Broken Cell Membrane Research Articles

Encapsulation of Zn/Co nanocrystals into sponge‐like 3D porous carbon for Escherichia coli inactivation coupled with ultrasound: Characterization, kinetics and inactivation mechanism

A sponge-like 3D Zn/Co co-doped porous carbon composite (Zn/Co-MPC) was successfully prepared using edible fungus residues as a template through a facile pyrolysis method in the current design. The optimized Zn/Co-MPC-0.3 composite was characterized by controllable release of bactericidal particles, fine sterilization ability and excellent magnetic recycle performance. The zinc oxide nanoparticles (ZnO NPs) and cobalt within the composite acted as an efficient killer of germs, while biochar could effectively achieve a controllable release of ZnO NPs and cobalt, endowing the Zn/Co-MPC composite with long-lasting antibacterial performance and eco-friendliness. High-frequency ultrasound, coupled with Zn/Co-MPC-0.3 composite, brought synergistic sterilization, contributing to the enhanced sterilization. The synergistic sterilization of Zn/Co-MPC-0.3 assisted with ultrasound caused a about 1.02-log CFU/mL reduction in Escherichia coli. Four kinetics models fitted well (R2 > 0.9), and the statistical parameters (R2 > 0.95 and smallest RMSE) of Log-linear and Weibull models fitting ultrasound-enhanced Zn/Co-MPC-0.3 treatment were fine. The sterilization mechanism of Escherichia coli by ultrasound-enhanced Zn/Co-MPC-0.3 treatment included the decreased enzyme activity, the broken cell membrane, and the loss of intracellular matter (protein, ATP, DNA). We argue that the ultrasound in combination with Zn/Co-MPC-0.3 treatment has a promising application in bacterial decontamination due to its advantages in eco-friendly characteristics and long-lasting antibacterial performance.

Antibacterial mechanism of thymol against Enterobacter sakazakii

Thymol has a broad-spectrum antibacterial effect, but few studies have elucidated the antibacterial mechanism of thymol on Enterobacter sakazakii to date. This study aimed to uncover its antimicrobial mechanism against E. sakazakii. The minimum inhibitory concentration (MIC) of thymol was determined using the broth microdilution. Membrane potential, intracellular ATP concentrations, and intracellular pH (pHi) were measured, and the membrane damage was observed using a confocal laser scanning microscopy (CLSM) and a field emission gun scanning electron microscope (FEGSEM) to discuss the antibacterial effect of thymol. The MIC of thymol against E. sakazakii BNCC 186088 was 1.25 mg/mL. Cell membrane depolarization, decreased intracellular ATP concentrations, and lower pHi were observed after treatment with thymol, which indicates broken cell membranes and disrupted intracellular homeostasis. These results suggest that thymol has the potential to prevent bacterial contamination by E. sakazakii in the food industry.

AutoIHC-Analyzer: computer-assisted microscopy for automated membrane extraction/scoring in HER2 molecular markers.

