Atrial natriuretic peptide does not affect corticotropin-releasing factor-, arginine vasopressin- and angiotensin II-induced adrenocorticotropic hormone release in vivo or in vitro

The effect of synthetic atrial natriuretic peptide (ANP) was examined on the in vivo and in vitro release of ACTH. Intravenous ANP (4 micrograms/kg body weight) administration did not affect the corticotropin releasing factor (CRF, 4 micrograms/kg body weight)-, arginine vasopressin (AVP, 2 micrograms/kg body weight)- and angiotensin II (A II, 4 micrograms/kg body weight)-induced ACTH release in unanesthetized freely moving rats. ANP did not inhibit the basal, CRF- and AVP-induced release of ACTH in pituitary cell cultures. ANP did not affect the CRF- and AVP-induced plasma corticosterone elevation, while it attenuated the AVP-induced corticosterone elevation. These results indicate that ANP does not affect the ACTH release at the pituitary level in vivo and in vitro.     

The biosynthesis and secretion of adrenocorticotropin by the ovine anterior pituitary is predominantly regulated by arginine vasopressin (AVP). Evidence that protein kinase C mediates the action of AVP

This study was undertaken to define the roles of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) in the regulation of adrenocorticotropin (ACTH) release and biosynthesis in cultured ovine anterior pituitary cells and to define the intracellular mechanisms responsible for their action. At 4 h, CRF and AVP increased both ACTH release and total ACTH content, with AVP clearly the more potent agonist (maximal ACTH release: AVP, 22.8-fold; CRF, 7.6-fold; maximal increment in total ACTH content: AVP, 1.9-fold; CRF, 1.1-fold; EC50 for ACTH release: AVP, 2.3 +/- 0.5 nM; CRF, 9.2 +/- 5.0 nM). The increase in total ACTH content was interpreted to reflect an augmentation of ACTH biosynthesis since it was abolished by 10 microM cycloheximide. Exposure of the anterior pituitary cells to increasing concentrations of forskolin or 8-bromo-cAMP elicited increases in ACTH release and total ACTH content that were similar to those caused by CRF. A 30-min incubation with phorbol 12-myristate 13-acetate (PMA) caused a dose-related translocation of protein kinase C from the cytosol to the cell membrane; after 4 h, the increases in ACTH release and total ACTH content in response to increasing concentrations of PMA were similar to those caused by AVP. Chronic (24 h) exposure to 150 nM PMA caused an almost total depletion of both cytosolic and membrane-bound protein kinase C activities. When protein kinase C-depleted cells were subsequently exposed to AVP, the increases in ACTH release and total ACTH content were markedly attenuated, but the responses to CRF were preserved. Finally, the combination of CRF and AVP, CRF and PMA, or AVP and 8-bromo-cAMP increased ACTH release and total ACTH content in a synergistic manner. We conclude that: 1) in ovine anterior pituitary cells, AVP is the predominant regulator of ACTH secretion and biosynthesis; 2) the action of AVP is predominantly mediated by activation of protein kinase C, whereas the action of CRF is likely to be mediated by activation of the cAMP-dependent protein kinase (protein kinase A); and 3) the ability of CRF and AVP to increase total ACTH content and secretion in a synergistic manner provides a demonstration in normal pituitary cells that protein kinases C and A may interact in a unidirectional manner to regulate ACTH biosynthesis in addition to ACTH release. This interaction may take place within, or between, individual corticotropes.     

In vivo and in vitro release of ACTH by synthetic CRF

The 41-residue corticotropin releasing factor (CRF) was synthesized by the solid phase method. The synthetic CRF and arginine vasopressin (AVP) were examined for ACTH releasing activity and effects on the release of 5 other pituitary hormones in vivo and in vitro. Injection of the CRF into pharmacologically blocked rats increased plasma corticosterone levels in a dose-related manner. The minimum effective dose was 1.6 x 10(-12) mol/100 g body weight. CRF also significantly stimulated release of ACTH-like immunoreactivity in a dose-related manner from rat pituitary quarters beginning at a concentration of 10(-9) M. AVP, a peptide known to have CRF activity, exhibited slightly lower corticotropin releasing activity than the CRF at equimolar dose levels. Secretion of other pituitary hormones was not appreciably altered by either the CRF or AVP.     

