Effects of the calcium channel activator BAY-K-8644 on in vitro secretion of calcitonin and parathyroid hormone

The recently discovered calcium (Ca) channel activator BAY-K-8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl) pyridine-5-carboxylate], an analog of the calcium channel blockers nifedipine and nitrendipine, was tested to determine its potential for altering hormone secretion in an in vitro system designed to study concurrent secretion of calcitonin (CT) and PTH. Addition of BAY-K-8644 (10(-4)-10(-5) M) to medium (1 mM Ca) bathing baby rat thyroparathyroids enhanced secretion of CT at least 2- to 4-fold and suppressed PTH release by as much as 75-85%. Addition of BAY-K-8644 alone to medium containing high (2.5 mM) Ca did not further enhance the already high rate of CT release, nor did it cause any further suppression of PTH secretion. BAY-K-8644 did not stimulate CT release or suppress PTH release in the absence of medium Ca. Addition of the Ca channel blocker nitrendipine (10(-5) M) inhibited CT release at either 1 or 2.5 mM Ca, and at 1 mM Ca, nitrendipine negated the simulatory effect of 10(-5) M BAY-K-8644 on CT release. However, at 2.5 mM Ca, 10(-5) M BAY-K-8644 reversed the marked inhibitory effect of 10(-5) M nitrendipine on CT release. At 1 mM Ca, PTH secretion was inhibited equally well by BAY-K-8644 and nitrendipine, and both agents together caused a further suppression of PTH release. The results indicate that Ca entry into the thyroid C-cell and parathyroid chief cell may occur via classical voltage-sensitive Ca channels and that the newly described Ca channel activator BAY-K-8644 should prove useful as a probe for studying hormone secretion in Ca-dependent secretory systems.     

Effects of high potassium concentration and dihydropyridine Ca2(+)-channel agonists on cytoplasmic Ca2+ and aldosterone production in rat adrenal glomerulosa cells.

The aldosterone secretory response of isolated rat adrenal glomerulosa cells to potassium was studied in a cell-perifusion system. Increasing the potassium concentration from 3.6 to 5.4 mM in the perifusion medium caused a rapid 40-fold stimulation of aldosterone production which was maintained during the 2 h period of stimulation. A dose of 8.4 mM potassium elicited a 100-fold increase of hormone production with rapid onset and a slowly decreasing plateau. When the potassium concentration was further increased to 18 mM, there was a rapid stimulation of aldosterone production comparable to that evoked by 8.4 mM potassium; however, the response declined very rapidly to levels still above basal. The dihydropyridine-agonist BAY-K 8644 (100 nM) greatly enhanced the aldosterone response to 5.4 mM potassium but did not significantly modify the response evoked by 8.4 mM potassium. The effect of BAY-K 8644 on the aldosterone response was inhibitory at 18 mM potassium concentration, suggesting that the character of dihydropyridine modulation of the secretory response was voltage dependent, showing reversal at relatively negative potentials. When the cytoplasmic Ca2+ concentration was monitored in glomerulosa cells by the fluorescent Ca2(+)-probe Fura-2, potassium evoked a rapid dose-dependent increase in free Ca2+, with elevated steady-state Ca2(+)-levels throughout stimulation, even at potassium concentrations higher than 18 mM. Moreover, BAY-K 8644 (100 nM) enhanced the cytoplasmic Ca2+ response to all potassium concentrations tested up to 30 mM. The initial 30 sec 45Ca2+ uptake, an indicator of potassium-stimulated voltage-sensitive Ca2+ influx into these cells, showed a fast increase and only an initial inactivation in response to 18 mM potassium. This response was enhanced by 100 nM BAY-K 8644, with no sign of enhanced inactivation or inhibition caused by the dihydropyridine agonist. These results indicate that the correlation between Ca2+ influx, cytoplasmic Ca2+ levels, and the secretory response in adrenal glomerulosa cells is lost at potassium concentrations higher than 8 mM, especially in the presence of the dihydropyridine agonist, BAY-K 8644.     

Involvement of sodium channels and two types of calcium channels in the regulation of adrenocorticotropin release

