Cytosolic free calcium in normal somatotropes: effects of forskolin and phorbol ester

1989 ◽  
Vol 256 (3) ◽  
pp. E375-E379
Author(s):  
R. W. Holl ◽  
M. O. Thorner ◽  
D. A. Leong
Keyword(s):  
Growth Hormone ◽  
Phorbol Ester ◽  
Plaque Assay ◽  
Extracellular Calcium ◽  
Free Calcium ◽  
Cytosolic Free Calcium ◽  
Gh Secretion ◽  
Free Calcium Concentration ◽  
Cytosolic Free Calcium Concentration ◽  
A Site

Digital imaging microscopy using the calcium-sensitive indicator probe fura-2 was combined with a reverse hemolytic plaque assay (RHPA) for growth hormone (GH) secretion. This technique allows dynamic measurements of the cytosolic free calcium concentration ([Ca2+]i) in individual pituitary somatotropes. Stimulation by growth hormone-releasing factor (GRF) increases, whereas somatostatin (SRIF) reduces [Ca2+]i in this cell type. [Ca2+]i increased in somatotropes when the cellular content of adenosine 3',5'-cyclic monophosphate (cAMP) was elevated by 1) activating cellular adenylate cyclase with forskolin (5 microM) and 2) treatment with the cAMP-analogues dibutyryl-cAMP (1 mM) or 8-bromo-cAMP (5 mM). The forskolin-induced calcium rise was abolished in the absence of extracellular calcium. This indicates that cAMP increases the influx of calcium into the cytosol and thereby stimulates hormone release. When forskolin was given in combination with SRIF (10 nM), [Ca2+]i decreased to the same level reached with SRIF treatment alone, indicating a site of action distal to the generation of cAMP. Activating protein kinase C with the phorbol ester 12,13-phorbol dibutyrate (PDB; 100 nM) increased [Ca2+]i as well. Again, this effect was dependent on extracellular calcium and blocked when PDB and SRIF were applied simultaneously. Combined stimulation with GRF plus PDB did not augment the response of [Ca2+]i over GRF treatment alone.

Platelet Cytosolic Free Calcium in Essential Hypertension: Responses to Vasopressin

1989 ◽  
Vol 77 (2) ◽  
pp. 183-188 ◽  
Author(s):  
A. F. Dominiczak ◽  
J. J. Morton ◽  
G. Murray ◽  
P. F. Semple
Keyword(s):  
Essential Hypertension ◽  
Arginine Vasopressin ◽  
Calcium Concentration ◽  
Systolic Pressure ◽  
Extracellular Calcium ◽  
Free Calcium ◽  
Cytosolic Free Calcium ◽  
Hypertensive Patients ◽  
Free Calcium Concentration ◽  
The Difference

1. Resting and stimulated free calcium concentrations have been measured in platelets loaded with the fluorescent probe quin2 from 30 patients with essential hypertension and from 30 age-matched controls. 2. Cytosolic free calcium concentrations were 94.6 ± 2.7 (mean ± sem) in the hypertensive group and 91.7 ± 2.8 nmol/l in the normotensive group, the difference was not significant. 3. Arginine vasopressin caused a transient increase in platelet free calcium concentration in all subjects. In the presence of extracellular calcium the increase was significantly higher in the control subjects than in the hypertensive patients (P = 0.005). In the absence of extracellular calcium, arginine vasopressin caused much smaller increases, and there was then no difference between the responses of the two groups. 4. Platelet free calcium concentrations were measured again in 13 patients after 8 weeks treatment with either verapamil (n = 6) or atenolol (n = 7). The reductions in systolic pressure after drug treatment were correlated with the changes in cytosolic free calcium concentrations (r = 0.75, P < 0.01).

scholarly journals Intracellular calcium mobilization and activation of the Na+/H+ exchanger in platelets

1993 ◽  
Vol 290 (2) ◽  
pp. 617-622 ◽  
Author(s):  
E Poch ◽  
A Botey ◽  
J Gaya ◽  
A Cases ◽  
F Rivera ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Fluorescent Dyes ◽  
Extracellular Calcium ◽  
Free Calcium ◽  
Regulatory Relationship ◽  
Cytosolic Ph ◽  
Intracellular Calcium Mobilization ◽  
Free Calcium Concentration ◽  
Cytosolic Free Calcium Concentration ◽  
Intracellular Alkalinization