Human epidermal growth factor receptor 2 (HER2) is one of the widely used Immunohistochemical (IHC) markers for prognostic evaluation amongst the patient of breast cancer. Accurate quantification of cell membrane is essential for HER2 scoring in therapeutic decision making. In modern laboratory practice, expert pathologist visually assesses the HER2-stained tissue sample under the bright field microscope for cell membrane assessment. This manual assessment is time consuming, tedious and quite often results in interobserver variability. Further, the burden of increasing number of patients is a challenge for the pathologists. To address these challenges, there is an urgent need with a rapid HER2 cell membrane extraction method. The proposed study aims at developing an automated IHC scoring system, termed as AutoIHC-Analyzer, for automated cell membrane extraction followed by HER2 molecular expression assessment from stained tissue images. A series of image processing approaches have been used to automatically extract the stained cells and membrane region, followed by automatic assessment of complete and broken membrane. Finally, a set of features are used to automatically classify the tissue under observation for the quantitative scoring as 0/1+, 2+ and 3+. In a set of surgically extracted cases of HER2-stained tissues, obtained from collaborative hospital for the testing and validation of the proposed approach AutoIHC-Analyzer and publicly available open source ImmunoMembrane software are compared for 90 set of randomly acquired images with the scores by expert pathologist where significant correlation is observed [(r = 0.9448; p < 0.001) and (r = 0.8521; p < 0.001)] respectively. The output shows promising quantification in automated scoring. LAY DESCRIPTION: In cancer prognosis amongst the patient of breast cancer, human epidermal growth factor receptor 2 (HER2) is used as Immunohistochemical (IHC) biomarker. The correct assessment of HER2 leads to the therapeutic decision making. In regular practice, the stained tissue sample is observed under a bright microscope and the expert pathologists score the sample as negative (0/1+), equivocal (2+) and positive (3+) case. The scoring is based on the standard guidelines relating the complete and broken cell membrane as well as intensity of staining in the membrane boundary. Such evaluation is time consuming, tedious and quite often results in interobserver variability. To assist in rapid HER2 cell membrane assessment, the proposed study aims at developing an automated IHC scoring system, termed as AutoIHC-Analyzer, for automated cell membrane extraction followed by HER2 molecular expression assessment from stained tissue images. The input image is preprocessed using modified white patch and CMYK and RGB colour space were used in extracting the haematoxylin (negatively stained cells) and diaminobenzidine (DAB) stain observed in the tumour cell membrane. Segmentation and postprocessing are applied to create the masks for each of the stain channels. The membrane mask is then quantified as complete or broken using skeletonisation and morphological operations. Six set of features were assessed for the classification from a set of 180 training images. These features are: complete to broken membrane ratio, amount of stain using area of Blue and Saturation channels to the image size, DAB to haematoxylin ratio from segmented masks and average R, G and B from five largest blobs in segmented DAB-masked image. These features are then used in training the SVM classifier with Gaussian kernel using 5-fold cross-validation. The accuracy in the training sample is found to be 88.3%. The model is then used for 90 set of unknown test sample images and the final labelling of stained cells and HER2 scores (as 0/1+, 2+ and 3+) are compared with the ground truth, that is expert pathologists' score from the collaborative hospital. The test sample images were also fed to ImmunoMembrane software for a comparative assessment. The results from the proposed AutoIHC-Analyzer and ImmunoMembrane software were compared with the expert pathologists' score where significant agreement using Pearson's correlation coefficient [(r = 0.9448; p < 0.001) and (r = 0.8521; p < 0.001) respectively] is observed. The results from AutoIHC-Analyzer show promising quantitative assessment of HER2 scoring.

Fabrication of Ag quantum dot/SnIn4S8 Schottky junction with enhanced photocatalytic inactivation of E. coli under visible light excitation

A highly efficient visible light driven Ag quantum dots (QDs)/SnIn4S8 Schottky junction photocatalyst was fabricated and applied for the inactivation of E. coli cells under visible light irradiation (λ > 420 nm). The 100 Ag QDs/SnIn4S8 composite displayed the highest inactivation ability with about 7.4 log cfu ml−1 of E. coli cells completely deactivated within 5 h visible light irradiation. The photocatalytic inactivation mechanism could be explained by the broken cell membrane, leakage and damage of biomolecules (proteins and DNA) as proved by scanning electron microscopy, electrophoresis, fluorescence-based stain and reactive species trapping experiments. The reactive species h+, and e− were involved in the bactericidal process over the Ag QDs/SnIn4S8 system. Moreover, the enhanced photoinactivation activity of Ag QDs/SnIn4S8 composites could be ascribed to the enhancement of light absorption by the surface plasmon resonance effect, the formation of the Schottky junction to facilitate the separation and migration of photoinduced charge carriers and the higher number of active sites provided by the enlarged specific surface area.

Thiodan stress on brain neurosecretory cells of the earthworm Eudichogaster kinneari: A Histological profile

Adult Eudichogaster kinneari were exposed to a safe concentration (0.003 ppm) of thiodan for twenty days to evaluate the effects on neurosecretory cells of cerebral and sub pharyngeal ganglion of brain. Brain was severely affected by exposure of above insecticide causing denatured neurosecretory cells due to vacuolization and liquification in cytoplasm, nucleoplasm, in neurosecretory material and in neuropile. Irregular shape of neurosecretory cells were seen, viz- uneven stain, disorderly thickened and broken cell membrane was seen at many places. Most of the neurosecretory material became fragmented and accumulated around the nucleus. Ultimate atrophy of whole histological architecture of neurosecretory cells in both the ganglion of brain was seen. Significant reduction was observed in diameter of cell area, nuclear area, cell length and axon length of neurosecretory cells (p

Antimicrobial activity and synergism of ursolic acid 3-O-α-L-arabinopyranoside with oxacillin against methicillin-resistant Staphylococcus aureus.