Recent electrophysiological studies from this laboratory demonstrated that anterior lobe corticotropes exhibited a tetrodotoxin-sensitive sodium current and two types of voltage-dependent calcium currents, consisting of low threshold (transient) and high threshold (long lasting) components. The present report describes cytophysiological and cytochemical studies that used specific blockers of each of these currents to assess their role in the regulation of CRF binding and ACTH secretion and storage. Two dihydropyridines, nimodipine and the pure antagonist enantiomer (-)R202-791, which block high threshold Ca2+ channels, decreased 1 h basal release by 54-74% and CRF-mediated (5 min or 3 h) release completely. Percentages of CRF-bound cells were reduced as much as 74%; however, the inhibitory effect on percentages of CRF-bound cells could be reversed by adding 10 nM Bay K 8644, (a pure dihydropyridine agonist) with the antagonists. CdCl2, which blocks both high and low threshold calcium currents, inhibited basal and CRF-stimulated ACTH release, but only the highest concentration (0.1 mM) reduced percentages of CRF-bound cells. Involvement of the low threshold Ca2+ channels could not be proved by adding dihydropyridine antagonists with 0.1 mM CdCl2. Basal and CRF-mediated ACTH release were blocked by the potent sodium channel blocker tetrodotoxin, and the highest concentration (3 microM) reduced percentages of CRF-bound cells. Basal (1 h) and CRF-stimulated (5 min) ACTH release were also inhibited in medium containing 1 mM EGTA and no Ca2+; however, percentages of CRF-bound cells were within the normal range. Densitometric analysis of stains for ACTH showed an increase in the concentration of stain per cell after a 1-h exposure to the highest concentrations of the inhibitors or to no Ca2+ and 1 mM EGTA coupled with a significant (10%) decrease in corticotrope cell area. Finally, in the last series of tests, the Bay K 8644 agonist or arginine vasopressin were used to study mechanisms of augmentation of basal or CRF-mediated ACTH release. Bay K 8644 augmented basal release in a concentration of 1 microM and CRF-mediated release in a concentration of 100 nM or 1 microM. After pretreatment with either Bay K 8644 or arginine vasopressin (10 nM) there was a significant (30%) increase in the percentage of CRF-bound cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Gonadotropin-releasing hormone stimulates luteinizing hormone secretion by extracellular calcium-dependent and -independent mechanisms

The dependence of LH responses to GnRH on extracellular calcium was investigated in cultured rat pituitary cells exposed to GnRH for 3 h in static culture or for 2 min during column perifusion. During static culture in normal medium, LH release was stimulated by GnRH with an ED50 of 0.3 nM and by K+ with an ED50 of 32 mM. Incubation in Ca2+-deficient (no added Ca2+) or Ca2+-free medium (containing 100 microM EGTA) substantially decreased, but did not abolish, the LH responses to 10 and 100 nM GnRH, whereas K+-induced LH release was almost completely abolished in Ca2+-deficient medium. The Ca2+ channel agonist (BK 8644) and antagonists (nifedipine, nicardipine, verapamil, and Co2+) respectively enhanced or reduced the LH responses to both GnRH and K+. However, the calcium antagonists completely abolished the LH response to depolarization by K+, but only partially inhibited the LH response to GnRH, confirming the existence of a significant component of GnRH action that is not dependent on extracellular Ca2+. In perifused pituitary cells, exposure to Ca2+-deficient medium or normal medium containing 5 mM EGTA or 5 mM EDTA, reduced the initial rapid LH response to 2-min pulses of 10 nM GnRH and abolished the second phase of LH release. Reintroduction of Ca2+-containing medium at the end of the GnRH pulse caused recovery of the second phase of LH secretion, demonstrating that influx of extracellular Ca2+ is not required for the early phase of the LH response to GnRH but, rather, appears to be essential for its prolongation. The release of LH in response to arachidonic acid, which has been implicated in the mechanism of the secretory action of GnRH, was completely independent of extracellular Ca2+ and unaffected by addition of 10 nM BK 8644. These observations indicate that the initiation of the secretory response to GnRH is largely independent of calcium entry, whereas the prolongation of gonadotropin secretion is maintained by calcium influx, in part through voltage-sensitive calcium channels. The role of arachidonic acid metabolites in GnRH action is probably related to the calcium-independent component of GnRH-induced LH secretion. Since GnRH is secreted episodically and for short periods, much of its physiological action on pulsatile gonadotropin release could be independent of calcium influx from the extracellular fluid.     

Fluoride stimulates the accumulation of inositol phosphates, increases intracellular free calcium, and inhibits parathyroid hormone release in dispersed bovine parathyroid cells