The aim of the present study was to evaluate the regulatory relationship between the cytosolic free calcium concentration ([Ca2+]i and cytosolic pH (pHi). [Ca2+]i and pHi were measured using the fluorescent dyes fura-2 and BCECF [2′,7′-bis-(carboxyethyl)-5,6-carboxyfluorescein] respectively. In a medium with 1 mmol/l extracellular calcium, thrombin (2.5 units/ml) induced an increment in [Ca2+]i of 638 +/- 31 nmol/l (n = 5) and an intracellular alkalinization of 0.14 +/- 0.01 pH units (n = 8). Both responses were dependent on the concentration of thrombin, displaying a sigmoidal dose-response pattern. The intracellular alkalinization was dependent upon extracellular Na+ and was amiloride-sensitive, indicating that it was mediated by activation of the Na+/H+ exchanger. When extracellular calcium was chelated with EGTA prior to the addition of thrombin, the intracellular alkalinization was not affected (0.15 +/- 0.02 at 2.5 units/ml thrombin, n = 8). Under these circumstances, the [Ca2+]i increment represents mobilization from internal stores, reaching 157 +/- 42 nmol/l at 2.5 units/ml thrombin. When platelets were preloaded with the intracellular calcium chelator MAPTAM (1,2-bis-5-methylaminophenoxylethane-NNN'-tetraacetoxymethyl acetate) to block the increase in [Ca2+]i induced by thrombin, no increment in pHi was observed. Moreover, MAPTAM-loaded calcium-depleted platelets had a basal pHi that was more acidic than in the presence of 1 mmol/l extracellular calcium (6.93 +/- 0.09 versus 7.14 +/- 0.01, n = 26, P < 0.001). Ionomycin induced an elevation of [Ca2+]i that was accompanied by a concomitant increase in pHi, which was Na(+)-dependent and amiloride-sensitive. [Ca2+]i and pHi increases induced by ionomycin were both dependent on the concentration of ionomycin. In conclusion, an increase in [Ca2+]i is necessary for the agonist-induced activation of the Na+/H+ exchanger in platelets. Non-agonist-induced increases in [Ca2+]i seems to prompt activation of the exchanger. In addition, Ca(2+)-depleted platelets have a more acidic basal pHi, indicating that the basal level of [Ca2+]i is also important for maintaining the basal pHi.

Angiotensin II causes sustained elevations in cytosolic calcium in glomerulosa cells

1988 ◽  
Vol 255 (3) ◽  
pp. E338-E346 ◽  
Author(s):  
R. E. Kramer
Keyword(s):  
Angiotensin Ii ◽  
Calcium Concentration ◽  
Cytosolic Calcium ◽  
Calcium Signal ◽  
Free Calcium ◽  
Cytosolic Free Calcium ◽  
Cytosolic Calcium Concentration ◽  
Free Calcium Concentration ◽  
Cytosolic Free Calcium Concentration ◽  
Glomerulosa Cells

Studies were conducted to examine the effects of angiotensin II on cytosolic free calcium concentration in bovine adrenal glomerulosa cells maintained in primary culture. The calcium indicator, fura-2, and discontinuous dual-wavelength fluorescence spectroscopy were used to measure cytosolic free calcium in superfused adherent cell monolayers. Basal cytosolic free calcium concentration was 63.7 +/- 3.3 nM. The threshold concentration for angiotensin II-stimulated increases in cytosolic calcium was 10(-14)-10(-13) M, and maximal elevation of cytosolic calcium was produced by 10(-9) M angiotensin II. Angiotensin II (10(-13) M) produced a gradual increase in cytosolic calcium concentration that plateaued after 3-5 min of superfusion at a level approximately 1.2 times that of control cells. The calcium signal invoked by a maximal concentration (10(-9) M) of angiotensin II, in contrast, was characterized by an immediate, intense (approximately 8-fold) increase in cytosolic calcium concentration that decayed within 5 min to a lower, but sustained, level 2.5-3 times that of control cells. The calcium signals invoked by intermediate concentrations (10(-12)-10(-10) M) of angiotensin II exhibited dose-dependent increases in magnitude and a gradual transition in nature between those invoked by threshold and maximal concentrations of the peptide. The effect of angiotensin II to increase cytosolic calcium concentration was accompanied by an increase in aldosterone output. The increase in steroidogenesis was most closely correlated with the magnitude of the initial calcium signal. At high concentrations (10(-10) and 10(-9) M) of angiotensin II, there was a clear dissociation between aldosterone output and the magnitude of the sustained calcium signal.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasopressin increases cytosolic free calcium in LLC-PK1 cells through a V1-receptor

1987 ◽  
Vol 253 (2) ◽  
pp. F328-F332 ◽  
Author(s):  
M. A. Burnatowska-Hledin ◽  
W. S. Spielman
Keyword(s):  
Parathyroid Hormone ◽  
Arginine Vasopressin ◽  
Calcium Concentration ◽  
Mdck Cells ◽  
Free Calcium ◽  
Base Line ◽  
Cytosolic Free Calcium ◽  
Free Calcium Concentration ◽  
Cytosolic Free Calcium Concentration ◽  
Dose Dependent

We examined the effects of arginine vasopressin (AVP), parathyroid hormone (PTH), and bradykinin (BK) on the cytosolic free calcium concentration ([Ca]i) in cultured LLC-PK1 and MDCK kidney cell lines by use of the fluorescent Ca chelator fura-2. In LLC-PK1 cells, the addition of AVP but not [1-desamino-8-D-arginine]vasopressin (dDAVP, V2 agonist), PTH, or BK (10(-6) M) caused a significant increase in [Ca]i. The AVP-induced increase in [Ca]i from 61 +/- 6 to 225 +/- 44 nM (n = 7, P less than 0.01) was rapid and transient, returning to base line in 2 to 3 min. The effect of AVP was dose dependent and was present at 1 (61% increase) but not 5 min after extracellular Ca was removed. The effect of 10(-6) M AVP could be blocked with the pressor (V1) antagonist, d(CH2)5Tyr(Me)AVP, but not dDAVP. In MDCK cells, BK, but not AVP and PTH, increased [Ca]i from 146 +/- 11 to 281 +/- 31 nM (n = 9, P less than 0.001). The removal of extracellular Ca (5 min), reduced but did not abolish this effect. These results indicate that [Ca]i mobilized by activation of V1-receptors may mediate AVP-regulated function in some transporting epithelia.

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