Role of calcium fluxes in the sustained phase of angiotensin II-mediated aldosterone secretion from adrenal glomerulosa cells

When angiotensin II stimulates aldosterone secretion, it causes a rapid but transient mobilization of calcium from an intracellular pool and a sustained increase in the influx of calcium in adrenal glomerulosa cells. The present studies were undertaken to determine the respective roles of the two angiotensin II-induced changes in cellular calcium metabolism in modulating events during the sustained phase of cellular response which is thought to be mediated by the C-kinase branch of the calcium messenger system. The sustained response to angiotensin II is only 50% of maximal in cells pretreated with dantrolene in a concentration sufficient to inhibit the angiotensin II-induced mobilization of intracellular calcium. Also, if A23187 is added to cells simultaneously with 1-oleoyl-2-acetylglycerol (OAG), the aldosterone secretory response is similar to that seen after angiotensin II. However, if A23187 is added first and the transient aldosterone secretory response allowed to decay, and OAG then added, the sustained aldosterone secretory response is only 45-50% of maximal. Addition of the calcium channel agonist, BAY K 8644, with OAG leads to an aldosterone secretory response which is only 50% of maximal, but if upon addition of OAG and BAY K 8644 the cells are also exposed for 5 min to media containing 8 mM K+, then the sustained secretory response is maximal. These data imply that the initial transient rise in the [Ca2+] of the cell cytosol plays a role in determining the extent to which C-kinase is shifted from its calcium-insensitive to its calcium-sensitive form. The second group of experiments examined the relationship between the sustained angiotensin II-induced increase in plasma membrane calcium influx and the sustained aldosterone secretory response. The results show that in the presence of 1 microM nitrendipine or 2 mM extracellular K+, angiotensin II causes no increase in calcium influx and only a transient rather than a sustained increase in the rate of aldosterone secretion indicating that the sustained phase of the response is dependent upon a continued high rate of Ca2+ influx which regulates the rate of turnover of the activated C-kinase.     

Temporal patterns of protein phosphorylation after angiotensin II, A23187 and/or 12-O-tetradecanoylphorbol 13-acetate in adrenal glomerulosa cells.

The temporal patterns of protein phosphorylation in the adrenal glomerulosa cell were analysed by two-dimensional electrophoresis after stimulation with 10 nM-angiotensin II or various agents [10 nM-12-O-tetradecanoylphorbol 13-acetate (TPA), 50 nM-A23187, 1 microM-nitrendipine], administered singly or in combination. These patterns were compared with the temporal patterns of aldosterone secretion induced by the same agonists and antagonists. After 1 and 30 min of stimulation with angiotensin II, different patterns of protein phosphorylation were observed. A comparison of these patterns reveals that: the phosphorylation of only one protein was persistently enhanced during the continuous incubation with angiotensin II; the phosphorylation of five proteins was transiently enhanced (at 1 min but not 30 min); and the phosphorylation of three proteins did not occur at 1 min but was seen at 30 min. Addition of the phorbol ester TPA alone, which at 30 min is without effect in enhancing aldosterone production, has no effect on protein phosphorylation. The combined addition of TPA and the Ca2+ ionophore, A23187, which, like angiotensin II, evokes a sustained increase in aldosterone production, reproduced the temporal patterns of protein phosphorylation seen after angiotensin II action. Manipulations (A23187 alone, angiotensin II plus nitrendipine) which evoke only a transient rise in aldosterone production rate induce a transient rise in cellular protein phosphorylation. The 1 min patterns of phosphorylation seen after A23187 or combined angiotensin II and nitrendipine (a Ca2+ channel antagonist) are similar to those observed after 1 min of angiotensin II stimulation. These results suggest that, when angiotensin II acts, the initial cellular response is mediated by a different mechanism than that responsible for the sustained response.     

