Properties of particle movement in the plasma membrane of 3T3 mouse fibroblasts

The effects of cytochalasin B, vinblastine and temperature on particle movement in the plasma membrane of several 3T3 mouse fibroblast lines were investigated. Preincubation of normal 3T3 cells for 24 h in 5–10 μg/ml cytochalasin B had no effect on the mean square relative displacement of marker particles in the membrane (motion constant), but preincubation for 4 h in 40 μg/ml vinblastine reduced the value of this constant by 70%. A 10 °C decrease in temperature decreased the motion constant in normal cells (Q₁₀ = 4) more than in virus-transformed 3T3 cells (Q₁₀ = 1.8). Interpreting the motion constant of the particles as an expression of the viscosity of the membrane material, values of 3 poise for normal 3T3 cells and 6 poise for the transformed cells are obtained for 37 °C and pH = 7.4.A method is suggested to quantitate aggregation of particles on the surface of cells. When this method is applied to gold particles on 3T3 cells, disaggregation of particles is seen to behave as an unordered process, whereas aggregation appears to express cellular control. This consideration and the effect of vinblastine indicate that the interpretation of particle movement as Brownian movement in a viscous membrane material does not cover all phenomena observed.The membrane movement of the flat revertant SVF1 101 [1] was investigated. This cell line occupies an intermediary position between normal 3T3 mouse fibroblasts and the polyoma and SV40 transformed 3T3 cell lines as judged by growth properties. Its membrane movement was found to occupy an intermediary position between the membrane movements of these cell lines, too.     

Imitation of contact inhibition by substrate-bound plasma membrane glycoproteins and lectins in serum-free hormone-supplemented cultures of GH3 cells

N-Acetylneuraminic acid-bearing glycoproteins and lectins isolated from the plasma membrane of GH₃ cells are coupled covalently to glass dishes and are used as substrates for cell culture. Under serum-free, hormone-supplemented culture conditions these coupled molecules inhibit the growth rate of GH₃ cells. This inhibition is non-toxic and is concentrationand time-dependent. For an inhibition of 50% 2 × 10⁶ coupled molecules contacting one cell are needed. The data suggest that specific plasma membrane-located glycoproteins and lectins are involved in the ‘density-dependent growth regulation’.     

Intrinsic tryptophan fluorescence of membranes of GH3 pituitary cells: quenching by thyrotropin-releasing hormone.

The intrinsic tryptophan fluorescence of membranes prepared from the GH₃ strain of hormone-producing pituitary cells was monitored by spectrofluorometry. Membranes of GH₃ cells have specific receptors which bind thyrotropin-releasing hormone (TRH). When TRH binds to GH₃ membranes there is quenching of tryptophan fluorescence. The kinetics of the change in fluorescence of GH₃ membranes and of TRH binding are similar. In addition, the concentration of TRH required to produce a half-maximum change in fluorescence is 10 nM, and that required for half-maximum binding of TRH to receptors is 11 nM. Inactive TRH analogs which do not bind to TRH receptors likewise do not alter GH₃ membrane fluorescence, and a pituitary cell strain which lacks TRH receptors does not change membrane fluorescence on incubation with TRH. We conclude that the TRH-receptor interaction in GH₃ membranes is associated with a change in membrane conformation that is readily measured by differential spectrofluorometry.     

Isolation of surface lectins of GH3 cells from whole cells and isolated plasma membranes

Lectins localized in the plasma membranes seem to be of special importance for the intercellular interaction mechanisms. We describe the isolation of mannose-binding proteins by Triton X-100 extraction and affinity chromatography on agarose-bound mannose. The isolation procedure was performed with whole GH₃ cells as well as with isolated plasma membranes. For the isolation of plasma membranes of GH₃ cells a mechanical pump was used for the disruption. After differential centrifugation an enriched plasma membrane fraction was achieved by discontinuous sucrose gradient centrifugation. The whole fractionation procedure was controlled by total balance sheets for the marker enzymes of the different cell organelles. The plasma membrane fraction was further characterized by gel electrophoresis and electron microscopy. The SDS gel electrophoresis patterns of the proteins, resulting from the Triton X-100 extraction and the affinity chromatography, are nearly identical for whole cells as well as for the enriched plasma membrane fraction. Therefore we presume these mannose-specific proteins to be plasma membrane bound, showing the molecular properties of integral proteins and having a molecular weight of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 67 000, 57 000, 47 000.     

