Regulation of changes in cytosolic Ca2+ and Na+ concentrations in rat submandibular gland acini exposed to carbachol and ATP.

The relationship between cytosolic concentrations of Ca2+ (Ca2i) and Na+ (Na+i) were studied in preparations of rat submandibular and pancreatic acini loaded with the Ca(2+)-sensitive dye Fura-2 or the Na(+)-sensitive dye SBFI. Pancreatic acini showed no changes in Na+i during either transient or persistent changes in Ca2+i. Increases in Ca2+i produced by exposure of submandibular gland acini to carbachol, a muscarinic cholinergic agonist, were followed by an increase in Na+i after a delay of 5-10 s. When Ca2+ stores were mobilized without Ca2+ influx Na+i also increased, but in acini loaded with BAPTA, a nonfluorescent Ca2+ chelator, the transient increase in Ca2+ caused by mobilization of stored Ca2+ was virtually abolished, as was the increase in Na+i. In the presence of inomycin, increases in Ca2+i were followed by increases in Na+i. Ca(2+)-dependent increases in Na+i were abolished in Na(+)-free buffer and by the presence of furosemide, a blocker of Na(+)-K(+)-2Cl- cotransport. In other studies, extracellular ATP (ATPo) produced an increase in Ca2+i and Na+i. The steady-state increase in Ca(i)2+ was reduced by increasing extracellular Na+ concentrations (Na+o in dose-dependent fashion (IC50 = 16.4 +/- 4.7 mM Na+). Likewise, increasing Na+o reduced ATPo-stimulated 45Ca2+ uptake at steady state (IC50 = 15.8 +/- 9.2 mM Na+). Changing Na+o had no effect on carbachol-stimulated increases in Ca2+i. We conclude that, in rat submandibular gland acini, ATPo promotes an increase in Ca2+i and Na+i via a common influx pathway and that, under physiologic conditions, Na+ significantly limits the ATPo-stimulated increase in Ca2+i. In the presence of carbachol, however, Na+i rises in Ca2+i-dependent fashion in submandibular gland acini via stimulation of Na(+)-K(+)-2Cl- cotransport.

Mobilization of Ca2+ influx, but not of stored Ca2+, by extracellular ATP in rat submandibular gland acini.

To determine its effect on cytosolic Ca2+ levels (Ca2+i) and to assess its role as an autonomic cotransmitter, ATP was added to suspensions of rat pancreatic or submandibular gland acini loaded with fura-2. ATP had no effect on pancreatic acinar Ca2+i at either high (2.5 mM) or low (< 10 nM) extracellular Ca2+ concentrations (Ca2+o). In submandibular acini, ATP had little effect on Ca2+i when Ca2+o was < 10 nM but, with Ca2+o equal to 2.5 mM, ATP increased Ca2+i to a steady-state level 2-4 times the resting level of 50-100 nM. Addition of carbachol or epinephrine further increased Ca2+i. The mean and standard deviation of the ATP4- concentration at half of maximal activity (K0.5 ATP4-) was 33 +/- 7 microM and the Hill coefficient was 1.5 +/- 0.7 (n = 9). The active species is apparently ATP4-, as adding Mg2+ increases the total concentration of ATP needed to elevate Ca2+i. GTP, UTP, ADP and adenosine produced no significant changes in Ca2+i. However, 3'-O-(4-benzoyl)benzoyl ATP (BZATP) increased Ca2+i with a K0.5 BZATP of 2.4 +/- 0.5 microM and a Hill coefficient of 2.8 +/- 0.4 (n = 8). ATP-induced changes in Ca2+i were not due to a generalized increase in acinar-cell permeability and were unaffected by voltage-dependent Ca2+ channel blockers or by the presence of depolarizing concentrations of K+.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular ATP prevents the release of stored Ca2+ by autonomic agonists in rat submandibular gland acini.

