Unsuitability of the 86Rb+ uptake method for estimation of (Na+ + K+)-ATPase activity in innervated tissues.

(Na+ + K+)-ATPase activity was estimated by 86Rb+ uptake in dog saphenous vein to determine the validity of the technique in tissues that have a sympathetic innervation. When saphenous vein rings were incubated at 37 degrees C in Krebs' solution containing 86Rb+, the cardenolide acetylstrophanthidin caused a concentration-dependent inhibition of Rb+ uptake. The threshold for inhibition was approx. 10 nM acetylstrophanthidin and the maximum effect was obtained at 9 microM. In the upper part of this concentration range (greater than 1 microM) acetylstrophanthidin released noradrenaline from the sympathetic nerve terminals associated with the tissue. In this upper part of the acetylstrophanthidin concentration range the alpha-adrenoceptor antagonist phentolamine (8 microM) reduced, by up to 25%, the degree of 86Rb+ uptake inhibition caused by the cardenolide. In other experiments, saphenous vein strips were loaded with 86Rb+ and perifused with Krebs' solution containing acetylstrophanthidin. At concentrations which release noradrenaline, acetylstrophanthidin increased the efflux of 86Rb+. Phentolamine (8 microM) prevented the acetylstrophanthidin-evoked efflux of the isotope as did prior in vitro denervation of 86Rb+ loaded strips with 6-hydroxydopamine. Exogenous noradrenaline (1-100 microM) added to the perifusing fluid also caused an efflux of 86Rb+ that was attenuated by phentolamine. The data indicate for dog saphenous vein that with low concentrations of acetylstrophanthidin the extent of 86Rb+ accumulation might accurately reflect prevailing (Na+ + K+)-ATPase activity. At higher concentrations of acetylstrophanthidin, however, noradrenaline is released from the nerve endings and causes 86Rb+ efflux from the smooth muscle cells consequent upon alpha-adrenoceptor activation. Since this efflux reduces the extent of Rb+ accumulation, measurement of the latter does not adequately reflect uptake mediated by the activity of (Na+ + K+)-ATPase. This is significant because in most applications of the 86Rb+ uptake method it is the estimate of Rb+ accumulation made in the presence of a high concentration of cardenolide that forms the basis of all subsequent calculations with respect to (Na+ + K+)-ATPase activity.

Histamine causes Ca2+ entry via both a store-operated and a store-independent pathway in bovine adrenal chromaffin cells.

The characteristics and properties of the increase in cytosolic [Ca2+] that occurs in bovine adrenal medullary chromaffin cells on exposure to histamine have been investigated. Specifically, these experiments were conducted to determine how much external Ca2+ enters the cell through a (capacitative) Ca2+ entry pathway activated as a consequence of intracellular Ca2+ store mobilization, relative to that which enters independently of store depletion via other channels activated by histamine. In Fura-2 loaded cells continued exposure to histamine (10 microM) caused a rapid but transient increase in cytosolic [Ca2+] followed by a lower plateau that was sustained as long as external Ca2+ was present. In the absence of external Ca2+, only the initial brief transient was observed. In cells previously treated with thapsigargin (100 nM) in Ca(2+)-free medium to deplete the internal Ca2+ stores, histamine caused no increase in cytosolic [Ca2+] when external Ca2+ was absent. Re-introduction of external Ca2+ to thapsigargin-treated store-depleted cells caused a sustained increase in cytosolic [Ca2+] that was further increased (P < 0.0002) upon exposure to histamine. The histamine-evoked increase was prevented by the H1-receptor antagonist, mepyramine (2 microM). A comparison was made between store-dependent Ca2+ entry consequent upon store mobilization with histamine in Ca(2+)-free medium and plateau phase Ca2+ entry resulting from stimulation with histamine in Ca(2+)-containing medium. The latter was found to be approximately 3 times greater in magnitude than the former (P << 0.0001) at the same concentration of histamine (10 microM). It is concluded that histamine causes Ca2+ entry not only via a capacitative entry pathway secondary to internal store mobilization, but also causes substantial Ca2+ entry through other pathways.

Neurotransmitter release from bovine adrenal chromaffin cells is modulated by capacitative Ca(2+)entry driven by depleted internal Ca(2+)stores.

