The somatotrope: an endocrine cell with functional calcium transients

1988 ◽  
Vol 139 (1) ◽  
pp. 169-179
Author(s):  
M. O. Thorner ◽  
R. W. Holl ◽  
D. A. Leong
Keyword(s):  
Intracellular Calcium ◽  
Calcium Concentration ◽  
Hormone Secretion ◽  
Extracellular Calcium ◽  
Calcium Oscillations ◽  
Calcium Transients ◽  
Male Rats ◽  
Free Calcium ◽  
Cytosolic Free Calcium ◽  
Gh Secretion

Growth hormone (GH) secretion by the somatotrope is under dual regulation by the hypothalamic peptides, somatostatin (SS) and GH-releasing hormone (GHRH). Cytosolic free calcium concentration and cumulative GH release were measured simultaneously in anterior pituitary cells from adult male rats. This was made possible using a combination of digital imaging video microscopy with the fluorescent calcium indicator Fura-2 and the reverse haemolytic plaque assay (RHPA) to identify the cell type and measure hormone secretion from the cells under study. This technique allows calcium measurements to be made at very short time intervals (less than 150 ms) in single cells. Spontaneous calcium transients were demonstrated in 85% of GH plaque-forming cells. These occurred at a frequency of 2–13 min-1 and had an amplitude of 50–500 nmoll-1. The somatotropes with the largest calcium fluctuations produced the largest plaques; thus, the calcium transients appeared to correlate with hormone release. Since the somatotrope alone shows these fluctuations, the mean intracellular calcium concentration is 238 +/− 18 nmoll-1 in somatotropes and 113 +/− 8 nmoll-1 in non-somatotropes. Upon exposure to SS (1 nmoll-1) intracellular calcium fell from 200–250 nmoll-1 to 50–100 nmoll-1 with an apparent reduction in oscillations. Withdrawal of SS increased the intracellular calcium level. GHRH increased intracellular calcium but 10 nmoll-1 GHRH given simultaneously with 1 nmoll-1 SS reduced intracellular calcium to that level observed during SS alone. Thus, the SS effect on intracellular calcium predominates. The effects of SS can be mimicked by removal of extracellular calcium, or by the addition of CoCl2 (2 nmoll-1) or by verapamil (100 mumoll-1), two agents which block calcium channels. The hormone secretion index (indicated by the area of the plaque formed in RHPA) enables us to demonstrate that GHRH in this system increases GH secretion, and SS inhibits it. In combination, GHRH and SS oppose one another. Spontaneous calcium oscillations are characteristic for normal somatotropes. These oscillations are related to spontaneous hormone secretion and due to influx of calcium through ion channels in the membrane. Intracellular signalling information may be encoded in both frequency and amplitude of calcium oscillations. The actions of GHRH and SS on regulation of GH secretion are proposed to be mediated, at least in part, by regulation of intracellular cytosolic free calcium. This modulation is dependent on extracellular calcium concentrations. We are now investigating the molecular mechanisms involved in this process.

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).

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.

V2-like vasopressin receptor mobilizes intracellular Ca2+ in rat medullary collecting tubules

1993 ◽  
Vol 265 (1) ◽  
pp. F35-F45 ◽  
Author(s):  
A. Champigneulle ◽  
E. Siga ◽  
G. Vassent ◽  
M. Imbert-Teboul
Keyword(s):  
Intracellular Calcium ◽  
Calcium Concentration ◽  
Free Calcium ◽  
Cytosolic Free Calcium ◽  
Free Calcium Concentration ◽  
Cytosolic Free Calcium Concentration ◽  
The Mean ◽  
V2 Antagonist ◽  
Collecting Tubules ◽  
Calcium Pool

