Muscarinic receptors on bovine chromaffin cells mediate a rise in cytosolic calcium that is independent of extracellular calcium

Although the mechanism by which nicotinic receptors on adrenal chromaffin cells regulate catecholamine secretion is reasonably well understood, that of the muscarinic receptors remains obscure. The effects of both acetylcholine and specific muscarinic agonists on cytosolic free calcium in isolated bovine adrenal chromaffin cells have been measured using the fluorescent probe Quin-2. Acetylcholine (0.1 mM) evokes a large increase in cytosolic free calcium from resting levels near 100 nM into the microM range, most of which is blocked by hexamethonium (0.5 mM) or removal of extracellular calcium. A small component of the acetylcholine-evoked rise in cytosolic free calcium (approximately 50-100 nM) is independent of extracellular calcium and is unaffected by 0.5 mM hexamethonium, but is totally blocked by 0.5 microM atropine. The muscarinic nature of this component is further confirmed by the fact that the muscarinic agonists, muscarine (0.1 mM) and methacholine (0.3 mM), stimulate a 50-100 nM rise in chromaffin cell cytosolic calcium which is blocked by 0.5 microM atropine and is largely independent of extracellular calcium. These results suggest that muscarinic receptors regulate cytosolic calcium in chromaffin cells by a new mechanism different from that of nicotinic receptors, a mechanism utilizing an intracellular calcium source. The small size of the muscarinic-induced rise in cytosolic calcium in the bovine chromaffin cell would explain why no secretion is evoked by muscarinic agonists in this species.     

Calcium mobilization and catecholamine secretion in adrenal chromaffin cells. A Quin-2 fluorescence study

To better understand the relation between cell calcium and exocytotic secretion, a quantitative dependence of adrenal catecholamine secretion on cytosolic free calcium has been determined for isolated, intact, bovine chromaffin cells, using the fluorescent probe Quin-2. The cells required a threshold of 250-300 nM cytosolic calcium to be reached before detectable secretion occurred and half-maximal secretion occurred near 2 microM cytosolic calcium. Nicotinic receptors mediated an increase of cytosolic calcium from resting levels near 100 nM to levels in the 1-10 microM range within seconds followed by a decay back to resting levels over several minutes. Muscarinic receptors mediated a smaller rise in cytosolic free calcium from 100 to about 200 nM, within seconds. The nicotinic response required extracellular calcium, while the muscarinic response was largely independent of extracellular calcium, suggesting the latter mobilizes intracellular calcium. The acetylcholine-evoked rise in cytosolic calcium decayed by at least two kinetically distinct processes with half-time constants: t1 = 0.6 min and t2 = 3.2 min. Extracellular Na+ deprivation caused a more prolonged elevation of the acetylcholine-evoked calcium transient, suggesting a possible role of Na+/Ca2+ exchange and/or other Na+ -dependent processes in lowering cytosolic calcium following stimulation. The possible perturbing effects of Quin-2 on resting and stimulated cytosolic calcium levels and on secretion were examined and a novel use of Quin-2 to measure membrane calcium flux was demonstrated.     

In vitro secretion of calcitonin from a rat C cell line: effect of repetitive stimulation with the calcium channel agonist BAY K 8644.

Calcium and BAY K 8644 acutely stimulate calcitonin secretion by influx of extracellular calcium (Ca) through voltage-dependent calcium channels, leading to an increase in cytosolic free Ca. Repetitive exposure to BAY K 8644 (10(-6) M) resulted in an increase in calcitonin (CT) secretion in the rat C-cell line (rMTC 6-23) lasting 9 hours, in comparison to that of 3 mM Ca2+ which lasted 6 hours. Equimolar concentration of nifedipine did not inhibit the stimulatory effect of BAY K 8644 as compared to the nifedipine only group. The decrease in stimulated CT secretion during long-term exposure to BAY K 8644 is due to desensitization of cells which may be attributed to down-regulation of dihydropyridine receptors. After 12 h exposures to 3 mM Ca2+ alone, BAY K 8644 (10(-6) M) alone or in combination with nifedipine (10(-6) M), CT content decreased below the control level, indicating a decrease in synthesis. Overall cellular protein content was not affected by the test agents. Repetitive exposure of C-cells to BAY K 8644 revealed a desensitization of the stimulatory effect on CT secretion and a decrease in CT cell content.     