Two potential mechanisms by which the intracellular Ca(2 stores might modulate catecholamine release from bovine adrenal chromaffin cells were investigated: (i) that the cytosolic Ca(2+)transient caused by Ca(2+)release from the intracellular stores recruits additional chromaffin granules to a readily releasable pool that results in augmented catecholamine release when this is subsequently evoked, and (ii) that the Ca(2+)influx that follows depletion of intracellular stores (i.e. store-operated Ca(2+)entry) triggers release per se thereby augmenting evoked catecholamine release. When histamine or caffeine were applied in Ca(2+)-free perfusion media, a transient elevation of intracellular free Ca(2+)occurred owing to mobilization of Ca(2+)from the stores. When Ca(2+)was later readmitted to the perfusing fluid there followed a prompt and maintained rise in intracellular Ca(2+)concentrations of magnitude related to the degree of store mobilization. In parallel experiments, increased catecholamine secretion was measured under the conditions when Ca(2+)influx following store-mobilization occurred. Furthermore, the size of the catecholamine release increment correlated with the degree of Ca(2+)influx. Store-operated Ca(2+)entry evoked by mobilization with histamine and/or caffeine did not augment nicotine-evoked secretion per se; that is, it augmented evoked catecholamine release only to the extent that it increased basal catecholamine release. The nicotine-evoked catecholamine release was sensitive to cytosolic BAPTA, which, at the concentration used (50 microM BAPTA-AM), reduced release by approximately 25%. However, the increment in basal catecholamine release which followed Ca(2+)influx triggered by Ca(2+)store mobilization was not reduced by intracellular BAPTA. This finding is inconsistent with the hypothesis that the elevated cytosolic Ca(2+)from store mobilization recruits additional vesicles of catecholamine to the sub-plasmalemmal release sites to augment subsequently evoked secretion. This position is supported by the observation that histamine (10 microM) in Ca(2+)-free medium caused a pronounced elevation of cytosolic free Ca(2+), but this caused no greater catecholamine release when Ca(2+)was re-introduced than did prior exposure to Ca(2+)-free medium alone, which caused no elevation of cytosolic free Ca(2+). It is concluded that intracellular Ca(2+)stores can modulate secretion of catecholamines from bovine chromaffin cells by permitting Ca(2+)influx through a store-operated entry pathway. The results do not support the notion that the Ca(2+)released from intracellular stores plays a significant role in the recruitment of vesicles into the ready-release pool under the experimental conditions reported here.

Calcium export by sodium-calcium exchange in bovine chromaffin cells.

Calcium efflux from bovine chromaffin cells in tissue culture has been examined after loading them with small amounts of Ca2+ by brief depolarization in media containing 20 mumol/l to 1 mmol/l Ca2+ and 45Ca2+ in trace amounts. In the presence of normal external Na+ and Ca2+ concentrations cells depolarized in media containing up to 200 mumol/l Ca2+ exported nearly 100% of their accumulated Ca2+ loads within 10 min and 20% within the first 5 s. In the absence of external Na+ and Ca2+ the proportion of a small (i.e., depolarization in 20 mumol/l calcium) Ca2+ load exported at any time point in the range to 10 min was approximately two thirds of the total efflux measured in their presence indicating that under these conditions the external Na+/Ca(2+)-dependent and Na+/Ca(2+)-independent mechanisms both contribute significantly to the export of calcium. At higher cellular loads of calcium (i.e., depolarization in 200 mumol/l to 1 mmol/l calcium) the Na+/Ca(2+)-dependent mechanism exported a progressively greater proportion of the accumulated Ca2+. Both sodium and calcium alone promoted a component of Ca2+ efflux; Ca2+ (i.e. calcium-calcium exchange) was as effective as Na+ (i.e. sodium-calcium exchange). The Km for Na+ stimulation of Ca(2+)-efflux (KNa) was approximately 65 mM. Increased external Mg2+ (from 1.2 to 10 mmol/l) increased the apparent KNa to 90 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Lanthanum can be transported by the sodium-calcium exchange pathway and directly triggers catecholamine release from bovine chromaffin cells.