Role of arginine vasopressin in control of ACTH and LH release during stress

To determine the role of arginine vasopressin (AVP) in stress-induced release of anterior pituitary hormones, AVP antiserum or normal rabbit serum (NRS) was micro-injected into the 3rd ventricle of freely-moving, ovariectomized (OVX) female rats. A single 3 microliter injection was given, and 24 hours later, the injection was repeated 30 min prior to application of ether stress for 1 min. Although AVP antiserum had no effect on basal plasma ACTH concentrations, the elevation of plasma ACTH induced by ether stress was lowered significantly. Plasma LH tended to increase following ether stress but not significantly so; however, plasma LH following stress was significantly lower in the AVP antiserum-treated group than in the group pre-treated with NRS. Ether stress lowered plasma growth hormone (GH) levels and this lowering was slightly but significantly antagonized by AVP antiserum. Ether stress also elevated plasma prolactin (Prl) levels but these changes were not significantly modified by the antiserum. To evaluate any direct action of AVP on pituitary hormone secretion, the peptide was incubated with dispersed anterior pituitary cells for 2 hours. A dose-related release of ACTH occurred in doses ranging from 10 ng (10 p mole)-10 micrograms/tube, but there was no effect of AVP on release of LH. The release of other anterior pituitary hormones was also not affected except for a significant stimulation of TSH release at a high dose of AVP. The results indicate that AVP is involved in induction of ACTH and LH release during stress. The inhibitory action of the AVP antiserum on ACTH release may be mediated intrahypothalamically by blocking the stimulatory action of AVP on corticotropin-releasing factor (CRF) neurons and/or also in part by direct blockade of the stimulatory action of vasopressin on the pituitary. The effects of vasopressin on LH release are presumably brought about by blockade of a stimulatory action of AVP on the LHRH neuronal terminals.     

Effect of ACTH 4-10 on passive avoidance of rats lacking vasopressin (Brattleboro strain).

The hypothesis that the effects of ACTH 4-10 on avoidance are mediated via the release of endogenous vasopressin was investigated. To test this hypothesis, we observed the effect of ACTH 4-10 on the passive avoidance of Brattleboro rats with diabetes insipidus resulting from a total genetic deficiency of vasopressin (DI) and Brattleboro rats without diabetes insipidus (HE). Normal Long-Evans rats (LE) were also included for comparison purposes. The results did not support the hypothesis. ACTH 4-10 did influence the passive avoidance of DI rats; this should not have occurred if the release of endogenous vasopressin is necessary for ACTH 4-10 to influence avoidance.     

In vivo regulation of adrenocorticotrophin secretion in the immature ovine fetus. Modulation by ovine corticotrophin releasing hormone and arginine vasopressin

This study investigates the in vivo regulation of ACTH secretion in the immature ovine fetus by AVP and oCRH. Previously we have demonstrated that whilst AVP-containing neurones are present from 42 days, oCRH-containing neurones cannot be detected in the fetal paraventricular nucleus or median eminence until after 90 or 100 days respectively. In acutely exteriorized fetuses aged between 64-90 days (n = 5), a haemorrhagic stress elicited a significant increase (P less than 0.01) in ACTH values. There was also a significant correlation between plasma ACTH and AVP concentrations in these fetuses. In chronically cannulated fetuses less than 100 days (n = 6) injection of AVP (200 ng) significantly elevated fetal plasma ACTH values at 10 min (P less than 0.01) post injection. Simultaneous injection of AVP (200 ng) and oCRH (10 micrograms) into these fetuses produced a plasma ACTH value that was significantly greater at 10 min (P less than 0.05) than the summed response obtained with separate injection of oCRH and AVP. When AVP and oCRH were injection in equimolar amounts to fetuses between 101-118 days, AVP (2 micrograms) was found to have a greater effect on ACTH than was oCRH (2 micrograms). Pretreatment with a specific vascular antagonist of AVP-d(CH2)5Tyr(Me)AVP failed to significantly inhibit the increase in mean arterial pressure associated with AVP (2 micrograms) injection but partially antagonized (P = 0.04) the decrease in fetal heart rate. The antagonist however completely abolished any effect of AVP on fetal plasma ACTH values. This study suggests that AVP most likely acts through receptors in the fetal pituitary with V1 characteristics.     