Biological actions of human and rat calcitonin and calcitonin gene-related peptide

We assessed the central and peripheral biological actions of human and rat calcitonin and calcitonin gene-related peptide (CGRP). After intravenous administration, human and rat calcitonin, but neither human nor rat CGRP significantly decreased plasma calcium and phosphorus concentrations in awake, freely moving rats. After intracerebroventricular as well as after intravenous administration, human and rat calcitonin and human and rat CGRP significantly inhibited gastric acid secretion in conscious rats. Intracerebroventricular administration of rat calcitonin did not alter plasma calcium and phosphorus concentrations. Linear, partially protected CGRP and calcitonin did not exhibit any biological effects. These studies indicate that calcitonin, but not CGRP, affects calcium and phosphorus homeostasis while both peptides decrease gastric acid secretion similarly. Furthermore, these studies support the hypothesis that the calcium and phosphorus lowering effects of calcitonin are peripheral while the gastric inhibiting actions of the calcitonin and CGRP are mediated by the central nervous system.     

Intracellular mediators of potassium-induced aldosterone secretion

We have investigated the intracellular messengers of potassium in eliciting aldosterone secretion in calf adrenal glomerulosa cells since there were unresolved issues relating to the role of phosphoinositides, cAMP and protein kinases. We observed no evidence of hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in 3H-inositol labeled alf adrenal cells or increase of cAMP in response to potassium. Addition of calcium channel blocker, nitrendipine after stimulating adrenal glomerulosa cells with potassium, markedly inhibited aldosterone secretion. A calmodulin inhibitor (W-7) produced greater reduction of aldosterone secretion than an inhibitor of protein kinase C (H-7). These results suggest that a rise in cytosolic free calcium concentration through voltage-dependent calcium channel and calmodulin are the critical determinants of aldosterone secretion stimulated by potassium.     

Phorbol ester stimulates calcitonin secretion synergistically with A23187, and additively with dibutyryl cyclic AMP in a rat C-cell line

The mechanism of action of 12-O-tetradecanoyl phorbol-13-acetate (TPA) on calcitonin secretion was studied in a rat C-cell line, rMTC 6–23. TPA stimulated calcitonin secretion at the concentration of 16nM. This effect was synergistically enhanced with calcium ionophore, A23187. Synthetic diacylglycerol, 1-oleoyl-2-acetyl-glycerol (OAG), also showed a synergism with A23187 on calcitonin secretion. When dibutyryl cyclic AMP was added with TPA, an additive effect was obtained. These data suggest that C-kinase might be a possible regulator of calcitonin secretion in addition to the cyclic AMP-mediated pathway.     

Decrease in glucose-stimulated insulin secretion following exposure to magnetic fields

We evaluated the effects of extremely low frequency magnetic field (ELFMF) on glucose-stimulated insulin secretion from HIT-T15 cells and investigated the mechanisms of these effects. We demonstrated that exposure to ELFMF at 5mT decreased glucose-stimulated insulin secretion by preventing the increases in cellular adenosine 5'-triphosphate/adenosine 5'-diphosphate, membrane depolarization, and cytosolic free calcium ion concentration. The glucose-induced upregulation of insulin mRNA expression was also attenuated by exposure to ELFMF, although cell viability was not affected. These findings demonstrate the potential of exposure to ELFMF for clinical use as a novel inhibitory method of insulin secretion.     

Inhibition of Ca(2+)-induced calcitonin secretion by somatostatin: roles of voltage dependent Ca2+ channels and G-proteins.

Somatostatin has recently been applied therapeutically for hypercalcitonemia in patients with calcitonin-producing tumours. Using calcitonin-secreting cells (C-cells) of the medullary thyroid carcinoma cell line rMTC 44-2, we investigated the inhibitory action of somatostatin on calcitonin release, cytosolic Ca2+ and Ca2+ channel currents. The Ca(2+)-induced rises of the cytosolic Ca2+ and calcitonin secretion were greatly inhibited by somatostatin or its stable analogue octreotide. The effects of somatostatin were pertussis toxin-sensitive. Under voltage clamp conditions, C-cells exhibited slowly inactivating Ca2+ channel currents. Bath application of 100 nM somatostatin reversibly reduced the Ca2+ channel current by about 30%. The Ca2+ channel current and its inhibition by somatostatin were not affected by intracellularly applied cyclic AMP. Moreover, pretreating the cells with pertussis toxin had no effect on the control Ca2+ channel currents but greatly abolished its inhibition by somatostatin. The data show that somatostatin suppresses the Ca(2+)-stimulated calcitonin secretion by inhibiting voltage-dependent Ca2+ channel currents and by lowering cytosolic Ca2+. These actions of somatostatin involve pertussis toxin-sensitive G-proteins and occur independently of changes in the cyclic AMP concentration.     

