Kinetics of 5-HT2B receptor signaling: profound agonist-dependent effects on signaling onset and duration.

The kinetics of drug-receptor interactions can profoundly influence in vivo and in vitro pharmacology. In vitro, the potencies of slowly associating agonists may be underestimated in assays capturing transient signaling events. When divergent receptor-mediated signaling pathways are evaluated using combinations of equilibrium and transient assays, potency differences driven by kinetics may be erroneously interpreted as biased signaling. In vivo, drugs with slow dissociation rates may display prolonged physiologic effects inconsistent with their pharmacokinetic profiles. We evaluated a panel of 5-hydroxytryptamine2B (5-HT2B) receptor agonists in kinetic radioligand binding assays and in transient, calcium flux assays, and inositol phosphate accumulation assays; two functional readouts emanating from Gαq-mediated activation of phospholipase C. In binding studies, ergot derivatives demonstrated slow receptor association and dissociation rates, resulting in significantly reduced potency in calcium assays relative to inositol phosphate accumulation assays. Ergot potencies for activation of extracellular signal-regulated kinases 1 and 2 were also highly time-dependent. A number of ergots produced wash-resistant 5-HT2B signaling that persisted for many hours without appreciable loss of potency, which was not explained simply by slow receptor-dissociation kinetics. Mechanistic studies indicated that persistent signaling originated from internalized or sequestered receptors. This study provides a mechanistic basis for the long durations of action in vivo and wash-resistant effects in ex vivo tissue models often observed for ergots. The 5-HT2B agonist activity of a number of ergot-derived therapeutics has been implicated in development of cardiac valvulopathy in man. The novel, sustained nature of ergot signaling reported here may represent an additional mechanism contributing to the valvulopathic potential of these compounds.

Latanoprost's effects on TIMP-1 and TIMP-2 expression in human ciliary muscle cells.

PURPOSE: To determine the effect of treatment with latanoprost on the tissue inhibitors of metalloproteinase (TIMP)-1 and -2 in cultured human ciliary muscle (HCM) cells. METHODS: Confluent serum-starved HCM cells were exposed to increasing concentrations of latanoprost acid (LA, 1 nM to 10 micro M) for 6, 18, and 24 hours. TIMP-1 and -2 mRNA transcripts were evaluated by RT-PCR. Gelatin zymography was used to measure changes in the amount of matrix metalloproteinase (MMP) in the culture medium. To evaluate the potential role of PKC, HCM cells were treated with phorbol 12-myrisate 13-acetate (PMA) in the absence or presence of the PKC inhibitor bisindolylmaleimide I (Bis I) or the PKA inhibitor KT5720. Data were quantitated by densitometry and statistically analyzed with the Student-Newman-Keuls test. RESULTS: TIMP-1 and -2 mRNA transcripts and proteins were detected in primary cultures of HCM cells. TIMP-1 mRNA levels were unchanged at 6 hours, but increased 45% +/- 17% and 54% +/- 13% in cultures exposed for 18 hours to 1 and 10 micro M LA, respectively (n = 3). In contrast, 6 hours of exposure to LA increased expression of TIMP-2 mRNA by up to 11.3% +/- 0.2% (n = 3). However, no significant induction of TIMP-2 mRNA was observed at either 18 or 24 hours (n = 3). TIMP-1 protein was significantly increased in cultures exposed to LA for 18 and 24 hours. In contrast, TIMP-2 protein expression was insignificantly different from control cultures at 6, 18, and 24 hours of treatment. HCM cells exposed to PMA for 24 hours produced similar increases in TIMP-1 mRNA levels, as seen with latanoprost (n = 5). However, no significant induction of TIMP-2 mRNA was observed. Zymographic analysis of the media from these cultures confirmed dose-dependent increases of MMP-1 at 6, 18, and 24 hours, whereas dose-dependent increases in MMP-2 were seen only after 24 hours' exposure to LA (n = 3). TIMP-1 protein levels were increased 27% +/- 9.3% and 15% +/- 11% in the media of cells exposed for 24 hours to 100 nM LA and 100 nM PMA, respectively (n = 5). The increases in TIMP-1 protein induced by LA were essentially eliminated by Bis I (n = 3) and unaffected by KT5720 (n = 3). CONCLUSIONS: For the most part, TIMP-1, and not TIMP-2, contributes to regulation of MMP within the uveoscleral outflow pathway after exposure to latanoprost. Moreover, this induction appears to be meditated by activation of PKC.

Differential regulation of Ca(2+)-dependent Cl- currents by FP prostanoid receptor isoforms in Xenopus oocytes.