A comparison of the effectiveness of the trivalent cation, lanthanum (La3+) relative to Ca2+ in causing catecholamine release from bovine chromaffin cells has been made, together with a determination of the pathway by which La3+ enters these cells. In chromaffin cells maintained in tissue culture and permeabilised with digitonin, both La3+ and Ca2+ caused 3H release from cells preloaded with [3H]-noradrenaline; La3+ and Ca2+ caused similar maximal release but the EC50 for La3+ was an order of magnitude less than that for Ca2+. At maximal release caused by either La3+ or Ca2+ (approximately 14% of cell 3H content in 15 min), the other cation caused a small, but significant, further release. At submaximal effective concentrations the effects of the two cations were exactly additive. Using 3H release as an indicator of cytosolic La3+, its route of entry into intact chromaffin cells was investigated. With La(3+)-containing medium there was no release evoked by nicotine or by K(+)-depolarisation indicating that La3+ does not enter either via the nicotinic receptor linked ion channel or via voltage-sensitive (Ca2+) channels. However, in sodium-loaded chromaffin cells (ouabain incubation in Ca(2+)-free medium for 15 min) exposure to bathing media containing either Ca2+ or La3+ caused 3H release. La3+ (0.1 mM) caused a release similar in magnitude to that caused by Ca2+ (about 1 mM). La3+ at low concentrations had an additive (0.1 mM La3+) or synergistic (0.25-0.45 mM La3+) action with Ca2+ (< 3.6 mM) on 3H release. At higher concentrations (> 0.9 mM) the effects of La3+ predominated and prevented the expected effects of Ca2+. In other experiments, La3+ (1 mM) blocked export of 45Ca2+ via both Nao-dependent and independent pathways, i.e. sodium-calcium exchange and the calcium pump. The results indicate that La3+ can enter bovine chromaffin cells via the Nai/Cao exchange pathway independently of, or together with, Ca2+ but, that concentrations above 0.9 mM block the influx or efflux of Ca2+. However, Ca2+, even at 3.6 mM, did not block the influx of La3+. The results further indicate that, within chromaffin cells, La3+ is at least as effective as Ca2+ in triggering catecholamine release and maintaining prolonged release. La3+ also appears to act cooperatively with Ca2+ at the release pathway.

Mn2+ can substitute for Ca2+ in causing catecholamine secretion but not for increasing tyrosine hydroxylase phosphorylation in bovine adrenal chromaffin cells.

The ability of the divalent cation manganese (Mn2+) to substitute for calcium (Ca2+) both in triggering catecholamine release and in stimulating catecholamine synthesis, as indicated by an increase in tyrosine hydroxylase (TOH) phosphorylation, has been determined in bovine adrenal medullary chromaffin cells maintained in tissue culture. Mn2+ was found to enter chromaffin cells through pathways activated by nicotinic receptor stimulation and potassium depolarisation, and via the Na1:Ca0 exchange mechanism in Na(+)-loaded cells. Like Ca2+, entry of Mn2+ through these pathways triggered immediate catecholamine release and, like Ca2+, maintained quantitatively comparable release at least up to 40 min. Unlike Ca2+, Mn2+ did not stimulate an increase in TOH phosphorylation in intact chromaffin cells, even over a prolonged time course, but Mn2+ did stimulate increased TOH phosphorylation in lysed cell preparations showing that its lack of effect in the intact cells was not due to inhibition of the specific phosphorylation pathway. In lysed cell preparations, Mn2+ stimulated also phosphorylation of a different spectrum of proteins to Ca2+, and of the same proteins to different extents. In particular, P80 (MARCKS protein) was more intensely phosphorylated in the presence of Mn2+ than in the presence of Ca2+. Since TOH phosphorylation always occurs when intracellular Ca2+ is increased, the absence of an increase with Mn2+ indicates that none of its intracellular effects could have occurred as a consequence of Mn2+ mobilisation of intracellular Ca2+. In summary, the data show that Mn2+ is a surrogate for Ca2+ in triggering and maintaining catecholamine release, but does not substitute for Ca2+ in stimulating TOH phosphorylation.

Mechanisms of synaptic plasticity. Changes in postsynaptic densities and glutamate receptors in chicken forebrain during maturation.