The eggs of the freshwater fish Epiplatys dageti have tight plasma membranes without intramembranous particles

Summary The plasma membrane of fish (Epiplatys dageti) eggs, which are capable of developing in salt-free water despite intracellular osmolarity corresponding to a pressure gradient of 7 to 8 atmospheres, is almost devoid of intramembrane particles. The specific membrane resistance is at least 3 orders of magnitude larger than that of nerve or muscle cells of different species indicating that the membrane is tight. These findings support the view that intramembranous particles are involved in transmembrane transport of ions, and indicate that the ionic concentration gradient in this cell is maintained by a tight membrane rather than by active transport.     

Molecular cytochemistry of CD3 and CD4 antigens in human lymphocytes as studied by label-fracture and by fracture-label

Label-fracture and fracture-label membrane immunocytochemistry are used to analyze the surface distribution, dynamics and partition on fracture of CD3 and CD4 antigens of human T lymphocytes. Redistribution of the antigens, induced by treatment at 37 degrees C with specific monoclonal antibodies, results in patching and capping of the labeling as observed in label-fractured specimens. Examination of platinum/carbon replicas of freeze-fractured plasma membranes of antibody-treated cells does not reveal recognizable domains of intramembrane particles. However, in cells where the aggregation of intramembrane particles is induced by incubation with glycerol, colloidal gold-labeled CD3 and CD4 molecules are seen confined to particulate domains of the membrane. Therefore, the lack of visible aggregation of intramembrane particles in patched or capped regions of the membrane implies that migration of CD3 and CD4 antigens with concentration in domains of the membrane is achieved contemporaneously with export of other non-capped integral membrane proteins from the same regions, in a process of diffusional equilibrium. Examination of fracture-labeled specimens shows that CD4 molecules partition on fracture with the inner protoplasmic face of the plasma membrane. This partition illustrates the transmembrane attitude of the antigen molecule and is a probable consequence of interaction of the protein with other components of the membrane or with the cytoskeleton.     

Effect of vinblastine sulfate, colchicine and lumicolchicine on membrane organization of normal and transformed cells

Differences in the distribution of plasma membrane intramembranous particles (PMP) have been demonstrated in normal and transformed fibroblasts using freeze fracture and electron microscopy. Transformed 3T3 cells contain randomly distributed PMP and contact-inhibited 3T3 cells have aggregated PMP when frozen in medium, glycerol, sucrose, or following stabilization in 1 % formaldehyde. To define some of the mechanisms controlling the organization of PMP in this system we have examined the effects of microtubule disruptive drugs including vinblastine sulfate and colchicine on SV3T3 cells. These drugs were observed to induce a dose- and time-dependent aggregation of PMP at concentrations between 10⁻⁹ and 10⁻⁵ M. These results suggest that modulation of PMP distribution in these cells may be influenced by an interaction of microtubules with plasma membrane components. However, the observation that lumicolchicine, a derivative of colchicine which does not disrupt microtubules, also promotes PMP aggregation, suggests that these drugs may also have a primary effect on the plasma membrane in addition to the disruption of microtubules. This is supported by the observation that reduced temperature (4 °C) which is known to disrupt microtubules fails to induce PMP aggregation in SV3T3 cells, suggesting the hypothesis that changes in the interaction of plasma membrane or plasma membrane associated constituents may control the distribution of PMP in this cell system.     

A model for de novo synthesis and assembly of tight intercellular junctions. Ultrastructural correlates and experimental verification of the model revealed by freeze-fracture