In dose-dependent fashion, extracellular ATP reduces the increase in cytosolic Ca2+ concentration ([Ca2+]o) due to mobilization of cellular Ca2+ stores by both epinephrine [half-maximal inhibitory concentration (IC50) = 35.7 +/- 12.9 microM; Hill coefficient (NH) = -2.0 +/- 0.7, n = 8] and by carbachol (IC50 = 27.0 +/- 7.0 microM, NH = -2.3 +/- 0.7, n = 9). Inhibition is due to ATP4- but does not result from any emptying or inaccessibility of Ca2+ stores, which are readily mobilized by thapsigargin in the presence of ATP4-. Reduction of Ca2+ mobilization is rapid but is not due to direct interference by ATP with the interaction of carbachol or epinephrine with their respective cell surface receptors. A benzoyl derivative of ATP, 3'-O-(4-benzoyl) adenosine 5'-triphosphate (BZATP) is more potent than ATP in reducing [Ca2+]i due to mobilization of stored Ca2+ by either carbachol or epinephrine (IC50 for carbachol = 3.9 +/- 0.4 microM, NH = -3.2 +/- 0.5; IC50 for epinephrine = 3.8 +/- 0.2, NH = -2.6 +/- 0.7, n = 3) but GTP, UTP, ADP, and adenosine do not inhibit mobilization of stored Ca2+ by either carbachol or epinephrine. Neither ATP nor BZATP prevents the influx of extracellular Ca2+ stimulated by carbachol or epinephrine. These results suggest that ATP inhibits Ca2+ mobilization by autonomic neurotransmitters after occupation of P2Z purinoceptors.

Differential regulation of agonist-stimulated Ca2+ influx in acini of rat pancreas and submandibular gland.

In order to characterize agonist-dependent Ca2+ influx pathways, changes in cystolic Ca2+ (Ca2+i) during stimulation with multiple Ca(2+)-mobilizing agents were measured in rat pancreatic and submandibular gland acini loaded with fura-2. In pancreatic acini, maximal levels of carbachol and cholecystokinin octapeptide (CCK-8) produced virtually identical changes in Ca2+i when added alone or together: an immediate increase to 4-5 times resting Ca2+i followed by a decline to a steady-state level 2-3 times resting, which was unchanged by another stimulus. In submandibular gland acini, maximal carbachol stimulation increased Ca2+i 3-4-fold followed by a plateau at 2-3 times resting, which was further increased by epinephrine. Epinephrine alone increased steady-state Ca2+i to 53 +/- 18% (n = 21) of that observed with carbachol. Stimulation with both agents increased the steady-state plateau level of Ca2+i to 144 +/- 28% of that during exposure to carbachol alone (n = 11, p < 0.05). When changes in Ca2+i due solely to Ca2+ influx were measured, carbachol and epinephrine together increased Ca2+i during the steady-state phase to 149 +/- 31% of that measured with carbachol alone (n = 8, p < 0.05). Atropine blocked only responses to carbachol, prazosin blocked only responses to epinephrine and L 364,718 blocked only CCK-8-induced changes in Ca2+i. Thus, in pancreatic acini, a single agonist-sensitive Ca2+ influx pathway is linked independently to muscarinic cholinergic and peptidergic (CCK-8) receptors. In contrast, submandibular gland acini contain functionally separate agonist-sensitive Ca2+ influx pathways, which are independently linked to muscarinic and to alpha-1 adrenergic receptors.

Changes of cytosolic Ca2+ interfere with measurements of cytosolic Mg2+ using mag-fura-2.