Mechanisms of action of corticotropin-releasing factor and other regulators of corticotropin release in rat pituitary cells

The role of cyclic AMP in the stimulation of corticotropin (ACTH) release by corticotropin-releasing factor (CRF), angiotensin II (AII), vasopressin (VP), and norepinephrine (NE) was examined in cultured rat anterior pituitary cells. Synthetic CRF rapidly stimulated cyclic AMP production, from 4- to 6-fold in 3 min to a maximum of 10- to 15-fold at 30 min. Stimulation of ACTH release by increasing concentrations of CRF was accompanied by a parallel increase in cyclic AMP formation, with ED50 values of 0.5 and 1.3 nM CRF for ACTH and cyclic AMP, respectively. A good correlation between cyclic AMP formation and ACTH release was also found when pituitary cells were incubated with the synthetic CRF(15-41) fragment, which displayed full agonist activity on both cyclic AMP and ACTH release with about 0.1% of the potency of the intact peptide. In contrast, the CRF(21-41) and CRF(36-41) fragments were completely inactive. The other regulators were less effective stimuli of ACTH release and caused either no change in cyclic AMP (AII and VP) or a 50% decrease in cyclic AMP (NE). Addition of the phosphodiesterase inhibitor, methylisobutylxanthine, increased the sensitivity of the ACTH response to CRF but did not change the responses to AII, VP, and NE. In pituitary membranes, adenylate cyclase activity was stimulated by CRF in a dose-dependent manner with ED50 of 0.28 nM, indicating that the CRF-induced elevation of cyclic AMP production in intact pituitary cells is due to increased cyclic AMP biosynthesis. The intermediate role of cyclic AMP in the stimulation of ACTH release by CRF was further indicated by the dose-related increase in cyclic AMP-dependent protein kinase activity in pituitary cells stimulated by CRF with ED50 of 1.1 nM. These data demonstrate that the action of CRF on ACTH release is mediated by the adenylate cyclase-protein kinase pathway and that the sequence requirement for bioactivity includes the COOH-terminal 27 amino acid residues of the molecule. The other recognized regulators of ACTH release are less effective stimuli than CRF and do not exert their actions on the corticotroph through cyclic AMP-dependent mechanisms.     

Stimulation of the hypothalamic-pituitary-adrenal axis by epidermal growth factor

The effects of mouse epidermal growth factor (mEGF) on the hypothalamic-pituitary-adrenocortical axis were studied in vivo in conscious male rats and in vitro with cultured anterior pituitary cells. Both intravenous (i.v.) and intracerebroventricular (i.c.v.) injections of mEGF (5-20 ng: 8.3-33.3 pmol) produced significant, dose-related increases in plasma ACTH and corticosterone concentrations. The potency of mEGF is 1/20-1/50 of that of rat corticotropin-releasing factor (rCRF), and pretreatment with 150 micrograms alpha-helical CRF (9-41) completely abolished the effects of the two peptides. mEGF in concentrations ranging from 10 pM to 10 nM did not significantly affect ACTH release from dispersed anterior pituitary cells. It also failed to alter ACTH secretion in response to rCRF. These results indicate that mEGF stimulates the pituitary-adrenocortical axis through a CRF-dependent mechanism.     

Intra-cerebroventricular captopril reduces plasma ACTH and vasopressin responses to hemorrhagic stress

The role of the brain renin-angiotensin system in mediating the peripheral hormone response to acute hemorrhagic stress (15 ml/kg over 10 min) was studied in 6 sheep during an intracerebroventricular infusion (2.8 micrograms/min) of the angiotensin-converting enzyme inhibitor, captopril. When compared with control experiments the plasma ACTH and vasopressin (AVP) response to hemorrhage was markedly reduced and delayed during icv captopril, which did not affect the response of plasma angiotensin II (AII). These results suggest that the normal and rapid response in ACTH and AVP secretion accompanying hemorrhagic stress is dependent on increased brain production of AII.     