Insulin secretion: Combined effects of phorbol ester and A23187

The effect of the ionophore, A23187, and/or the phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), on insulin secretion were compared with those of glucose. Glucose induces a biphasic pattern of insulin secretion; A23187 a comparable initial spike but no second phase; and TPA a slowly progressive increase. Combined A23187 and TPA evoke a pattern similar to that induced by glucose. Forskolin enhances both phases of glucose-induced and of TPA-A23187-induced insulin secretion. These results are interpreted in terms of a model of cell activation in which two branches of the calcium messenger system, the calmodulin branch and the C-kinase branch, control, respectively, the initial and sustained phases of insulin secretion.     

The temporal integration of the aldosterone secretory response to angiotensin occurs via two intracellular pathways

Angiotensin II (AII) regulates the secretion of aldosterone from adrenal glomerulosa cells by a calcium-dependent mechanism which involves both the uptake of calcium from the extracellular pool, and the release of calcium from a dantrolene-sensitive intracellular pool. In the present study, it was shown that AII induces the rapid (10 s) hydrolysis of phosphatidylinositol 4-phosphate and -4,5-bisphosphate, leading to the sustained production of inositol bis- and trisphosphate (Ins-P3), and diacylglycerol rich in arachidonic acid. Saponin-permeabilized glomerulosa cells accumulate calcium into a nonmitochondrial pool by an ATP-dependent manner. Ins-P3 (0.5-5 microM) induces a release of Ca2+ from this pool. This release was blocked by dantrolene (10 microM). Adrenal glomerulosa cells were shown to contain the calcium-activated, phospholipid-dependent protein kinase (C-kinase). Perfusion of glomerulosa cells with combined 12-O-tetradecanoyl phorbol 13-acetate and A23187 induced an immediately developing, sustained, maximal secretory response similar to that induced by AII. These data are interpreted in terms of a model in which, after AII addition, there is a flow of information through two separate branches of the calcium messenger system, each with its unique temporal role: a calmodulin branch activated by the transient rise in the [Ca2+] in the cell cytosol, which is largely responsible for the initial transient cellular response; and a C-kinase branch activated by the increase in both cytosolic [Ca2+] and the diacylglycerol content of the plasma membrane, which is largely responsible for the sustained phase of the cellular response. The temporal integration of these two phases underlies the observed pattern of cellular response.     

Intracellular calcium and adenosine 3'',5''-cyclic monophosphate as mediators of potassium-induced aldosterone secretion.

We compared the action of K+ on aldosterone secretion from isolated bovine adrenal glomerulosa cells with that of ionophore A23187. Addition of either 50 nM-A23187 or 8 mM-K+ to perifused cells induces a similar initial aldosterone-secretory responses, and a similar sustained increases in Ca2+ entry. However, K+-induced secretion is more sustained than is A23187-induced secretion, even though each agonist appears to act by increasing Ca2+ entry into the cells. When [3H]inositol-labelled cells are stimulated by 8 mM-K+, a small decrease in phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is observed. This decrease is not accompanied by an increase in inositol trisphosphate (InsP3) concentration. Also, if [3H]arachidonic acid-labelled cells are exposed to 8 mM-K+, there is no increase in [3H]diacylglycerol production. When [3H]inositol-labelled cells are stimulated by 50 nM-A23187, a small decrease in PtdIns(4,5)P2 is observed. This decrease is not accompanied by an increase in InsP3. The cyclic AMP content of K+-treated cells was approximately twice that in A23187-treated cells. If cells are perifused simultaneously with 50 nM-forskolin and 50 nM-A23187, the initial aldosterone-secretory response is similar to that induced by A23187 alone, and the response is sustained rather than transient, and is similar to that seen during perifusion of cells with 8 mM-K+. This dose of forskolin (50 nM) causes an elevation of cyclic AMP concentration in A23187-treated cells, to a value similar to that in K+-treated cells. These results indicate that, in K+-treated cells, a rise in cyclic AMP content serves as a positive sensitivity modulator of the Ca2+ message, and plays a key role in mediating the sustained aldosterone-secretory response.     