The stimulation of polyphosphoinositide (PPI) turnover is associated with cellular activation and hormone secretion in numerous systems. GTP-binding proteins appear to couple receptors to phospholipase-C-mediated PPI breakdown. We assessed the effects of fluoride, an activator of GTP-binding proteins, on inositol phosphate accumulation, intracellular free Ca2+ [(Ca2+)i], cAMP content, and PTH release in dispersed bovine parathyroid cells. Sodium fluoride (5-30 mM) produced marked dose-dependent increases in inositol phosphates. With anion exchange HPLC, we confirmed that 30 mM fluoride stimulated a rapid increase in 1,4,5-inositol trisphosphate, a potent Ca2+-mobilizing compound. Using the Ca2+-sensitive probe fura-2, we determined that 30 mM fluoride increased [Ca2+]i from 339 +/- 9 to 650 +/- 39 nM (n = 8) within 30-60 sec at 1 mM extracellular Ca2+. After the depletion of extracellular Ca2+ by the addition of 1 mM EGTA, 30 mM fluoride increased [Ca2+]i 45 +/- 9% (n = 4), indicating that fluoride can mobilize intracellular Ca2+ stores. Fluoride (1-30 mM) also inhibited PTH release in dose-dependent fashion. Fluoride (30 mM) produced 72.8 +/- 4.2% suppression of maximal low Ca2+-stimulated PTH release comparable to the 83.7 +/- 3.7% inhibition by 2.0 mM extracellular Ca2+. Since changes in both [Ca2+]i and cAMP regulate PTH release, we measured the effect of fluoride on intracellular cAMP. Fluoride did not detectably change basal cAMP content, but it reduced forskolin-stimulated increases in cAMP. We conclude that fluoride may activate at least two GTP-dependent processes in parathyroid cells, resulting in PPI breakdown and cAMP accumulation. While both may contribute to the fluoride-induced suppression of PTH release, our findings suggest that the stimulation of PPI turnover leads to inhibition of PTH secretion.     

High osmolality: a potent parathyroid hormone secretogogue in dispersed parathyroid cells

We have examined possible mechanisms by which osmolality might modulate PTH secretion in dispersed bovine parathyroid cells. Increasing medium osmolality by adding sodium chloride causes a marked, dose-dependent increase in PTH release. The maximum effect (4-fold increase) is observed when osmolality is around 650 mosM, with half-maximal stimulation at about 400 mosM. When osmolality is increased to a similar extent with either sucrose or sodium chloride, PTH secretion is enhanced to a comparable degree, suggesting that osmolality itself, rather than ionic strength, is responsible for the increase in PTH secretion. Time course experiments show that the increased secretion of PTH with high osmolality occurs very rapidly (in less than 5 min). In contrast to the suppressive effects of high Ca2+ on PTH release, increasing calcium concentration in the incubation media does not inhibit the stimulation of PTH secretion by high osmolality. Moreover, the effects of dopamine (10(-5) M) and high osmolality on PTH release are additive, further suggesting that high osmolality and cAMP modulate PTH release by different mechanisms. In fact, direct measurement of cellular cAMP in cells exposed to high osmolality shows no change relative to control cells incubated with normal osmolality, 127 +/- 20 vs. 146 +/- 21 fmol/10(5) cells, respectively. Cytosolic Ca2+ increases from 257 +/- 29 nM to 703 +/- 50 nM after 200 mM NaCl is added to the incubation medium at low Ca2+ (0.5mM). Prior removal of extracellular calcium abolished this effect. Increasing the osmolality to a similar level by adding sucrose to the medium does not demonstrate any increase in cytosolic calcium. We conclude that high osmolality is a potent secretogogue in dispersed bovine parathyroid cells. Unlike dopamine and isoproterenol, high osmolality does not act through changes in intracellular cAMP. It also prevents the normal inhibitory effect of high Ca2+ on PTH release. Change of cytosolic Ca2+ is variable and suggests that the effect of high osmolality on PTH release cannot be explained by cytosolic Ca2+ alone. Further understanding of the mechanisms by which osmolality affects PTH release, therefore, may provide clues to the unusual inverse relationship between extracellular and cytosolic calcium and PTH release.     

The effects of phorbol myristate acetate on the intracellular degradation of bovine parathyroid hormone

The suppression of PTH release by high extracellular calcium (Ca2+) has been associated with secretion of biologically inactive carboxyl-terminal fragments of PTH (C-PTH), while relatively more intact PTH is released under low extracellular Ca2+ conditions. In the presence of high extracellular Ca2+, phorbol myristate acetate (PMA) has been shown to stimulate PTH release to levels observed at low Ca2+, suggesting that protein kinase-C (PKC) is involved in the regulation of PTH secretion. We have examined the effect of PMA on PTH secretion and the release of PTH fragments at high and low calcium concentrations. Primary cultures of bovine parathyroid cells were incubated for 90 min in 0.5 mM (low) or 2.0 mM (high) Ca2+ with or without 1.6 microM PMA. Reverse phase HPLC using an 18-60% gradient of acetonitrile in 0.1% trifluoroacetic acid was performed on the medium from these incubations, and the eluant fractions were analyzed with a carboxyl (C)-terminal-specific PTH RIA. Medium from cultures exposed to low Ca2+ exhibited two large peaks of PTH immunoreactivity, coeluting with intact PTH-(1-84) and a synthetic human C-PTH-(39-84). PMA treatment at low Ca2+ resulted in the secretion of a greatly reduced amount of intact PTH, suggesting that PKC may increase the production of PTH fragment. At high extracellular Ca2+ PMA caused an increase in total immunoreactive PTH release similar to that seen at low Ca2+. However, on HPLC analysis, proportionally more PTH eluted in the position of the C-PTH fragment than was seen with low Ca2+ stimulation of PTH secretion. It, therefore, appears that the degradation of PTH to C-PTH may be linked to activation of PKC and can be separated from the Ca2+ regulation of PTH release occurring at the cell membrane.     

