Effect of cytoplasmic Ca2+ on (1,4,5)IP3 formation in vasopressin-activated hepatocytes.

The role of cytoplasmic calcium as a regulator of phospholipase C in vasopressin-activated hepatocytes was examined. According to models in which calcium spiking arises because of a positive feedback by calcium on phospholipase C, Ca2+ is seen as a positive modulator of phospholipase C under conditions of submaximal receptor activation. However, in hepatocytes whose precursor lipids had been labeled by incubation in [3H]-inositol, no increase in [3H]-(1,4,5)IP3 was detected in response to thapsigargin, in either unstimulated cells, or in cells stimulated with 1 nM vasopressin. Addition of a maximal concentration of vasopressin (1 microM) caused a rapid and substantial increase in [3H]-(1,4,5)IP3. These results indicate that changes in cytoplasmic calcium do not influence phospholipase C activity in hepatocytes, even under conditions of submaximal agonist activation. These findings also support models that provide for calcium spiking at constant levels of (1,4,5)IP3 at least in the case of the rat hepatocyte.

Role of cyclic GMP in the control of capacitative Ca2+ entry in rat pancreatic acinar cells.

We have investigated the possible roles of cyclic GMP (cGMP) in initiating or regulating capacitiative Ca2+ entry in rat pancreatic acinar cells. In medium containing 1.8 mM external Ca2+, thapsigargin activated Ca2+ entry and slightly but significantly increased intracellular cGMP concentration. This rise in cGMP levels was prevented by pretreating the cells with the guanylate cyclase inhibitor, LY-83583, or by omitting Ca2+ during stimulation by thapsigargin or methacholine. LY-83583 and NG-nitro-L-arginine (L-NA, an inhibitor of NO synthase) both had a small inhibitory effect on Ca2+ entry when they were added after thapsigargin in Ca2(+)-containing medium, and they reduced by 32 and 48% respectively the thapsigargin-induced capacitative Ca2+ entry when added to the cells during a 20 min preincubation period. However, neither dibutyryl cGMP (Bt2cGMP) nor sodium nitroprusside, an NO mimic, affected either basal intracellular Ca2+ concentration [Ca2+]i or thapsigargin-induced capacitative Ca2+ entry. Further, the inhibitory effects observed after preincubation with LY-83583 or L-NA could not be prevented by preincubation with Bt2cGMP, nor could they be reversed by adding Bt2cGMP, 8-bromo-cGMP or sodium nitroprusside acutely after activation of capacitative Ca2+ entry by thapsigargin. Finally, pretreatment of cells with LY-83583 or L-NA did not affect Ca2+ signalling in response to 1 microM methacholine, including the pattern of [Ca2+]i oscillations. In conclusion, in pancreatic acinar cells, the rise in cellular cGMP levels appears to depend on, rather than cause, the increase in [Ca2+]i with agonist stimulation.

Sinusoidal oscillations in intracellular calcium requiring negative feedback by protein kinase C.

Stimulation of mouse lacrimal acinar cells with submaximal concentrations of the muscarinic agonist, methacholine, resulted in an increase in intracellular calcium ([Ca2+]i), which took the form of sinusoidal oscillations. These oscillations were relatively constant (approximately 4-5/min) regardless of the methacholine concentration, suggesting that the oscillations arise from an oscillating negative feedback in the signal transduction pathway. This negative feedback appears to involve oscillations in protein kinase C activity because the oscillations were prevented by activation, inhibition, or down-regulation of protein C. Activation of protein kinase C with phorbol esters inhibited the methacholine-induced [Ca2+]i signal and formation of the Ca2+ mobilizing messenger, inositol 1,4,5-trisphosphate. [Ca2+]i signals elicited by intracellular introduction of inositol phosphates did not oscillate and were not affected by activators or inhibitors of protein kinase C. Thus, the constant frequency [Ca2+]i oscillations appear to result from a negative feedback loop involving inhibition of inositol trisphosphate production by protein kinase C.

The interconversion of inositol 1,3,4,5,6-pentakisphosphate and inositol tetrakisphosphates in AR4-2J cells.

