Calcium Dependence of Vasoactive Intestinal Peptide-Stimulated Cyclic AMP Accumulation in Rat Hippocampal Slices

Previous work has shown that incubation of hippocampal slices in medium without added calcium markedly attenuates the capacity of vasoactive intestinal peptide (VIP) to elevate cyclic AMP levels. The present studies examined the mechanism that confers calcium dependence on VIP stimulation of cyclic AMP accumulation in hippocampal slices. Calcium dependence was apparent immediately on slice preparation and was reversible only if calcium ions were added back very early during slice incubation (within 5 min). The cyclic AMP response to VIP was not abolished by preincubating slices in 100 microM adenosine, suggesting that calcium-dependent, VIP-induced release of adenosine does not mediate VIP elevation of cyclic AMP. VIP-stimulated cyclic AMP accumulation was not decreased by agents that block calcium influx (verapamil, nifedipine, magnesium ions), or by calmodulin antagonists (trifluoperazine, calmidozolium). In fact both verapamil (100 microM) and magnesium (14 mM) augmented VIP stimulation of cyclic AMP generation. Incubation of slices with the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX) did not affect VIP activation of cyclic AMP accumulation if slices were incubated without added calcium, but MIX did enhance VIP elevation of cyclic AMP content in slices incubated with calcium. Thus calcium dependence of the cyclic AMP response to VIP in hippocampal slices is unlikely to result from VIP-dependent calcium influx, from interactions with calmodulin, or from calcium-inhibited phosphodiesterase(s).(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of calcium channel blockers on isoproterenol induced cyclic adenosine 3',5'-monophosphate generation in intact human lymphocytes

We have investigated the effects of clinically available calcium channel blockers (nifedipine, verapamil and diltiazem) on isoproterenol stimulated cyclic adenosine 3',5'-monophosphate (cyclic AMP) generation in intact human lymphocytes. After preincubation of various calcium antagonists with intact lymphocytes at 37 degrees C for 15 minutes, 10 microM nifedipine or verapamil partially inhibited isoproterenol induced cyclic AMP generation in the presence of cyclic AMP phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine) while they alone had no effect on cyclic AMP level at a concentration of up to 100 microM. In contrast, 10 nM-1.0 microM nifedipine, verapamil or diltiazem potentiated cyclic AMP generation induced by isoproterenol in a dose dependent manner. Similar results were observed in the time course studies of cyclic AMP generation. These effects are somewhat similar to the effect of phenothiazine, a calmodulin inhibitor, which, at 10 microM (close to IC50), also potentiated the effects of isoproterenol. In contrast, lanthanum chloride (LaCl3), an extracellular inorganic calcium antagonist, at 1.0 mM, inhibited isoproterenol induced cyclic AMP generation. The biochemical mechanisms underlying these potentiating effects are unknown. It may be partly related to the effect of calcium channel blockers (at least for nifedipine) on preventing beta 2 adrenergic receptor desensitization. This may provide a potential mechanism for the synergistic effect between calcium channel blockers and beta 2 adrenoceptor agonists on bronchial dilatation.     

Involvement of calcium and cyclic AMP in controlling ixodid tick salivary fluid secretion.

Catecholamine-stimulated salivary fluid secretion (in vitro) by ixodid ticks is reduced by deletion or lowering the concentration of exogenous bathing medium Ca++. The Ca++ antagonist, verapamil, reversibly inhibits dopamine-stimulated secretion. Ionophore A-23187 is unable to induce glands to secrete. Studies in which labeled and unlabeled Ca++ flux were measured indicate that catecholamines induce release of calcium from intracellular stores during secretion. Cyclic AMP/theophylline-stimulated secretion is inhibited by verapamil, and the exclusion of calcium from the support medium. It is concluded that the primary catecholamine stimulus induces cyclic AMP formation and mobilization of Ca++ (intra- and extracellular). Cyclic AMP and calcium are thought to interact to control secretion within the fluid transporting cells of types II and III alveoli.     