Correlation between spontaneous oscillations of cytosolic free Ca2+ and tumor necrosis factor-induced degranulation in adherent human neutrophils

By using a hemolytic plaque assay to detect release of lactoferrin and myeloperoxidase, tumor necrosis factor (TNF) was shown previously to induce secretion of these granule proteins from single adherent neutrophils. Secretion was inhibited by loading neutrophils with calcium chelators, indicating a crucial role of cytosolic free [Ca2+] in the signal transduction mechanism of TNF. In the present study, using a microfluorometer technique to follow changes in the cytosolic free [Ca2+] in single adherent neutrophils, we were not able to detect any TNF-induced [Ca2+] transients. However, these adherent cells exhibited spontaneous oscillations of their cytosolic free [Ca2+], as previously reported (Jaconi, M.E.E., Rivest, R.W., Schlegel, W., Wollheim, C.B., Pittet, D., and Lew, P.D. (1988) J. Biol. Chem. 263, 10557-10560). A close correlation was found between a reduced oscillatory activity of cytosolic free [Ca2+] and a reduced ability of TNF to induce degranulation, by reducing the extracellular [Ca2+] or loading the cells with a calcium chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). In addition, when the cells were incubated at 37 degrees C for 3 h there was a parallel decline in the spontaneous oscillatory activity of cytosolic free [Ca2+] and TNF-induced secretion of lactoferrin. Control experiments showed that phorbol 12-myristate 13-acetate-induced secretion was not affected under the same conditions, indicating that the secretory process per se was not disturbed. We conclude that TNF by itself does not give rise to any changes of the cytosolic free [Ca2+] but that the spontaneous oscillatory activity of cytosolic free [Ca2+] in adherent neutrophils is necessary for TNF-induced degranulation.     

The Adenosine Analogue N6-L-Phenylisopropyladenosine Inhibits Catecholamine Secretion from Bovine Adrenal Medulla Cells by Inhibiting Calcium Influx

We reported earlier that adenine nucleotides and adenosine inhibit acetylcholine-induced catecholamine secretion from bovine adrenal medulla chromaffin cells. In this article, we used an adenosine analogue, N6-L-phenylisopropyladenosine (PIA), to study the mechanism underlying inhibition of catecholamine secretion by adenosine. PIA inhibits secretion induced by a nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium, or by elevated external K+. The half-maximal effect on 1,1-dimethyl-4-phenylpiperazinium-induced secretion occurred at approximately 5 x 10(-5) M. The inhibition is immediate and reversible. Fura-2 measurements of cytosolic free Ca2+ indicate that PIA inhibits Ca2+ elevation caused by stimulation; measurements of 45Ca2+ influx show that PIA inhibits uptake of Ca2+. PIA does not inhibit calcium-evoked secretion from digitonin-permeabilized cells, nor does PIA cause any significant change in the dependence of catecholamine secretion on calcium concentration. These data suggest that inhibition by PIA occurs at the level of the voltage-sensitive calcium channel.     

Physiological studies in heterozygous calcium sensing receptor (CaSR) gene-ablated mice confirm that the CaSR regulates calcitonin release <Emphasis Type="Italic">in vivo</Emphasis>

Background  

The calcium sensing receptor (CaSR) regulates serum calcium by suppressing secretion of parathyroid hormone; it also regulates renal tubular calcium excretion. Inactivating mutations of CaSR raise serum calcium and reduce urine calcium excretion. Thyroid C-cells (which make calcitonin) express CaSR and may, therefore, be regulated by it. Since calcium stimulates release of calcitonin, the higher blood calcium caused by inactivation of CaSR should increase serum calcitonin, unless CaSR mutations alter the responsiveness of calcitonin to calcium.     