Simultaneous determination of intracellular free calcium and aldosterone production in bovine adrenal zona glomerulosa

Angiotensin II (AII) induces an initial rapid but transient rise in [Ca2+]i detected with aequorin in bovine adrenal capsule strips. The rise in [Ca2+]i begins immediately after AII addition, reaches a peak in 30 seconds, and returns to near basal values within 5 minutes. The [Ca2+]i transient is receptor-mediated and its height is dose-dependent. The increase in [Ca2+]i is largely due to the release of Ca2+ from an intracellular pool. The uncorrected peak rise in [Ca2+]i after 1 X 10(-6) M beta-[asp1]-AII stimulation is approximately 3 fold, from 110 nM to 300 nM; the peak rise, corrected for diffusion and nonsynchronous cellular response, is from 110 nM to 1.2 microM. Perifusion of aequorin-loaded strips with beta-[asp1]-AII, an aminopeptidase-resistant analog of AII, allows the simultaneous measurement of [Ca2+]i and aldosterone production rate. Levels of agonist which generate a transient rise in [Ca2+]i also produce a sustained increase in aldosterone production rate, but the two events are temporally separated: the transient rise in [Ca2+]i precedes the increase in aldosterone production rate. However, there is a strong correlation, r = 0.94, between the amplitude of the initial [Ca2+]i transient and the magnitude of the sustained increase in steroid production rate.     

Synergistic stimulation of prolactin release by phorbol ester, A23187 and forskolin

The effects of 12-0-tetradecanoyl-phorbol-13-acetate (TPA), A23187, forskolin and thyrotropin-releasing hormone (TRH) on prolactin release from GH4C1 cells were compared. TPA caused a 2-fold release, maximum after 6 or more min, that was sustained for 30 min or more. A23187 caused only a small and variable response that peaked within 4 to 6 min. Combination of TPA and A23187 caused a rapid 3- to 5-fold increase in release that declined slowly. TRH increased prolactin release 3- to 5-fold, reaching a maximum within 4 min, followed by sustained release at lower rates. Forskolin had little effect by itself, but potentiated release caused either by combined TPA and A23187, or by TRH. These data are consistent with a model in which two branches of the Ca2+ messenger system participate in the action of TRH, a calmodulin branch and a C-kinase branch that interact to cause large amounts of sustained release. Forskolin, by regulating the cyclic AMP content of the cell determines the set point around which the Ca2+ messenger system operates.     

Angiotensin II effect on cytosolic pH in cultured rat vascular smooth muscle cells

This study investigated fluctuations of cytosolic pH (pHi) of cultured rat vascular smooth muscle cells (VSMCs) in reaction to metabolic alterations induced by angiotensin II (AII). Serially passed VSMCs from Wistar rat aortae were grown on coverslips and loaded with the pH-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. A biphasic reaction was seen after exposure of these cells to AII (1 nM to 1 microM); an initial and relatively brief phase of acidification was followed by sustained alkalinization. The rate of acidification and magnitude of alkalinization were dose-dependent. This biphasic effect of AII was also demonstrated in Ca2+-free medium and was mimicked by subjecting VSMCs to the calcium ionophore A23187 (5 microM) in Ca2+-containing medium but not in Ca2+-free medium. Verapamil (10 microM) almost entirely eliminated the AII-induced acidification, whereas amiloride analogues 5-(N-methyl-N-isobutyl)amiloride and 5-(N-ethyl-N-isopropyl)amiloride (100 microM) as well as Na+-deficient medium abolished the subsequent (alkalinization) phase produced by the hormone. Activation of the Na+/H+ antiport by subjecting VSMCs to phorbol 12-myristate 13-acetate (100 nM) prevented a subsequent effect of AII on the pHi profile. This resistance to a further action of the hormone was not mediated via cytoplasmic alkalinization. AII produced a dramatic redistribution in the cellular compartments of 45Ca2+ associated with accelerated 45Ca2+ washout. These findings suggest that the AII-induced acidification phase may relate to activation of the Ca2+ pump (Ca2+/H+ exchange) and that this process can take place in the presence and absence of extracellular Ca2+. The alkalinization phase is the consequence of stimulation of the Na+/H+ antiport, which in cultured VSMCs can be activated by a rise in cytosolic free Ca2+ as well as other mechanisms.     