The structure and function of intercellular tight (occluding) junctions, which constitute the anatomical basis for highly regulated interfaces between tissue compartments such as the blood-testis and blood-brain barriers, are well known. Details of the synthesis and assembly of tight junctions, however, have been difficult to determine primarily because no model for study of these processes has been recognized. Primary cultures of brain capillary endothelial cells are proposed as a model in which events of the synthesis and assembly of tight junctions can be examined by monitoring morphological features of each step in freeze-fracture replicas of the endothelial cell plasma membrane. Examination of replicas of non-confluent monolayers of endothelial cells reveals the following intramembrane structures proposed as 'markers' for the sequential events of synthesis and assembly of zonulae occludentes: development of surface contours consisting of elongate terraces and furrows (valleys) orientated parallel to the axis of cytoplasmic extensions of spreading endothelial cells, appearance of small circular PF face depressions (or volcano-like protrusions on the EF face) that represent cytoplasmic vesicle-plasma membrane fusion sites, which are positioned in linear arrays along the contour furrows, appearance of 13-15 nm intramembrane particles at the perimeter of the vesicle fusion sites, and alignment of these intramembrane particles into the long, parallel, anastomosed strands characteristic of mature tight junctions. These structural features of brain endothelial cells in monolayer culture constitute the morphological expression of: reshaping the cell surface to align future junction-containing regions with those of adjacent cells, delivery and insertion of newly synthesized junctional intramembrane particles into regions of the plasma membrane where tight junctions will form, and aggregation and alignment of tight junction intramembrane particles into the complex interconnected strands of mature zonulae occludentes. The distribution of filipin-sterol complex-free regions on the PF intramembrane fracture face of junction-forming endothelial plasmalemmae corresponds precisely to the furrows, aligned vesicle fusion sites and anastomosed strands of tight junctional elements.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of calcium and cyclic nucleotides in thyroliberin-stimulated prolactin release

The object of the present study was to determine the relative importance of Ca++ and cyclic nucleotides as “second messengers” in thyroliberin (TRH)-mediated prolactin (PRL) release in the GH₃ and GH₄ rat pituitary tumor cell lines. PRL, cyclic adenosine 3': 5'-monophosphate (cAMP), and cyclic guanosine 3': 5'-monophosphate (cGMP) were measured by radioimmunoassay (RIA) following TRH stimulation. TRH increased PRL release and cAMP levels in GH₃ and GH₄ cells, but cGMP increases were variable. Treatment with 1 mM theophylline increased PRL release and raised cAMP and cGMP. Addition of TRH to theophylline-pretreated cells produced further significant increases in PRL release without any additional increases in cAMP and cGMP. Co++, a Ca++ antagonist, abolished TRH-induced PRL release in a dose-dependent manner. The Co++ inhibition was partially reversed by Ca++ in GH₃ or GH₄ cells. Furthermore, the Ca++ ionophore A23187 stimulated PRL release. We conclude that Ca++ is the primary “second messenger” for TRH-mediated PRL release from GH₃ or GH₄ cells.     

Stereological and biochemical analysis of prolactin and growth hormone secreting rat pituitary cells in culture

Summary The secretion of prolactin is increased by treatment of prolactin producing rat pituitary cells with the hypothalamic tripeptide thyroliberin. To investigate the underlying mechanisms we used three closely related rat pituitary tumor cell strains (GH₁2C₁, GH₃ and GH₄C₁), which synthesize and spontaneously secrete prolactin and/or growth hormone. Growth hormone and prolactin released into the culture medium over a period of 24 h were measured by radioimmunoassay. Initial rates of synthesis were measured by immunoprecipitation of intracellular growth hormone and prolactin after incubation of cell cultures with ³H-leucine. The observed increase in prolactin synthesis and release was correlated with morphological effects of thyroliberin treatment. The volume density of Golgi complexes and the volume and surface densities of rough endoplasmic reticulum were compared in untreated cells and thyroliberin treated cells. As normal distribution could not be assumed, the non-parametric rank test of Wilcoxon was used whereby the densities calculated for each cell section were ranked. Alle three morphological parameters increased after thyroliberin treatment in cells secreting prolactin only (GH₄C₁), implying that the increase of prolactin secretion, at lest in part, is due to increased prolactin synthesis.     

Freeze-fracture studies on isolated sperm cells ofSpinacia oleracea L.