In rat pancreatic and submandibular gland acini during exposure to carbachol, changes in the fluorescence emission intensity ratio (R) of acini loaded with mag-fura-2 resemble changes in cytosolic Ca2+ concentration (Ca2+i) in acini loaded with fura-2. Furthermore, changes of R depend on the presence of extracellular Ca2+ (Ca2+o) but are much less influenced by changes in extracellular Mg2+ (Mg2+o). To evaluate interference with measurement of cytosolic Mg2+ (Mg2+i) by changes in Ca2+i, we determined the dissociation constant (Kd) and Hill coefficient (NH) of the Ca(2+)-mag-fura-2 and Mg(2+)-mag-fura-2 complexes in standard solutions, in intact acini after loading with the acetoxymethyl ester of mag-fura-2 (mag-fura-2/AM), and in lysates derived from acini loaded with mag-fura-2/AM. The Kd of the Ca(2+)-mag-fura-2 complex in acini (determined with ionomycin) was 20.49 +/- 5.20 microM, and NH was 1.44 +/- 0.16 (n = 24). Kd of the Mg(2+)-mag-fura-2 complex in acini was 2.25 +/- 0.98 mM, and NH was 1.20 +/- 0.20 (n = 25). Mean Kd values were slightly lower in acinar lysates and in solutions of standard mag-fura-2. In acini from either gland, 1,2-bis(2-aminophenoxy)ethan-N,N,N',N'-tetraacetic acid (BAPTA) suppressed carbachol-induced Ca2+i transients. The R value in stimulated acini loaded with BAPTA and mag-fura-2 increased slightly when Mg2+o was increased from less than 10 nM to 1.2 mM, suggesting that Mg2+ influx contributes to the maintenance of Mg2+i during exposure to carbachol. Under these conditions, pancreatic acinar Mg2+i is 0.53 +/- 0.14 mM (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Differing significance of Na(+)-Ca2+ exchange in the regulation of cytosolic Ca2+ in rat exocrine gland acini and cardiac myocytes.

In order to compare the importance of Na(+)-Ca2+ exchange in the regulation of cytosolic Ca2+ concentration (Ca2+i), acini obtained from rat pancreas and submandibular glands as well as cardiac myocytes were loaded with Na+ by inhibition of Na(+)-K+ ATPase activity then loaded with fura-2. In the exocrine tissues, incubation in K(+)-free buffer or with ouabain had no substantial effect on resting Ca2+i or on the changes in Ca2+i following exposure to carbachol as compared with acini incubated under control conditions. In contrast, rat cardiac myocytes, treated identically, showed marked changes in Ca2+i under resting and stimulated conditions as compared with controls. We conclude that the Na(+)-Ca2+ exchange systems of rat pancreatic and submandibular gland acini contribute little to the overall regulation of Ca2+i at rest during cholinergic stimulation.

Regulating transient and sustained changes of cytosolic Ca2+ in rat pancreatic acini.

The regulation of changes in cytosolic Ca2+ concentration (Cai2+) during exposure to carbachol was studied in rat pancreatic acini loaded with fura-2. With an extracellular Ca2+ concentration (Cao2+) of 2.5 mM, resting Cai2+ is 185 +/- 48 (SD) nM (n = 23), which rises to 696 +/- 222 nM, then falls over 2 min to a stable plateau of 401 +/- 106 nM. With Cao2+ less than 10 nM, carbachol produces an immediate threefold increase in Cai2+ that dissipates over 2-3 min, and Cai2+ steadily falls below prestimulation levels. Atropine prevents all responses to carbachol, and when it is added during a response to carbachol, Cai2+ drops to resting values within seconds. Ca2+ influx is required for the prolonged elevation of Cai2+ during carbachol exposure, but Ca2+ entry is not regulated by an increase in Cai2+ itself nor does Ca2+ enter via voltage-gated L-type channels. The muscarinic receptor-operated Ca2+ entry mechanism is sensitive to Cao2+, since sustained elevations in Cai2+ are maximal at 2.5 mM Cao2+ but are much less pronounced at lower external Ca2+ concentrations.

Regulation of cytosolic Ca2+ in resting and stimulated rat submandibular salivary gland acini.