Cytosolic free calcium concentration ([Ca2+]i) was measured in single microdissected rat medullary collecting tubules [outer (OMCD) and inner (IMCD)] to identify receptors involved in vasopressin (AVP)-induced [Ca2+]i increases. In both segments, [Phe2,Orn8]vasotocin ([Phe2,Orn8]VT), a specific V1 agonist, as well as the V2 agonist 1-desamino-8-D-AVP (dDAVP) triggered [Ca2+]i variations. In OMCD, the mean response to 10 nM AVP roughly corresponded to the sum of V1 and V2 agonists effects. In IMCD, dDAVP (10 nM) alone reproduced the calcium response to AVP (delta[Ca2+]i = 243 +/- 34 nM, n = 6, and 248 +/- 27 nM, n = 8, with dDAVP and AVP, respectively). Furthermore, in the same experiments V1 and V2 maximal effects were not additive ([Phe2,Orn8]VT = 154 +/- 21 nM, n = 6; dDAVP + [Phe2,Orn8]VT = 233 +/- 23 nM, n = 9). As AVP, dDAVP released intracellular calcium (delta[Ca2+]i in calcium-free medium = 182 +/- 24 nM, n = 8, vs. 182 +/- 14 nM, n = 6 with 10 nM dDAVP and AVP, respectively). Neither 8-(4-chlorophenyl-thio)-adenosine 3',5'-cyclic monophosphate nor forskolin modified [Ca2+]i. A cross-reaction of dDAVP with an oxytocin (OT) receptor can be excluded since 1) the specific OT agonist [Thr4,Gly7]OT (10 nM) increased only slightly [Ca2+]i (delta-[Ca2+]i = 20 +/- 5 nM, n = 11); 2) the dDAVP response was not altered by the specific OT antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylene propionic acid),2-(O-methyl)tyrosine,4-threonine, 8-ornithine,9-tyrosylamide]vasotocin [d(CH2)5(1),O-Me-Tyr2,Thr4,Tyr-NH2(9)]OVT; 3) it was insensitive to V1 antagonists but was totally blocked by the V1/V2 antagonist [d(CH2)5(1),O-Et-Tyr2,Val4]AVP ([delta[Ca2+]i = 18 +/- 4 nM, n = 6). These results indicate that in IMCD AVP increases [Ca2+]i via both V1 and V2 receptors. [Ca2+]i variations due to V2 receptors involve a mechanism independent of adenylate cyclase and coupled to the same intracellular calcium pool as V1 and V2 receptors.

Intracellular calcium and phospholipid turnover are not involved in the inhibition of iodothyronine 5'-deiodinase type II activity by T4

1991 ◽  
Vol 124 (1) ◽  
pp. 67-75
Author(s):  
Cristiana Juge-Aubry ◽  
Pierre Dôme ◽  
Catherine A. Siegrist-Kaiser ◽  
Alessandro M. Capponi ◽  
Albert G. Burger
Keyword(s):  
Intracellular Calcium ◽  
Calcium Concentration ◽  
Enzyme Inactivation ◽  
Free Calcium ◽  
Type Ii ◽  
Cytosolic Free Calcium ◽  
Experimental Conditions ◽  
Phospholipid Turnover ◽  
Free Calcium Concentration ◽  
Cytosolic Free Calcium Concentration

Abstract. In glial cell cultures, iodothyronine 5'-deiodinase type II is stimulated by dibutyryl cAMP. Serum-free medium increases enzyme activity and prolongs the half-life of the enzyme. T4 and rT3 specifically inhibit this activity. We tested whether enzyme inactivation by T4 was mediated by changes in cytosolic free calcium concentration and/or phospholipid turnover. Intracellular calcium concentration was decreased either by chelation of extracellular calcium or by chelation of extracellular and intracellular calcium. Neither basal hypothyroid 5'-deiodinase activity nor its inactivation by T4 were modified in such experimental conditions, compared with control cells incubated in normal calcium-containing medium. T4 by itself had no effect on the cytosolic free calcium concentration for up to 20 min. Studies on phospholipid turnover included norepinephrine in parallel to T4 as positive stimulation control. While norepinephrine clearly accelerated phosphoinositide turnover, there was no effect of T4 on any phospholipid turnover. These results suggest that neither cytosolic free calcium nor phospholipid turnover is involved in T4-dependent modulation of 5'-deiodinase type II activity in astrocytes in culture.

189 CHARACTERIZATION OF THE FIRST SPERM-INDUCED CALCIUM TRANSIENT IN PIG OOCYTES

2016 ◽  
Vol 28 (2) ◽  
pp. 225
Author(s):  
C. Wang ◽  
Z. Machaty
Keyword(s):  
Calcium Concentration ◽  
Calcium Transient ◽  
Calcium Oscillations ◽  
Calcium Transients ◽  
Intracellular Free Calcium ◽  
Calcium Signal ◽  
Free Calcium ◽  
Essential Information ◽  
Intracellular Free Calcium Concentration ◽  
Free Calcium Concentration