Relationship between parathyroid hormone secretion and cytosolic calcium concentration in dispersed bovine parathyroid cells.

The parathyroid cell is unusual among exocytotic systems in that low extracellular Ca2+ concentrations stimulate, while high Ca2+ concentrations inhibit, parathyroid hormone (PTH) release, suggesting that this cell might have unique secretory mechanisms. In the present studies, we used the Ca2+-sensitive fluorescent dye QUIN -2 to examine the relationship between cytosolic Ca2+ concentration and PTH release in dispersed bovine parathyroid cells. The secretagogue dopamine, which enhances PTH release 2- to 3-fold in association with 20- to 30-fold increases in cellular cAMP, had no effect on the cytosolic Ca2+ level (261 +/- 28 vs. 236 +/- 22 nM for control cells at 1 mM extracellular Ca2+; P greater than 0.05). Dibutyryl-cAMP, which produces a comparable stimulation of PTH release, likewise did not modify the level of cytosolic Ca2+. Removal of extracellular Ca2+ produced a further decrease of the cytosolic Ca2+ to 82 +/- 10 nM. However, PTH secretion persisted at a near maximal rate despite this decrease of extracellular and cytosolic Ca2+ and was 95 +/- 2.5% of the rate of hormonal release at 0.5 mM extracellular Ca2+. In contrast, addition of the divalent cation ionophore ionomycin to parathyroid cells at 1.0 mM extracellular Ca2+ inhibited PTH secretion in association with an increase in cytosolic Ca2+ from 230 +/- 13 nM to 570 +/- 50 nM. Moreover, the magnitude of the ionomycin-induced reduction in PTH secretion (64 +/- 4% relative to the secretory rate at 0.5 mM Ca2+) was equivalent to the inhibition of PTH release caused by 1.5 mM extracellular Ca2+ (64 +/- 6%), which increased the cytosolic Ca2+ to similar levels (450 +/- 48 nM). Thus, the parathyroid cell differs from secretory cells thought to operate by stimulus-secretion coupling in the following ways: changes in PTH release can occur without detectable alterations in the cytosolic Ca2+ concentration, maximal rates of PTH secretion occur at cytosolic Ca2+ concentrations that fail to support exocytosis in other cell types, and increases in the cytosolic Ca2+ concentration due to ionomycin inhibit rather than stimulate PTH release. Therefore, the control of PTH secretion by Ca2+ and other secretagogues may involve previously undefined mechanisms whereby hormonal release is relatively independent of the cytosolic Ca2+ at low levels of this parameter and is inversely related to cytosolic Ca2+ at higher levels of intracellular Ca2+.     

Influence of calcium on the metabolism of intact parathyroid hormone by isolated perfused rat kidney and liver

The metabolism of synthetic human PTH [PTH-(1-84)] 10(-9) M was studied in isolated rat kidneys and livers, perfused at a calcium concentration of 1 mM or 4 mM. Clearances were measured by an assay specific for intact PTH, and by assays specific for NH2-terminal, mid-molecule, and COOH-terminal immunoreactive PTH (iPTH). Production of PTH fragments was analyzed by HPLC. The kidneys cleared PTH mainly by filtration. The glomerular filtration rate was not lower at 4 mM calcium than at 1 mM calcium, and no significant differences were found between the clearance of PTH at 4 mM and at 1 mM calcium. At 1 mM calcium the kidneys cleared intact PTH without release of detectable fragments. At 4 mM calcium there was significant (P less than 0.05) accumulation of mid-molecule and COOH-terminal iPTH in the perfusate. Both at low and at high calcium the livers cleared NH2-terminal iPTH at the same rate as intact PTH, whereas mid-molecule and COOH-terminal iPTH was cleared significantly (P less than 0.005) slower. In the livers, metabolic clearance of PTH was 60% faster at 4 mM calcium than at 1 mM calcium (P less than 0.001). Assuming that the hepatic metabolism of PTH represents degradation of the biologically active hormone and hormone fragments, rather than activation of the hormone, the present results suggest a homeostatic control of PTH degradation in the liver to enhance inactivation of the hormone at high serum levels of calcium.     