The objective of the present study was to investigate the antibacterial activity of a single constituent, ursolic acid 3-O-α-L-arabinopyranoside (URS), isolated from the leaves of Acanthopanax henryi (Oliv.) Harms, alone and in combination with oxacillin (OXA) against methicillin-resistant Staphylococcus aureus (MRSA). A broth microdilution assay was used to determine the minimal inhibitory concentration(MIC). The synergistic effects of URS and OXA were determined using a checkerboard dilution test and time-kill curve assay. The mechanism of action of URS against MRSA was analyzed using a viability assay in the presence of a detergent and an ATPase inhibitor. Morphological changes in the URS-treated MRSA strains were evaluated via transmission electron microscopy(TEM). In addition, the producing penicillin-binding protein2a (PBP2a) protein level was analyzed using western blotting. The MIC value of URS against MRSA was found to be 6.25µg/ml and there was a partial synergistic effect between OXA and URS. The time-kill growth curves were suppressed by OXA combined with URS at a sub-inhibitory level. Compared to the optical density at 600 nm (OD600) value of URS alone (0.09µg/ml), the OD600 values of the suspension in the presence of 0.09µg/ml URS and 0.00001% TritonX-100 or 250µg/ml N,N'-dicyclohexylcarbodiimide reduced by 56.6 and 85.9%, respectively. The TEM images of MRSA indicated damage to the cell wall, broken cell membranes and cell lysis following treatment with URS and OXA. Finally, an inhibitory effect on the expression of PBP2a protein was observed when cells were treated with URS and OXA compared with untreated controls. The present study suggested that URS was significantly active against MRSA infections and revealed the potential of URS as an effective natural antibiotic.

Curcumin reverse methicillin resistance in Staphylococcus aureus.

Curcumin, a natural polyphenolic flavonoid extracted from the rhizome of Curcuma longa L., was shown to possess superior potency to resensitize methicillin-resistant Staphylococcus aureus (MRSA) to antibiotics. Previous studies have shown the synergistic activity of curcumin with β-lactam and quinolone antibiotics. Further, to understand the anti-MRSA mechanism of curcumin, we investigated the potentiated effect of curcumin by its interaction in diverse conditions. The mechanism of anti-MRSA action of curcumin was analyzed by the viability assay in the presence of detergents, ATPase inhibitors and peptidoglycan (PGN) from S. aureus, and the PBP2a protein level was analyzed by western blotting. The morphological changes in the curcumin-treated MRSA strains were investigated by transmission electron microscopy (TEM). We analyzed increased susceptibility to MRSA isolates in the presence of curcumin. The optical densities at 600 nm (OD600) of the suspensions treated with the combinations of curcumin with triton X-100 and Tris were reduced to 63% and 59%, respectively, compared to curcumin without treatment. N,N'-dicyclohexylcarbodiimide (DCCD) and sodium azide (NaN3) were reduced to 94% and 55%, respectively. When peptidoglycan (PGN) from S. aureus was combined with curcumin, PGN (0–125 μg/mL) gradually blocked the antibacterial activity of curcumin (125 μg/mL); however, at a concentration of 125 µg/mL PGN, it did not completely block curcumin. Curcumin has a significant effect on the protein level of PBP2a. The TEM images of MRSA showed damage of the cell wall, disruption of the cytoplasmic contents, broken cell membrane and cell lysis after the treatment of curcumin. These data indicate a remarkable antibacterial effect of curcumin, with membrane permeability enhancers and ATPase inhibitors, and curcumin did not directly bind to PGN on the cell wall. Further, the antimicrobial action of curcumin involved in the PBP2a-mediated resistance mechanism was investigated.

Pathological characteristics of liver allografts from donation after brain death followed by cardiac death in pigs.