Greater importance of Ca(2+)-calmodulin in maintenance of ang II- and K(+)-mediated aldosterone secretion: lesser role of protein kinase C.

In this study we have investigated various components of the stimulus-secretion coupling process leading to aldosterone secretion from the calf adrenal glomerulosa cells as evoked by angiotensin II (AII) and potassium (K+). The roles of Ca2+, calmodulin and protein kinase C in the sustained phase rather than initiation of aldosterone secretion were of special interest. Our investigations revealed that the reduction of extracellular Ca2+ by EGTA or interruption of Ca2+ influx by nitrendipine at various time points after stimulation with either AII or K+ markedly compromised aldosterone secretion. Calmodulin inhibitors, calmidazolium and W-7 reduced aldosterone secretion profoundly. Agonists of protein kinase C, phorbol ester or diacylglycerol analogues failed to stimulate aldosterone secretion while the protein kinase C inhibitor, H-7, only partially inhibited aldosterone secretion at a concentration which completely inhibited protein kinase C activity. Calmodulin inhibitors produced significantly greater inhibition of aldosterone secretion than inhibitors of protein kinase C.     

Insulin secretion: Combined tolbutamide, forskolin and TPA mimic action of glucose

We have proposed that the two phases of glucose-induced insulin secretion are regulated by two distinct branches of the calcium messenger system: the initial phase by a calmodulin branch, and the sustained phase by a C-kinase branch. To provide further support for this concept, we examined the separate and combined effects of tolbutamide, TPA, and forskolin upon insulin secretion from rat islets perifused in the absence of added fuels. Addition of 200 μM tolbutamide to the perifusate induces only a first phase of insulin secretion, addition of 200 nM TPA only a second phase, and addition of 10 μM forskolin only a small elevation in the basal rate of secretion. The combination of tolbutamide and TPA induces a biphasic secretory response qualitatively and quantitatively similar to that evoked by an increase in glucose concentration from 2.75 to 7 mM. The combination of TPA, tolbutamide, and forskolin evokes a biphasic pattern of insulin secretion qualitatively and quantitatively similar to that evoked by an increase in glucose concentration from 2.75 to 10 mM.     

Effects of an inhibitor of myosin light chain kinase on amylase secretion from rat pancreatic acini

Ca(2+)/calmodulin-dependent protein (CaM) kinases play an important role in Ca(2+)-mediated secretory mechanisms. Previously, we demonstrated that a CaM kinase II inhibitor KN-62 had a small inhibitory effect on amylase secretion stimulated by CCK. In the present study, we investigated the effects of a myosin light chain kinase (MLCK) inhibitor on amylase secretion and Ca(2+) signaling in rat pancreatic acini. A specific inhibitor of MLCK, wortmannin, inhibited amylase secretion stimulated by CCK-8 (30 pM) in a concentration-dependent manner. Wortmannin (10 microM) had no effects on basal secretion but reduced amylase secretion stimulated by CCK-8 (30 pM) by 67 +/- 3%. Wortmannin inhibited amylase secretion stimulated by calcium ionophore (A23187) and phorbol ester (TPA). Wortmannin also inhibited amylase response to thapsigargin by 76 +/- 8% and to both thapsigargin and TPA by 52 +/- 10%. Ca(2+) oscillations evoked by CCK-8 (10 pM) were inhibited by wortmannin (10 microM). Wortmannin had a little inhibitory effect on an initial rise in [Ca(2+)](i), and abolished a subsequent sustained elevation of [Ca(2+)](i) evoked by 1 nM CCK-8. In conclusion, MLCK plays a crucial role in amylase secretion from pancreatic acini and regulates Ca(2+) entry from the extracellular space.     