Data from several cell types have indicated that activation of hormone receptors promotes the metabolism of inositol 1,3,4,5,6-pentakisphosphate (IP5) to inositol 3,4,5,6-tetrakisphosphate ((3,4,5,6)IP4). However, to date, metabolism of IP5 by cell-free preparations has resulted in the formation of only inositol 1,4,5,6-tetrakisphosphate ((1,4,5,6)IP4). Thus, the metabolic relationships of IP5 with various inositol tetrakisphosphate (IP4) isomers have been investigated in both intact cells and cell homogenates of the rat pancreatoma cell line, AR4-2J. The steady-state concentration of IP5 was estimated to be 65 microM, while the combined concentration of (3,4,5,6)IP4 and (1,4,5,6)IP4 was approximately 1.0 microM. AR4-2J cell homogenates converted (1,3,4,6)IP4, (3,4,5,6)IP4, and (1,4,5,6)IP4 to IP5. (1,4,5,6)IP4 previously has not been demonstrated to be a precursor of IP5. To alter steady-state levels of inositol phosphates that were maintained by phosphorylation-dephosphorylation cycles, intact cells were treated with 10 microM antimycin A which reduced ATP levels by > 90% within 10 min. Following 2 h of treatment with antimycin A, there was a 6-fold increase in both (3,4,5,6)IP4 and (1,4,5,6)IP4, presumably derived from IP5. Experiments with cell-free systems determined that IP5 was dephosphorylated to (1,4,5,6)IP4 by a predominantly particulate Mg(2+)-independent, Li(+)-insensitive IP5 3-phosphatase. However, in the presence of 5 mM MgATP, IP5 also was metabolized to (3,4,5,6)IP4. Therefore, our data demonstrate novel and complex relationships between IP5, (3,4,5,6)IP4, and (1,4,5,6)IP4.

Functional homogeneity of the non-mitochondrial Ca2+ pool in intact mouse lacrimal acinar cells.

In the absence of extracellular Ca2+, treatment of mouse lacrimal acinar cells with maximal concentrations of methacholine released Ca2+ from intracellular stores. No additional Ca2+ was mobilized by subsequent application of the intracellular Ca(2+)-ATPase inhibitor, thapsigargin, the stable inositol 1,4,5-trisphosphate ((1,4,5)IP3) analog, inositol 2,4,5-trisphosphate ((2,4,5)IP3) (by microinjection), or the Ca2+ ionophore, ionomycin. However, following prolonged activation of cells by methacholine in the presence of extracellular Ca2+, Ca2+ accumulated into a pool which was released by ionomycin but not by thapsigargin. This latter accumulation was blocked by prior microinjection of ruthenium red, indicating that it represents mitochondrial uptake. In saponin-permeabilized lacrimal cells, two Ca(2+)-sequestering pools were detected: (i) a ruthenium red-sensitive, thapsigargin-insensitive pool, presumed to be the mitochondria; and (ii) a ruthenium red-insensitive, thapsigargin-sensitive pool. Only the thapsigargin-sensitive pool accumulated Ca2+ at concentrations similar to those in unstimulated cells. The thapsigargin-sensitive Ca2+ pool was sensitive to (1,4,5)IP3; however, in contrast to findings in intact cells, only 44% of this pool was releasable by (1,4,5)IP3 or (2,4,5)IP3. These data indicate that, in intact lacrimal acinar cells, all exchangeable (ionomycin-sensitive) Ca2+ residues in a pool which responds homogeneously to agonists, (1,4,5)IP3, and thapsigargin. Prolonged elevation of [Ca2+]i results in Ca2+ accumulation into a second, ruthenium red-sensitive pool, presumably mitochondria. Finally, permeabilization of the cells fragments the non-mitochondrial pool, resulting in two pools, one sensitive and one insensitive to (1,4,5)IP3.

Sustained Ca2+ signaling in mouse lacrimal acinar cells due to photolysis of "caged" glycerophosphoryl-myo-inositol 4,5-bisphosphate.

