The differential expression of the $\beta\sb2$-adrenergic receptor modifies agonist-dependent adenylylcyclase activation

There have been multiple reports which indicate that variations in $\beta$AR expression affect the V$\sb{\rm max}$ observed for the agonist-dependent activation of adenylylcyclase. This observation has been ignored by most researchers when V$\sb{\rm max}$ values obtained for wild type and mutant receptors are compared. Such an imprecise analysis may lead to erroneous conclusions concerning the ability of a receptor to activate adenylylcyclase. Equations were derived from the Cassel-Selinger model of GTPase activity and Tolkovsky and Levitzki's Collision Coupling model which predict that the EC$\sb{50}$ and V$\sb{\rm max}$ for the activation of adenylylcyclase are a function of receptor number. Experimental results for L cell clones in which either hamster or human $\beta$AR were transfected at varying levels showed that EC$\sb{50}$ decreases and V$\sb{\rm max}$ increases as receptor number increases. Comparison of these results with simulations obtained from the equations describing EC$\sb{50}$ and V$\sb{\rm max}$ showed a close correlation. This documents that the kinetic parameters of adenylylcyclase activation change with the level of receptor expression and relates this phenomenon to a theoretical framework concerning the mechanisms involved in $\beta$AR signal transduction.^ One of the terms used in the equations which expressed the EC$\sb{50}$ and V$\sb{\rm max}$ as a function of receptor number is coupling efficiency, defined as $\rm k\sb1/k\sb{-1}$. Calculation of $\rm k\sb1/k\sb{-1}$ can be accomplished for wild type receptors with the easily measured experimental values of agonist K$\sb{\rm d}$, EC$\sb{50}$ and receptor number. This was demonstrated for hamster $\beta$AR which yielded a coupling efficiency of 0.15 $\pm$ 0.003 and human $\beta$AR which yielded a coupling efficiency of 0.90 $\pm$ 0.031. $\rm k\sb1/k\sb{-1}$ replaces the traditional qualitative evaluation of the ability to activate adenylylcyclase, which utilizes V$\sb{\rm max}$ without correction for variation in receptor number, with a quantitative definition that more accurately describes the ability of $\beta$AR to couple to G$\sb{\rm s}$.^ The equations which express the EC$\sb{50}$ and V$\sb{\rm max}$ for adenylylcyclase activation as a function of receptor number and coupling efficiency were tested to determine whether they could accurately simulate the changes seen in these parameters during desensitization. Data from original desensitization experiments and data from the literature (24,25,52,54,83) were compared to simulated changes in EC$\sb{50}$ and V$\sb{\rm max}$. In a variety of systems the predictions of the equations were consistent with the changes observed in EC$\sb{50}$ and V$\sb{\rm max}$. In addition reductions in the calculated value of $\rm k\sb1/k\sb{-1}$ was shown to correlate well with $\beta$AR phosphorylation and to be minimally affected by sequestration and down-regulation. ^

Fluorophotometry is a sensitive method/technique to evaluate epithelial permeability in murine experimental dry eye

Purpose. Fluorophotometry is a well validated method for assessing corneal permeability in human subjects. However, with the growing importance of basic science animal research in ophthalmology, fluorophotometry’s use in animals must be further evaluated. The purpose of this study was to evaluate corneal epithelial permeability following desiccating stress using the modified Fluorotron Master™. ^ Methods. Corneal permeability was evaluated prior to and after subjecting 6-8 week old C57BL/6 mice to experimental dry eye (EDE) for 2 and 5 days (n=9/time point). Untreated mice served as controls. Ten microliters of 0.001% sodium fluorescein (NaF) were instilled topically into each mouse’s left eye to create an eye bath, and left to permeate for 3 minutes. The eye bath was followed by a generous wash with Buffered Saline Solution (BSS) and alignment with the Fluorotron Master™. Seven corneal scans using the Fluorotron Master were performed during 15 minutes (1 st post-wash scans), followed by a second wash using BSS and another set of five corneal scans (2nd post-wash scans) during the next 15 minutes. Corneal permeability was calculated using data calculated with the FM™ Mouse software. ^ Results. When comparing the difference between the Post wash #1 scans within the group and the Post wash #2 scans within the group using a repeated measurement design, there was a statistical difference in the corneal fluorescein permeability of the Post-wash #1 scans after 5 days (1160.21±108.26 vs. 1000.47±75.56 ng/mL, P<0.016 for UT-5 day comparison 8 [0.008]), but not after only 2 days of EDE compared to Untreated mice (1115.64±118.94 vs. 1000.47±75.56 ng/mL, P>0.016 for UT-2 day comparison [0.050]). There was no statistical difference between the 2 day and 5 day Post wash #1 scans (P=.299). The Post-wash #2 scans demonstrated that EDE caused a significant NaF retention at both 2 and 5 days of EDE compared to baseline, untreated controls (1017.92±116.25, 1015.40±120.68 vs. 528.22±127.85 ng/mL, P<0.05 [0.0001 for both]). There was no statistical difference between the 2 day and 5 day Post wash #2 scans (P=.503). The comparison between the Untreated post wash #1 with untreated post wash #2 scans using a Paired T-test showed a significant difference between the two sets of scans (P=0.000). There is also a significant difference between the 2 day comparison and the 5 day comparison (P values = 0.010 and 0.002, respectively). ^ Conclusion. Desiccating stress increases permeability of the corneal epithelium to NaF, and increases NaF retention in the corneal stroma. The Fluorotron Master is a useful and sensitive tool to evaluate corneal permeability in murine dry eye, and will be a useful tool to evaluate the effectiveness of dry eye treatments in animal-model drug trials.^