratridine cause only depolarization of Em and inhibition of Na-K pump activity.

Sodium channel comodification with full activator reveals veratridine reaction dynamics.

Veratridine association and dissociation rates were determined at single sodium channels in outside-out patches of cultured ventricular myocytes obtained from late-fetal rat hearts. In single cardiac sodium channels depolarized from -110 to -30 mV, intracellular veratridine induced a long lasting (tau = 0.48 sec) open state with small current amplitude (-0.3 pA, i.e., 1/4 of normal) and frequent closing transitions, giving it a burstlike appearance, in agreement with reports on other types of sodium channel. Veratridine-associated and veratridine-free states of a single sodium channel were monitored by comodifying it with an allosteric activator, BDF 9145 (1 microM), that induced a burst with normal open channel current amplitude (-1.2 pA at -30 mV) upon veratridine dissociation. Veratridine and BDF 9145 interacted with reciprocal synergism at the single sodium channel such that veratridine-induced bursts (called P-bursts for partially activated) alternated with BDF 9145-induced bursts (called F-bursts for fully activated) many times following a single depolarization to -30 mV. P-bursts and F-bursts within such trains of bursts had exponentially distributed durations. The reciprocal time constant for F-bursts, tau F-1, increased linearly with veratridine concentration (0.3-30 microM), whereas tau P was insensitive. We conclude, therefore, that P-bursts reflect veratridine occupancy and F-bursts reflect the veratridine-free state; if veratridine and BDF 9145 bind to a sodium channel simultaneously, veratridine exerts conformational dominance, i.e., retains its property to reduce channel conductance. For the single cardiac sodium channel activated (i.e., deprived of inactivation) by BDF 9145, we have determined a veratridine association rate constant k1 = 4.3 x 10(6) M0-1 sec-1, dissociation rate constant K-1 = 2.2 sec-1 and equilibrium dissociation constant KD = 5.1 x 10(-7) M (20 degrees, -30 mV membrane potential).

Ovarian steroids increase veratridine-induced release of amino acid neurotransmitters in preoptic area synaptosomes.

In vivo treatment of ovariectomized rats with estradiol benzoate plus progesterone, but not with either steroid alone, produced a large increase in veratridine-induced release of radiolabeled glutamate and newly synthesized GABA from preoptic area synaptosomes in vitro. Neither basal nor KCl-evoked release of amino acids was altered. Thus gonadal steroids appear to be involved in the control of amino acid neurotransmitter release in a brain region of importance for regulation of female reproductive physiology and behavior.

Depolarizing influences regulate somatostatin synthesis and processing in cultured cerebral cortical cells.

There is increasing evidence that persistent depolarization plays a critical role not only in excitation-secretion coupling, but also in the mechanisms linking excitation of neuronal cells to long-term adaptative changes in biosynthesis of neuropeptides. Somatostatin (SRIF) release and synthesis are affected by numerous agents, such as high concentrations of potassium that cause depolarization of cellular membrane. In the present work, we tried to determine whether prolonged exposure to veratridine (VTD) regulates SRIF synthesis. We found that exposure to VTD (100 microM) resulted in the stimulation of total (cell content + media) immunoreactive SRIF (IR-SRIF). This effect was calcium- and sodium-dependent, since it was prevented when verapamil (VPM) 20 microM or tetrodotoxin (TTX) 1 microM were added simultaneously with VTD. Cerebral cortical cells were exposed to high potassium concentrations, and the nature of the IR-SRIF was characterized by high-pressure liquid chromatography (HPLC) or gel filtration. It was evident that chronic exposure to high potassium concentrations modified the elution profile of medium IR-SRIF on HPLC and gel filtration, causing an increase in somatostatin-28 (S-28) and a decrease in somatostatin-14 (S-14). The results indicate that chronic exposure to VTD or high potassium concentration increases immunoreactive somatostatin and augments synthesis of its high-molecular-weight forms. This suggests that chronic membrane depolarization activating sodium and calcium channels initiates the entry of calcium ions, which triggers somatostatin release and causes a depletion of its intracellular stores. The stimulation of somatostatin secretion could be coupled to synthesis of the peptide.

Veratridine stimulation of sodium influx in carotid body cells from newborn rabbits in primary culture.