In saponin-permeabilized mouse lacrimal acinar cells, glycerophosphoryl-myo-inositol 4,5-bisphosphate (GPIP2) activated the release of sequestered Ca2+ to the same extent as inositol 1,4,5-trisphosphate ((1,4,5)IP3) but with a potency about 1/10 that of (1,4,5)IP3. In lacrimal gland homogenates, [3H]GPIP2 was metabolized to two compounds which upon anion exchange high performance liquid chromatography eluted at positions indicating that they were [3H]GPIP and [3H]GPIP3. The rate of metabolism of [3H]GPIP2 was much slower than that of [3H](1,4,5)IP3, and its rate of phosphorylation was less than 1% of that of [3H] (1,4,5)IP3. In intact lacrimal cells, photolysis of a microinjected "caged" derivative of GPIP2, 1-(alpha-glycerophosphoryl)-myo-inositol 4,5-bisphosphate P4(5)-1-(2-nitrophenyl)ethyl ester, resulted in sustained activation of Ca2+ signaling; i.e. intracellular Ca2+ release followed by increased entry of Ca2+ across the plasma membrane. These findings indicate that caged GPIP2 should provide a useful tool for producing photolytically initiated, sustained activation of intracellular (1,4,5)IP3 receptors. They also provide strong support for the idea that sustained Ca2+ signaling can be achieved in lacrimal acinar cells by activation of intracellular receptors for (1,4,5)IP3 in the absence of stimulated production of inositol 1,3,4,5-tetrakisphosphate.

Role of inositol (1,4,5)trisphosphate in epidermal growth factor-induced Ca2+ signaling in A431 cells.

The effects of epidermal growth factor on Ca2+ signaling in A431 cells were investigated. Epidermal growth factor induced a transient Ca2+ signal in the absence of external Ca2+ and a sustained response in the presence of extracellular Ca2+, indicating an ability to mobilize intracellular Ca2+ as well as the ability to increase Ca2+ entry from the extracellular space. The Ca(2+)-ATPase inhibitor thapsigargin also activated Ca2+ entry, and neither epidermal growth factor nor the guanine nucleotide-dependent protein-linked receptor agonist bradykinin activated additional Ca2+ entry over that due to thapsigargin. In nominally Ca(2+)-free medium, the addition of bradykinin to A431 cells rapidly but transiently increased inositol 1,4,5-trisphosphate and, in parallel fashion, transiently increased cytosolic Ca2+. Unexpectedly, under these experimental conditions, epidermal growth factor elicited a small but significant Ca2+ signal after the addition of bradykinin. Experiments were designed to determine whether the Ca2+ response to epidermal growth factor after bradykinin results from mobilization of Ca2+ by an inositol 1,4,5-trisphosphate-independent mechanism. Epidermal growth factor stimulated additional inositol 1,4,5-trisphosphate formation in bradykinin-treated cells. Furthermore, the Ca2+ signals elicited by both bradykinin and epidermal growth factor were blocked in cells microinjected with the inositol 1,4,5-trisphosphate receptor antagonist heparin, whereas the intracellular Ca(2+)-ATPase inhibitor thapsigargin still mobilized Ca2+. Finally, histamine, a less efficacious guanine nucleotide-dependent protein-linked receptor agonist, as well as photolyzed, microinjected, caged inositol 1,4,5-trisphosphate, also mobilized Ca2+ after bradykinin. The results of this study show (i) that epidermal growth factor activates intracellular Ca2+ release as well as Ca2+ entry, the latter most likely resulting from an indirect effect due to the depletion of intracellular Ca2+ pools, (ii) that the actions of epidermal growth factor on Ca2+ homeostasis can be fully accounted for by inositol 1,4,5-trisphosphate formation, and (iii) that the ability of A431 cells to produce Ca2+ signals when epidermal growth factor is applied after bradykinin can be explained by the rapid and complete desensitization of the bradykinin stimulated phospholipase C activity.

The mechanism for synergism between phospholipase C- and adenylylcyclase-linked hormones in liver. Cyclic AMP-dependent kinase augments inositol trisphosphate-mediated Ca2+ mobilization without increasing the cellular levels of inositol polyphosphates.