Role of calcium in histamine release from rat mast cells activated by various secretagogues; intracellular calcium mobilization correlates with histamine release

Anti-IgE, con A or antigen caused an increase in the intracellular calcium concentration, [Ca2+]i, of mast cells. The increase occurred in two stages: a rapid initial rise caused by Ca-mobilization from intracellular Ca-stores and a second sustained rise caused by an influx of extracellular calcium (White, J.R., Pluznik, D.V., Ishizaka, K. & Ishizaka, T. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 8193-8197). The rapid initial rise was followed by a release of histamine, which seemed to coincide with the second rise. A23187 and compound 48/80 induced a rapid initial rise in [Ca2+]i, followed by a gradual decrease in [Ca2+]i, GMCHA-OPhBut, a specific pH 7 tryptase inhibitor (Muramatu, M., Ito, T., Takei, M. & Endo, K. (1988) Biol. Chem. Hoppe-Seyler 369, 617-625), strongly inhibited both the initial and second rises of [Ca2+]i, as well as histamine release by these secretagogues, and its effects on the initial rise were closely correlated with those on histamine release. Addition of GMCHA-OPhBut immediately after the initial rise strongly inhibited the second rise, thereby decreasing the final [Ca2+]i. These results strongly suggested a possible involvement of pH 7 tryptase, not only in Ca-mobilization leading to the initial rise in [Ca2+]i, but also in the second rise. Trapping of extracellular calcium by 3mM EGTA decreased both the initial rise in [Ca2+]i and histamine secretion induced by anti-IgE or con A; the magnitude of this effect depended on the time between induction and EGTA addition. Histamine release was closely correlated with the initial rise in [Ca2+]i. Similar results were obtained with A23187, but even 5 min after the addition of EGTA an initial rise of [Ca2+]i could still be induced, and histamine (30% of total histamine) was still released. However, A23187 did not induce a rise in [Ca2+]i in mast cells which had been exhaustively washed with Tyrode/Hepes solution containing 3mM EGTA, followed by suspension in the same solution. Even at 20 min after depletion of the extracellular calcium, compound 48/80 still caused an initial rise in [Ca2+]i to above half the maximal value, and histamine secretion was even less affected. The above results indicated that the initial rise in [Ca2+]i, due to Ca-mobilization, correlates with the histamine release promoted by the secretagogues described. On the other hand, isoproterenol strongly induced histamine secretion with no change of [Ca2+]i, while EGTA treatment prior to isoproterenol stimulation had no effect on histamine release, indicating a different secretion mechanism.     

Epidermal growth factor (EGF), tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) and calcium ionophore A23187 increase cytoplasmic free calcium and stimulate arachidonic acid release and PGE2/6-keto PGF1 alpha production in cultured porcine thyroid cells

Epidermal growth factor (EGF), 12-O-tetradecanoylphorbol 13-acetate (TPA) and calcium ionophore A23187 increase cytoplasmic free calcium ([Ca2+]i) and stimulate arachidonic acid release and production of PGE2 and 6-keto PGF1 alpha, an end metabolite of PGI2, in cultured porcine thyroid cells. Addition of EGF, TPA or A23187 to the cells loaded with fura-2, a fluorescent Ca2+ indicator, causes an immediate increase in [Ca2+]i, which is the earliest event after mitogen stimulation. This [Ca2+]i response occurs immediately, reaching a maximum within several seconds. EGF, TPA and A23187 stimulate arachidonic acid release and PGE2 and 6-keto PGF1 alpha production; the maximum effects are obtained after 2-4 h incubation. EGF, TPA and A23187 increase [Ca2+]i and then stimulate arachidonic acid release and PG production.     

Quantitative analysis of the cytosolic-free-Ca2+-dependency of aldosterone production in bovine adrenal glomerulosa cells. Different requirements for angiotensin II and K+.