It was examined whether or not 22Na+ influx into cultured cells of the carotid body (CB) from newborn rabbits might be stimulated by veratridine (VRT), using superior cervical ganglion (SCG) cells as a standard, showing the VRT-stimulating effects on 22Na+ influx. In a CB glomus cell-rich culture, VRT induced a 22Na+ influx increase, as seen in a SCG neuronal cell-rich culture, which was entirely inhibited by tetrodotoxin (TTX). In contrast, in a CB non-glomus cell culture as well as in a SCG non-neuronal cell culture, the VRT-stimulating effect was not seen. This indicates that the VRT-stimulating effect found in the CB glomus cell-rich culture was evoked from only glomus cells. It is concluded that glomus cells have TTX-sensitive voltage-dependent sodium channels, which might be indirectly involved in the chemotransduction mechanism in the CB.

Potentiation of [3H]inositol phosphate formation by receptor activation and membrane depolarization in brain cortical slices (I).

Potentiation of [3H]inositol phosphate ([3H]IP) accumulation by receptor agonists combined with depolarizing agents was studied in rat brain cortical slices, prelabeled with [3H]inositol. Muscarinic agonists, alpha 1-adrenergic, histaminergic and serotonergic agonists remarkably enhanced (2-7-fold) the accumulation of [3H]IP in the presence of KCl (30 mM). The potentiated levels of [3H]IP were strongly dependent on K+ concentration and displayed a dose-response relationship with the agonist. Other depolarizing agents such as veratridine and ouabain induce potentiation of [3H]IP formation similarly to that observed by KCl, but to a lesser extent. The production of elevated levels of [3H]IP is Ca2+-dependent with maximal effect at 0.6 mM which is similar to the Ca2+ dependency observed for the agonist and the depolarizing agent alone. Enhanced [3H]IP levels induced by agonists in the presence of depolarizing agents affect Vmax values only, since the apparent half maximal effective concentration of carbachol (CCh)-induced-IP-formation (1.2 x 10(-4) M) and of the phenylephrine-induced IP-formation (8 x 10(-6) M), were not affected in the presence of either K+ or veratridine. In addition the efficacy of various muscarinic agonists as inducers of IP-accumulation was conserved under depolarizing conditions as compared to IP accumulation under normal conditions. In the presence of KCl (30 mM) the maximal degree of potentiation was at a range of 5-7-fold, with order efficacy of ACh greater than CCh greater than Oxo M greater than arecoline much greater than pilocarpine.(ABSTRACT TRUNCATED AT 250 WORDS)

The cardiac sodium channel shows a regular substate pattern indicating synchronized activity of several ion pathways instead of one.

Cardiac sodium channel substates were induced by using different gating modifiers, namely S-DPI 201-106 (s), toxin II from Anemonia sulcata (a), veratridine (v) and mixtures of these agents (s + v, a + v). Current ratios (normalized substate currents), slope conductances, reversal potentials and saturation characteristics were evaluated for the individual channel substates. The results can be summarized as follows: (i) Current ratios fell into a pattern of six equidistant values (I to VI) irrespective of the modification applied (0.20, 0.34, 0.51, 0.69, 0.85, 1.00). Slope conductances, determinable for substates II, V and VI (4.8, 11.7 and 14.0, respectively), are also consistent with six conductance substates which are integer multiples of a smallest conductance (state I). (ii) The permeability ratio PNa+/PK+ (i.e., reversal potential of substate currents) of the sodium channel was conserved both for different modifications, i.e., by s, a, s + v and a + v, and for the different substates (at least for II, IV and VI) observed for each modification. (iii) Sodium binding to the channel is substate independent. Analysis of slope conductances of states II and VI for three sodium chloride concentrations (71.5, 140 and 303 mM) revealed different maximal conductances (geVImax = 2.9.geIImax) but similar apparent affinities for sodium (KNa + VI = 286 mM; KNa + II = 303 mM). These findings are shown to seriously challenge the commonly unquestioned conception that 'single-current events' reflect ion passage through only one single pathway. The alternative view, that not one pore, but either six or three pores with synchronized gating ('oligochannel') underlie 'single-channel events', is shown to readily account for the observed substate properties and appears not to contradict known properties of 'the sodium channel'. This fundamentally new view of the sodium channel aims to invoke further efforts to distinguish between conceptually distinct models of structure-function relationships for a variety of channels which show multiple substates and conserved ion selectivity.

Veratridine modification of the purified sodium channel alpha-polypeptide from eel electroplax.