Donation after brain death followed by circulatory death (DBCD) is a unique practice in China. The aim of this study was to define the pathologic characteristics of DBCD liver allografts in a porcine model. Fifteen male pigs (25-30 kg) were allocated randomly into donation after brain death (DBD), donation after circulatory death (DCD) and DBCD groups. Brain death was induced by augmenting intracranial pressure. Circulatory death was induced by withdrawal of life support in DBCD group and by venous injection of 40 mL 10% potassium chloride in DCD group. The donor livers were perfused in situ and kept in cold storage for 4 h. Liver tissue and common bile duct samples were collected for hematoxylin and eosin staining, TUNEL testing and electron microscopic examination. Spot necrosis was found in hepatic parenchyma of DBD and DBCD groups, while a large area of necrosis was shown in DCD group. The apoptosis rate of hepatocytes in DBD [(0.56±0.30)%] and DBCD [(0.50 ± 0.11)%] groups was much lower than that in DCD group [(3.78±0.33)%] (P<0.05). And there was no significant difference between DBD group and DBCD group (P>0.05)). The structures of bile duct were intact in both DBD and DBCD groups, while the biliary epithelium was totally damaged in DCD group. Under electron microscope, the DBD hepatocytes were characterized by intact cell membrane, well-organized endoplasmic reticulum, mild mitochondria edema and abundant glycogens. Broken cell membrane, mild inflammatory cell infiltration and sinusoidal epithelium edema, as well as reduced glycogen volume, were found in the DBCD hepatocytes. The DCD hepatocytes had more profound cell organelle injury and much less glycogen storage. In conclusion, the preservation injury of DBCD liver allografts is much less severe than that of un-controlled DCD, but more severe than that of DBD liver allografts under electron microscope, which might reflect post-transplant liver function to some extent.

Hypocrellin B Enhances Ultrasound-Induced Cell Death of Nasopharyngeal Carcinoma Cells

Hypocrellin B, a natural pigment from a traditional Chinese herb, has been attracting extensive attention. The present study aims to investigate whether hypocrellin B can enhance cell death induced by ultrasound sonification on nasopharyngeal carcinoma cells in vitro. The sonodynamic action of hypocrellin B was investigated on nasopharyngeal carcinoma cell line CNE2 cells as tumor model cells. In the experiments, the hypocrellin B concentration was kept constant at 2.5 μM and the cells were subject to ultrasound exposure for 15 s at an intensity of 0.65 W/cm 2. Cytotoxicity was investigated 24 h after ultrasound sonification. Apoptosis was evaluated using flow cytometry with annexin V-FITC and propidium iodine staining and nuclear staining with Hoechst 33258. Cell ultrastructure morphology was observed using transmission electron microscopy (TEM). No significant dark cytotoxicity of hypocrellin B in the CNE2 cells was observed at the concentration of 2.5 μM. The cell death rate induced by ultrasound sonification was significantly higher in the presence of hypocrellin B than in the absence of hypocrellin B. Flow cytometry showed that ultrasound exposure in the presence of hypocrellin B significantly increased the early and late apoptotic rate, 18.64% and 22.57%, respectively, compared with the controls. Nuclear condensation was observed in the nuclear staining and swollen mitochondria and more vacuolar and broken cell membrane were found in TEM after the treatment of hypocrellin B and ultrasound. Our findings demonstrated that the presence of hypocrellin B significantly enhanced the cytotoxicity of ultrasound radiation in CNE2 cells, suggesting that hypocrellin B is a novel sonosensitizer and hypocrellin B-mediated sonodynamic therapy is a potential therapeutic modality in the management of malignant tumors. (E-mail: xcshan@163.com or awnleung@cuhk.edu.hk)

VPAC 1 receptors have different agonist efficacy profiles on membrane and intact cells

The vasoactive intestinal peptide receptor VPAC 1 is preferentially coupled to G αs protein but also increases [Ca 2+] i through interaction with G αi/G αq protein. We evaluated a panel of full, partial and null agonists for their capability to stimulate adenylate cyclase activity in both intact cells and membrane and [Ca 2+] i in intact cells transfected with the reporter gene aequorin. In intact cells, the agonists efficacy for cAMP and calcium increase were well, but not linearly correlated: VPAC 1 receptors activated G αs protein more efficiently but with the same pharmacological profile as the other G proteins. In contrast, there was a difference between cAMP increase in intact and broken cell membranes: EC 50 values were generally lower in intact cells whereas the efficacy was higher. There was, however, no correlation between the shift in the EC 50 value and the intrinsic activity. Of interest, the (4–28) fragment, a reported antagonist on cell membrane, was a full agonist in intact cells. We concluded that the active states of the VPAC 1 receptor resulting from the coupling to different effector are undistinguishable by the VIP analogs tested but that receptor properties are different when evaluated in intact cells or cell membranes.

Cytological alterations in isolated hepatocytes from common carp (Cyprinus carpio L.) exposed to microcystin-LR.

