Calcium responses to thyrotropin-releasing hormone, gonadotropin-releasing hormone and somatostatin in phospholipase css3 knockout mice.

These studies examined the importance of phospholipase Cbeta (PLCbeta) in the calcium responses of pituitary cells using PLCbeta3 knockout mice. Pituitary tissue from wild-type mice contained PLCbeta1 and PLCbeta3 but not PLCbeta2 or PLCbeta4. Both Galphaq/11 and Gbetagamma can activate PLCbeta3, whereas only Galphaq/11 activates PLCss1 effectively. In knockout mice, PLCbeta3 was absent, PLCbeta1 was not up-regulated, and PLCbeta2 and PLCbeta4 were not expressed. Since somatostatin inhibited influx of extracellular calcium in pituitary cells from wild-type and PLCbeta3 knockout mice, the somatostatin signal pathway was intact. However, somatostatin failed to increase intracellular calcium in pituitary cells from either wild-type or knockout mice under a variety of conditions, indicating that it did not stimulate PLCbeta3. In contrast, somatostatin increased intracellular calcium in aortic smooth muscle cells from wild-type mice, although it evoked no calcium response in cells from PLCbeta3 knockout animals These results show that somatostatin, like other Gi/Go-linked hormones, can stimulate a calcium transient by activating PLCbeta3 through Gbetagamma, but this response does not normally occur in pituitary cells. The densities of Gi and Go, as well as the relative concentrations of PLCbeta1 and PLCbeta3, were similar in cells that responded to somatostatin with an increase in calcium and pituitary cells. Calcium responses to 1 nM and 1 microM TRH and GnRH were identical in pituitary cells from wild-type and PLCbeta3 knockout mice, as were responses to other Gq-linked agonists. These results show that in pituitary cells, PLCbeta1 is sufficient to transmit signals from Gq-coupled hormones, whereas PLCbeta3 is required for the calcium-mobilizing actions of somatostatin observed in smooth muscle cells.

Genetic alteration of phospholipase C beta3 expression modulates behavioral and cellular responses to mu opioids.

Morphine and other micro opioids regulate a number of intracellular signaling pathways, including the one mediated by phospholipase C (PLC). By studying PLC beta3-deficient mice, we have established a strong link between PLC and mu opioid-mediated responses at both the behavioral and cellular levels. Mice lacking PLC beta3, when compared with the wild type, exhibited up to a 10-fold decrease in the ED(50) value for morphine in producing antinociception. The reduced ED(50) value was unlikely a result of changes in opioid receptor number or affinity because no differences were found in whole-brain B(max) and K(d) values for mu, kappa, and delta opioid receptors between wild-type and PLC beta3-null mice. We also found that opioid regulation of voltage-sensitive Ca(2+) channels in primary sensory neurons (dorsal root ganglion) was different between the two genotypes. Consistent with the behavioral findings, the specific mu agonist [D-Ala(2),(Me)Phe(4),Gly(ol)(5)]enkephalin (DAMGO) induced a greater whole-cell current reduction in a greater proportion of neurons isolated from the PLC beta3-null mice than from the wild type. In addition, reconstitution of recombinant PLC protein back into PLC beta3-deficient dorsal root ganglion neurons reduced DAMGO responses to those of wild-type neurons. In neurons of both genotypes, activation of protein kinase C with phorbol esters markedly reduced DAMGO-mediated Ca(2+) current reduction. These data demonstrate that PLC beta3 constitutes a significant pathway involved in negative modulation of mu opioid responses, perhaps via protein kinase C, and suggests the possibility that differences in opioid sensitivity among individuals could be, in part, because of genetic factors.

Novel fluorescent indicator proteins for monitoring free intracellular Ca2+.

We have recently described a fluorescent indicator protein in which red- and blue-shifted variants of green fluorescent protein are joined by the calmodulin-binding sequence from smooth muscle myosin light chain kinase [Romoser V.A., Hinkle P.M., Persechini A. Detection in living cells of Ca(2+)-dependent changes in the fluorescence of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators. J Biol Chem 1997; 272: 13270-13274]. The fluorescence emission of this protein at 505 nm (380 nm excitation) is reduced by approximately 65% when (Ca2+)4-calmodulin is bound, with a proportional increase in fluorescence emission at 440 nm. We have found that fusion of an engineered calmodulin, in which the C- and N-terminal EF hand pairs have been exchanged, to the C-terminus of this protein results in a novel indicator that responds directly to changes in the Ca2+ ion concentration, with an apparent Kd value of 100 nM for Ca2+ in the presence of 0.5 mM Mg2+. The affinity of the indicator for Ca2+ can be decreased by altering the amino acid sequence of the calmodulin-binding sequence to weaken its interaction with the intrinsic calmodulin domain. The fluorescence emission of this indicator can be used to monitor physiological changes in the free Ca2+ ion concentration in living cells.

Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators.

We have designed a novel fluorescent indicator composed of two green fluorescent protein variants joined by the calmodulin-binding domain from smooth muscle myosin light chain kinase. When (Ca2+)4-calmodulin is bound to the indicator (Kd = 0.4 nM), fluorescence resonance energy transfer between the two fluorophores is attenuated; the ratio of the fluorescence intensity measured at 505 nm to the intensity measured at 440 nm decreases 6-fold. Images of microinjected living cells demonstrate that emission ratios can be used to monitor spatio-temporal changes in the fluorescence of the indicator. Changes in indicator fluorescence in these cells are coupled with no discernible lag (<1 s) to changes in the cytosolic free Ca2+ ion concentration, ranging from below 50 nM to approximately 1 microM. This observation suggests that the activity of a calmodulin target with a typical 1 nM affinity for (Ca2+)4-calmodulin is responsive to changes in the intracellular Ca2+ concentration over the physiological range. It is likely that the indicator we describe can be modified to detect the levels of ligands and proteins in the cell other than calmodulin.