The interaction of stimulants on the function of isolated canine parietal cells

With isolation, the parietal cell is removed from the effects of the many endogenous substances that may modulate its function in intact mucosa, even in the basal state. The isolated canine parietal cell responds to the major endogenous regulators of secretion: histamine, acetylcholine and gastrin. These agents act on specific receptors as evidenced by (1) the specificity of antagonist (H2 antagonists, atropine, and dibutyryl cyclic GMP respectively), (2) the binding of radiolabelled ligands, and (3) the existence of separate second messenger systems (cyclic AMP for histamine, calcium influx for acetylcholine, and an unidentified mechanism for gastrin). Potentiating interactions, which occur between histamine and acetylcholine or histamine and gastrin, do not involve extra production of second messenger. When histamine and acetylcholine are given together, the amounts of cyclic AMP generated and of calcium entering the cell are not greater than when each is acting alone. The apparent non-specific effects of inhibitors acting in vivo, such as the inhibition of all forms of stimulation by H2 antagonists, could reflect withdrawal of the potentiating action of the background histamine always present in the mucosa.     

Dopaminergic Inhibition of Prolactin Release and Calcium Influx Induced by Neurotensin in Anterior Pituitary Is Independent of Cyclic AMP System

The present study demonstrates that 3,4-dihydroxyphenylethylamine (DA, dopamine) prevents neurotensin (NT) stimulation of both prolactin (PRL) release and calcium influx by interacting with specific receptors that are functionally linked to calcium channels. As shown by the studies with dispersed cells from rat anterior pituitary, the pharmacology of the control of PRL release and calcium influx, both induced by NT, was found to be typical of a DAergic process. This was demonstrated by the order of potency of agonists in inhibiting PRL release and calcium influx (DA greater than epinephrine greater than norepinephrine much greater than isoproterenol); by the high affinity of antagonists such as haloperidol and fluphenazine for this process; and by the high degree of stereoselectivity of sulpiride. Specific D2 receptor agonists, such as bromocriptine and lisuride, and the specific D2 receptor antagonist (-)-sulpiride were found to be highly potent on the DA receptors negatively coupled with calcium channels and PRL release. DA was found to lack the capacity to change the influx of calcium induced by either the sodium channel activator veratridine or high extracellular potassium levels, thus indicating a specific action of this amine on calcium channels sensitive to NT. In a range of concentrations that are effective in inhibiting either the calcium influx or the PRL release, both induced by NT, DA did not alter the cyclic AMP generating system. DA (from 1.0 nM to 50 nM) did not affect adenylate cyclase activity in rat pituitary gland homogenates and did not modify intracellular cyclic AMP levels in pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of Neurotensin Receptors Associated with Calcium Channels and Prolactin Release in Rat Pituitary

Neurotensin (NT) is now reasonably well established as a neurotransmitter or neuromodulator candidate in the CNS. In the present study, we characterized the NT receptors in dispersed cells from the anterior lobe of rat pituitary and investigated the involvement of both cyclic AMP and calcium in the release of prolactin (PRL) induced by NT receptor stimulation. The [3H]NT binding to membranes from anterior pituitary dispersed cells was found saturable and stereospecific. Scatchard analysis of the data gave a straight line indicating a Bmax value of 121 +/- 11 fmol/mg protein and a KD value of 1.4 +/- 0.2 nM. The calculated IC50 values for [3H]NT binding were 5.8 nM for NT, 7.8 nM for L-Phe-NT, and 3,000 nM for the pharmacologically inactive form D-Phe-NT. NT, up to a concentration of 1 microM, did not affect the cyclic AMP generating system in homogenates of anterior pituitary from male or lactating female rats. The same pattern of results was obtained for cyclic AMP formation in intact cells. NT and its analogs stereospecifically enhanced the influx of calcium into dispersed cells from rat anterior pituitary. The effect was time- and dose-dependent. It appeared to be associated with neurotransmitter-operated calcium channels since: preincubation of the cells with tetrodotoxin did not affect the increase in calcium influx induced by NT; concentrations of verapamil that counteract the influx of calcium induced by potassium lacked the capacity to modify the influx of calcium induced by NT; and NT lost its capacity to release PRL in the absence of extracellular calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antagonism of alpha- and beta-adrenergic-mediated accumulations of cyclic AMP in rat cerebral cortical slices by the beta-antagonist (-)alprenolol.

