scholarly journals Melanocortin receptor-mediated mobilization of intracellular free calcium in HEK293 cells

2001 ◽  
Vol 5 (1) ◽  
pp. 11-19 ◽  
Author(s):  
KATHLEEN G. MOUNTJOY ◽  
PHILIP L. KONG ◽  
JOHN A. TAYLOR ◽  
DERRIL H. WILLARD ◽  
WILLIAM O. WILKISON
Keyword(s):  
Intracellular Free Calcium ◽  
Melanocortin Receptor ◽  
Hek293 Cells ◽  
Melanocortin Receptors ◽  
Calcium Signal ◽  
Free Calcium ◽  
Agouti Protein ◽  
Transient Rise ◽  
Physiological Relevance ◽  
Free Calcium Concentration

Mouse melanocortin receptors, MC1-R, MC3-R, MC4-R, and MC5-R, when expressed in HEK293 cells and stimulated with either α-melanocyte-stimulating hormone (α-MSH) or desacetyl-α-MSH, mediate increases in intracellular free calcium concentration ([Ca2+]i) with EC50 values between 0.3 and 4.3 nM. The increase in [Ca2+]i is cholera toxin sensitive and pertussis toxin insensitive. The mechanism involves calcium mobilization from intracellular stores without a transient rise in inositol trisphosphate. Mouse agouti protein (55 nM) is a competitive antagonist of α-MSH (6-fold) and desacetyl-α-MSH (8-fold), coupling the mMC1-R to increased [Ca2+]i. Agouti protein (55 nM) significantly increased the EC50 for α-MSH (3-fold), and 550 nM agouti protein significantly increased the EC50 for desacetyl-α-MSH (4-fold), coupling the mMC4-R to a rise in [Ca2+]i. However, agouti protein antagonism of the MC4-R may not be competitive since there was a trend for the maximum response to also increase. There was no significant antagonism of the MC3-R and MC5-R by agouti protein (55 nM). Understanding the physiological relevance of the transduction of a calcium signal by melanocortin peptides may be important for future development of therapeutic targeting of the melanocortin receptors.

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.

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)

Cholinergic effects on intracellular free calcium concentration in renal corpuscle: role of parietal sheet

1992 ◽  
Vol 262 (2) ◽  
pp. F248-F255
Author(s):  
F. Lebrun ◽  
F. Morel ◽  
G. Vassent ◽  
J. Marchetti
Keyword(s):  
Calcium Release ◽  
Intracellular Free Calcium ◽  
Free Calcium ◽  
Response Curves ◽  
Glomerular Function ◽  
Intracellular Free Calcium Concentration ◽  
Free Calcium Concentration ◽  
External Calcium ◽  
Cholinergic Effects

To investigate a possible effect of cholinergic agonists on the renal glomerular function, fura-2 microfluorometric measurements of intracellular free calcium [( Ca2+]i) were performed on single intact glomeruli, single isolated parietal sheets of the Bowman's capsule and single parietal sheet-deprived glomeruli (PS-D glomerulus). Carbachol (10(-4) M), in the presence of 2 mM external calcium, induced a biphasic increase in [Ca2+]i characterized by a sharp initial peak followed by a sustained plateau in the whole glomerulus (delta [Ca2+]i = 177 +/- 13 and 70 +/- 7 nM, respectively; n = 21) and in the parietal sheet (418 +/- 30 and 111 +/- 13 nM, respectively; n = 21). In the PS-D glomerulus (n = 9), the response was less marked and included a barely visible peak (77 +/- 13 nM) and a relatively low plateau (49 +/- 11 nM). In the absence of external calcium, the peak phase was preserved in the three structures, indicating a calcium release from intracellular pools, whereas the plateau, due to the entry of external calcium, was suppressed. These effects were fully inhibited by 10(-4) M of either atropine or pirenzepine, demonstrating the muscarinic nature of the receptors. Dose-response curves showed that the parietal sheet was more sensitive to the physiological agonist (acetylcholine) than to carbachol. A still unexplained difference in sensitivity was noted between peak and plateau, respectively (half-maximal responses were 5 x 10(-6) vs. 5 x 10(-7) M for carbachol and 2 x 10(-7) vs. 3 x 10(-8) M for acetylcholine).(ABSTRACT TRUNCATED AT 250 WORDS)

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