Search

The properties of Ca2+ channels in cultured rat pituitary gonadotrophs were analyzed by the patch-clamp technique. The inward Ca2+ currents, recorded in the presence of 5.2 mM Ca2+ or Ba2+, included a fast, transient component with activation-inactivation kinetics and a delayed component with slower activation. The midpoint of the activation curve lay at -30 mV for the transient component and at -12 mV for the delayed component. At the midpoint, changes in potential of 9.5 and 13 mV induced an e-fold change in the activation of the transient and delayed components, respectively. The rate of inactivation of the first component was strongly voltage dependent. At -43 mV, a 7.4-mV change in potential induced an e-fold change in the fraction of Ca2+ channels available to conduct Ca2+ current. During long-lasting (100-200 ms) low-frequency depolarizing voltage-clamp pulses, the size of the delayed component of the Ca2+ current remained constant. The differential effects of membrane potential on inactivation and the different time constants for activation of the two components of the Ca2+ conductance indicate the presence of two types of Ca2+ channels in the membrane of the gonadotroph: the rapidly inactivating current appears to be attributable to a T-type channel, and the noninactivating current corresponds to the L-type channel described in many other cell types.

Gonadotropin-releasing hormone (GnRH) stimulates calcium mobilization and influx in pituitary gonadotrophs, and agonist-induced calcium entry through voltage-sensitive channels (VSCC) is required for the maintenance of gonadotropin secretion. However, prolonged or frequent exposure to GnRH attenuates the extracellular Ca2+-dependent cytosolic Ca2+ signal and diminishes hormone secretion. Measurements of membrane Ca2+ currents revealed significant impairment of VSCC activity in gonadotrophs during desensitization by GnRH. VSSC were also inactivated in a calcium-dependent manner during exposure to high K+. Prolonged inactivation of such Ca2+ channels by high K+ reduced the calcium and secretory responses to GnRH and vice versa. The calcium-dependent inactivation of VSCC during GnRH action appears to be a primary factor in the onset of desensitization in pituitary gonadotrophs. This mechanism could also account for the development of agonist-induced refractoriness in other calcium-regulated target cells.

Synexin induces chromaffin granule ghosts to fuse one to another, a process which is followed continuously and quantitatively by monitoring the mixing of the intragranular aqueous compartments. A freeze-thaw technique was used for preparing chromaffin granule ghosts loaded with a self-quenching concentration of the fluorescent, high molecular weight probe FITC-Dextran. When the loaded ghosts were mixed with empty ghosts in the presence of synexin, the two compartments fused, resulting in the dilution of the probe with the concomitant increase in fluorescence. So as to suppress possible leakage signals, anti-fluorescein antibodies which quench probe fluorescence were present in the reaction media. Synexin-mediated fusion of freeze-thaw (F/Th) ghosts and binding of 125 I-synexin to these membranes were found to be dependent on Ca 2+ concentration, but only in a partial manner. However, these two synexin-mediated properties were demonstrably sensitive to [H + ] in the medium. A detailed pH profile of fusion revealed an apparent midpoint of activation at approx. pH 5.2, with asymptotic value at pH 4 (maximum) and pH 7.2 (minimum). In our attempt to determine whether the pH effect was on the synexin or on the membranes, we found that fusion was blocked only by treatment of the membranes with the membrane-impermeant carboxyl group modifier 1-ethyl-3(4-azonia-4,4-dimethylpentyl)carbodiimide. These data suggest that membrane fusion evoked by synexin seems to be promoted by rendering the F/Th membranes relatively less negatively charged while the synexin becomes more positively charged. The fusion process was entirely dependent upon synexin concentration; the k 1/2 under optimal conditions of p C a and pH was 85 nM. Similar to what has been previously found with intact granules, an anti-synexin polyclonal antibody partially (48%) blocked fusion, as did pretreatment of the chromaffin granules ghosts with trypsin (30%). We conclude that the coincident p Ca and pH sensitivity of synexin-mediated binding to chromaffin granule membranes and their subsequent fusion might be associated with physiological changes in the concentration of both cations in the cytoplasm of secreting chromaffin cells.