(?)Alprenolol, a compound reported to bind with a high degree of specificity and stereoselectivity to β-adrenergic receptors from rat cerebral cortex completely inhibited the accumulations of cyclic AMP elicited by maximally effective concentrations of norepinephrine and epinephrine at antagonist concentrations as low as 10^?5M. Other β-adrenergic antagonists such as (?)propranolol, (±)sotalol, and (+)alprenolol only partially antagonized accumulations of cyclic AMP elicited by these catecholamines even at 10-fold higher concentrations. α-Adrenergic antagonists such as phentolamine, phenoxybenzamine and clonidine only partially antagonized inhibited the accumulation of cyclic AMP elicited by methoxamine, a compound shown to stimulate the accumulation of cyclic AMP by interaction with α-adrenergic receptors. The results indicate that in brain tissue containing a mixed population of α- and β- adrenergic linked cyclic AMP generating systems, (?)alprenolol does not exhibit absolute specificity for β-receptors.     

The role of calcium ions in the mechanism of ACTH stimulation of cortisol synthesis

Removal of free calcium ions from the incubation medium of isolated bovine adrenocortical cells with EGTA reduced basal cortisol synthesis and blocked the effects of ACTH; additional calcium restored normal steroid synthesis. Calcium channel blockers, verapamil and nitrendipine and the calmodulin antagonist, trifluoperazine inhibited ACTH-stimulated cortisol synthesis in a dose-dependent manner (IC50s of 6.2, 10 and 5.2 microM, respectively). Steroidogenic effects of dibutyryl cyclic AMP were prevented with 50 microM verapamil or trifluoperazine. Calcium ionophore A23187 at 1 microM increased cortisol synthesis 2-3 fold which was less than the normal response to ACTH. Stimulatory effects of ionophore and cyclic AMP or ACTH were not additive. ACTH-stimulation of cortisol synthesis appears to involve cyclic AMP-dependent uptake of extracellular calcium ions, possibly by a mechanism requiring calmodulin. Increases in intracellular calcium ions cannot wholly mimic ACTH actions.     

Influence of electrically induced tachyarrhythmia on the release of cyclic AMP and PGE in canine coronary sinus blood and on the level of cyclic AMP in myocardial tissue

In anaesthetized open-chest dogs tachyarrhythmia (TA) was electrically induced by above-threshold stimuli via the right ventricle. During TA a significant increase in the release of PGE and cyclic AMP of 20% and 40% of the control levels, respectively, was observed in the canine coronary sinus blood (CSB), whereas the level of PGF2 alpha remained nearly unchanged under these conditions. The efflux of cyclic AMP corresponded with a concomitant increase in the left ventricular tissue level of this nucleotide by 59% during TA. Pretreatment with the beta-adrenergic blocking agent propranolol (1.0 mg/kg i.v.) prevented the TA induced changes in the level of PGE as well as cyclic AMP in the CSB and in the tissue levels of cyclic AMP. Propranolol alone was without any effect on the efflux of cyclic AMP, but decreased significantly the efflux of PGE by 32%. There was an increase in the activity of phosphorylase a in the myocardial tissue from 10% to 20% of the total (a + b) activity of this enzyme during TA, which could be abolished by propranolol pretreatment. The results suggest possible interrelationships between catecholamines, cyclic AMP and PGE.     

Effect of substance P and eledoisin on K+ efflux, amylase release and cyclic nucleotide levels in slices of rat parotid gland.

The undecapeptides, substance P and eledoisin, caused a rapid, concentration-dependent increase in K+ efflux and amylase release from parotid tissue slices. The effects were not blocked by beta-adrenergic, alpha-adrenergic, or cholinergic antagonists. Incubation buffer calcium was required for stimulation of K efflux and amylase release. The action of the undecapepides was independent of any effects on parotid cyclic AMP or cyclic GMP levels. Since the actions of the undecapeptides were Ca2+ dependent and no effects on cyclic nucleotide levels were discerned it was concluded that Ca2+ plays a primary role in agonist regulation of K+ efflux from the parotid.     

Dibutyryl-cAMP increases functions of 5-hydroxytryptamine2 receptors, but not of beta 2-adrenergic receptors, in a clonal cell line of rat neurotumor RT4.