Fertilization in mammals is associated with repetitive elevations in the oocytes’ intracellular free calcium concentration. The elevations are triggered by the fertilizing sperm and are responsible for stimulating embryo development. In mouse oocytes, the sperm-induced calcium signal starts with a calcium rise that is larger and longer in duration than any succeeding transients. It also has unique characteristics: it begins with a rapid increase for 2–3 s followed by a shoulder, which is an inflection point that represents a brief decline in the rise of calcium levels. Once calcium level reaches its maximum, it decreases but remains elevated for several minutes while it is superimposed by several smaller calcium spikes. In bovine oocytes the situation is somewhat different. In this species, the first sperm-induced calcium transient is larger than the additional spikes but it lacks the sustained elevation phase and is not superimposed by small calcium rises. In the present study our purpose was to characterise the first sperm-induced calcium transient in pig oocytes. Oocytes were obtained from ovaries of prepubertal gilts collected at an abattoir and matured in vitro for 44 h. Mature oocytes were loaded with the calcium indicator dye fura-2; subsequently, they were either IVF or used for intracytoplasmic sperm injection (ICSI). Changes in their intracellular free calcium concentration were then immediately monitored using InCyt Im2, a dual-wavelength fluorescence imaging system. Characteristics of the first transients (including amplitude and duration) were compared to those of the additional ones using Student’s t-test. We found that in oocytes that underwent IVF (n = 11), the oscillations started 83.4 ± 23.2 min after adding the sperm to the oocytes. In the ICSI group (n = 10 oocytes) the calcium oscillations started sooner, 27.1 ± 17.7 min after injection. The average peak amplitude and the mean interval between the calcium transients varied among individual oocytes, but no significant differences were found between the IVF and ICSI groups (which on average were fluorescence ratio of 2.6 ± 1.1 and 23.5 ± 11.4 min, respectively; P > 0.1). The oscillation patterns showed slight differences between individual oocytes in terms of spike frequency, which has been described before and may be due to variations in the amount of sperm-derived activating factor present in the ooplasm. Most importantly, in all oocytes measured, the initial calcium spike showed no differences when compared to subsequent calcium transients: its amplitude and duration was similar to the additional transients. This points at potential species-specific differences in the regulation of calcium signalling in oocytes and provides essential information for the better understanding of the fertilization process. This work was supported by Agriculture and Food Research Initiative Competitive Grant 2011–67015–30006 from the USDA National Institute of Food and Agriculture.

Differential effects of passive immunization with somatostatin antiserum on adenohypophysial hormone secretions in starved rats

1986 ◽  
Vol 109 (2) ◽  
pp. 169-174 ◽  
Author(s):  
J. N. Hugues ◽  
A. Enjalbert ◽  
E. Moyse ◽  
C. Shu ◽  
M. J. Voirol ◽  
...  
Keyword(s):  
Binding Capacity ◽  
Hormone Secretion ◽  
Plasma Concentrations ◽  
Passive Immunization ◽  
Negative Control ◽  
Prolactin Secretion ◽  
Male Rats ◽  
Minimal Effective Dose ◽  
Gh Secretion

ABSTRACT The role of somatostatin (SRIF) on adenohypophysial hormone secretion in starved rats was reassessed by passive immunization. Because of the absence of pulsatile GH secretion in starved rats, the effects of the injection of SRIF antiserum on GH levels can be clearly demonstrated. To determine whether starvation modifies the sensitivity of the adenohypophysis to SRIF, we measured 125I-labelled iodo-N-Tyr-SRIF binding. There was no difference in the dissociation constant (Kd) nor in the maximal binding capacity (Bmax) in fed (n = 15) and starved (n = 15) animals (Kd = 0·38 ± 0·09 (s.e.m.) and 0·45 ± 0·09 nmol; Bmax = 204 ± 39 and 205 ± 30 fmol/mg protein respectively). Administration of SRIF antiserum resulted in a dose-dependent increase in plasma concentrations of GH, TSH and prolactin. The minimal effective dose of SRIF antiserum was 50 μl for GH, 100 μl for TSH and 200 μl for prolactin. Our results show that: (1) starvation does not modify adenohypophysial SRIF-binding sites, (2) in starved male rats endogenous SRIF exerts a negative control on prolactin secretion in vivo and (3) sensitivity to endogenous SRIF seems to be different for each hypophysial cell type. J. Endocr. (1986) 109, 169–174

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