Summary With freeze-fracturing sperm cells appear to be fractured preferentially through the plasma membranes. Only few fracture planes through the cytoplasm are found. Both the PF as well as the EF side of the sperm cell plasma membranes show a slightly undulating surface and contain intramembrane particles. The particle distribution is irregular and does not show any clustering. The EF side of the plasmamembrane contains approximately 3 times more particles per m² than the PF side.Abbreviations EF extraplasmatic fracture face - IMP intramembrane particles - FDA fluorescein diacetate - PF plasmatic fracture face     

A Freeze-Fracture Study of the Microcyst Wall,Plasma Membrane,and Intramembrane Components of Physarum polycephalum1

ABSTRACT. Freeze-cleave replicas of encysted cells of the myxamoebae of Physarum polycephalum were examined to determine the intrawall substructure and to compare the intramembrane structural components. Cleaved areas of the cyst wall revealed a laminated substructure devoid of the macromolecular interruptions (intramembrane particles or IMP) visible in the cleaved cell membranes. The cyst wall adjacent to the cell membrane appears laminated, and a loose branching network of fibers and fibrils occurs at the wall periphery. The absence of intrawall particles is interpreted as a lack of protein or polypeptide components, thus suggesting additional support for the conclusion that polysaccharides are the major wall constituents. When cleaved cell membranes of encysted cells were examined, more intramembrane particles per unit area were observed on the extracellular membrane leaflet than on the protoplasmic membrane leaflet. In addition, homogeneous as well as aggregated particle distributions were visible on cleaved membrane leaflets. Moreover, the presence of aggregated and unaggregated particles on the same membrane leaflets similarly suggests asynchrony of the cell population. This paper examines and compares biological processes involving the cell membrane that may be related to different stages in the cell cycle or to periods of temporary stasis during the cell cycle.     

Effect of transient overexpression of Gqα on soluble and polymerized tubulin pools in GH3 and AtT-20 cells

In order to study G_q-tubulin interaction in the cytosol, GH₃ and AtT-20 cells (stably expressing TRH receptor) were transiently transfected with G_qα cDNA. Forty-eight hours after transfection, thyrotropin-releasing hormone (TRH)-stimulated prolactin (PRL) secretion by G_qα-transfected GH₃ cells increased by 90% compared to mock-transfected cells. In addition, using immunocytochemistry it was observed that G_qα-specific staining was much more prominent in G_qα-transfected GH₃ and AtT-20 cells (also transfected with G_qα) compared to mock-transfected cells. Thus, transfection resulted in successful overexpression of functional G_qα. Forty-eight hours after transfection, cells were processed to obtain soluble and polymerized tubulin fractions. Tubulin levels were determined in these fractions by immunoblotting using polyclonal anti-tubulin antibodies. Compared to mock-transfected cells soluble tubulin levels decreased in G_qα-transfected GH₁ and AtT-20 cells, by 33 and 52%, respectively. Moreover, compared to mock-transfected cells a 50% reduction in the ratio (an index of the flux between tubulin pools) of soluble and polymerized tubulin levels was observed in G_qα-transfected GH₃ and AtT-20 cells. To determine whether these effects on tubulin were mediated by G_q directly, we examined the influence of purified G_q on tubulin polymerization. G_q (0.5 μM) inhibited polymerization of crude tubulin (present in GH₃ cell cytosol) by 53%. In contrast to its effects on GH₃ cell cytosol tubulin, G_q stimulated purified tubulin polymerization by 160%. These results suggest that G_q modulates the polymerization and depolymerization cycles of tubulin and that this modulation is in turn influenced by other unknown cellular components. © 1996 Wiley-Liss, Inc.     

Freeze-fracture study of the pavement cell in the lamprey gill epithelium. Analogy of membrane structure with the granular cell in the amphibian urinary bladder

Freeze-fracture studies of the lamprey gill epithelium reveal structural differences of the luminal and basolateral plasma membrane of the pavement cells. The luminal membrane is characterized by only a few intramembrane particles on the P face and numerous large (10-13 nm) particles on the E face, whereas the basolateral membrane shows the majority of intramembrane particles (6-8 nm) on the P face. The structural specialization of the luminal membrane and the differences between the luminal and basolateral membranes of the pavement cell are similar to those previously demonstrated for the unstimulated granular cell of the amphibian urinary bladder. Because of this similarity, it is suggested that the 2 cell types are analogous and that the luminal membrane of the pavement cell in the lamprey gill epithelium is functionally characterized by a low water permeability. A possible role of sodium uptake by the pavement cells from freshwater and putative differences of osmoregulatory mechanisms in the gills of lampreys and teleosts in freshwater environments are discussed.     

