Intermediate role of adenosine 3':5'-cyclic monophosphate and protein kinase during gonadotropin-induced steroidogenesis in testicular interstitial cells

1. The serotonergic modulation of pleural sensory neurons in Aplysia is mediated via two second messenger systems: the adenosine cyclic monophosphate/protein kinase A (cAMP/PKA) and diacylglycerol/protein kinase C systems. Often membrane permeable derivatives of cAMP, such as 8-(4-parachlorophenylthio)-cAMP (pcpt-cAMP), have been used to investigate the role of cAMP/PKA in modulating sensory neurons. In light of recent findings that pcpt-cAMP may have cAMP-independent actions, we have reexamined the effects of pcpt-cAMP on the action potential and membrane currents of the sensory neurons. 2. Although pcpt-cAMP (500 microM to 1 mM) and serotonin (5-HT; 10 microM) induced comparable measures of spike broadening (an average increase above baseline of 29 and 40%, respectively), the broadening produced by the two was qualitatively different. Serotonin-induced broadening developed slowly over 9-12 min, was most prominent during later phases of the spike repolarization, and reduced the spike afterhyperpolarization. In contrast, pcpt-cAMP-induced broadening developed rapidly, was rather uniform throughout the repolarization phase of the spike, delayed the peak of the action potential, and increased the afterhyperpolarization. 3. Preexposure of sensory neurons to 5-HT did not occlude further spike broaden by subsequent application of pcpt-cAMP. Indeed the effects of the two were additive. In addition, the effects of pcpt-cAMP were not mimicked by another analogue of cAMP, 8-bromo-cAMP. Interestingly, most of the effects of pcpt-cAMP on the action potential were mimicked by 8-(4-parachlorophenyl-thio)-guanosine cyclic monophosphate (pcpt-cGMP), but not by 8-bromo-cGMP. 4. During voltage-clamp pulses to 20 mV, pcpt-cAMP reduced the membrane current throughout the voltage-clamp pulse, which was qualitatively different from the modulation of the membrane current by 5-HT. In addition, the pcpt-cAMP-induced reduction in the membrane current at the beginning of the pulse was much greater than that induced by 5-HT. Moreover, preexposure of sensory neurons to 5-HT did not occlude further reduction in the membrane current by subsequent application of pcpt-cAMP. 5. These results suggest that pcpt-cAMP has some mechanisms of action that are not shared by 5-HT or cAMP but are shared by pcpt-cGMP. In addition, these findings provide further evidence that results obtained with this compound should be interpreted with caution.

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Effects of cAMP on serotonin evoked calcium transients in cultured rat airway smooth muscle cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.272.5.l865 ◽

1997 ◽

Vol 272 (5) ◽

pp. L865-L871 ◽

Cited By ~ 1

Author(s):

B. Tolloczko ◽

Y. L. Jia ◽

J. G. Martin

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

Airway Smooth Muscle ◽

Airway Smooth Muscle Cells ◽

Intracellular Camp ◽

Dependent Protein Kinase ◽

Cyclic Monophosphate ◽

Dependent Protein ◽

High Concentrations

Agents increasing intracellular adenosine 3',5'-cyclic monophosphate (cAMP) cause relaxation of airway smooth muscle. However, the mechanisms of their action are not fully understood. We investigated the role of cAMP in the modulation of intracellular Ca2+ concentration ([Ca2+]i) transients evoked by serotonin (5-HT) in cultured rat tracheal smooth muscle (TSM) cells. Forskolin (10(-7) M) caused a significant elevation of intracellular cAMP and a 60% relaxation of tracheal rings contracted with 5-HT but did not affect [Ca2+]i in TSM cells. Forskolin (10(-5) M) completely relaxed tracheal rings and significantly decreased [Ca2+]i during the sustained phase of the 5-HT response. Forskolin-induced relaxation was attenuated by the cAMP-dependent protein kinase A (PKA) inhibitor Rp diastereomer of cAMP (Rp-cAMPS; 10(-4) M) and by the guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor [Rp isomer of 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphorothioate, 10(-4) M]. The effects of forskolin on [Ca2+]i were not altered by the PKA inhibitor but were abolished by the PKG inhibitor and thapsigargin. These results indicate that, in rat TSM, the relaxant effects of high concentrations of cAMP may be mediated, at least in part, by facilitating the sequestration of Ca2+ into intracellular stores by a mechanism involving PKG.

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Role of protein kinase A in the regulation of cytosolic free calcium in human osteoblast-like SaOS-2 cells

AJP Cell Physiology ◽

10.1152/ajpcell.1993.264.2.c464 ◽

1993 ◽

Vol 264 (2) ◽

pp. C464-C470 ◽

Cited By ~ 19

Author(s):

S. Fukayama ◽

A. H. Tashjian ◽

F. R. Bringhurst

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Resistant Mutant ◽

Free Calcium ◽

Wild Type ◽

Cyclic Monophosphate ◽

Type Pattern ◽

High Concentrations ◽

Kinase A

We have used wild-type and adenosine 3',5'-cyclic monophosphate (cAMP)-resistant mutant osteoblast-like SaOS-2 cells to investigate the role of protein kinase A (PKA) in the regulation of cytosolic free Ca2+ concentration ([Ca2+]i). Basal levels of [Ca2+]i were the same in wild-type (127 +/- 6.1 nM) and transfected (117 +/- 6.8 nM) SaOS-2 cells, although 45Ca2+ efflux was slower in the transfected cells. In wild-type cells, thapsigargin (TG, > or = 200 nM), an inhibitor of the Ca(2+)-ATPase activity of the endoplasmic reticulum, acutely increased [Ca2+]i (by up to 2-fold), which then returned promptly to basal [Ca2+]i. In cAMP-resistant cells, TG elicited a significantly greater acute rise in [Ca2+]i, which then decayed to an elevated plateau level. In mutant cells, high concentrations of dibutyryladenosine 3',5'-cyclic monophosphate, which overcome the PKA blockade, restored the changes in [Ca2+]i to the wild-type pattern. In cAMP-resistant, TG-blocked cells, ionomycin (or alpha-thrombin) induced a further elevation in [Ca2+]i, which then declined rapidly to the original basal level. We conclude that basal PKA activity is involved actively in regulation of [Ca2+]i in SaOS-2 cells by promoting Ca2+ efflux from the cell and, possibly, by inhibiting Ca2+ release from or stimulating net Ca2+ sequestration into the ER. We have also obtained evidence for an alternate Ca(2+)-triggered Ca2+ reuptake mechanism in SaOS-2 cells that is not dependent on either Ca(2+)-ATPase or PKA.

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