Effects of pH on murine insulinoma betaTC3 cells.

Confluent monolayer cultures of betaTC3 cells were exposed for 4 h to acidic, neutral, or alkaline pH media. Studies determined the impact of pH on viability, insulin secretion rate, glucose consumption rate, lactate production rate, and ATP content. Cell viability was not affected by exposure to media of different pH (>95% for all groups). Insulin release from cells exposed to acidic media (pH of 6.4) was approximately 75% higher than that from cells exposed to either neutral (pH of 7.1) or alkaline (pH of 7.8) conditions. Conversely, ATP content was significantly reduced in cultures exposed to acidic conditions, although there was no statistical difference between neutral and alkaline conditions. Glucose consumption and lactate production rates increased linearly with increasing pH.

SP1/SP3-binding sites and adjacent elements contribute to basal and cyclic adenosine 3',5'-monophosphate-stimulated transcriptional activation of the rhesus growth hormone-variant gene in trophoblasts.

Transcriptional activation of the rhesus monkey GH-variant gene in syncytiotrophoblasts is developmentally regulated by trophoblast-specific and cAMP-responsive mechanisms. Progressive deletions of 5'-flanking DNA defined the most proximal 140 bp as the minimal region retaining full cAMP-stimulated mGH-V transcription. To identify the regions of this promoter critical for transcription, transient transfections of reporter plasmids containing systematic 10 base mutations throughout this proximal region were performed. Mutation of the region from -140/-131 decreased transcription in syncytiotrophoblasts by 50%, and gel mobility-shift analyses demonstrated that Sp1 and Sp3 bound to a region containing a GGGAGG motif at -136/-131. Mutation of the -62/-53 region decreased transcriptional activation by 66-99%, and Sp1 and Sp3 bound to a GGTGGG motif overlapping this region (at -65/-60). Selective mutation of this Sp1/Sp3 site decreased basal transcription by approximately 80%, and cAMP-stimulated transcription by up to 75% (with the greatest effect in primary syncytiotrophoblast cultures), indicating that the Sp1/Sp3 site is critical for transcriptional activation. Mutations in the regions adjacent to the Sp1/Sp3 sites (-130/-111 and -52/-43) also dramatically reduced (by 75%) transcriptional activation in trophoblasts. We conclude that two Sp1/Sp3 sites as well as additional elements directly adjacent to these sites contribute to trophoblast-specific cAMP-responsiveness of the mGH-V proximal promoter.

Molecular orbital calculations of proton transfer energetics in model systems: the interaction of opiate agonists and antagonists with opiate receptors.

Semiempirical (CNDO) molecular orbital calculations, based on a previously investigated morphine-receptor clastic-binding system, were performed using a series of ethyl and propyl amines as models for the analgesic receptor. Trimethyl and dimethyl amines were chosen to represent the opiate and noropiate agonist molecules. The opiate antagonist molecules, levallorphan, naloxone, nalorphine, and pentazocine were represented by a series of allyl and dimethylallyl amines. The results using these systems paralleled those of our previous investigations. The potential energy curves for all the systems studied had two minima at an internuclear distance of greater than 0.275 nm. At 0.2731 nm (the optimized N-N distance), the potential energy curve of some systems had single minima. The agonist systems studied had optimum energy curves for the conformer in which the (drug nitrogen-hydrogen) bond is equatorial. The noropiate model had the greatest transfer potential, an optimal condition for analgesic activity. The antagonist drug receptor models had energy curves that indicated a decreased proton transfer barrier that was similar to the noropiate case (although no delta E was obtained). The two largest models studied had energy curves that indicated decreased or inhibited proton transfer. The systems investigated had small deprotonation barriers, indicating deprotonation would most likely occur following protonation of the receptor.

Molecular orbital calculations of proton transfer involving amines as models for the clastic binding of opiates with their receptor.

Semi-empirical (CNDO) molecular orbital calculations, based on a previously reported ammonia-amine model system, were performed on an extended series of methyl-, ethyl-, and propylamines as models for the analgesic receptor. Methyl-, dimethyl-, and trimethylamines were chosen to represent the opiate molecules. Interatomic distances were varied within normally expected biological values. The results for the larger systems are similar to more elaborate calculations previously reported using smaller molecules. At internuclear distances of greater than 0.275 nm, the potential energy curves had two minima. At 0.2731 nm, the optimized N-N distance, the "depth" of the minima in the potential energy curve were not as great. Energy differences as well as population differences suggest deviation from the currently stated clastic binding theories mechanism for the analgesic response of the tertiary amines. The dimethylamine energy profile and population data indicate that the hypothesis of N-demethylated opiate as the active molecule needs further consideration and investigation. Investigation of larger systems is also indicated to develop increasingly realistic models for the analgesic response.