Reversible desensitization of calcitonin secretion by repetitive stimulation with calcium

The extracellular ionized calcium concentration (Ca2+) is a main regulator of calcitonin (CT) release. Calcium-induced CT secretion differs for acute versus long-term alterations of Ca2+. Using the rat C cell line rMTC 6-23 we have investigated the effect of repetitive stimulation by Ca2+ on CT release. After a Ca-induced initial rise of CT secretion, repetitive Ca stimulation led to a decline of CT release to unstimulated levels (after about 4 h). Reversing the high Ca2+ concentration (2.0 mM) to basal (1.1 mM) for 2 h and then increasing Ca2+ again resulted in a restored stimulatory action of Ca2+ (about 100% increase above the control). In contrast, repetitive stimulation with the dihydropyridine Ca channel agonist Bay K-8644 showed an unchanged stimulatory effect, as observed for the cAMP analog 8-bromo-cAMP, too. The results indicate that the reversible desensitization of Ca-induced CT secretion might be due to a modification of the voltage-dependent Ca channels proximal to or at the site of Bay K-8644 action.     

Blood pressure-dependent inhibition of Renin secretion requires A1 adenosine receptors

Renal perfusion pressure (RPP) regulates renin release with a reduction of RPP stimulating and an elevation inhibiting renin secretion. The precise sensing and effector mechanisms by which changes in arterial pressure are linked to the exocytosis of renin are not well-defined. The present experiments were designed to study the potential role of adenosine as a mediator of this renal baroreceptor mechanism. In isolated perfused mouse kidneys a stepwise reduction of RPP from 90 mm Hg to 65 and 40 mm Hg stimulated renin secretion rates (RSR) 1.4-fold and 3.6-fold, whereas stepwise elevations of RPP from 90 mm Hg to 115 and 140 mm Hg suppressed RSR to 64% or 40% of baseline. Inactivation of A1 adenosine receptors by either pharmacological blockade (DPCPX 1 micromol/L) or genetic deletion (A1AR(-/-) mice) did not modify the stimulation of renin release by a low RPP, but completely prevented the suppression of renin secretion by higher perfusion pressures. In vivo, the induction of arterial hypertension by either acute (single subcutaneous injection) or chronic (osmotic minipump for 72 hours) application of phenylephrine significantly reduced plasma renin concentration (PRC) in wild-type mice to approximately 40% of control, whereas it did not significantly affect PRC in A1AR(-/-) mice. Together these data demonstrate that A1 adenosine receptors are indispensable for the inhibition of renin secretion by an increase in blood pressure, suggesting that formation and action of adenosine is responsible for baroreceptor-mediated inhibition of renin release. In contrast, the stimulation of the renin system by a low blood pressure appears to follow different pathways.     

The role of calcium in luteinizing hormone/human chorionic gonadotrophin stimulation of Leydig cell immunoactive inhibin secretion in vitro.

The mechanism by which luteinizing hormone (LH) stimulates Leydig cell immunoactive inhibin (I-inhibin) secretion was investigated using Percoll-purified adult rat Leydig cells. Using a maximally stimulating dose of LH (16 ng/ml). Leydig cell I-inhibin secretion was non-detectable at 1-2 h of incubation, but subsequently increased at all time points during a 25 h incubation period. LH stimulated both Leydig cell content and release of I-inhibin. Increasing concentrations of LH stimulated both inhibin and testosterone immunoactivity in the incubation media over a similar dose-response range, with a 2- to 4-fold rise in I-inhibin secretion at maximal doses of LH. Dibutyryl cAMP stimulated testosterone secretion in a manner similar to that of LH, but I-inhibin secretion was less sensitive than testosterone and a significant stimulation was observed only at the highest doses (200-1000 micrograms/ml). LH-stimulated I-inhibin secretion was significantly decreased when Leydig cells were incubated in calcium-depleted (0.15 mM Ca2+ + 1 mM EGTA) or low [Ca2+] media (0.15 mM) as compared to normal (1.15 mM) or high [Ca2+] (2-5 mM) media. In contrast, LH-stimulated testosterone secretion remained unchanged by altering extracellular [Ca2+], and although decreased in the presence of EGTA, testosterone secretion remained significantly greater than basal levels. Furthermore both diltiazem and verapamil completely blocked the LH and dibutyryl cAMP-stimulated increase in Leydig cell I-inhibin, but did not reduce either LH or dibutyryl cAMP-stimulated testosterone production to basal levels. We conclude that LH stimulates both I-inhibin synthesis and release by adult rat Leydig cells in culture, by mechanisms involving calcium.     