To determine the range over which cytosolic Ca2+ concentration (Cai2+) is regulated, Cai2+ was measured in salivary acini loaded with Fura-2. With extracellular Ca2+ (Cao2+) = 2.5 mM, mean resting Cai2+ (+/- SD) was 103 +/- 20 nM (n = 29). Carbachol increased Cai2+ immediately to 288 +/- 81 nM, which then declined to a sustained level of 253 +/- 58 nM. When Cao2+ was less than 10 nM, the immediate response to carbachol was little affected but the sustained response was abolished. Atropine abolished all responses to carbachol. Changes in Cai2+ were not due to Ca2+-induced Ca2+ release or to Ca2+ influx via voltage-dependent channels. With Cao2+ = 2.5 mM, epinephrine raised Cai2+ initially to 196 +/- 57 nM (n = 6), which soon declined to stable levels of 178 +/- 40 nM. When Cao2+ was less than 10 nM the prolonged response to epinephrine was abolished but the initial increase was relatively unaffected. All epinephrine-induced changes in Cai2+ were abolished by prazosin. The range of Cai2+ measured corresponds to that over which non-mitochondrial Ca2+ transport is stimulated in permeabilized submandibular gland acini suggesting that such a Ca2+ transport system may regulate physiological changes in Cai2+ in these acini in the rat.

Kinetics of high-affinity Ca2+ sequestration in permeabilized rat pancreatic acini.

Energy-dependent subcellular Ca2+ sequestration was studied in the presence of ruthenium red using rat pancreatic acini, which had been permeabilized by exposure to medium nominally free of Ca2+. The initial rate of Ca2+ uptake (approximately 2,800 pmol.min-1.mg acinar protein-1) quickly slowed, and a mean steady-state Ca2+ content of approximately 3,000 pmol/mg was reached after 5-10 min of incubation at 37 degrees C. Ca2+ uptake was stimulated by submicromolar Ca2+ concentrations (K0.5 = 156 nM); required Mg2+-ATP (K0.5 = 0.78 mM) was greatest at a pH of 7.0 and was abolished by the Ca2+ ionophore A23187. Other nucleotide phosphates as well as p-nitrophenylphosphate were relatively poor substrates, supporting Ca2+ uptake at initial rates that were 6-14% of those measured in the presence of ATP. These results show that pancreatic acini permeabilized without detergents possess a nonmitochondrial Ca2+ transporting system not located in the plasma membrane but with the properties expected of a major regulator of acinar cytosolic Ca2+ concentration.

The control of cytosolic Ca2+ concentration: studies of high affinity Ca2+ transport in permeabilized acini of rat submandibular glands.

Active Ca2+ transport was studied in acini that had been permeabilized by incubation in buffer nominally free of Ca2+ in order to avoid any contribution to measured Ca2+ uptake by elements of the plasma membrane. In most experiments, ruthenium red was used to inhibit mitochondrial Ca2+ uptake. Non-mitochondrial Ca2+ transport was greatest at pH 7.0-7.5 and required Mg2+-ATP at concentrations typical of the cytosol (K0.5 = 1.36 +/- 0.53 mM). Other substrates were much less effective than ATP. Ca2+ uptake was stimulated by Ca2+ at concentrations near those measured in intact cells (K0.5 = 0.43 +/- 0.17 microM). Hill coefficients at subsaturating concentrations of Ca2+ (1.08 +/- 0.27) and Mg2+-ATP (0.81 +/- 0.22) indicated that cooperative interactions are not characteristic of the major cationic regulators of Ca2+ transporting activity. Na+ did not release Ca2+ from acinar mitochondria, but consistently reduced non-mitochondrial Ca2+ uptake by about 20% as compared to uptake in the presence of an equimolar K+ concentration. The properties of non-mitochondrial Ca2+ uptake in permeabilized acini are similar to those of high affinity Ca2+ uptake which, in broken cell preparations, has been found distributed in parallel with elements of the endoplasmic reticulum. The Ca2+-sequestering properties of a non-mitochondrial organelle in permeabilized submandibular gland acini are those expected of a principal regulator of cytosolic Ca2+ and could account for the ionized Ca2+ concentration measured in resting salivary acinar cells.

Differential effects of chronic reserpine exposure on Ca2+ sequestering mechanisms in rat submandibular gland vesicles.