We have shown that the synapse maturation phase of synaptogenesis is a model for synaptic plasticity that can be particularly well-studied in chicken forebrain because for most forebrain synapses, the maturation changes occur slowly and are temporally well-separated from the synapse formation phase. We have used the synapse maturation phase of neuronal development in chicken forebrain to investigate the possible link between changes in the morphology and biochemical composition of the postsynaptic density (PSD) and the functional properties of glutamate receptors overlying the PSD. Morphometric studies of PSDs in forebrains and superior cervical ganglia of chickens and rats have shown that the morphological features of synapse maturation are characteristic of a synaptic type, but that the rate at which these changes occur can vary between types of synapses within one animal and between synapses of the same type in different species. We have investigated, during maturation in the chicken forebrain, the properties of the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptors, which are concentrated in the junctional membranes overlying thick PSDs in the adult. There was no change in the number of NMDA receptors during maturation, but there was an increase in the rate of NMDA-stimulated uptake of 45Ca2+ into brain prisms. This functional change was not seen with the other ionotropic subtypes of the glutamate receptor and was NMDA receptor-mediated. The functional change also correlated with the increase in thickness of the PSD during maturation that has previously been shown to be due to an increase in the amount of PSD associated Ca(2+)-calmodulin stimulated protein kinase II (CaM-PK II). Our results provide strong circumstantial evidence for the regulation of NMDA receptors by the PSD and implicate changing local concentrations of CaM-PK II in this process. The results also indicate some of the ways in which properties of existing synapses can be modified by changes at the molecular level.

Alpha 2-adrenoceptor blockade prevents cardiac glycoside-evoked neurotransmitter release from sympathetic nerves in dog saphenous vein.

1 The effect of alpha-adrenoceptor antagonists upon neurotransmitter release evoked by cardiac glycosides from sympathetic nerve terminals has been investigated in dog saphenous vein. 2 In rings of saphenous vein preloaded with [3H]-noradrenaline, acetylstrophanthidin (ACS) caused a concentration-dependent efflux of 3H (EC50 ca. 4.4 microM) that was attenuated by phentolamine and yohimbine but not by prazosin. 3 In helical strips of saphenous vein superfused with ACS at EC50 the efflux of 3H-compounds in general, and of [3H]-noradrenaline in particular, occurred after a short delay and increased with time to a maximum reached at 75 min. Phentolamine and phenoxybenzamine, but not prazosin reduced the efflux of [3H]-noradrenaline and of total 3H-compounds throughout the time-course of the ACS-evoked effect. 4 In helical strips of saphenous vein the glycoside ouabain also caused an increase in [3H]-noradrenaline and in total 3H-efflux that was attenuated by phentolamine. 5 By contrast with the above, in bovine adrenal medullary chromaffin cells, which appear to have no functional alpha-adrenoceptors, ACS caused a small, but significant increase in 3H-efflux which was not prevented by phentolamine. 6 Phentolamine, at concentrations that attenuate markedly the ouabain- or ACS-evoked increase in 3H-efflux from dog saphenous vein, did not cause significant inhibition of cocaine-sensitive [3H]-noradrenaline uptake nor did it reduce the extent of the 3H-efflux evoked either by tyramine or by reduced extracellular Na+. These findings imply that phentolamine does not affect ACS-evoked neurotransmitter release by an action on the catecholamine uptake mechanism. 7. It is concluded that the cardiac glycoside-evoked increase in neurotransmitter release from noradrenergic nerve terminals ofdog saphenous vein is modulated by a mechanism that involves an alpha2- adrenoceptor.

Involvement of pertussis toxin-sensitive and -insensitive mechanisms in alpha-adrenoceptor modulation of noradrenaline release from rat sympathetic neurones in tissue culture.