The ability of cAMP-dependent hormones to modulate the actions of Ca2(+)-mobilizing hormones was studied in single fura-2-injected guinea pig hepatocytes. In 91% of cells the cAMP-linked hormone, isoproterenol, applied alone, did not alter cytosolic Ca2+ concentration. In 78% of cells which had been pre-exposed to a low concentration of angiotensin II, isoproterenol was able to increase cytosolic Ca2+. Isoproterenol did not, however, increase inositol 1,4,5-trisphosphate or inositol tetrakisphosphate on its own, or in the presence of angiotensin II. Isoproterenol was also able to raise cytosolic Ca2+ concentration in cells microinjected with inositol 2,4,5-trisphosphate or a photoactivatable derivative of inositol 1,4,5-trisphosphate. The elevation of cytosolic Ca2+ concentration induced by isoproterenol in angiotensin II-treated cells and cells injected with caged inositol 1,4,5-trisphosphate was blocked by heparin, implying that the effect was mediated by an inositol 1,4,5-trisphosphate receptor agonist. In permeabilized hepatocytes, inositol 1,4,5-trisphosphate-induced Ca2+ release was enhanced by 8-bromo-cAMP and the catalytic subunit of cAMP-dependent kinase. Cyclic AMP-dependent kinase shifted the dose-response curve for inositol 1,4,5-trisphosphate-mediated Ca2+ release to the left by a factor of 4 and increased the total amount of Ca2+ released by 25%. These results indicate that increased sensitivity of the intracellular Ca2+ releasing organelle to inositol 1,4,5-trisphosphate is responsible for synergism between phospholipase C- and adenylylcyclase-linked hormones in the liver.

Origins of myo-inositol tetrakisphosphates in agonist-stimulated rat pancreatoma cells. Stimulation by bombesin of myo-inositol 1,3,4,5,6-pentakisphosphate breakdown to myo-inositol 3,4,5,6-tetrakisphosphate.

In the rat pancreatoma cell line, AR4-2J, three inositol tetrakisphosphate isomers were identified, (1,3,4,6), (1,3,4,5), (3,4,5,6), which were increased during activation of phospholipase C by bombesin. Two other isomers were identified, (1,4,5,6) and a fifth isomer which was either (1,2,3,4) or (1,2,3,6), which have not previously been detected in any cell type. To study the metabolic interrelationships between these compounds and inositol 1,3,4,5,6-pentakisphosphate in the intact cell, their turnover was assessed under different protocols of [3H]myo-inositol labeling; the inositol phosphates were labeled to near steady state or under conditions where either rapidly or slowly turning over inositol polyphosphates were preferentially labeled. The relative specific radioactivities of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, inositol 1,3,4-trisphosphate, and inositol 1,3,4,6-tetrakisphosphate were very similar in bombesin-stimulated cells, consistent with the pathway for the conversion of inositol 1,4,5-trisphosphate to the other three inositol polyphosphates. Compared with these inositol phosphates, the turnover of inositol 1,3,4,5,6-pentakisphosphate was slow. An accumulation of radioactivity into inositol 1,3,4,5,6-pentakisphosphate was observed only under labeling conditions where its relative specific radioactivity was substantially below that of inositol 1,3,4,6-tetrakisphosphate. This indicated that the precursor for de novo synthesis of inositol 1,3,4,5,6-pentakisphosphate was inositol 1,3,4,6-tetrakisphosphate. Bombesin stimulated the net breakdown of inositol 1,3,4,5,6-pentakisphosphate and increased the level of inositol 3,4,5,6-tetrakisphosphate; the relative specific radioactivities of these two compounds were similar under all conditions. These data led to the novel proposal that inositol 3,4,5,6-tetrakisphosphate is the product of inositol 1,3,4,5,6-pentakisphosphate breakdown. This reaction was apparently stimulated by a regulated change in the enzyme(s) which interconvert inositol 1,3,4,5,6-pentakisphosphate and inositol 3,4,5,6-tetrakisphosphate.

Effects of MeCh, thapsigargin, and La3+ on plasmalemmal and intracellular Ca2+ transport in lacrimal acinar cells.