Effect of hormones on cytosolic free calcium in adipocytes

Some studies have indicated that insulin was able to increase the level of free cytosolic calcium in adipocytes [e.g. 7]. The present study was designed to examine this phenomenon. Insulin did not increase free cytosolic calcium, however oxytocin, vasopressin, alpha-adrenergic agonists and ATP did increase free cytosolic calcium in adipocytes. Other agonists which also did not alter calcium were epidermal growth factor, angiotensin II, glucagon, and beta-adrenergic agonists. The effect of oxytocin at increasing free cytosolic calcium was inhibited by activation of protein kinase C with phorbol 12-myristate 13-acetate and by ADP ribosylation of a Gi like protein with islet activating protein. The hormones that did increase cytosolic free calcium did so by mobilizing internal calcium and by promoting calcium influx. Even though insulin did not increase free cytosolic calcium, it was able to attenuate the alpha-adrenergic mediated increase in cytosolic free calcium. The fact that certain hormones can increase the level of the second messenger calcium in adipocytes implies that it may be a key intracellular regulator of adipocyte function as it is in many other tissues.     

Calcitonin secreting property of ipriflavone in the presence of estrogen

Calcitonin secretion is influenced by estrogen. In the present study, basal and calcium-stimulated serum calcitonin levels were reduced in ovariectomized rats, and replacement estrogen administered for 3 weeks increased both calcitonin levels to those in intact rats. Ipriflavone, 7-isopropoxy-3-phenyl-4H-1-benzopyran-4-one, alone did not influence either calcitonin level. However, ipriflavone and subeffective doses of estrogen administered simultaneously increased both levels; the increase depended upon the dose of ipriflavone. Furthermore, pretreatment with estrone resulted in greater calcitonin release in response to various doses of calcium and pretreatment with estrone and ipriflavone caused an even greater release. These findings indicate that ipriflavone increases the sensitivity of the thyroid gland to estrogen to secrete calcitonin in response to calcium.     

Stimulatory and inhibitory effects of TMB-8 on pancreatic enzyme secretion

The putative intracellular calcium antagonist 3,4,5-trimethoxybenzoate 8-(diethylamino)-octyl ester (TMB-8) affects carbachol-induced enzyme secretion from rabbit pancreatic acini in a different way than it does that induced by either the C-terminal octapeptide of cholecystokinin (CCK-8), the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) or the calcium ionophore, A23187. In the presence of TMB-8 the dose-response curve for carbachol-induced amylase release shifts to the right, suggesting competitive antagonism at the muscarinic receptor. The hypothesis that TMB-8 acts as a muscarinic receptor antagonist is supported by the observation that TMB-8 dose-dependently inhibits the carbachol-, but not CCK-8-induced increases in cytosolic free calcium, measured in acinar cells by means of the fluorescent calcium indicator quin2. At a concentration of 100 microM, TMB-8 maximally potentiates the secretory response to suboptimal, but not (supra)optimal, concentrations of CCK-8. At the same concentration the drug also potentiates TPA- and A23187-induced enzyme secretion. Cytosolic free calcium levels and CCK-8-induced increases in cytosolic free calcium remain unaffected by 100 microM TMB-8. The above results strongly suggest that potentiation occurs at or beyond the site of interaction between the diacylglycerol- and the Ca2+-activated pathways. At concentrations beyond 100 microM the potentiating effect of TMB-8 declines and, finally, at a concentration of 500 microM the drug completely abolishes the secretory response to CCK-8 and TPA. Basal enzyme secretion, however, remains unaffected. At 500 microM severe side effects are observed as is shown by Trypan blue uptake, lactic dehydrogenase release and release of trapped quin2. It is concluded that at lower concentrations TMB-8 does not act as a specific intracellular calcium antagonist in pancreatic enzyme secretion and that inhibitory effects obtained with rather high concentrations of this drug should be treated with caution.     

Serotonin-induced cytosolic free calcium transients in cultured vascular smooth muscle cells

Serotonin induced a transient elevation in the levels of cytosolic calcium in cultured rat vascular smooth muscle cells. Ketanserin, a selective antagonist of serotonin 2 receptors, dose-dependently inhibited the elevation of cytosolic calcium induced by serotonin, and ultimately unmasked a serotonin-induced decrease in the levels of cytosolic calcium. These observations show that serotonin has direct and dual effects, that is, it increases and decreases cytosolic free calcium concentrations in vascular smooth muscle cells, in culture. Knowledge of such events is important because serotonergic inhibitors may prove to be useful drugs for treating clinical hypertension and vasospastic disorders.     