Microcystin-LR (MC-LR) is the most commonly encountered of the toxic cyclic peptide hepatotoxins occurring in China. It is a model compound for toxicological studies. In this study, the toxicity of MC-LR (50 and 500 micrograms/L) on isolated carp hepatocytes was determined and the ultrastructural alterations of cells induced by MC-LR were observed. The alterations noted when hepatocytes were exposed to 50 micrograms/L MC-LR were blebbing of cell membrane, shrinking and deformation of nuclei, vesiculation and transformation into concentric membrane whorls of RER, and swelling and rearrangement of the cytoskeleton. However, when the cells were exposed to 500 micrograms/L MC-LR, broken cell membranes, nuclei and cytoskeleton could be observed. These ultrastructural changes paralleled the pathological events, which lead to apoptosis or necrosis of hepatocytes. These results suggest that disruption of the cytoskeletal structures could account for the blebbing of cell membrane and apoptosis induced by MC-LR.

The Alternatively Spliced Type II Corticotropin-Releasing Factor Receptor, Stably Expressed in LLCPK-1 Cells, Is Not Well Coupled to the G Protein(s)

Two alternatively spliced corticotropin-releasing factor receptor (CRF-R) cDNAs, type I and type II, were recently isolated from a human cDNA library. The two cDNAs are identical except that the type II cDNA encodes an additional 29 amino acid inserted in the first putative cytoplasmic loop. Since the first cytoplasmic loop is highly conserved in all the members of the hCRF receptor family we have examined whether the presence of the 29 amino acid cassette in CRF-RII influences G protein coupling in LLCPK-1 cells stably expressing the type I and type II hCRF receptors. Whether measured in intact cells or in membrane preparations, LLCPK-1 cells stably expressing CRF-RII have a 4-5-fold lower binding affinity. Maximal CRF-stimulated cAMP accumulation in LLCPK-1 cells stably expressing CRF-RT was 10-15-fold higher than that in LLCPK-1 cells expressing CRF-RII. The EC50 for CRF-stimulated cAMP accumulation in hCRF-RI-expressing cells was in the range of 0.5 ± 0.2 nM. In contrast, the EC50 for CRF-stimulated cAMP accumulation in hCRF-RII expressing cells was 7.7 ± 0.2 nM. hCRF increased phosphoinositide turnover in LLCPK-1 cells stably expressing CRF-RI but not in those expressing CRF-RII; this effect required hCRF concentrations of 100 nM and higher. In membrane preparations, GTP-γ-S inhibited hCRF binding to CRF-RII and shifted the binding Kd from 4.5 nM to 16.7 nM. Conversely, GTP-γ-S did not influence hCRF binding to CRF-RII in broken cell membranes. Additionally, CRF-stimulated adenylate cyclase activity in cell membranes expressing CRF-RI was potentiated by GTP, whereas CRF-stimulated adenylate cyclase activity in cell membranes expressing CRF-RII was insensitive to GTP. These data indicate that CRF-RII is not well coupled to the G protein. Since the only difference between the CRF-RII and CRF-RI is the insert in the first putative cytoplasmic loop, these data indicate that the first cytoplasmic loop plays a crucial role in hCRF receptor coupling to the G protein.

Investigation of stroke in sickle cell disease by 1H nuclear magnetic resonance spectroscopy.

Localized proton nuclear magnetic resonance spectroscopy (MRS), obtained with stimulated echo and spin echo sequences, MR imaging (MRI) and MR angiography (MRA) were used to study the brain in 13 children and adolescents with sickle cell disease. Regions of interest (ROI) studied by MRS included regions appearing normal on MRI as well as regions showing complications of sickle cell disease, including focal deep white matter areas of high signal intensity (deep white matter ischemia, DWMI) seen on long TR images, focal atrophic brain areas, and infarcts. The findings in these studies are summarized as follows: Normal-appearing regions on MRI have normal MRS. In ROI including small areas of DWMI, lactate elevation was not detected, but the levels of N-acetyl-aspartate (NAA) appeared slightly elevated. In areas of DWMI 1-2 cm in size, reduced blood flow could be seen on MRA and lactate elevation could be detected with MRS. When blood flow to a DWMI region was normal, NAA was reduced and there was little lactate elevation, as cell death had already occurred. ROI consisting of atrophic tissue had reduced NAA levels but total creatine levels were not changed. Sometimes lipids, presumably from broken cell membrane, could be detected. In regions of past massive stroke, all metabolites were absent except for small amounts of lactate or lipids.