The Ca2(+)-mobilizing actions of the muscarinic receptor agonist, methacholine (MeCh), and the microsomal Ca2+ pump inhibitor, thapsigargin, were investigated in lacrimal acinar cells. As previously shown for parotid cells (J. Biol. Chem. 264: 12266-12271, 1989), thapsigargin activates both internal Ca2+ release and Ca2+ entry from the extracellular space without increasing cellular inositol phosphates. The inorganic Ca2+ antagonist La3+ inhibited MeCh- or thapsigargin-activated Ca2+ entry. However, when added before MeCh or thapsigargin, La3+ inhibited the extrusion of Ca2+ at the plasma membrane. This phenomenon was exploited in protocols designed to investigate the pathways for filling agonist-sensitive Ca2+ stores in lacrimal cells. The results show that, in contrast to previous suggestions that external Ca2+ is required to replenish agonist-regulated Ca2+ stores, the inhibition of Ca2+ extrusion permits recycling of Ca2+ released by MeCh back into an MeCh- and thapsigargin-sensitive pool. Thus, although extracellular Ca2+ is the major source for refilling the intracellular Ca2+ stores under physiological conditions, the pathway by which this Ca2+ enters the pool need not be a direct one. These results are consistent with the recently revised capacitative model for the refilling of intracellular Ca2+ stores through Ca2+ influx subsequent to Ca2+ depletion, according to which refilling of intracellular Ca2+ stores occurs via a cytoplasmic route rather than a direct channel between intracellular Ca2+ stores and the extracellular space.

Two modes of regulation of the phospholipase C-linked substance-P receptor in rat parotid acinar cells.

In rat parotid acinar cells prelabelled with [3H]inositol, substance P (100 nM) induced the formation of [3H]inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Ins(1,4,5)P3 reached a maximum 7 s after substance P stimulation, and thereafter decreased and reached a stable value at 60 s. When the cells were exposed to substance P for 10, 30, 60, or 300 s, washed, and re-exposed to this peptide, the formation of [3H]inositol trisphosphate (InsP3) was attenuated in a time-dependent manner. In the cells pretreated as described above, the number of [3H]substance-P-binding sites (Bmax) was also decreased. Possible role(s) of Ca2+ and protein kinase (protein kinase C) control mechanisms in regulating substance P responses were investigated. Desensitization of substance P-induced InsP3 was not affected by the Ca2+ ionophore ionomycin, nor was it dependent on Ca2+ mobilization. On the other hand, in the presence of 4 beta-phorbol 12,13-dibutyrate (PDBu) and 12-O-tetradecanoyl-4 beta-phorbol 13-acetate, known activators of protein kinase C, substance P-induced InsP3 formation was inhibited. However, PDBu had no effect on [3H]substance P binding, whether present during the assay or when cells were pretreated. The persistent desensitization of InsP3 formation induced by substance P was not affected by PDBu. These results suggest that the persistent desensitization of InsP3 formation induced by substance P is a homologous process involving down-regulation of the substance P receptor; the mechanism does not appear to involve, or to be affected by, the Ca2+ or protein kinase C signalling systems. Protein kinase C activation can, however, inhibit substance P-induced InsP3 formation, which may indicate the presence of a negative-feedback control on the substance P pathway.

Secretion of calcitonin in the genetically obese Zucker rat (fa/fa).

Previously we found that adult Zucker fatty rats have C-cell hyperplasia and increased thyroidal calcitonin (CT) compared to lean controls. In this study we have evaluated both secretion of CT and responsiveness to CT in order to see whether they, too, were altered. Fat rats and lean littermates, 13-15 months old, were used. CT secretion was provoked by (1) feeding for 2 hr after an 18-hr fast, (2) giving pentagastrin iv, and (3) injecting CaCl2 iv. CT was measured by radioimmunoassay. Responsiveness to CT was examined by giving porcine or salmon CT iv and measuring serum Ca 1-3 hr later. For CT secretion, compared to leans the fat rats showed (1) higher fasting serum Ca and CT and a greater rise in CT after feeding, (2) a similar 5- to 10-fold increase in CT after iv pentagastrin, and (3) a greater rise in both serum Ca and CT at various times between 5 min and 3 hr after iv CaCl2. For CT responsiveness, fat and lean rats were equally responsive to iv CT in terms of the fall in plasma Ca 1-3 hr later. The results show that fat rats can secrete as much or more CT in response to provocative stimuli as lean rats and that they appear normally responsive to injected CT. Therefore, inability to release CT and insensitivity to CT do not underly the C-cell hyperplasia, increased thyroidal CT, and increased circulating CT in the fat rat.

Ability of calcitonins to alter food and water consumption in the rat.