The effect of serotonin agonist 1-(trifluoromethylphenyl)-piperazine on corticotropin releasing factor and arginine vasopressin in rat hypothalamic nuclei

Effects of 1-(m-trifluoromethylphenyl)-piperazine, a serotonin agonist, were examined on rat plasma levels of adrenocorticotropin (ACTH) and arginine vasopressin (AVP), and on hypothalamic contents of corticotropin releasing factor (CRF) and AVP, to investigate the role of brain serotonin in ACTH regulation. Both plasma ACTH and AVP levels increased markedly 30 min after injection of the compound and were still elevated at 80 min. CRF and AVP contents in the median eminence decreased 30 min after injection but returned to the basal levels by 80 min. The AVP content in the supraoptic nucleus was elevated 80 min after injection. The CRF and aVP content did not significantly change in the paraventricular, suprachiasmatic and arcuate nuclei. Serotonin or 1-(m-trifluoromethylphenyl)-piperazine did not stimulate the release of ACTH in pituitary cell cultures. These results suggest that both CRF and AVP were secreted into the portal vessels by 1-(m-trifluoromethylphenyl)-piperazine to release ACTH from the anterior pituitary and that both the ACTH and AVP release were stimulated via the brain serotonergic mechanism.     

Oxytocin potentiates the ACTH-releasing activity of CRF(41) but not vasopressin

The effects of CRF(41), oxytocin (OT), and arginine vasopressin (AVP) on ACTH secretion were studied alone and in combination in an in vitro system of superfused rat hemipituitaries. CRF(41) (10(-9)M) and AVP (10(-8)M) alone produced a significant increase in ACTH secretion while OT (10(-8)M) alone had no effect. However the same concentration of OT markedly potentiated the ACTH response to CRF(41) while having no effect on the ACTH response to AVP. The data support a physiologic role for OT in the regulation of ACTH secretion.     

Corticotropin-releasing factor (CRF) and vasopressin concentration in major brain regions after adrenalectomy and their involvement in adrenalectomy-induced ACTH elevation

CRF and vasopressin concentrations in major brain regions after bilateral adrenalectomy and their involvement in adrenalectomy-induced ACTH secretion were investigated. At 5, 14 and 28 days after bilateral adrenalectomy, the plasma ACTH level was greatly elevated, whereas hypothalamic CRF content was reduced at 5 days and was not changed at 14 and 28 days after adrenalectomy. The CRF concentration in the medulla oblongata was reduced at 2-4 weeks after adrenalectomy. On the other hand, the arginine vasopressin (AVP) concentration was significantly elevated 2-4 weeks after adrenalectomy. An intrajugular administration of anti-ovine or anti-rat CRF serum significantly suppressed the elevated plasma ACTH level in adrenalectomized, freely moving rats, whereas anti-AVP serum or antipressor AVP antagonist, dpTyr(Me)AVP did not suppress the ACTH level. These results indicate that CRF played an important role in the adrenalectomy-induced ACTH elevation but that vasopressin was not involved.     

Analysis of the dual mechanism of ACTH release by arginine vasopressin and its analogs in conscious rats

Plasma ACTH and/or corticosterone levels were measured in conscious rats 30 min after subcutaneous administration of arginine vasopressin (AVP), oxytocin (OT) and various analogs with a large range of activity on the vasopressor (V1), antidiuretic (V2) or oxytocic receptors. The comparison of their dose-response curves indicated that two different mechanisms are involved in the release of ACTH by neurohypophysial peptides and their analogs. AVP itself and a specific vasopressor agonist (Phe2, Orn8, OT) displayed a similar, high slope dose-response curve. Non-vasopressor analogs, such as dDAVP were characterized by a low slope dose-response curve. Furthermore, dDAVP potentiated CRF and neither its own ACTH-releasing action nor its potentiation of CRF were sensitive to previous VI- or V2-receptor blockade. These results, together with other available data, are interpreted as indicative of the existence of two mechanisms of action for ACTH release by AVP and its analogs in vivo: an indirect action via endogenous CRF release, mediated by a VI receptor mechanism, and a direct action on the pituitary, shared by dDAVP and other non-vasopressor analogs, with receptor characteristics different to both the V1 and the V2 classical types.     