To determine the effects of exposure to reserpine on subcellular Ca2+ transporting systems, active Ca2+ uptake was measured with and without ruthenium red in submandibular gland vesicles obtained from rats after chronic treatment with reserpine. The properties of ruthenium red-sensitive Ca2+ uptake were similar to those measured in submandibular gland vesicles from untreated rats: it was abolished by the dye, was relatively low at 1 microM Ca2+ but increased markedly at millimolar Ca2+ levels and was positively and significantly correlated with the mitochondrial membrane marker, cytochrome-C oxidase activity, in membrane subfractions obtained by differential centrifugation (r = 0.67, p = 0.0005, n = 29). On the other hand, ruthenium red-insensitive Ca2+ uptake, though stimulated at submicromolar Ca2+ concentrations, was reduced by a mean of 54% compared to preparations from untreated animals and particulate RNA content was 18% of that found in control preparations. Moreover, the distributions of ruthenium red-insensitive Ca2+ uptake and particulate RNA (which are closely correlated in vesicles from untreated rats) were not significantly related when measured in vesicles of submandibular glands from reserpine treated rats. Other membrane markers and overall membrane protein content were not significantly altered after chronic reserpine exposure. We conclude that reserpine treatment has little effect on mitochondrial Ca2+ uptake capacity but abolishes or drastically reduces the high affinity Ca2+-sequestering activity which, in submandibular gland vesicles from untreated rats, is apparently associated with the endoplasmic reticulum.

Characterization and localization of two forms of active Ca2+ transport in vesicles derived from rat submandibular glands.

Energy-dependent Ca2+ uptake was characterized in vesicles derived from rat submandibular salivary glands. Ca2+ transport was stimulated by submicromolar levels of Ca2+, reached a plateau at 1-20 microM Ca2+ then again increased as the Ca2+ concentration rose to millimolar levels. Ruthenium red (2.5 microM) was used to resolve this pattern of uptake into two components: ruthenium red-insensitive Ca2+ transport occurs in the presence of the dye, is stimulated by submicromolar Ca2+ concentrations and reaches a maximum steady state at about 1 microM Ca2+. The distribution of ruthenium red-insensitive Ca2+ uptake in membrane subfractions obtained by differential centrifugation is positively (r = 0.717) and significantly (p = 0.001) correlated with the distribution of membrane-bound RNA in the same subfractions. Ca2+ uptake which is abolished by ruthenium red is greatest at millimolar Ca2+ concentrations. Its distribution is positively (r = 0.828) and significantly (p = 0.0001) correlated with the cytochrome-c oxidase activity of the membrane subfractions but is unrelated to the distribution of particulate RNA and is negatively correlated with Na+-K+ ATPase activity. We conclude that vesicles derived from the endoplasmic reticulum of rat submandibular glands actively transport Ca2+ by a ruthenium red-insensitive mechanism which is stimulated at Ca2+ concentrations typical of the cytosol. Membranes derived from mitochondria also sequester Ca2+ but by a mechanism which is inhibited by ruthenium red and which reaches its maximum steady state capacity at relatively high Ca2+ concentrations.

Characterization of the kinetic and regulatory properties of high-affinity Ca2+-ATPase activity in acinar preparations of rat submandibular salivary glands.

High-affinity Ca2+-ATPase activity was characterized in the total particulate fraction of acinar preparations from rat submandibular glands. The Ca2+ concentration (as Ca2+-ATP) at half of maximal activity was 82 +/- 17 nM, the Hill coefficient was 2.36 +/- 0.6 and activity reached a steady level at approximately 200 pmol Pi min-1 microgram of membrane protein-1 from 1 to 20 microM Ca2+-ATP. High-affinity Ca2+-ATPase required micromolar concentrations of Mg2+ and was inhibited approximately 60 per cent by the phenothiazine derivative, fluphenazine, but was unaffected by ouabain, Na+, K+, La3+, ruthenium red, oligomycin and added calmodulin. Kinetics of adenosine diphosphate, guanosine triphosphate, uridine triphosphate and inosine triphosphate hydrolysis were similar to those of Ca2+-ATP but p-nitrophenylphosphate was a poor substrate. In the heavy microsomal fraction of whole glands, active Ca2+ uptake (ATP-dependent, oxalate-enhanced and abolished by A23187) was measurable in the absence of added Mg2+, was inhibited by fluphenazine and was stimulated by submicromolar concentrations of Ca2+-ATP. Thus rat submandibular glands contain the enzymic basis of active Ca2+ transport and can actively transport Ca2+. Both activities are stimulated at Ca2+ concentrations typical of the cytosol, appear to be positively cooperative and may be regulated, in part, by calmodulin.

