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An important focus in cell biology is understanding how different feedback mechanisms regulate G protein-coupled receptor systems. Toward this end we investigated the regulation of endogenous [[beta].sub.2] adrenergic receptors ([beta]2ARs) and phosphodiesterases (PDEs) by measuring cAMP signals in single HEK-293 cells. We monitored cAMP signals using genetically encoded cyclic nucleotide-gated (CNG) channels. This high resolution approach allowed us to make several observations. (a) Exposure of cells to 1 pM isoproterenol triggered transient increases in cAMP levels near the plasma membrane. Pretreatment of cells with 10 pM rolipram, a PDE4 inhibitor, prevented the decline in the isoproterenol-induced cAMP signals. (b) 1 tiM isoproterenol triggered a sustained, twofold increase in phosphodiesterase type 4 (PDE4) activity. (c) The decline in isoproterenol-dependent cAMP levels was not significantly altered by including 20 nM PKI, a PKA inhibitor, or 3 [micro]M 59-74E, a GRK inhibitor, in the pipette solution; however, the decline in the cAMP levels was prevented when both PKI and 59-74E were included in the pipette solution. (d) After an initial 5-min stimulation with isoproterenol and a 5-min washout, little or no recovery of the signal was observed during a second 5-min stimulation with isoproterenol. (e) The amplitude of the signal in response to the second isoproterenol stimulation was not altered when PKI was included in the pipette solution, but was significantly increased when 59-74E was included. Taken together, these data indicate that either GRK-mediated desensitization of [beta]2ARs or PKA-mediated stimulation of PDE4 activity is sufficient to cause declines in cAMP signals. In addition, the data indicate that GRK-mediated desensitization is primarily responsible for a sustained suppression of [beta]2AR signaling. To better understand the interplay between receptor desensitization and PDE4 activity in controlling cAMP signals, we developed a mathematical model of this system. Simulations of cAMP signals using this model are consistent with the experimental data and demonstrate the importance of receptor levels, receptor desensitization, basal adenylyl cyclase activity, and regulation of PDE activity in controlling cAMP signals, and hence, on the overall sensitivity of the system.

Many studies of [Ca.sup.2+] signaling use PC12 cells, yet the balance of [Ca.sup.2+] clearance mechanisms ill these cells is unknown. We used pharmacological inhibition of [Ca.sup.2+] transporters to characterize [Ca.sup.2+] clearance after depolarizations in both undifferentiated and nerve growth factor-differentiated PC12 cells. Sarco-endoplasmic reticulum [Ca.sup.2+] ATPase (SERCA), plasma membrane [Ca.sup.2+] ATPase (PMCA), and [Na.sup.+]/[Ca.sup.2+] exchanger (NCX) account for almost all [Ca.sup.2+] clearance in both cell states, with NCX and PMCA making the greatest contributions. Any contribution of mitochondrial uniporters is small. The ATP pool in differentiated cells was much more labile than that of undifferentiated cells in the presence of agents that dissipated mitochondrial proton gradients. Differentiated PC12 cells have a small component of [Ca.sup.2+] clearance possessing pharmacological characteristics consistent with secretory pathway [Ca.sup.2+] ATPase (SPCA), potentially residing on Golgi and/or secretory granules. Undifferentiated and differentiated cells are similar in overall [Ca.sup.2+] transport and in the small transport due to SERCA, but they differ in the fraction of transport by PMCA and NCX. Transport in neurites of differentiated PC 12 cells was qualitatively similar to that in the somata, except that the ER stores in neurites sometimes released [Ca.sup.2+] instead of clearing it after depolarization. We formulated a mathematical model to simulate the observed [Ca.sup.2+] clearance and to describe the differences between these undifferentiated and NGF-differentiated states quantitatively. The model required a value for the endogenous [Ca.sup.2+] binding ratio of PC 12 cell cytoplasm, which we measured to be 268 [+ or -] 85. Our results indicate that [Ca.sup.2+] transport in undifferentiated PC12 cells is quite unlike transport ill adrenal chromaffin cells, for which they often are considered models. Transport in both cell states more closely resembles that of sympathetic neurons, for which differentiated PC12 cells often are considered models. Comparison with other cell types shows that different cells emphasize different [Ca.sup.2+] transport mechanisms.

For over a decade, phosducin's interaction with the [beta][gamma]subunits of the G protein, transducin, has been thought to contribute to light adaptation by dynamically controlling the amount of transducin heterotrimer available for activation by photoexcited rhodopsin. In this study we directly tested this hypothesis by characterizing the dark- and light-adapted response properties of phosducin knockout (Pd-/-) rods. Pd-/- rods were notably less sensitive to light than wild-type (WT) rods. The gain of transduction, as measured by the amplification constant using the Lamb-Pugh model of activation, was 35% lower in Pd-/- rods than in WT rods. This reduced amplification correlated with a 36% reduction in the level of transducin [beta][gamma]-subunit expression, and thus available heterotrimer in Pd-/- rods. However, commonly studied forms of light adaptation were normal in the absence of phosducin. Thus, phosducin does not appear to contribute to adaptation mechanisms of the outer segment by dynamically controlling heterotrimer availability, but rather is necessary for maintaining normal transducin expression and therefore normal flash sensitivity in rods.

Ghosts developed from human red blood cells were studied to explore the link between K-Cl retransport activity and ATP. ATP enhances K-Cl cotransport activity in swollen ghosts. This enhancement most probably results from the phosphorylation of some membrane component, as it needs Mg. Neither phosphrylation nor dephosphorylation nor any other known messenger are concerned with signal transduction between the cell volume sensor and the cotransporter, although both these processes alter the activity of the cotransproter in shrunken and swollen ghosts.