The FP(A) and FP(B) prostanoid receptor isoforms are G-protein-coupled receptors that are activated by prostaglandin F(2alpha) (PGF(2alpha)). Differences in their carboxyl termini prompted us to examine the intracellular calcium (Ca(2+)) signaling of these receptor isoforms using the Xenopus oocyte expression system. Protein expression was determined by immunofluorescence microscopy and whole cell binding with [3H]PGF(2alpha). Positive immunolabeling was observed on the outer membranes of oocytes expressing FLAG-tagged FP receptor isoforms, but not on control (water-injected) oocytes. Intracellular signaling was examined using a two-electrode voltage clamp. Specific whole-cell binding was also detected for both receptor isoforms. Bath application of 10 microM PGF(2alpha) to FP(A)-expressing oocytes produced a chloride (Cl-) current response similar to that of an injection of inositol 1,4,5-trisphosphate (InsP(3)) (5.76+/-0.6 microA, peak current; N=23) that returned to control levels within 25 min. In FP(B)-expressing oocytes the activation of the Cl- current was delayed or completely absent (1.38+/-0.2 microA, peak current; N=18). Control oocytes were not responsive to the application of PGF(2alpha) (0.87+/-0.1 microA, peak current; N=10). Activation of Cl- currents for both FP receptor isoforms was dependent upon intracellular Ca(2+) stores as a 30-min pretreatment with thapsigargin (1 microM; N=5) blocked the PGF(2alpha) induction of the Cl- current. These data indicate that the FP prostanoid receptor isoforms differ in their ability to activate Ca(2+)-dependent Cl- channels when expressed in Xenopus oocytes. The difference appears to be in the ability of the two FP prostanoid receptor isoforms to mobilize intracellular calcium.

Intraocular distribution of 70-kDa dextran after subconjunctival injection in mice.

PURPOSE: To investigate the intraocular distribution kinetics of 70-kDa dextran after subconjunctival injection. METHODS: The right eye of 15 mice received a single subconjunctival injection of a 1.5-microL solution of 0.25% 70-kDa tetramethylrhodamine-dextran (TMR-D). The distribution of fluorescent labeling in eye sections was examined by fluorescence microscopy at 0.25, 1, 4, 24, or 72 hours after the injection. The brightness and homogeneity of fluorescence in the sclera, choroid, and retina were scored near the injection site, on the side of the globe opposite the injection site, and adjacent to the optic nerve head. Fluorescence intensity within the sclera and choroid adjacent to the optic nerve was assessed quantitatively by imaging densitometry. RESULTS: TMR-D readily diffused transsclerally and dispersed throughout a large portion of the sclera, uvea, and cornea. Shortly after the injection, homogenous fluorescence was observed in the sclera and choroid on the same meridian as that of the injection site. This fluorescence gradually decreased in intensity with distance from the injection site. At the opposite meridian, fluorescence in the choroid was more intense than in the adjacent sclera and could be traced up to the ciliary muscle. TMR-D was also observed in the retinal and optic nerve vessels. The intensity of scleral and choroidal fluorescence adjacent to the optic nerve reached a maxima at 1 hour, and then decreased slowly, with half-lives of approximately 16 and 100 hours, respectively. Visible fluorescence was maintained at least until 72 hours in the sclera, choroid, iris, and cornea. Specific fluorescent labeling was never found in the contralateral eyes. CONCLUSIONS: Macromolecular 70-kDa dextran can be readily delivered to the mouse retina and uveal tissues by subconjunctival injection through transscleral diffusion, local hematogenous spread, and possibly movement through the uveoscleral outflow pathway. Subconjunctival injection may be a useful approach for delivering macromolecules to the retina and uvea.

Expression of alpha(2)-adrenergic receptor subtypes in prenatal rat spinal cord.

The results of molecular cloning have revealed three subtypes of the alpha(2)-adrenergic receptors (alpha(2) AR) that have been defined alpha(2)C10 (alpha(2A)), alpha(2)C2 (alpha(2B)) and alpha(2)C4 (alpha(2C)). The differential expression of alpha(2) AR subtypes is affected by developmental factors in rat submandibular gland, lung and brain. In the spinal cord of postnatal and adult rats, alpha(2A) and alpha(2C) AR subtypes are expressed and appear to mediate pain perception. However, the relative expression of alpha(2) AR subtypes in the prenatal spinal cord is unknown. In the present study subtype-specific antibodies and reverse transcription-polymerase chain reaction (RT-PCR) were used to determine the expression and localization of the alpha(2) AR subtypes in sections of embryonic day 14 rat spinal cords and primary cultures of cells isolated from these cords. Spinal cords were removed from day 14 embryos, and were sectioned or used for the preparation of cell cultures. After 9 days in culture, neurons were examined by immunofluorescence microscopy or used for preparation of total RNA. In both intact spinal cords and isolated cells, positive immunoreactivity was detected with antibodies against alpha(2A) and alpha(2B) subtypes, but not with antibodies against the alpha(2C) subtype. Using a dual-labeling approach, anti-alpha(2A) and anti-alpha(2B) immunoreactivity was present on the same population of neurons. RT-PCR results were consistent with immunofluorescence studies, and showed that mRNA encoding the alpha(2A) and alpha(2B) subtypes was present in total RNA prepared from primary cultures of rat spinal cord neurons. In contrast to spinal cords of postnatal or adult rats that express alpha(2A) and alpha(2C) AR subtypes on different neurons, prenatal spinal cords contain alpha(2A) and alpha(2B) AR subtypes, and these two subtypes appear to be co-expressed in the same cells.