Interactions of lectins with specific cell types in toad urinary bladder. Surface distribution revealed by colloidal gold probes and label fracture

Colloidal gold probes were used in conjunction with pre-embedding labeling and label-fracture to show the plasma membrane distribution of Helix pomatia lectin (HPL) and wheat germ lectin (WGL) binding sites on different epithelial cell types of toad urinary bladder. Mitochondria-rich cells were virtually unlabeled with HPL, but showed a strong affinity for WGL. Granular cells were weakly labeled with WGL but had a variable affinity for HPL. Strongly labeled granular cells were arranged in either chains or clusters that were surrounded by poorly-stained granular cells. By label-fracture, the distribution of gold-labeled lectins was related to other membrane features seen in freeze-fracture. Neither HPL nor WGL binding sites appeared to be specifically related to the large intramembrane particles that characterize granular cells, or to the rod-shaped intramembrane particles that are a feature of membranes of mitochondria-rich cells. The preferential lectin binding affinity of these functionally distinct cell types provides an important starting point for their isolation and the characterization of their plasma membranes. Furthermore, the label-fracture approach can now be used to examine the plasma membrane modifications that occur in these cells under different physiologic conditions affecting epithelial transport processes.     

Hormonal growth control of cells in culture

Summary Serum is the last undefined component in cell culture media. Our results indicate that the primary role of serum is to provide hormones and that serum can be replaced by a group of hormones. A rat pituitary cell line, GH₃, can grow in serum-free medium if the medium is supplemented with 3,3′,5-triiodothyronine, TSH-releasing hormone, transferrin, parathyroid hormone, insulin and three isoelectric focusing fractions of blood meal. The blood-meal components can be replaced by fibroblast growth factor and somatomedin C. The growth rate of GH₃ cells in hormone-supplemented serum-free medium is equal to that in serum-supplemented medium, and subculture in such medium is also possible. These results indicate that the replacement of the serum component is complete in the GH₃ system. The hormonal requirements of GH₃ cells and those of HeLa and mouse melanoma, M2R, were compared. Two generalizations could be made: (a) All three cell lines require insulin and transferrin. (b) There is a requirement for a hormone which localizes in the nucleus for each cell line. These generalizations seem to hold true for most of the other cell lines for which the hormonal requirements have been partially worked out. Since insulin is one of the universally required hormones, its effects on GH₃, HeLa and M2R were compared. Insulin stimulates glycogen synthesis in all three cell lines and facilitates fatty-acid synthesis in GH₃ and M2R. However, there is a difference in the effect of insulin on growth among the three cell lines. Insulin is an absolute requirement for GH₃ cells without which the cells cannot survive, whereas this is not the case for HeLa and M2R. The most stringent requirement for HeLa cells is for hydrocortisone, and for M2R, it is for transferrin. These results indicate that even though the necessity for some hormones is common, the degree of requirement may vary from one cell line to another. Whether this difference reflects the difference in the primary mode of action of the hormone on each cell type needs further investigation. Presented in the Opening Symposium on Nutritional Factors and Differentiation at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, June 6–9, 1977. This work was supported by NIH Grant GM 17019. J. Larner was supported by Josiah Macy Foundation     

Quenching of intrinsic tryptophan fluorescence in membranes of rat pituitary cells

The GH₄C₁ strain of hormone-producing rat pituitary cells has specific receptors for the tripeptide thyrotropin-releasing hormone (TRH). Membranes prepared from GH₄C₁ cells show intrinsic tryptophan fluorescence which was quenched by low concentrations (10–100 nM) of TRH and N^τ-methyl TRH but not by biologically inactive analogs of TRH. Membranes from GH₄C₁ cells were subjected to thermal denaturation. A conformational transition was noted above 40°C and an irreversible denaturation was observed at 52°C. TRH-induced quenching of intrinsic fluorescence was lost completely in membranes previously incubated for 10 min at 30°C while loss of [³H]-TRH binding was only about 20% at this temperature. Collisional quenching by iodide revealed that about 38% of the tryptophanyl residues in GH₄C₁ membranes were exposed to solvent. Quenching by TRH occurred with a shift in wavelength maximum from 336 to 342 nm suggesting that few of the tryptophanyl residues quenched by the tripeptide are totally exposed. Membranes prepared from cells preincubated with 20 nM TRH for 48 h, in which TRH receptors were decreased to 30% of control values, showed no quenching of tryptophan fluorescence in response to freshly added TRH. We conclude that the TRH-receptor interaction in GH₄C₁ cells is associated with a change in membrane conformation that can be measured by differential spectrofluorometry of intrinsic tryptophan fluorescence.     