A peripheral nervous system cell line RT4-B, established by Imada and Sueoka (Dev. Biol., 66:97-108, 1978), was shown to respond to serotonin [5-hydroxytryptamine (5-HT)] and catecholamines. 5-HT induced a small and transient increase in cytosolic free Ca2+ concentration ([Ca2+]i) in the RT4-B cells. The increase was effectively blocked by 5-HT2 receptor antagonists (spiperone, ritanserin and mianserin), but not by a 5-HT3 receptor antagonist (MDL72222), or a alpha 1-adrenergic receptor antagonist (prazosin), indicating that RT4-B cells express 5-HT2 receptors. On the other hand, catecholamines increased cyclic AMP production by RT4-B. The order of potency for stimulating cyclic AMP synthesis was isoproterenol greater than epinephrine much greater than norepinephrine much greater than dopamine, and the stimulation was effectively inhibited by the nonselective beta-adrenergic receptor antagonist propranolol, but not by the beta 1-adrenergic receptor antagonist atenolol, suggesting that RT4-B cells express beta 2-adrenergic receptors. The differentiating agent N6,2'-O-dibutyryladenosine 3',5'-monophosphate (dibutyryl-cAMP) enhanced the 5-HT-induced [Ca2+]i increase, but not the catecholamine-induced cyclic AMP production. The increase in the 5-HT response paralleled the increase in the density of 5-HT2 receptors. n-Butyric acid (2 mM) and 8-bromoadenosine 3',5'-monophosphate (1 mM) also increased the 5-HT response, and the sum of these increases was nearly equal to that induced by dibutyryl-cAMP. These results indicate that RT4-B is a novel model cell line for the study of 5-HT2 and beta 2-adrenergic receptors and their second messenger responses and for the analysis of the mechanisms how 5-HT2 receptor gene expression is controlled.     

Serotonin stimulates calcium influx in isolated rat adrenal zona glomerulosa cells.

To investigate the role of calcium as a second messenger in serotonin-stimulated aldosterone secretion, radiolabelled calcium influx studies were carried out in purified rat adrenal zona glomerulosa cells using 45CaCl2. The results show that serotonin caused calcium influx within 45 seconds of addition and this continued for up to 105 seconds. Angiotensin II also caused calcium influx; however, the effect was significantly smaller than that of serotonin. Serotonin-stimulated calcium influx could be inhibited by the calcium antagonist verapamil and by methysergide, a selective serotonin receptor type-1/2 antagonist. The data indicate that serotonin directly stimulates calcium uptake in zona glomerulosa cells via calcium channels which are coupled to specific serotonin receptors.     

Interaction of calcium antagonists with cyclic AMP phosphodiesterases and calmodulin

The calcium antagonists, nimodipine and nicardipine, competitively inhibited calmodulin-sensitive and calmodulin-insensitive forms of cyclic AMP phosphodiesterase, with IC_50's in the micromolar range. Verapamil showed similar inhibitory potency against calmodulin-insensitive phosphodiesterases, but in marked contrast, it was a very weak inhibitor (30–100 times less potent) against calmodulin-sensitive forms of the enzyme. Verapamil and nimodipine both antagonized the calmodulin stimulation of phosphodiesterase. Through use of hydrophobic fluorescent probes, verapamil, and another calmodulin antagonist, proadifen, were shown to interact directly with calmodulin in a manner that differed from the interaction of calmodulin with trifluoperazine.     

Effects of verapamil on lipolysis due to dibutyryl cyclic AMP.

The effects of verapamil, a calcium antagonist, on lipolysis in isolated rat adipocytes were studied. Verapamil (100 microM) potentiated lipolysis due to dibutyryl cyclic AMP (Bt2cAMP) at submaximal concentrations, with or without extracellular Ca2+. Lipolysis due to 0.5 mM-Bt2cAMP was potentiated by verapamil in a dose-dependent manner up to 200 microM, whereas at concentrations higher than 100 microM the stimulatory effect of verapamil was progressively diminished with or without extracellular Ca2+. Verapamil showed only an inhibitory effect on lipolysis due to adrenaline (0.1-10 microM) or 3-isobutyl-1-methylxanthine (IBMX; 25-200 microM). The stimulatory effect of verapamil on lipolysis due to Bt2cAMP was not blocked by alpha-adrenergic antagonists. These results suggest (i) that verapamil has a biphasic effect on lipolysis due to Bt2cAMP and only an inhibitory effect on that due to adrenaline or IBMX, and (ii) that extracellular Ca2+ or alpha-adrenergic receptors are not involved in the action of verapamil.     