In the interest of continuing structure-function studies, highly purified sodium channel preparations from the eel electroplax were incorporated into planar lipid bilayers in the presence of veratridine. This lipoglycoprotein originates from muscle-derived tissue and consists of a single polypeptide. In this study it is shown to have properties analogous to sodium channels from another muscle tissue (Garber, S. S., and C. Miller. 1987. Journal of General Physiology. 89:459-480), which have an additional protein subunit. However, significant qualitative and quantitative differences were noted. Comparison of veratridine-modified with batrachotoxin-modified eel sodium channels revealed common properties. Tetrodotoxin blocked the channels in a voltage-dependent manner indistinguishable from that found for batrachotoxin-modified channels. Veratridine-modified channels exhibited a range of single-channel conductance and subconductance states. The selectivity of the veratridine-modified sodium channels for sodium vs. potassium ranged from 6-8 in reversal potential measurements, while conductance ratios ranged from 12-15. This is similar to BTX-modified eel channels, though the latter show a predominant single-channel conductance twice as large. In contrast to batrachotoxin-modified channels, the fractional open times of these channels had a shallow voltage dependence which, however, was similar to that of the slow interaction between veratridine and sodium channels in voltage-clamped biological membranes. Implications for sodium channel structure are discussed.

Release of [3H]L-glutamate and [3H]L-glutamine in rat cerebellum slices: a comparison of the effect of veratridine and electrical stimulation.

Depolarization-elicited release of neurotransmitter glutamate was studied in rat cerebellar slices previously loaded with either [3H]L-glutamate or [3H]L-glutamine. Both depolarization conditions used (e.g. long-lasting tonic depolarization elicited by veratridine, or short repetitive electrical pulses) increased 6 to 8 folds the release of labelled glutamate and of another compound, presumably alpha-ketoglutarate, without modifying the release of labeled glutamine. Because of the position of the label in the precursor radioactive molecules, GABA was weakly labeled and aspartate was unlabeled. The properties of the evoked glutamate release from cerebellar slices were those of a neurotransmitter since it was inhibited by tetrodotoxin and was Ca2+-dependent. Alpha-ketoglutarate is either coreleased from nerve terminals or is released from astrocytes and could participate in glutamate recycling. The data confirm the generally accepted model implying the presence of two neurotransmitter glutamate pools, a neuronal pool of newly synthesized glutamate and an astrocytic storage pool, but in addition indicate that the former is in rapid isotopic equilibrium with the extracellular compartment. Our present results also indicate that the glutamate/glutamine cycle is not activated in depolarizing conditions.

Synergy between membrane depolarization and muscarinic receptor activation leads to potentiation of neurotransmitter release (II)

Amplification of muscarinic agonist-induced [3H]noradrenaline ([3H]NE)-release by depolarizing agents, was studied in rat brain cortical slices. [3H]NE basal outflow was enhanced by either K+ (25 mM) or veratridine (2 microM) in a Ca2+-dependent manner and was potentiated beyond additivity in the presence of muscarinic agonists. Facilitation of the [3H]NE-induced release by the simultaneous presence of muscarinic agonists and depolarizing agents is calcium-dependent with a maximal effective concentration of 0.6-0.8 mM. The efficacy of muscarinic agonist to induce basal outflow of [3H]NE is as follows: CCh greater than arecoline greater than oxotremorine M greater than bethanechol greater than pilocarpine, which is similar to their potentiatory effects observed in the presence of depolarizing agents. Potentiation of muscarinic agonist-induced release of [3H]NE by elevated K+ is more pronounced (up to 7-fold) in comparison to potentiation by veratridine (up to 4-fold), irrespective of the various muscarinic agonists. The sequential presence of muscarinic agonists followed by depolarizing agents is not sufficient for eliciting a synergy of [3H]NE outflow, whether receptor activation was initiated prior to depolarization or depolarization was initiated prior to receptor activation. Receptors which do not mediate phosphatidyl inositol (PI) turnover such as nicotine induced [3H]NE-release which is not affected by the presence of depolarizing agents and yielded in their presence additive fractional release only. In this report we establish synergy of [3H]NE release by muscarinic agonists under depolarizing conditions, similar to synergism of inositol phosphate (IP) production which was observed by muscarinic agonists and depolarization agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+-mediated neuronal death in rat brain neuronal cultures by veratridine: protection by flunarizine.