Erythropoietin increases cytosolic free calcium concentration and thrombin induced changes in cytosolic free calcium in platelets from spontaneously hypertensive rats

Using fura-2 cytosolic free calcium concentrations were measured in intact washed platelets from 9 spontaneously hypertensive rats (SHR) and from 9 age-matched normotensive Wistar-Kyoto rats (WKY). In resting platelets cytosolic free calcium concentration was significantly higher in SHR than in WKY (171.8 +/- 64.4 nM vs 93.1 +/- 59.0 nM, p less than 0.05). After preincubation with erythropoietin cytosolic free calcium concentration was significantly higher in SHR than in WKY (197.5 +/- 83.2 vs 93.0 +/- 60.1, p less than 0.01). Using platelets from SHR erythropoietin increased mean resting cytosolic free calcium concentration by 14.9% (p less than 0.05) and mean thrombin induced changes of cytosolic free calcium by 58.3% (p less than 0.01). In contrast, erythropoietin caused no significant increase in the resting calcium concentration or in thrombin induced changes of cytosolic free calcium in platelets from WKY. It is concluded that erythropoietin is involved in the pathogenesis of hypertension by elevating cytosolic free calcium concentration.     

The effects of haloalkene cysteine conjugates on cytosolic free calcium levels in suspensions of rat renal proximal tubules

Disturbances in intracellular calcium homeostasis may play a role in the injury induced by various haloalkene cysteine conjugates. The effects of S-(1,2,3,4,4-pentachloro-1,3-butadienyl)-L-cysteine (PCBC), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) on cytosolic free calcium levels were examined in suspensions of rat renal proximal tubules. Cytosolic free calcium levels, measured with fura 2, in control tubules, were 112 +/- 3 nM and increased more than 200% within 1 minute after exposure to the calcium ionophore ionomycin (0.005 mM). PCBC (0.1 mM) increased cytosolic free calcium levels 18% after 5 minutes, while tubular oxygen consumption was unaffected. DCVC (1 mM) did not alter tubular cytosolic free calcium levels or oxygen consumption under similar conditions. TFEC (1 mM) increased cytosolic free calcium levels 36%, had no effect on basal oxygen consumption, and decreased nystatin-stimulated oxygen consumption 30% after 5 minutes. TFEC increased cytosolic free calcium levels in tubules incubated in a nominally calcium-free buffer but not in a calcium containing buffer in the presence of EGTA. The data suggest that the TFEC-induced increase in cytosolic free calcium levels may result from an influx of extracellular calcium or from inhibition of calcium efflux. The increase in cytosolic free calcium levels preceded changes in basal oxygen consumption in tubules exposed to PCBC and TFEC. This study shows that an increase in cytosolic free calcium levels is an early event following PCBC and TFEC but not DCVC exposure.     

3H]nitrendipine binding to adrenal capsular membranes

The physiologic regulation of aldosterone secretion is dependent on extracellular calcium and appears to be mediated by increases in cytosolic free calcium concentration in the zona glomerulosa cell. A specific role for voltage-dependent calcium channels was suggested by previous studies with the calcium channel antagonist verapamil. We therefore studied the [3H]nitrendipine calcium channel binding site in adrenal capsules. These studies revealed a single class of saturable, high affinity sites with KD = .26 +/- .04 nM and Bmax = 105 +/- 5.7 fmol/mg protein. Specific binding of [3H]nitrendipine was inhibited by calcium channel antagonists with potencies nitrendipine = nifedipine much greater than verapamil, while diltiazem had no inhibitory effect. In the rat, binding sites for [3H]nitrendipine were located in the adrenal capsule and medulla and were undetectable in the zona fasciculata. Physiologic studies with collagenase-dispersed adrenal glomerulosa cells demonstrated that nifedipine selectively inhibited angiotensin-II and potassium-stimulated steroidogenesis. These observations suggest both a pharmacologic and physiologic role for the nitrendipine binding site in aldosterone production.     

Bisphenol A influences the plasma calcium level and inhibits calcitonin secretion in goldfish

In teleosts, it is well known that plasma calcium levels increase as a result of treatment with estrogen for at least during 2 weeks and that calcitonin secretion is induced by estrogen. The present study examined the influence of bisphenol A on calcium homeostasis in goldfish and compared the above known estrogenic action. In goldfish kept in water containing bisphenol A (10(-6) M), the plasma calcium concentration increased significantly (P<0.001) at 4 days but decreased significantly (P<0.05) at 8 days. By the treatment of bisphenol A, calcitonin secretion was not induced until 4 days. At 8 days, however, plasma calcitonin, as well as calcium, decreased significantly (P<0.05), although vitellogenin was detected in the plasma. Therefore, bisphenol A influences plasma calcium levels, but its action is different from that of estrogen, which indicates that bisphenol A affects the calcium homeostasis and might bring about abnormal conditions in teleosts.