Adrenergic versus VIPergic control of cyclic AMP in human colonic crypts

The actions of catecholamines on VIP-induced cyclic AMP is studied in human colon. We show that: (1) Epinephrine in the 10(-7)-10(-3) M concentration range (ED50 = 11.10(-6) M) inhibits VIP-induced cyclic AMP rise in isolated colonic epithelial cells; the maximal inhibition reaches 30% of VIP effect; epinephrine alters the efficacy of the peptide and does not modify its potency; epinephrine also reduces the basal cyclic AMP level. (2) The inhibition is found with other alpha adrenergic agonists with the order of potencies epinephrine greater than norepinephrine greater than phenylephrine. Clonidine has a poor intrinsic activity but antagonizes the action of epinephrine. (3) The inhibition of VIP action by epinephrine is reversed by the alpha antagonists dihydroergotamine, phentolamine and the alpha 2 antagonist yohimbine, while unaffected by the beta antagonist propranolol and the alpha 1 antagonist prazosin, (4) Epinephrine inhibits VIP-stimulated adenylate cyclase activity in preparations of colonic plasma membranes. Thus catecholamines exert through an alpha 2 adrenoreceptor a negative control on basal and VIP-stimulated cyclic AMP formation in human colon. We suggest that colonic cyclic AMP metabolism undergoes a dual control: VIPergic, activator and adrenergic, inhibitor.     

Forskolin potentiates calcium-dependent amylase secretion from rat pancreatic acinar cells

The cellular and molecular effects of forskolin, a direct, nonhormonal activator of adenylate cyclase, were assessed on the enzyme secretory process in dispersed rat pancreatic acinar cells. Forskolin stimulated adenylate cyclase activity in the absence of guanyl nucleotide. It promoted a rapid and marked increase in cellular accumulation of cyclic AMP alone or in combination with vasoactive intestinal peptide (VIP) but was itself a weak pancreatic agonist and did not increase the secretory response to VIP or other cyclic AMP dependent agonists. Somatostatin was a partial antagonist of forskolin stimulated cyclic AMP synthesis and forskolin plus cholecystokinin-octapeptide (CCK-OP) induced amylase release. Forskolin potentiated amylase secretion in response to calcium-dependent agonists such as CCK-OP, carbachol and A-23187, but did not affect the ability of CCK-OP and (or) carbachol to mobilize 45Ca from isotope preloaded cells; forskolin alone did not stimulate 45Ca release. In calcium-poor media, the secretory response to forskolin and CCK-OP was reduced in a both absolute and relative manner. The data suggests that calcium plays the primary role as intracellular mediator of enzyme secretion and that the role of cyclic AMP may be to modulate the efficiency of calcium utilization.     

Norepinephrine acts as D1-dopaminergic agonist in the embryonic avian retina: late expression of beta1-adrenergic receptor shifts norepinephrine specificity in the adult tissue

Dopamine is the main catecholamine found in the chick retina whereas norepinephrine is only found in trace amounts. We compared the effectiveness of dopamine and norepinephrine in promoting cyclic AMP accumulation in retinas at embryonic day 13 (E13) and from post-hatched chicken (P15). Dopamine (EC(50)=10microM) and norepinephrine (EC(50)=30microM), but not the beta(1)-adrenergic agonist isoproterenol, stimulated over seven-fold the production of cyclic AMP in E13 retina. The cyclic AMP accumulation induced by both catecholamines in embryonic tissue was entirely blocked by 2microM SCH23390, a D(1) receptor antagonist, but not by alprenolol (beta-adrenoceptor antagonist). In P15 retinas, 100microM isoproterenol stimulated five-fold the accumulation of cAMP. This effect was blocked by propanolol (10microM), but not by 2microM SCH23390. Embryonic and adult retina display beta(1) adrenergic receptor mRNA as detected by RT-PCR, but the beta(1) adrenergic receptor protein was detected only in post-hatched tissue. We conclude that norepinephrine cross-reacts with D(1) dopaminergic receptor with affinity similar to that of dopamine in the embryonic retina. In the mature retina, however, D(1) receptors become restricted to activation by dopamine. Moreover, as opposed to the embryonic tissue, norepinephrine seems to stimulate cAMP accumulation via beta(1)-like adrenergic receptors in the mature tissue.     