Monoamine and amino acid content in brain regions of Brattleboro rats

Monoamine and amino acid content were measured in brain regions from 12 week old male, homozygous Brattleboro (DI,n=12) and Long-Evans control (LE,n=12) rats. Norepinephrine (NE) content was significantly elevated (16–25%) in the spinal cord, pons-medulla and anterior hypothalamus of DI rats when compared to LE controls. NE content of the neurointermediate lobe of pituitary in DI rats was almost twice that of LE controls. Serotonin content was also significantly elevated in the spinal cord, pons-medulla, anterior hypothalamus and forebrain of DI rats relative to the LE controls. Taurine content in DI rats was increased (31–42%) above that of LE rats in the anterior hypothalamus, striatum and forebrain. Glutamine content was also greater in DI rats than LE in the spinal cord, pons-medulla, anterior hypothalamus, striatum, hippocampus and forebrain. The changes in monoamine and amino acid content were discussed in relation to the cardiovascular and osmoregulatory deficits that are present in DI rats due to arginine vasopressin (AVP) deficiency. The possible role of AVP in modulating NE turnover was also discussed. The increase in brain TAU content in DI rats may be a physiological response to hypernatremia.     

The cellular actions of vasopressin on corticotrophs of the anterior pituitary: resistance to glucocorticoid action

The cellular actions of vasopressin (AVP) in the anterior pituitary were investigated. HPLC analysis of [3H]inositol-labeled cells indicated that AVP stimulated a rapid increase in inositol-1,4,5 trisphosphate (IP3), inositol-1,4 bisphosphate, and inositol-4 monophosphate levels. While CRF had no effect on basal IP3 levels, it blocked their stimulation by AVP. CRF-stimulated ACTH secretion and cAMP accumulation were potentiated by AVP. AFter dexamethasone (DEX) treatment (20 nM, 18 h), CRF-dependent ACTH secretion and cAMP accumulation were attenuated but AVP was still able to potentiate both of these actions of CRF suggesting that cellular actions of AVP may be resistant to DEX effects. Therefore, [3H]AVP binding was determined in control and DEX-treated cells. Pretreatment with DEX had no effect on either AVP receptor affinity or on the number of available binding sites. Consistently, stimulation of IP3 production by AVP in DEX-treated cells was comparable to that of control cells. Protein kinase C activators such as 12-O-tetradecanoyl-phorbol-13-acetate and dioctanoylglycerol were either near additive with CRF or also potentiated the action of CRF on ACTH secretion, respectively, even after DEX pretreatment. These results indicate that, in the anterior pituitary, distinct intracellular signaling pathways mediate the actions of CRF and AVP; cAMP mediates CRF actions and IP3/protein kinase C mediate the effects of AVP. Neuromodulation of ACTH secretion by dual effector mechanisms which exhibit a complex mode of interaction and only one of which is negatively influenced by glucocorticoids, provides these cells a mechanisms by which appropriate responses can be elicited under various physiological states.     

Functional compartmentalization of arginine-vasopressin-activated cyclic AMP in anterior pituitary gland: the presence of a compartment activated by prostaglandin E2

AVP(10(-8)-10(-6)M) increased ACTH as well as PGE2 release from rat anterior pituitary quarters in vitro in a concentration dependent manner. IBMX (0.1 mM), a phosphodiesterase inhibitor, increased the ACTH response to AVP. The cAMP content in pituitary tissue was increased by AVP. Cyclooxygenase inhibition by indomethacin(1.4 X 10(-5) M) or diclofenac (1.8 X 10(-5)M) led to a potentiation of AVP-evoked ACTH secretion and to a decrease in AVP-stimulated cAMP formation. PGE2(10(-6)M) significantly increased pituitary cAMP content and indomethacin did not affect cAMP levels activated by PGE2. PGE2 attenuated the AVP-induced ACTH release. These results indicate that at least two functional compartments of AVP-activated cAMP responses are involved in the AVP-induced ACTH release. One compartment is directly activated by AVP and participates in the propagation of AVP-induced ACTH release. The second compartment is activated by PGE2. The contribution of the second compartment to the regulation of ACTH secretion is not well understood since PGE2 shows an inhibitory effect on AVP-induced ACTH secretion.     

In vitro potentiation of the activity of synthetic ovine corticotropin-releasing factor by arginine vasopressin

The ability of arginine vasopressin (AVP) to potentiate the actions of synthetic ovine corticotropin-releasing factor (CRF) was examined using anterior pituitary fragments. Marked potentiation of ACTH release was observed upon incubating the fragments with a combination of 2 nM AVP and 1 nM CRF. Potentiation of CRF-induced ACTH release was also observed when the fragments were incubated with a combination of 1 nM AVP and 0.5 nM CRF. These results suggest that AVP may play a role in the release of ACTH from the adenohypophysis.     