Insulin-like growth factor-II/mannose 6-phosphate receptor is incapable of activating GTP-binding proteins in response to mannose 6-phosphate, but capable in response to insulin-like growth factor-II

We previously reported that insulin-like growth factor-II (IGF-II) stimulates both calcium influx and DNA synthesis by acting on the cell surface IGF-II receptor (IGF-IIR) in a manner sensitive to pertussis toxin, and recently demonstrated that IGF-II binding to the IGF-IIR gives rise to functional changes of purified Gi-2, a GTP-binding protein (G protein) in phospholipid vesicles as well as in broken cell membranes. On the other hand, a variety of evidence indicates that the IGF-IIR binds mannose 6-phosphate (man6P) with high affinity probably at a receptor extracellular region different from the IGF-II-binding site. In the present study, we examined whether man6P stimulation of the IGF-IIR evokes the activation of Gi-2 in phospholipid vesicles and in native cell membranes. In vesicles reconstituted with purified rat IGF-IIR and bovine Gi-2, man6P did not stimulate GDP dissociation from Gi-2 even in concentrations up to 10 mM, while IGF-II dose-dependently facilitated GDP release from Gi-2 with an EC50 of 6 nM. The stimulatory effect of IGF-II was not observed in vesicles reconstituted with Gi-2 alone. In addition, also in a native environment of cell membranes, man6P did not affect an endogenous 40-kDa protein or exogenously added purified Gi-2 as assessed with reduction of the pertussis toxin-catalyzed ADP-ribosylation. These results indicate that the IGF-IIR does not activate Gi-like proteins upon man6P binding in phospholipid vesicles and in native cellular membranes, whereas the receptor activates Gi-like proteins upon IGF-II binding in both environments. Thus, we postulate that the IGF-IIR dissimilarly responds to the two structurally unrelated ligands, IGF-II and man6P, in the linkage function with G proteins.

Protein kinase C stimulates adenylate cyclase activity in prolactin‐secreting rat adenoma (GH4C1) pituicytes by inactivating the inhibitory GTP‐binding protein Gi

The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and thyroliberin exerted additive stimulatory effects on prolactin release and synthesis in rat adenoma GH4C1 pituicytes in culture. Both TPA and thyroliberin activated the adenylate cyclase in broken cell membranes. When combined, the secretagogues displayed additive effects. TPA did not alter the time course (time lag) of adenylate cyclase activation by hormones, guanosine 5'-[beta,gamma-imino]triphosphate or forskolin, nor did it affect the enzyme's apparent affinity (basal, 7.2 mM; thyroliberin-enhanced, 2.2 mM) for free Mg2+. The TPA-mediated adenylate cyclase activation was entirely dependent on exogenously added guanosine triphosphate. ED50 (dose yielding half-maximal activation) was 60 microM. Access to free Ca2+ was necessary to express TPA activation of the enzyme, however, the presence of calmodulin was not mandatory. TPA-stimulated adenylate cyclase activity was abolished by the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, by the protein kinase C inhibitor polymyxin B and by pertussis toxin, while thyroliberin-sensitive adenylate cyclase remained unaffected. Experimental conditions known to translocate protein kinase C to the plasma membrane and without inducing adenylate cyclase desensitization, increased both basal and thyroliberin-stimulated enzyme activities, while absolute TPA-enhanced adenylate cyclase was maintained. Association of extracted GTP-binding inhibitory protein, Gi, from S49 cyc- murine lymphoma cells with GH4C1 cell membranes yielded a reduction of basal and hormone-stimulated adenylate cyclase activities, while net inhibition of the cyclase of somatostatin was dramatically enhanced. However, TPA restored completely basal and hormone-elicited adenylate cyclase activities in the Gi-enriched membranes. Finally, TPA completely abolished the somatostatin-induced inhibition of adenylate cyclase in both hybrid and non-hybrid membranes. These data suggest that, in GH4C1 cells, protein kinase C stimulation by phorbol esters completely inactivates the n alpha i subunit of the inhibitory GTP-binding protein, leaving the n beta subunit functionally intact. It can also be inferred that thyroliberin conveys its main effect on the adenylate cyclase through activation of the stimulatory GTP-binding protein, Gs.