ATP-dependent mechanism for coordination of intercellular Ca2+ signaling and renin secretion in rat juxtaglomerular cells

A change in intracellular Ca2+ is considered to be the common final signaling pathway through which renin secretion is governed. Therefore, information relating to the generation, control, and processing of Ca2+ signaling in juxtaglomerular cells (JG) will be critical for understanding JG cell behavior. In this study, we investigated the means by which JG cells harmonize their intracellular Ca2+ signals and explored the potential role of these mechanisms in renin secretion. Mechanical stimulation of a single JG cell initiated propagation of an intercellular Ca2+ wave to up to 11.9+/-4.1 surrounding cells, and this was prevented in the presence of the ATP-degrading enzyme, apyrase (1.7+/-0.7 cells), or by desensitization of purinergic receptors via pretreatment of cells with ATP (1.8+/-0.9 cells), thus implicating ATP as a mediator responsible for the propagation of intercellular Ca2+ signaling. Consistent with this, JG cells were demonstrated not to express the gap junction protein connexin43, and neither did they possess functional gap junction communication. Furthermore, massive mechanical stretching of JG cells elicited a 3-fold increase in ATP release. Administration of ATP into isolated perfused rat kidneys induced a rapid, potent, and persistent inhibition of renin secretion, together with a transient elevation of renal vascular resistance. ATP (1 mmol/L) caused up to 79% reduction of the renin secretion activated by lowering the renal perfusion flow (P<0.01). Taken together, our results indicate that under mechanical stimulation, ATP functions as a paracellular mediator to regulate renin secretion, possibly through modulating intra- and intercellular Ca2+ signals.     

The role of calcium in the control of renin release

The effects of removing external calcium and inhibiting entry of calcium into the cell by treatment with D-600 on renin release from renal cortical slices of male Sprague-Dawley rats were examined. Baseline renin release, angiotensin II (AII)-induced inhibition, and isoproterenol-induced stimulation of renin release were studied. Removal of external calcium by chelation with 5 mM EGTA inhibited basal renin release while treatment with 1 mM EGTA stimulated basal renin release slightly. Incubation of slices with zero calcium medium containing 1 mM EGTA had no effect on isoproterenol-induced stimulation of renin release. In contrast, similar treatment reduced the inhibitory effect of AII from 58.7% of baseline to 85.3% (p less than 0.001). Similarly, blockage of calcium entry into cells with D-600 had no effect on isoproterenol-induced stimulation of renin release but abolished AII-induced inhibition. Replacement of sodium in the bathing medium with choline had no effect on baseline renin release or on AII-induced inhibition of renin release, ruling out the possibility that D-600 altered renin release via an effect on sodium influx. Taken together, the data strongly suggest that AII-induced inhibition of renin release is partially dependent on the presence of external calcium but that isoproterenol-induced stimulation of renin release is not.     

Differences in the actions of calcium versus lanthanum to influence parathyroid hormone release

PTH release from bovine parathyroid cells is inhibited by increasing concentrations of extracellular calcium (Ca2+). We have proposed that this inhibition is mediated by Ca2+ channels via a G-protein. To further test this hypothesis, we evaluated the effect of lanthanum (La3+), a potent Ca2+ channel antagonist that does not cross the cell membrane. PTH release was determined in dispersed bovine parathyroid cells by radioimmunoassay: extracellular Ca2+ concentration was 0.2 mM. PTH release was inhibited by maximal concentrations of La3+ to a greater extent than by Ca2+: 93% inhibition by La3+ vs. 40% by Ca2+. La3+ was more potent (set-point = 0.12 mM) than Ca2+ (set-point = 1.2 mM). Incubation of parathyroid cells with pertussis toxin, which inactivates a G-protein(s) and blocks inhibition by Ca2+, did not block the inhibition of PTH release by La3+ at the concentrations tested. The Ca2+ ionophore A23187, which potentiates the effect of Ca2+, did not enhance the inhibition of PTH release by La3+. Increasing concentrations of calcium enhanced the inhibition of PTH release by the Ca2+ channel agonist, (+)202-791. The Ca2+ channel antagonist, (-)202-791, shifted the Ca2+ inhibition curve to the right. La3+ did not alter the inhibition of PTH release by the Ca2+ channel agonist but blocked the stimulatory effect of the Ca2+ channel antagonist, (-)202-791. In summary: 1) La3+, which blocks Ca2+ channels and does not cross cell membranes, effects a greater inhibition of PTH release than Ca2+; 2) La3+, like Ca2+, overrides the effect of Ca2+ channel antagonist (-)202-791; and 3) La3+, unlike Ca2+, inhibits PTH release by a mechanism that is independent of a pertussis toxin-sensitive G-protein. There may be two cell surface sites that recognize La3+ and Ca2+ independently.     