The effects of ghrelin/GHSs on AVP mRNA expression and release in cultured hypothalamic cells in rats

Ghrelin, the endogenous ligand for growth hormone secretagogues (GHSs) receptor (GHS-R), increases adrenocorticotropin (ACTH) and cortisol (corticosterone) as well as GH secretion in humans and animals. However, the site of GHSs action to induce ACTH secretion is not fully understood. To clarify the mechanisms of the action of ghrelin/GHSs on ACTH secretion, we analyzed the effects of KP-102 and ghrelin on the mRNA expression and release of corticotropin releasing factor (CRF) and arginine vasopressin (AVP), ACTH secretagogues, in monolayer-cultured hypothalamic cells of rats. Incubation of cells with KP-102 for 4 h and 8 h and with ghrelin for 4 h significantly increased AVP mRNA expression and release without changing CRF mRNA expression. CRF levels in culture media were undetectable. Suppression of GHS-R expression by siRNA blocked ghrelin- and KP-102-induced AVP mRNA expression and release. NPY significantly increased AVP mRNA expression and release. Furthermore, treatment of cells with anti-NPY IgG blocked KP-102-induced AVP mRNA expression and release. We previously reported that KP-102 significantly increases NPY mRNA expression in cultured hypothalamic cells. Taken together, these results suggest that ACTH secretion by ghrelin/GHSs is induced mainly through hypothalamic AVP, and that NPY mediates the action of ghrelin/GHSs.     

Effects of CRF, AVP and opioid peptides on pituitary-adrenal responses in sheep

Intravenous administration of equimolar doses of CRF (30 μg) and AVP (6 μg) to mature female sheep resulted in elevated plasma concentrations of ACTH and cortisol. Simultaneous administration of equimolar amounts of CRF and AVP resulted in a greater ACTH response compared with the sum of the responses to CRF or AVP given independently. Intravenous bolus administration of the endogenous opioid, Met-enkephalin (2.5 mg), and its potent and long-acting analogue, [D-Ala²,N-Phe⁴,Met(O)ol⁵]-enkephalin [FK33–824 (250 μg)], did not alter ACTH or cortisol secretion. Furthermore, naloxone, an opioid receptor antagonist given alone or concurrently with Met-enkephalin or FK33–824, was without effect. Pituitary-adrenal responses to CRF were unaltered by simultaneous administration of Met-enkephalin, FK33–824 or naloxone. These results suggest that in the sheep, opioid involvement in the tonic regulation of pituitary-adrenal function is absent. However, CRF and AVP may act alone or in synergy to control the release of biologically active ACTH from the sheep pituitary gland.     

Corticotropin-releasing factor, but not arginine vasopressin, stimulates concentration-dependent increases in ACTH secretion from a single corticotrope. Implications for intracellular signals in stimulus-secretion coupling.

The two fundamental parameters of corticotropin (ACTH) secretion are the number of secreting corticotropes and the amount of ACTH secreted by each cell. We have measured these parameters in rat corticotropes in response to increasing concentrations of corticotropin-releasing factor (CRF) or arginine vasopressin (AVP). Increasing concentrations of AVP stimulated more corticotropes to secrete, while the amount of ACTH each cell secreted remained relatively fixed (nongraded secretory response). Conversely, increasing concentrations of CRF stimulated more ACTH secretion per cell (graded secretory response), while the number of secretory cells remained relatively constant. When viewed from the perspective of a single corticotrope, it was clear that CRF and AVP induced completely distinct specific responses. We have previously shown, and provide further evidence here, that secretory responses to CRF or AVP occur in the same cell. It is therefore apparent that a single corticotrope is able to generate either a graded, or a nongraded secretory response. We have also considered the potential intracellular changes that must direct graded or nongraded secretion. It is generally accepted that CRF stimulates activation of adenylate cyclase, whereas AVP activates phosphoinositidase in pituitary corticotropes. Our findings, and others surveyed here, suggest that the activation of adenylate cyclase results in graded secretion, while the activation of phosphoinositidase induces the nongraded secretion. Graded or nongraded secretion may therefore be linked to specific second messengers. It is hypothesized that the inositol 1,4,5-trisphosphate-mediated release of an intracellular Ca2+ store constitutes a mechanism whereby phosphoinositidase-coupled hormones set in motion the nongraded secretory response. These findings suggest novel functions for individual second messengers.