Recruitment of Ca2+ channels by protein kinase C during rapid formation of putative neuropeptide release sites in isolated Aplysia neurons.

Activation of protein kinase C (PKC) in Aplysia bag cell neurons causes the recruitment of voltage-dependent calcium channels. Using imaging techniques on isolated cells, we have now found that an activator of PKC, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), promotes the rapid appearance of new sites of calcium influx associated with a change in the morphology of neurite endings. In untreated cells, calcium influx triggered by action potentials occurs along neurites and in the central region of growth cones, but does not usually occur at the leading edge of lamellipodia. TPA produces extension of the lamellipodium, and action potentials now trigger calcium influx at the distal edge of the newly extended endings. Cotreatment with TPA and a cyclic AMP analog promotes movement of secretory organelles toward the new sites of calcium influx. Our results suggest that these second messenger systems promote the rapid formation of morphological structures that contribute to the potentiation of peptide release.     

Effects of calcium channel antagonists on the corpora allata of adult male loreyi leafworm Mythimna loreyi: juvenile hormone acids release and intracellular calcium level

The effects of voltage-dependent calcium channel (VDCC) antagonists and the non-specific calcium channel antagonists on both juvenile hormone acids (JHA) release and cytosolic free calcium concentration ([Ca2+]i) are investigated in the corpora allata (CA) of the adult males loreyi leafworm Mythimna loreyi. The VDCC antagonists used in this study are: the L-type antagonists diltiazem, nifedipine, and verapamil, the N-type antagonist omega-Conotoxin (CgTx) GVIA, the N- and P/Q-type antagonist omega-CgTx MVIIC, and the T-type antagonist amiloride. The non-specific calcium channel antagonists used in this study were cadmium (Cd2+), cobalt (Co2+), nickle (Ni2+), and lanthanum (La3+). The results show that both the DHPs-sensitive L-type antagonist nifedipine and the N-type antagonist omega-CgTx GVIA were able to inhibit JHA release, but only omega-CgTx GVIA was able to reduce the [Ca2+]i. Among the non-specific calcium channel antagonists, Cd2+ is the most potent in reducing JHA release but without obvious effect on the [Ca2+]i, La3+ significantly increases the [Ca2+]i but without effect on JHA release.     

The release of slow-reacting substance of anaphylaxis from guinea pig lung: effects of calcium antagonists

The effects of three calcium antagonists, verapamil, lanthanum, and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) were studied on the release of slow-reacting substance of anaphylaxis (SRS-A) from ovalbumin-sensitized chopped guinea pig lung parenchyma in calcium-containing and calcium-free media. The SRS-A levels (mean +/- SEM) obtained from tissues incubated in normal and calcium-free Krebs-bicarbonate buffer were 51 +/- 8 (N = 19) and 21 +/- 4 (N = 14) U/mL, respectively. TMB-8 (0.1-10 microM), a reported intracellular calcium antagonist, reduced antigen-stimulated SRS-A release from lung tissue incubated in calcium-containing, but not calcium-free, medium; A23187-induced SRS-A release from normal guinea pig lung was not significantly altered by TMB-8 at concentrations up to 10 microM. Verapamil and lanthanum consistently reduced SRS-A release only at high concentrations (100 microM and 1mM, respectively). The quantities of SRS-A released from lung tissue incubated in the presence of verapamil in normal medium were similar to those obtained in calcium-free medium. Tissues incubated in the presence of potassium chloride (60 and 100 mM) did not release significant quantities of SRS-A, and release which did occur was not blocked by verapamil, suggesting that antigen-induced SRS-A release is not dependent on membrane depolarization and that verapamil was not exerting inhibition via blockade of voltage-dependent calcium channels. These data suggest that although intracellular calcium is important for the regulation of SRS-A secretion from guinea pig lung tissue, extracellular calcium is necessary for optimal release of SRS-A.