1. Sympathetic neurones derived from superior cervical ganglia of neonatal rats and maintained in tissue culture were used to investigate the modulation of neurotransmitter release by presynaptic receptors. Three week old cultures of neurones were loaded with [3H]-noradrenaline to label endogenous neurotransmitter stores. Release of noradrenaline was evoked by depolarization with raised extracellular K+ in the presence of desipramine and corticosterone to prevent uptake of released catecholamine. 2. Potassium (55 mmol l-1) depolarization for 30 s caused more than a four fold increase in 3H overflow from basal levels but this increase was reduced by up to 40% in the presence of exogenous noradrenaline (1 mumol l-1). The inhibition by noradrenaline of depolarization-evoked overflow was blocked by the alpha 1/alpha 2-adrenoceptor antagonist, phentolamine. Phentolamine alone did not increase K(+)-evoked 3H overflow. 3. The alpha 2-adrenoceptor antagonist, yohimbine, produced a concentration-dependent block of the inhibition by noradrenaline of K(+)-evoked overflow, while the alpha 1-adrenoceptor antagonist, prazosin, was without effect at concentrations up to 0.1 mumol l-1. 4. The beta-adrenoceptor antagonist, propranolol, neither reduced K(+)-evoked overflow nor increased the degree of inhibition caused by the addition of 1 mumol l-1 noradrenaline. 5. The alpha 2-adrenoceptor agonist, clonidine (1 mumol l-1) was less effective than noradrenaline at inhibiting K(+)-evoked overflow, while the alpha 1-adrenoceptor agonist, phenylephrine (1 mumol l-1) had no significant effect. 6. The L-channel calcium blocker, nicardipine (1 mumol l-1) significantly inhibited 3H overflow evoked by K+. In the presence of L-channel block, however, noradrenaline still inhibited residual evoked overflow.7. In the presence or absence of nicardipine, pertussis toxin pretreatment (1 nmol 1-1) reduced, but did not prevent, the effect of noradrenaline (1 micromol 1-1). Pertussis toxin alone caused a significant enhancement of K+-evoked 3H overflow.8. The data indicate that on postganglionic neurones of cultured rat sympathetic ganglia there are alpha 2-adrenoceptors that modulate neurotransmitter release, but no functional beta-adrenoceptors that mediate an enhancement of transmitter release. The data suggest further that in this preparation the mechanism of alpha2-adrenoceptor modulation may involve pertussis toxin sensitive and insensitive G-proteins and effects on calcium channels other than L-type.

The subdivision of beta-adrenoceptors in the cardiovascular system of the rat.

1 The antagonism by the beta-adrenoceptor blocking drugs, propranolol (non-selective) and practolol (beta-selective), of the cardiovascular actions of isoprenaline has been investigated in the rat. 2 All doses of practolol (0.1, 1 and 3 mg/kg) blocked the cardio-accelerator action of isoprenaline but only the largest dose blocked the vasodilator effect. 3 All doses of propranolol (0.01, 0.03 and 0.1 mg/kg) blocked the vasodilator effect of isoprenaline but only the largest dose diminished the tachycardia. 4 It is concluded that in the rat, as in other species, beta-adrenoceptors may be subdivided into beta 1 (cardiac) and beta 2 (peripheral vascular) types.

Effect of Gd3+ on bradykinin-induced catecholamine secretion from bovine adrenal chromaffin cells.

1. The effects of Gd3+ on bradykinin- (BK-) induced catecholamine secretion, 45Ca2+ efflux and cytosolic [Ca2+] were studied using bovine adrenal chromaffin cells. 2. BK increased secretion in a Ca2+-dependent manner. From 1 - 100 microM, Gd3+ progressively inhibited secretion induced by 30 nM BK to near-basal levels, however from 0.3 - 3 mM Gd3+ dramatically enhanced BK-induced secretion to above control levels. Gd3+ also increased basal catecholamine secretion by 2 - 3 fold at 1 mM. These effects were mimicked by Eu3+ and La3+. 3. Gd3+ enhanced secretion induced by other agonists that mobilize intracellular Ca2+ stores, but simply blocked the response to K+. 4. Gd3+ still enhanced basal and BK-induced secretion in Ca2+-free solution or in the presence of 30 microM SKF96365, however both effects of Gd3+ were abolished after depleting intracellular Ca2+ stores. 5. Gd3+ (1 mM) reduced the rate of basal 45Ca2+ efflux by 57%. In Ca2+-free buffer, BK transiently increased cytosolic [Ca2+] measured with Fura-2. The [Ca2+] response to BK was substantially prolonged in the presence of Gd3+ (1 mM). 6. The results suggest that Gd3+ greatly enhances the efficacy of Ca2+ released from intracellular stores in evoking catecholamine secretion, by inhibiting Ca2+ extrusion from the cytosol. This suggests that intracellular Ca2+ stores are fully competent to support secretion in chromaffin cells to levels comparable to those evoked by extracellular Ca2+ entry. Drugs that modify Ca2+ extrusion from the cell, such as lanthanide ions, will be useful in investigating the mechanisms by which intracellular Ca2+-store mobilization couples to Ca2+-dependent exocytosis.