High affinity, calcium-stimulated adenosine triphosphatase activity in the particulate fraction of rat pancreatic acini.

Adenosine triphosphatase activity which is Mg2+-dependent and stimulated by submicromolar concentrations of Ca2+ (as Ca . ATP) was identified in the total particulate fraction of rat pancreatic acini. Half-maximal activity (V0.5) is obtained at 100.1 +/- 6 nM Ca . ATP with a Hill coefficient of 2.2 +/- 0.1 (mean +/- S.E.; n = 4). Maximal activity was 75 +/- 19 pmol of Pi released from ATP minute-1 microgram of membrane protein-1 (mean +/- S.E.; n = 7). High affinity Ca2+-ATPase activity was unaffected by ouabain, Na+, K+, La3+, and added calmodulin. Activity was slightly reduced by ruthenium red (0.1 mM) and by oligomycin (80 micrograms/ml) but was reduced almost 50% by the phenothiazine derivative fluphenazine in a dose-related and Ca2+-dependent manner. Hydrolysis of p-nitrophenyl phosphate was 9% of the rate of ATP hydrolysis and was independent of Ca2+ concentration. However, ADP, GTP, UTP, and ITP were hydrolyzed at 76-93% the rate that ATP was hydrolyzed with V0.5 values and Hill coefficients similar to those of Ca . ATP. We conclude that rat pancreatic acini contain an enzyme for active Ca2+ translocation: ATPase activity that is Mg2+-dependent and stimulated by submicromolar concentrations of Ca . ATP. Substrate hydrolysis appears to involve positive cooperative interactions of multiple ligand-binding sites and may be regulated in part by calmodulin.

Stimulation of ovine placental lactogen secretion by arginine infusion.

Arginine has been demonstrated to be a potent stimulus to GH and PRL secretion. To determine the effect of arginine on plasma ovine placental lactogen (oPL) concentrations, arginine (50 g in 350 ml distilled water, pH 7.4) or hypertonic saline of identical volume, osmolality, and pH was infused iv over a 30-min period into nine pregnant ewes, and blood samples from chronic indwelling venous catheters were obtained at frequent intervals before and for 8 h after the infusions. After the infusion of hypertonic saline, plasma oPL concentrations (measured by homologous RIA) decreased 20--50% over 1--2 h and then returned to baseline concentrations. After the infusion of arginine, plasma oPL concentrations also decreased by 20--50% for 1--2 h. However, 2--3 h after the infusion, plasma oPL concentrations in creased 79--115% (delta = 204--700 ng/ml) over preinfusion concentrations in seven ewes and 454% (2930 ng/ml) and 1142% (2042 ng/ml) in two ewes and remained elevated for the remainder of the 8-h interval. When the amount of arginine infused was reduced from 50 to 25 g, an increase in plasma oPL concentrations occurred in only one of five ewes. Plasma oPL concentrations increased by 8--58% after infusions of 50 g alanine or glycine but did not increase after 50 g glutamic acid. The delayed oPL response to arginine suggests that the increase in plasma oPL concentrations is not caused directly by arginine but rather by changes in the synthesis, secretion, and/or degradation of oPL induced indirectly by arginine.

Ovine placental lactogen induces somatomedin: a a possible role in fetal growth.

Ovine placental lactogen (oPL) and bovine growth hormone (bGH) increase the circulating concentration of somatomedin in hypophysectomized rats. This finding indicates that some of the somatotropic properties of oPL may be due to the induction of somatomedin and raises the possibility that oPL has a role in the control of fetal growth.