Cytosolic Ca2+ during atrial natriuretic peptide secretion from cultured neonatal cardiomyocytes

Mechanisms of atrial natriuretic peptide (ANP) release were studied in neonatal rat heart atrial and ventricular myocytes cultured on Cytodex 3 microcarriers. For simultaneous observations of cytosolic free calcium concentration ([Ca2+]f) and ANP secretion, the culture was packed in a chromatography column, inserted into the cell holder of a spectrofluorometer was perifused with a buffer solution. [Ca2+]f was measured by the fluorescent calcium indicator Fura-2 and ANP in the effluent perfusate by radioimmunoassay. No cell damage was observed and the basal ANP secretion rate and [Ca2+]f were comparable with values obtained by other methods. K(+)-induced depolarization raised [Ca2+]f by 50%, but it rapidly declined again to a steady level 10-20% above the baseline. The calcium channel agonist Bay k8644 elicited a similar temporal pattern of [Ca2+]f changes and 1 microM ionomycin induced a 100-fold increase in [Ca2+]f with a slow re-establishment of the original baseline. None of these stimuli increased the ANP secretion rate of the atrial or ventricular myocytes. Protein kinase C activation by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulated ANP secretion from the atrial myocytes, while the ventricular myocytes were unresponsive to TPA. It is concluded that Ca2+ is not the main mediator in the regulation of ANP release in cultured neonatal heart cells.     

Selective enhancement of angiotensin II- and potassium-stimulated aldosterone secretion by the calcium channel agonist BAY K 8644

Recent studies with dihydropyridine calcium channel antagonists have indicated that voltage-sensitive calcium channels (VSCC) play a major role in the control of aldosterone secretion. The modulation of VSCC by physiological regulators of zona glomerulosa function was further evaluated by analysis of the actions of the dihydropyridine calcium channel agonist BAY K 8644 (BK 8644) on basal and stimulated aldosterone production in isolated rat glomerulosa cells. In the presence of normal K+ concentrations (3.5-4.5 mM), only high concentrations of BK 8644 (greater than or equal to 100 nM) stimulated aldosterone secretion. However, addition of 10 nM BK 8644 markedly enhanced steroid production (70% over control) in cells stimulated by incubation in 7.5 mM K+ or 0.1 nM angiotensin II (AII). Greater enhancement was achieved with 1 microM BK 8644, with aldosterone secretion 150% and 300% above control levels for K+ and AII, respectively. In AII-stimulated cells, 30 nM BK 8644 enhanced aldosterone secretion at all peptide concentrations studied, including a 70% increase in the maximum steroid response, with no change in sensitivity to AII. In K+-stimulated cells, the effects of BK 8644 were dependent on the medium concentration of K+. At submaximally stimulating K+ concentrations (less than 9 mM), 30 nM BK 8644 increased the sensitivity of glomerulosa cells to K+ with no change in the maximal aldosterone response. However, at supramaximally stimulating concentrations of K+ (greater than 10 mM), BK 8644 reduced aldosterone production by 50%. In contrast to the effects of BK 8644 on cells stimulated with K+ or AII, the channel agonist had no effect on the action of ACTH. The ability of BK 8644 to enhance the maximum aldosterone response to AII suggests that AII, unlike K+, does not fully activate the Ca2+ influx pathway that leads to aldosterone secretion. Since BK 8644 is believed to facilitate Ca2+ influx primarily through previously activated channels, these results suggest that VSCC in the rat glomerulosa cell are partially operative under basal conditions, and that the same types of channels are further activated by AII and K+.     

Phorbol esters enhance stretch-induced atrial natriuretic peptide secretion.

Stretching of atrial myocytes stimulates atrial natriuretic peptide (ANP) secretion, but the cellular processes linking mechanical distention to ANP release are unknown. We studied whether or not protein kinase C activation by phorbol ester affects atrial stretch-induced ANP secretion using the modified perfused rat heart preparation that enabled stepwise distention of the right atrium as an experimental model for stretch-stimulated ANP release. The increase in right atrial pressure (2.65 +/- 0.13 mm Hg) was accompanied by an increase in the perfusate immunoreactive ANP (IR-ANP) concentration (from 8.3 +/- 1.1 ng/5 min to 13.9 +/- 2.0 ng/5 min, P less than 0.05, n = 14). During stretch, a slight inotropic response was observed, while heart rate and perfusion pressure remained unchanged. Increase in right atrial pressure in the presence of a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), known to stimulate protein kinase C activity in heart cells, resulted in a significantly greater increase in the perfusate IR-ANP concentration than after vehicle infusion. The calculated ANP increase corresponding to the 2 mm Hg increase in the right atrial pressure was 1.52-fold in the control group and 1.84-fold when 10 nM TPA was infused (P less than 0.05). Infusion of TPA at a dose of 24 nM further increased the stretch-induced ANP release by causing 2.22-fold (P less than 0.01) increase in IR-ANP secretion. As judged by gel filtration chromatography, abnormal release of the large mol wt stored ANP could not account for the secretory response to phorbol ester. Additionally, a phorbol ester analog, 4 alpha-phorbol 12,13-didecanoate, which is incapable of binding to and activating protein kinase C, was inactive as an ANP secretagogue. In contrast, drugs known to increase the concentration of intracellular Ca2+ in myocytes, Bay K8644 (3 and 6 microns) and forskolin (0.14 microM), significantly inhibited the stretch-stimulated ANP release. This study shows that phorbol ester enhances atrial stretch-stimulated ANP secretion from the isolated perfused heart, suggesting that protein kinase C activity is positively coupled to the stretch-induced ANP release. The results further demonstrate the negative effect of increase in intracellular Ca2+ on stretch-induced ANP release.     