Responses of the cardiovascular system of the rat to alpha-and beta-adrenoceptor agonists.

1 The effects of intravenously infused phenylephrine and isoprenaline upon the cardiovascular system of the rat anaesthetized with pentobarbitone, have been investigated.2 Phenylephrine produces a dose-dependent rise in mean arterial blood pressure (MABP) that is due mainly to an increase in total peripheral vascular resistance (TPR), though at all doses tested cardiac output was invariably raised.3 The increase in cardiac output was due in each instance to an increase in stroke volume, heart rate being unchanged. This increase in cardiac output is probably brought about by effects of phenylephrine on the capacitance vessels rather than by an effect on the heart.4 Evidence is presented to show that the effects of phenylephrine are mediated largely by alpha-adrenoceptors, but that beta-adrenoceptors which affect TPR are also stimulated by the amine.5 Isoprenaline produces a dose-dependent fall in MABP that is due entirely to a fall in TPR since the cardiac output increases.6 Unlike phenylephrine, the increase in cardiac output obtained with isoprenaline was achieved by an increase in heart rate while stroke volume remained close to control values. It is contended that the augmented venous return required for the elevated cardiac output results in this case mainly from the isoprenaline-induced fall in TPR which enhances transfer of blood from arteries to the veins.7 Evidence is presented to show that the effects of isoprenaline are mediated mainly by beta-adrenoceptors.8 Under the present experimental conditions the adrenoceptor-mediated cardiovascular changes are little modified reflexly by the arterial baroreceptors.

Responses of the cardiovascular system of the rat to noradrenaline infusions and their modification by adrenoceptor blocking agents.

1 The effects of noradrenaline upon the cardiovascular system of the rat, anaesthetized with pentobarbitone, have been investigated.2 Noradrenaline produces a dose-dependent increase in mean arterial blood pressure (MABP) which is due entirely to an increase in cardiac output; total peripheral vascular resistance (TPR) remains unchanged.3 Following beta-adrenoceptor blockade the pressor response to infused noradrenaline is enhanced and is now due mainly to an increase in TPR; the increment in cardiac output is reduced.4 After alpha-adrenoceptor blockade the pressor response is greatly reduced; the residual increase in MABP is due solely to an increase in cardiac output.5 After ganglion blockade resting cardiac output and TPR both fall, resulting in a reduction in MABP. The pressor response to noradrenaline is enhanced and is now due to increases in both TPR and in cardiac output.6 The cardiovascular response of the anaesthetized rat to noradrenaline can be explained in terms of classical alpha- and beta-adrenoceptor stimulation by the amine; the unusual form of the response may be due to an effective predominance of beta-adrenoceptor-mediated effects in this species.7 It is suggested that the failure of exogenous noradrenaline to produce a rise in TPR results from a balance between the alpha-adrenoceptor-mediated increase and beta-adrenoceptor-mediated decrease in this variable. However, this proposed balance is lost if resting vasoconstrictor tone is reduced by ganglion blockade.

Stimulation of neuronal Na+, K+-ATPase by calcium.

The effect of calcium on ATP-phosphohydrolase activity of rat brain homogenates has been investigated. In both the presence and absence of the chelating agent EDTA, free calcium within the concentration range 1.2 x 10(-7) to 5.0 x 10(-4) moles/l consistently affected only the activity of Na+, K+-ATpase; the activities of Mg2+-ATPase and Na+-ATPase were essentially unchanged by Ca2+; Ca2+-ATPase could not be demonstrated. In either the presence or absence of EDTA, concentrations of free-Ca2+ above 3 x 10(-6) moles/l caused an inhibition of Na+,K+-ATPase activity. In the presence of EDTA, concentrations of free-Ca2+ below 3 x 10(-6) moles/l were ineffective at altering Na+, K+-ATPase activity but, in the absence of EDTA, free-Ca2+ in this concentration range caused a marked stimulation of the enzyme. Evidence is presented to show that the stimulation of Na+, K+-ATPase by calcium is modulated by the regulatory protein calmodulin. Since the stimulation occurs over the range of concentrations at which calcium would be expected to be encountered within the cell, it is suggested that this is the major physiological effect of calcium on Na+, K+-ATPase.

Effects of alpha- and beta-adrenoceptor agonists and antagonists on ATP and catecholamine release and desensitization of the nicotinic response in bovine adrenal chromaffin cells.