Synthetic salmon calcitonin (sCT) given SC (30 MRC U/kg) or ICV (1.4 U, 300 ng) inhibited 24-hr food consumption in the rat by 50-100%. Furthermore, ICV doses of sCT ranging from 75 ng (0.35 U) to 300 ng (1.4 U) lowered both food and water intake in a dose-dependent manner. Use of various forms of CT give an apparent order of potency of salmon greater than porcine greater than or equal to human with sCT being at least 40 fold more potent than the mammalian forms. Measurement of food and water intake over a brief (30-90 min) period showed that ICV sCT was effective in reducing food and water consumption regardless of whether it had been given 1, 12, or 23 hr previously. Daily administration of sCT for 5 days caused marked suppression of food and water intake for 2 days; thereafter, consumption returned toward normal, becoming equivalent to vehicle injected controls by day 3 or 4 despite continued daily injections of sCT. The results show that CT can act centrally to modify certain types of behavior and are of special interest since CT-like peptides have been described in the pituitary and hypothalamus and since CT receptors have been reported in hypothalamic and other brain regions.

Stimulation of in vitro release of calcitonin from rat and human C-cells by cimetidine.

Previous reports have suggested that the H2-receptor blocker, cimetidine, can inhibit parathyroid hormone (PTH) release. The present studies were designed in an attempt to see whether cimetidine affects secretion of calcitonin (CT) as well. Entire thyroparathyroid glands from 8-day-old baby rats or small pieces of a human medullary thyroid carcinoma were incubated at 37 C for up to 8 h in chemically defined culture medium gassed with 95% 02-5% CO2. With rat thyroparathyroids, both CT and PTH released into medium were measured using RIAs that detect that rat hormones. CT secreted from human C-cells was measured using an RIA for human CT. The results were: (A) As we had found previously, cimetidine at doses of 3 x 10(-3) M and 9 x 10(-3) M inhibited PTH release from rat thyroparathyroids by as much as 50-60% (p less than 0.05 - less than 0.001). In these same experiments, cimetidine produced increases of approximately 1- to 5-fold in the CT levels found in the medium (p less than 0.05 - less than 0.001). (B) Fragments of human medullary thyroid carcinoma incubated for 3 h in the presence of 10(-5) M cimetidine released considerably more CT than tissue incubated in control medium (48 +/- 9.3 vs 21 +/- 3.6 ng CT/microgram tissue protein, p less than 0.05). Our results show that cimetidine in vitro not only can inhibit secretion of PTH but also apparently can promote the release of CT from both rat and human C-cells. Whether this effect is mediated by H2 receptors and whether it is of any physiological significance awaits clarification.

Calcitonin-like immunoreactivity in rat and human pituitary glands: histochemical, in vitro, and in vivo studies.

This study was designed to determine whether pituitary glands contain an immunoreactive material which reacts with antisera to calcitonin (CT) and, if so, whether secretion of the material could be demonstrated. Testing 15 antisera to rat and human CT and using an immunoperoxidase method, we found 2 antisera to human CT which stained rat pituitaries and several which stained human pituitaries. Essentially all cells in the rat intermediate lobe and scattered cells in the rat and human anterior lobes showed staining, and staining was not entirely abolished by prior adsorption of antisera with rat or human CT. The 2 antisera which stained rat pituitaries showed cross-reactivity with several synthetic human CT fragments (1-18, 11-23 and 22-32) but not with ACTH-(1-39), ACTH-(1-24), beta-endorphin, alpha- or beta MSH, or bovine lipotropin. Crude extracts of pituitaries from 2 strains of young rats showed CT-like immunoreactivity which could be measured easily by RIA (0.2-0.3 ng/gland). In vivo, an antiserum which stained pituitaries and 1 which did not were compared using young rats made hypercalcemic (15-20 mg/dl) with iv Ca. In rats with thyroids, both antisera showed an increase in serum CT of more than 15-fold whether the pituitary was present or absent. In thyroidectomized rats, serum CT remained undetectable (less than 50 to 100 pg/ml) during hypercalcemia even if the pituitary was present. In vitro, rat pituitaries in a serum-free medium did not release measurable amounts of immunoreactive CT-like material even when medium contained high Ca (2.5 mM), high K (25 mM), or TRH (10(-6) M). Therefore, the findings agree with other reports of a CT-like material in the pituitary, but no secretion of the material could be demonstrated. We hypothesize that the material is not authentic CT but is, rather a related peptide sequence probably contained in the 31 K precursor protein of ACTH-beta-lipotropin.