Purification and characterization of plasma membrane fractions from cultured pituitary cells

Highly purified plasma membrane fractions have been prepared from GH₃ pituitary cells grown in suspension cultures. These membrane fractions have been obtained by differential and sucrose gradient centrifugation and were characterized in terms of their lipid content, marker enzyme analysis and the binding of ³H-labelled thyrotropin-releasing hormone (TRH) to its receptor. Alkaline phosphatase and 5′-nucleotidase activities were enriched 12- to 15-fold in the plasma membrane fraction with somewhat greater enrichment (28-fold) of the specific binding component for [³H]TRH, with a specific activity of 2286 fmol [³H]TRH bound per mg protein. A single class of binding sites for TRH was observed with an apparent dissociation constant of 18 nM, a value similar to that observed for intact cells. No detectable TRH binding to the nuclear fraction was observed that could not be ascribed to residual plasma membrane contamination. By electron microscopy, these fragments appeared to be sealed vesicles with an average diameter of approximately 1800 Å. The binding of ¹²⁵I-labelled wheat germ agglutinin was used as a marker for plasma membrane purification. In addition to specific binding to this membrane fraction, specific binding was also observed in the nuclear fraction. Studies with fluorescein-labelled wheat germ agglutinin revealed that, in fixed cells, fluorescence was restricted to the plasma membrane. However, if the cells were treated with Triton before labelling, most of the fluorescence was then associated with the cell nucleus. Hence, the use of wheat germ agglutinin binding as a specific plasma membrane marker must be reevaluated.     

Alterations in intramembrane particle distribution during interaction of erythrocyte-bound ligands with immunoprotein receptors

Freeze fracture studies have been performed on rabbit pulmonary alveolar macrophages and a nonphagocytic murine lymphoblastoid cell line, PU-5 Fc+, incubated with sheep erythrocytes, sheep erythrocyte-IgG Forssman antibody complex, sheep erythrocyte-IgG Forssman antibody-C complexes and aggregated IgG. Alveolar macrophages show redistribution of intramembrane particles after interaction with (EIgG) and E(IgM)C. The murine lymphoblastoid cell line shows intramembrane particle redistribution consequential to binding of E(IgG) and aggregated IgG. The results demonstrate that after specific immunoprotein receptor-ligand interaction, there is extensive plasma membrane reorganization which results in a redistribution and loss of intramembrane particles. Changes are observed in the protoplasmic face of the plasma membrane after the binding of ligand to the outer membrane surface. The findings suggest that interaction of erthrocyte-bound ligands with specific lymphoid and macrophage plasma membrane receptors leads to a generalized redistribution of integral membrane components in the membrane.     

Growth hormone-releasing factor reduces voltage-gated Ca2+ channel current in Rat GH3 cells

Summary The action of GRF on GH₃ cell membrane was examined by patch electrode techniques. Under current clamp with patch elecrtrode, spontaneous action potentials were partially to totally eliminated by application of GRF. In the case of partial elimination, the duration of remaining spontaneous action potentials was prolonged and the amplitude of afterhyperpolarization was decreased. The evoked actiion potential in the cells which did not show spontaneous action potentials was also eliminated by GRF. In order to examine what channels were affected by GRF, voltage-clamp analysis was performed. It was revealed that voltage-gated Ca²⁺ channel current and Ca²⁺-induced K⁺ channels current were decreased by GRF, while voltage-gated Na⁺ channel and delayed K⁺ channel current was considered to be a consequence of he decrease of voltage-gated Ca²⁺ channels current. Therefore it is likely that the effect of GRF on GH₃ cells was due to the block of voltage-gated Ca²⁺ channels. The elimination of action potential under current clamp corresponded to the block of voltage-gated Ca²⁺ channels and the prolongation of action potential could be explained by the decrease of Ca²⁺-induced K⁺ channel current. The amplitude decrease of afterhyperpolarization could also be explained by the reduction of Ca²⁺-induced K⁺ channel current. Thus the results under current clamp well coincide with the results under voltage clamp. Hormone secretion from GH₃ cells was not stimulated by GRF. However, the finding that GRF solely blocked voltage-gated Ca²⁺ channel suggested the specific action of GRF on GH₃ cell membranes.