Neuronal cell degeneration was studied in vitro in primary rat brain neuronal cultures grown in serum-free, chemically defined, CDM R12 medium, by measuring lactate dehydrogenase (LDH) released in the culture medium. A Ca2+-dependent neuronal cell degeneration was observed after prolonged and transient exposure 30 microM veratridine. The release of LDH occurred gradually and could be completely prevented by 2 mM ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 0.1 microM tetrodotoxin, and 1 microM flunarizine. Flunarizine was without effect on neuronal cell loss induced by 1 mM glutamate, 1 mM kainic acid, and 5 mM KCN. The lack of effect on neurotoxicity induced by 1 mM glutamate differentiates flunarizine from N-methyl-D-aspartate antagonists such as MK-801. The latter protected at nanomolar concentrations against glutamate-induced neuronal cell death but had a maximal effect only at 0.1 mM on the veratridine-induced released LDH. It is suggested that, besides the excitatory amino acid receptor pathway, prolonged opening of the veratridine-sensitive Na+ channel can be neurotoxic. The latter can be prevented by flunarizine. The role of Na+ channel blockers as therapeutic agents in cerebral ischemia is discussed.

Polypeptide neurotoxins modify gating and apparent single-channel conductance of veratridine-activated sodium channels in planar lipid bilayers.

The effects of scorpion and sea anemone polypeptide toxins on partially purified veratridine (VER)-activated Na channels from rat brain were studied at the single-channel level in planar lipid bilayers. The probability of the VER-activated channel being open (Po) increased with depolarization; Po was 0.5 at -40 to -50 mV. Saxitoxin (STX) blocked VER-activated channels with an apparent dissociation constant of about 1 nM at -45 mV. The apparent single-channel conductance was approximately 9 pS, similar to that seen in VER-activated Na channels from skeletal muscle transverse tubules. Addition of sea anemone or scorpion polypeptide toxins to VER-activated Na channels resulted in a 19% increase in apparent single-channel conductance and a hyperpolarizing shift in the Po vs. Vm relation such that the channels were more likely to be open at potentials less than 40 mV. These effects of the polypeptide toxins on the single-channel properties of VER-activated Na channels may account for the previously described potentiation of VER action by polypeptide toxins.

High-affinity uptake of 67Cu into a veratridine-releasable pool in brain tissue.

We have previously characterized two saturable, ligand-dependent processes for 67Cu uptake by hypothalamic slices: a high- and low-affinity process (22). In this study, we wished to ascertain if veratridine, a secretagogue that mimics a physiological release process, stimulates the release of newly taken up 67Cu and whether uptake of 67Cu into the releasable pool of copper is dependent on the process of 67Cu uptake. Hypothalamic or caudate slices from male rats were loaded for 30 min with 67Cu complexed to histidine (His) under conditions favoring high- or low-affinity uptake. First, we assessed the stability of the newly taken up 67Cu and found that, regardless of the mode of 67Cu entry into the tissue, greater than or equal to 85% of the 67Cu is retained in tissues incubated for 3 h in 67Cu-free buffer. Moreover, the 67Cu taken up by the high-affinity process was not displaced by 15-fold molar excess of nonradiolabeled Cu2+, histidine, albumin, or Zn2+, and only 20-30% of the 67Cu taken up by the low-affinity process was displaced by 10-fold excess Cu2+ or albumin. Next, we assessed veratridine stimulation of 67Cu release and found that 67Cu release occurred only from tissues loaded with the high- but not with the low-affinity process. This effect of veratridine was calcium dependent and was blocked by Tetrodotoxin, a specific blocker of the voltage-sensitive Na+ channel. In addition, we confirmed our earlier observation that a depolarizing concentration of K+ stimulates 67Cu release.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantal stores of excitatory transmitter in nerve-muscle synapses of crayfish evaluated from high-frequency asynchronous quantal release induced by veratridine or high concentrations of potassium.