Angiotensin II (AII) and K+ raise the cytosolic free Ca2+ concentration [( Ca2+]i) and stimulate aldosterone production in isolated bovine adrenal glomerulosa cells. The mechanisms leading to an elevation of [Ca2+]i were analysed with the fluorescent Ca2+ probe quin 2. (1) Whereas [Ca2+]i rose transiently and returned to basal values within 5 min in response to AII, the effect of K+ was sustained for at least 15 min. (2) AII released Ca2+ from intracellular stores, whereas the [Ca2+]i response to K+ depended solely on extracellular [Ca2+]. (3) When added after K+ stimulation, AII provoked a dramatic decrease in [Ca2+]i to below the resting value. The role of [Ca2+]i in stimulating steroidogenesis was determined by manipulating the concentration of this cation. (4) In a cell superfusion system, the aldosterone response to AII is biphasic. Suppressing the transient [Ca2+]i elevation triggered by AII resulted in the disappearance of the initial secretory peak, but the final production rate was similar to that of control cells. (5) Normal basal [Ca2+]i levels were, however, necessary to maintain continuous AII-induced steroidogenesis. (6) When added after AII, the antagonist analogue [Sar1,Ala8]AII suppressed steroidogenesis without affecting [Ca2+]i levels. (7) In contrast, continuously elevated [Ca2+]i values were required for the initiation and the maintenance of K+-stimulated aldosterone production. These results demonstrate important differences in the mechanisms through which AII and K+ activate the Ca2+ messenger system. Moreover, functional correlations have shown that K+, but not AII, depends solely on a sustained [Ca2+]i response for its steroidogenic effect. However, the AII-induced effect is also a Ca2+-requiring process: the initial [Ca2+]i transient accelerates the onset of steroidogenesis, which is subsequently extremely sensitive to [Ca2+]i decreases below normal basal levels.     

An acute release of Ca2+ from sequestered intracellular pools is not the primary transduction mechanism causing the initial burst of PRL and TSH secretion induced by TRH in normal rat pituitary cells.

With 1.5 mM [Ca2+]e, 10 nM TRH induced a prompt high-amplitude burst of hormone secretion and an initial high-amplitude [Ca2+]i burst (first phase) followed by a sustained low-amplitude [Ca2+]i increment (second phase) in both tumor-derived GH4C1 and normal adenohypophyseal (AP) cells. With less than 2 microM [Ca2+]e, in both cell types the TRH-induced first phase rise in [Ca2+]i was suppressed 30% while the second phase rise was completely abolished; however, hormone secretion was inhibited only 20-30% in GH4C1 but greater than 80% in AP cells. Thapsigargin induced a first-phase rise in [Ca2+]i in AP cells equal to that induced by 10 nM TRH but only 20% as much first-phase hormone secretion. Blocking Ca2+ channels with nifedipine inhibited TRH-induced secretion in AP cells significantly more than in GH4C1 cells. Our data indicate that the TRH-induced first-phase spike in [Ca2+]i from intracellular Ca2+ stores may play a major transduction role in hormone secretion in GH4C1 cells but not in normal AP cells. Transduction mechanisms coupled to Ca2+ influx through Ca2+ channels in the plasmalemma are apparently a much more important component of TRH-induced secretion in normal than in tumor-derived pituitary cells.     

Tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) can reduce the Ca-transporting ability of Ca-ionophores in T lymphocytes: the involvement of intracellular heavy metal ions

12-0-tetradecanoyl-phorbol-13-acetate (TPA) can significantly reduce the Ca-ionophore-induced rise in the intracellular calcium concentration (Cai) of T lymphocytes measured by quin2 or fura-2 fluorescence. This counteraction of TPA is maximal at a preincubation of 90 min at TPA concentrations higher than 20 nM. 45Ca uptake and efflux measurements directly indicate that TPA does not activate the calcium extrusion systems in thymocytes but impairs the Ca-transporting ability of Ca-ionophores. TPA causes no immobilization of the Ca-ionophores as it is demonstrated by the lack of significant changes in fluorescence and fluorescence polarisation of A23187 during TPA incubation. Similarly the energy transfer between the Tyr, Try groups of membrane proteins and A23187 shows no significant difference in control and TPA treated thymocytes. This indicates that A23187 is not in a membrane protein-bound form after TPA preincubation. The intracellular heavy metal chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) restores the ionophoretic ability of Ca-ionophores in TPA pretreated cells to the control level. Diacyl-glycerols also impair the Ca-transporting ability of Ca-ionophores. TPEN prevents this effect as well. These findings suggest that TPA and diacyl-glycerols may cause an increase in the availability of intracellular heavy metal ions. Our results may reflect a new, physiologically important mechanism of the action of diacyl-glycerols and phorbol esters.     