Effects of phosphatidic acid on parathyroid hormone release, intracellular free Ca2+, and inositol phosphates in dispersed bovine parathyroid cells

The observation that increases in extracellular Ca2+ or the addition of divalent cations, such as Ba2+, Mg2+, Mn2+, or Sr2+, stimulate the accumulation of inositol trisphosphate (InsP3) and its breakdown products in parathyroid cells strongly supports the idea that polyphosphoinositides are hydrolyzed under these conditions. Since phosphatidic acid is produced as a result of polyphosphoinositide hydrolysis, and it has been proposed that phosphatidic acid may be a second messenger for Ca2+ mobilization, we examined the effects of this compound on parathyroid cells. We assessed PTH release, intracellular free Ca2+ ([Ca2+]i), and inositol polyphosphate accumulation in response to phosphatidic acid. Natural phosphatidic acid reduced PTH release at 1.0 mM extracellular Ca2+ by 18 +/- 6%, 48 +/- 5%, 59 +/- 10%, and 79 +/- 6% at concentrations of 1, 10, 50, and 100 micrograms/ml, respectively (n = 5-11). The effect was not dependent on the presence of extracellular Ca2+, since phosphatidic acid (100 micrograms/ml) inhibited PTH secretion by 39 +/- 3% in medium with no added Ca2+ and 1.0 mM EGTA (n = 3). This agent rapidly and transiently increased [Ca2+]i in a dose-dependent manner, as determined by fura-2 fluorescence. At 1.0 mM extracellular Ca2+, [Ca2+]i rose from 309 +/- 8 to a peak of 356 +/- 26, 454 +/- 22, and 587 +/- 57 nM with the addition of 1, 10, and 100 micrograms/ml phosphatidic acid, respectively (n = 2-14). In the absence of extracellular Ca2+ (i.e. medium with 1 or 2 mM EGTA and no added Ca2+), phosphatidic acid produced a quantitatively smaller peak increment of 38 +/- 4% in [Ca2+]i, indicating that this compound could mobilize Ca2+ from intracellular stores (n = 3). At 1.0 mM extracellular Ca2+, phosphatidic acid (200 micrograms/ml) stimulated the accumulation of Inositol trisphosphate (InsP3), Inositol bisphosphate (InsP2), and Inositol monophosphate (InsP1) by 46 +/- 9%, 37 +/- 9%, and 59 +/- 11% after 60 sec, respectively (n = 5-7). Phosphatidic acid had no significant effect on forskolin-stimulated cAMP accumulation. We further determined whether the specific fatty acid composition of phosphatidic acid might influence its effects in parathyroid cells by testing several synthetic compounds. Dipalmitoyl phosphatidic acid (greater than or equal to 50 micrograms/ml) inhibited PTH release in a dose-dependent manner without significantly changing [Ca2+]i. Dioleoyl phosphatidic acid had modest biphasic effects on secretion, with 20 +/- 5% inhibition observed at lower doses (10 micrograms/ml) and a 27 +/- 8% stimulation of secretion at 100 micrograms/ml (n = 6).(ABSTRACT TRUNCATED AT 400 WORDS)     

Kallikrein secretion by the isolated perfused rat kidney. Role of perfusion pressure and the renin-angiotensin system

The isolated perfused rat kidney (IPRK) releases kallikrein in urine and renin in perfusate. We have previously shown (Kidney Int 24: 58-65, 1983) and confirm here that kallikrein, as well as renin releases are influenced by changes in renal hemodynamics in this model: a rise in perfusion pressure (PP) from 80 to 98 mmHg increases renal perfusate flow (RPF) by 48 +/- 3 p. 100, inhibits renin release and stimulates kallikrein secretion to 234 +/- 84 p. 100 of control values (n = 8). Since the perfusate lacks angiotensinogen, we decided to study the effect on kallikrein of the reconstitution of the renin-angiotensin system in the IPRK by adding angiotensinogen + angiotensin converting enzyme (AG + ACE) to the perfusion medium. After AG + ACE, PP rose to 107 +/- 4 mmHg, RPF decreased by 82 +/- 3 p. 100 as a consequence of the vasoconstrictor effect of angiotensin II, and renin release was suppressed. Again kallikrein secretion was stimulated and increased to 333 +/- 153 p. 100 of control values (n = 4). It is concluded 1) that kallikrein release is influenced by changes in PP but not in RPF on the IPRK. 2) that reconstitution of the renin-angiotensin system by addition of AG + ACE to the perfusate leads to vasoconstriction, suppression of renin release and a marked increase in kallikrein secretion.