The effects of a number of alpha- and beta-adrenoceptor agonists and antagonists on the modulation of secretion from bovine adrenal chromaffin cells were investigated. Secretion was induced by nicotine, 56 mM K+, histamine or Ba2+ and was detected by the ATP luciferin-luciferase bioluminescence technique or by the measurement of endogenous catecholamines (CA) by HPLC coupled with electrochemical detection. ATP release from freshly isolated cells by 5 microM nicotine was only weakly inhibited by adrenaline and noradrenaline and even then required high concentrations (greater than 500 microM), while dopamine (1 microM-1 mM) and isoproterenol (100 microM) had no effect. Clonidine (100 microM), oxymetazoline (100 microM), yohimbine (100 microM), and propranolol (5 microM) all produced inhibition of nicotine-induced ATP release with the order of potency:propranolol greater than oxymetazoline greater than clonidine = yohimbine. The inhibitory effect by propranolol could not be reversed by high concentrations of adrenaline or isoproterenol. In chromaffin cell monolayer cultures, all alpha 2-adrenoceptor agents tested (clonidine, oxymetazoline and yohimbine), produced a dose-dependent, Na+-sensitive, non-competitive inhibition of nicotine-induced catecholamine release with little effect on the catecholamine release induced by K+ (56 mM), histamine (10 microM) or Ba2+ (2.2 mM). (+/-)Propranolol caused a similar pattern of inhibition, however, this inhibition was also observed by (+)propranolol, an isomer with little beta-adrenoceptor antagonist activity. The effects of clonidine and propranolol on desensitization of nicotine-induced CA secretion were also investigated. The degree of desensitization of the nicotinic response was dependent on the concentration of nicotine to which the cells were pre-exposed. Desensitization was detected as the decrease in response to a near EC50 concentration of nicotine (5 microM) following pre-incubation of cells to nicotine in the range of 0.3-300 microM. The desensitization had a threshold of 1 microM nicotine and was maximal at 3 microM nicotine in the pre-incubation. Both clonidine (50 microM) and (+/-)propranolol (5 microM) inhibited CA secretion induced by nicotine (0.3 microM-300 microM) during the pre-incubation period. However, regardless of this inhibition of secretion, neither clonidine nor propranolol had an effect on either the onset, or the rate of nicotine-evoked desensitization subsequently observed. These data suggest that inhibition of the nicotinic response and desensitization of the nicotinic response are regulated independently.(ABSTRACT TRUNCATED AT 400 WORDS)

Tyrosine hydroxylase phosphorylation in bovine adrenal chromaffin cells. Clonidine stimulates basal but inhibits nicotinic receptor evoked phosphorylation.

Clonidine inhibited the uptake of calcium and the overall phosphorylation of tyrosine hydroxylase induced by nicotinic receptor activation in bovine adrenal medullary chromaffin cells in culture. However, clonidine did not inhibit the increase in these parameters that accompanied K+ depolarisation of the cells. There was also no effect of clonidine on the overall phosphorylation of tyrosine hydroxylase when cells were stimulated by muscarine. Nicotinic receptor activation increased the phosphorylation of Ser-19, Ser-31, and Ser-40 on tyrosine hydroxylase, and this was inhibited by clonidine in a concentration-dependent manner. On the other hand, clonidine had no effect on calcium uptake, yet increased the phosphorylation of Ser-19 under basal conditions. Using calcium and calmodulin-stimulated protein kinase II obtained from rat brain clonidine increased the autophosphorylation of the alpha-subunit of the kinase by 37%, and also its activity against an exogenous peptide substrate by 29%. These data are consistent with the hypothesis that clonidine inhibits nicotinic receptor-induced tyrosine hydroxylase phosphorylation by decreasing calcium influx into chromaffin cells, perhaps by an action at the nicotinic receptor. Clonidine also increases the basal phosphorylation of tyrosine hydroxylase at Ser-19, perhaps by directly activating calcium and calmodulin-stimulated protein kinase II.

Ca(2+) influx stimulated phospholipase C activity in bovine adrenal chromaffin cells: responses to K(+) depolarization and histamine.