A new technique has been developed to study fusion of biological membrane vesicles. Bovine chromaffin granule ghosts (CGG) were loaded with fluorescein isothiocyanate-dextran (FITC-dextran) at self-quenching concentrations. Loaded ghosts were then made to fuse with empty CGG. Fusion was induced by synexin, a protein previously proposed to be involved in exocytosis. The fusion process was monitored by measuring the dequenching of the fluorescence. Dequenching occurred as FITC-dextran was diluted into the increased volume due to fusion with empty ghosts. Spurious signals from leakage or breakage of vesicles were removed by including a specific anti-fluorescein antibody in the reaction medium. This new technique may prove to be of more general use for studying membrane fusion processes in other systems.

Calcium is only one of a number of second messengers that are becoming increasingly implicated in processes regulating hormone secretion by exocytosis. The most completely studied calcium-dependent secretory system is the chromaffin cell from the adrenal medulla, a system with many operational similarities to the insulin secreting β-cell from islets of Langerhans. Inasmuch as we have had the opportunity to compare these two systems in detail we will attempt to make a number of hopefully interesting comparisons between data obtained from the chromaffin cell and certain data of relevance from the β-cell system.

Fusion of chromaffin granule ghosts was induced by synexin at pH 6, 37 degrees C, in the presence of 10(-7) M Ca2+. To study the kinetics and extent of this fusion process we employed two assays that monitored continuously mixing of aqueous contents or membrane mixing by fluorescence intensity increases. In both assays chromaffin granule ghosts were either labeled on the membrane or in the included aqueous phase. The ratios of blank to labeled chromaffin granule ghosts were varied from 1 to 10. The results were analyzed in terms of a mass action kinetic model, which views the overall fusion reaction as a sequence of a second-order process of aggregation followed by a first-order fusion reaction. The model calculations gave fare simulations and predictions of the experimental results. The rate constants describing membrane mixing are more than 2-fold larger than those for volume mixing. The analysis also indicated that the initial aggregation and fusion processes, up to dimer formation, were extremely fast. The rate constant of aggregation was close to the limit in diffusion-controlled processes, whereas the fusion rate constant was about the same as found in fastest virus-liposome fusion events at pH 5. A small increase in volume was found to accompany the fusion between chromaffin granule ghosts. Using ratios of synexin to chromaffin granule ghost protein of 0.13, 0.41 and 1.15 indicated that the overall fusion rate was larger for the intermediate (0.41) case. The analysis showed that the main activity of synexin was an enhancement of the rate of aggregation. At intermediate or excessive synexin concentrations it, respectively, promoted moderately, or inhibited the actual fusion step.

The kinetic and steady-state characteristics of calcium currents in cultured bovine adrenal chromaffin cells were analyzed by the patch-clamp technique. Whole cell inward Ca2+ currents, recorded in the presence of either 5.2 or 2.6 mM Ca2+ exhibited a single, noninactivating component. To analyze the effects of Ca2+ and Bay K-8644 on the kinetics of the Ca2+ currents, we used a modified version of the Hodgkin-Huxley empirical model. At physiological [Ca2+] (2.5 mM) the midpoint of the steady-state Ca2(+)-channel activation curve lay at -6.9 mV. Increasing the [Ca2+] to 5.2 mM shifted the midpoint by -4.3 mV along the voltage axis. At the midpoint, changes in potential of 7.8 mV (for 5.2 mM Ca2+) and 9.2 mV (for 2.5 mM Ca2+) induced an e-fold change in the activation of the current. Increasing [Ca2+]o from 2.5 to 5.2 mM induced a marked increase in the rate constant for turning on the Ca2+ permeability. Conductances were estimated from the slope of the linear part of the current-voltage relationships as 8.7 and 4.2 nS in the presence of 5.2 and 2.5 mM Ca2+, respectively. Incubation of the cells in the presence of Bay K-8644 at increasing concentrations from 0.001 to 0.1 microM increased the slope conductance from 4.2 to 9.6 nS. Further increases in the concentration of Bay K-8644 from 1 to 100 microM induced a marked reduction in the conductance to 1.1 nS. In the presence of Bay K-8644 (0.1 microM) the midpoint of the activation curve was shifted by 6.1 mV towards more negative potentials, i.e., from -6.9 to -13 mV. At the midpoint potential of -13 mV, a change in potential of 6.9 mV caused an e-fold change in Ca2+ permeability. The kinetic analysis showed that Bay K-8644 significantly reduced the size of the rate constant for turning off the Ca2+ permeability.