At single voltage-clamped opener muscle fibres of crayfish claw, 10-100 mumol/l veratridine increased within a few seconds the rate of asynchronous quantal release, ñ, of excitatory transmitter from ñ less than 1 quantum/s to ñ congruent to 10,000 quanta/s. Thereafter ñ declined exponentially either with a single, tau(2) congruent to 50 s, or with two time constants tau(1) congruent to 19 s, tau(2) congruent to 50 s. In total (t----infinity), about 0.3 million quanta were released by veratridine in a single short fibre of about 1 mm length. These values were estimated by means of the noise analysis technique and they agreed with equivalent parameters of release when 100 mmol/l K+ were used as release stimulus. Strong quantal release could be elicited only once in a single muscle by veratridine. Furthermore, the effect of veratridine on quantal release could be completely prevented by pretreatment with tetrodotoxin. In another nerve-muscle preparation of crayfish, the abdominal superficial extensor muscle, up to 3 million excitatory quanta could be released by veratridine in a single fibre. In the latter muscle veratridine-induced asynchronous quantal release was strongly dependent on the extracellular concentration of Ca2+ whereas in the claw opener dependence of quantal release on extracellular Ca2+ was negligible.

Benzodiazepines facilitate the stimulatory action of gamma-aminobutyric acid (GABA) on basal and veratridine-evoked catecholamine release from cultured bovine adrenal chromaffin cells.

Effects of benzodiazepines were investigated on the gamma-aminobutyric acid-induced modulation of the basal and veratridine-evoked catecholamine release from cultured bovine adrenal chromaffin cells. GABA by itself, caused catecholamine release and facilitated veratridine-evoked catecholamine release. Midazolam enhanced the GABA-evoked catecholamine release in a dose-related fashion and further facilitated the enhancement by GABA of the veratridine-evoked catecholamine release. Clonazepam, a selective central-type benzodiazepine receptor agonist, also enhanced the GABA-induced catecholamine release, whereas ethyl-beta-carboline-3-carboxylate, an inverse agonist of the benzodiazepine receptor, reduced the GABA-evoked catecholamine release. The dose-response curve of the GABA-evoked catecholamine release was shifted to the left by midazolam without affecting the maximal response to GABA. Facilitation by midazolam and clonazepam of the GABA action or inhibition by ethyl-beta-carboline-3-carboxylate was antagonized by RO15-1788, which by itself had no effect on the basal or GABA- and veratridine-evoked catecholamine release. These results suggest that the central-type benzodiazepine receptor participates in the GABAergic modulation of the catecholamine release from adrenal chromaffin cells.

Depolarization and insulin-like growth factor-I (IGF-I) differentially regulate the mitotic cycle in cultured rat sympathetic neuroblasts.

Although neuroblast generation is highly reproducible temporospatially, the underlying regulation is undefined. Employing a fully defined sympathetic neuroblast culture system, we previously found that insulin growth factors regulated the mitotic cycle. We now report that depolarizing stimuli, including elevated K+ and veratridine, also increased the proportion of mitotic neuroblasts in vitro. Moreover, Na+ channel blockade prevented effects of depolarization, but not that of insulin-like growth factor I, suggesting that these influences stimulate mitosis by different membrane transduction mechanisms.

Veratridine-induced phosphorylation and activation of tyrosine hydroxylase, and synthesis of catecholamines in cultured bovine adrenal medullary cells.

The mechanism of the synthesis of catecholamines by veratridine was studied in cultured bovine adrenal medullary cells. (1) Veratridine increased the phosphorylation and activity of tyrosine hydroxylase as well as the synthesis of [14C]catecholamines from [14C]tyrosine, all of which were inhibited by tetrodotoxin. Veratridine-induced activation of tyrosine hydroxylase and synthesis of [14C]catecholamines were reduced in 20 mmol/l extracellular Na+ or in Ca2+-free medium. (2) 12-O-Tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, increased the synthesis of [14C]catecholamines. In the presence of TPA, veratridine did not produce any additional increase in [14C]catecholamine synthesis. In protein kinase C-deficient cells which were prepared by pretreatment with 1 mumol/l TPA for 24 h, TPA failed to increase [14C]catecholamine synthesis and veratridine-induced [14C]catecholamine synthesis was suppressed by 50%. (3) Polymyxin B, an inhibitor of protein kinase C and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), an inhibitor of calmodulin, inhibited veratridine-stimulated synthesis of [14C]catecholamines as well as veratridine-induced influx of 22Na+ and 45Ca2+ with similar potencies. (4) In digitonin-permeabilized cells, polymyxin B attenuated the activation of tyrosine hydroxylase caused by Ca2+. These results suggest that veratridine-induced synthesis of catecholamines and activation of tyrosine hydroxylase were mediated by Ca2+-dependent phosphorylation of this enzyme, and protein kinase C may be responsible, at least in part, for this process.