The role of Ca(2+) influx in activating phospholipase C in bovine adrenal chromaffin cells has been investigated. Phospholipase C activity in response to K(+) depolarization (56 mM) was blocked by the L-type Ca(2+) channel antagonist nifedipine and partially inhibited by the omega-conotoxins GVIA and MVIIC. In contrast, phospholipase C activity in response to histamine receptor activation was unaffected by omega-conotoxin GVIA and partially inhibited by omega-conotoxin MVIIC or nifedipine. This response was however markedly inhibited by the non-selective Ca(2+) channel antagonists La(3+) or 1-[beta-[3-(4-Methoxyphenyl)propoxy]-4-methoyphenethyl]-H-imidazol e (SKF-96365). Despite this Ca(2+) dependence phospholipase C activity was not increased during periods of "capacitative" Ca(2+) inflow generated by histamine-, caffeine- or thapsigargin-mediated depletion of internal Ca(2+) stores. Thus, while Ca(2+) influx in response to K(+) depolarization or G-protein receptor activation can increase phospholipase C activity in these cells, in the latter case it appears to be ineffective unless there is concurrent agonist occupation of the receptor.

A difference in the cellular mechanisms of secretion of adrenaline and noradrenaline revealed with lanthanum in bovine chromaffin cells.

A comparison of the effectiveness of the trivalent cation, lanthanum (La3+) relative to Ca2+ in causing adrenaline and noradrenaline release from bovine adrenal medullary chromaffin cells has been made. In cells maintained in tissue culture and permeabilised with digitonin, both La3+ and Ca2+ triggered catecholamine release. La3+ was more effective than Ca2+: the EC50 for La3+ was shifted to the left of that for Ca2+ by close to one order of magnitude for both adrenaline and noradrenaline. With respect to adrenaline, the same maximal release was triggered by the two cations, but with respect to noradrenaline, La3+ triggered a significantly greater release than did Ca2+. Mixtures of experimental media containing both La3+ and Ca2+ caused release of adrenaline and noradrenaline in amounts that approximated closely the sum of the releases caused by Ca2+ and La3+ alone. The data strongly imply that either the release mechanisms for adrenaline and for noradrenaline from their respective chromaffin cells are different, or the cellular mechanisms that regulate release from the two cells are different.

Depleted internal store-activated Ca2+ entry can trigger neurotransmitter release in bovine chromaffin cells.

A potential role of the intracellular Ca2+ stores in modulating catecholamine release has been investigated in bovine chromaffin cells maintained in tissue culture. Pharmacological depletion of the stores with a combination of caffeine, histamine and thapsigargin in Ca2+-free media resulted in a significantly greater release of catecholamines on re-exposure to Ca2+-containing media compared with that from non-store depleted cells. The increase in catecholamine release was prevented by intracellular BAPTA indicating that the increase was caused by a rise in Ca2+. Measurement of intracellular free Ca2+ concentration with the fluorescent indicator, fura-2, over the same time-course as the catecholamine release experiments showed that upon restoration of external Ca2+ there was an immediate, substantial and maintained increase in cytosolic Ca2+. It is most probable that the increase in catecholamine release was a consequence of an increase in Ca2+ influx triggered by prior depletion of the internal Ca2+ stores. However, the data suggest that capacitative Ca2+ entry is poorly linked to catecholamine release; although Ca2+ entry on restoration of external Ca2+ was immediate and substantial, the increase in catecholamine release, although quantitatively significant, was slowly realised.

Histamine-stimulated phospholipase C activity in bovine adrenal medullary chromaffin cells: the effect of chloride-channel antagonists and low extracellular chloride concentrations.

Histamine activates phospholipase C (PLC) in a number of cell-types including those of neuronal and neuroendocrine origin. We report here that Cl(-)-channel antagonists of the niflumic acid-, but not stilbene disulphonic acid-class, produced a concentration-dependent inhibition of histamine-stimulated PLC activity in bovine adrenal medullary chromaffin cells. Low extracellular [Cl-] (10 mM) produced a similar degree of inhibition. While the mechanism(s) responsible for this inhibition are not resolved it may be significant that low extracellular Cl- also reduced the magnitude of the histamine-induced Ca2+ signal. Thus, PLC inhibition may be secondary to a reduction in Ca2+-inflow, a conclusion consistent with the known actions of niflumic acid-type compounds and the previously reported importance of Ca2+-influx in supporting histamine-stimulated PLC activity.