Atrial natriuretic factor inhibits angiotensin-induced aldosterone secretion: not through cGMP or interference with phospholipase C

ANF did not prevent the formation of [3H] inositol trisphosphate in response to AII but inhibited aldosterone secretion in calf adrenal glomerulosa cells. 8-bromo cGMP did not affect either inositol phosphate formation or aldosterone secretion. Changes in cytosolic Ca++ concentration induced by AII, as measured by Quin 2 fluorescence, were also unaffected by ANF. No difference in adrenal cell protein phosphorylation with AII or AII + ANF was observed. The results suggest that ANF may inhibit aldosterone secretion through a non-guanyl cyclase linked receptor system not involving the formation of phosphoinositide-derived second messengers. Interference with protein kinase C activity cannot be ruled out.     

Cholinergic desensitization of pepsinogen secretion and calcium mobilization of dispersed guinea pig chief cells

When dispersed chief cells from guinea pig stomach were first incubated with carbachol, washed, and then reincubated with carbachol in fresh incubation solution, the stimulation of pepsinogen secretion and the rise in intracellular calcium concentration during the second incubation were reduced. Carbachol did not cause residual enzyme secretion, but the same range of concentrations that causes enzyme secretion caused desensitization that was rapid, temperature dependent, and reversible with time. Preincubation with carbachol caused approximately a 65% reduction in enzyme secretion stimulated during a subsequent incubation with this agonist, but the potency of carbachol was unaffected. Prior exposure to carbachol also reduced subsequent stimulation caused by cholecystokinin (CCK-8), gastrin I, ionophore A23187, or 12-O-tetradecanoylphorbol 13-acetate but did not alter stimulation by any agonist that increases cellular cAMP. Carbachol pretreatment of Fura-loaded chief cells caused a threefold increase in the EC50 for carbachol-stimulated [Ca2+]i and approximately a 30% reduction in the maximal rise in [Ca2+]i in response to carbachol or CCK-8. Inhibition of [N-methyl-3H] scopolamine binding by carbachol following carbachol pretreatment indicated that modulation of receptor affinity or number did not account for functional desensitization. These data indicate that carbachol causes heterologous desensitization of pepsinogen secretion stimulated by agonists that mobilize cellular Ca2+ or activate protein kinase C through a postreceptor action and suggest that an attenuated rise in chief cell calcium is one mechanism mediating the desensitization of enzyme secretion.     

Phorbol ester inhibits angiotensin-induced activation of phospholipase C in adrenal glomerulosa cells. Its implication in the sustained action of angiotensin.

The present study was undertaken to determine whether an agonist-induced activation of C-kinase leads to an inhibition of phospholipase C in adrenal glomerulosa cells. When cells are treated with 100 nM-TPA (12-O-tetradecanoylphorbol 13-acetate), subsequent angiotensin ('angiotensin II')-induced aldosterone secretion is greatly inhibited. Treatment with TPA completely inhibits the angiotensin-induced increase in both inositol trisphosphate and the cytosolic Ca2+ concentration. The dose-response curve for TPA-induced inhibition reveals that quite a high concentration of TPA is necessary to block angiotensin action compared with that needed to stimulate aldosterone secretion. 1-Oleoyl-2-acetylglycerol has a weak inhibitory effect, whereas neither 4 alpha-phorbol 12,13-didecanoate or 4 beta-phorbol inhibits angiotensin action. When the time course of changes in inositol trisphosphate and diacylglycerol is measured, angiotensin action is sustained for up to 30 min. In addition, 100 nM-TPA added after 20 min of angiotensin addition attenuates production of both inositol trisphosphate and diacylglycerol. These results suggest that high dose of TPA inhibits angiotensin-induced activation of phospholipase C by acting, at least partly, on C-kinase, but that an inhibitory effect of TPA may be a pharmacological effect with little physiological significance in this system.