Activation of alpha-protein kinase C leads to association with detergent-insoluble components of GH4C1 cells

TRH and phorbol dibutyrate (PDBu) stimulate PRL secretion and synthesis from GH4C1 rat pituitary cells through activation of protein kinase C (PKC). TRH responses are mediated by increases in cellular levels of two PKC activators, Ca2+ and diacylglycerol (DAG), whereas PDBu acts as a DAG analog. We conducted experiments to compare the effects of Ca2+ and PDBu/DAG on alpha-PKC redistribution and to determine to what components of the particulate fraction activated alpha-PKC associates. Subcellular fractionation experiments demonstrated that TRH and PDBu both caused chelator-stable association of alpha-PKC with the particulate fraction. In contrast, Ca2+-mediated association with the particulate fraction was not chelator stable. Immunocytofluorescence experiments also demonstrated that TRH, PDBu, and increased cytosolic Ca2+ (due to ionomycin or K+ depolarization) caused redistribution. The effect of TRH was rapid and transient, similar to TRH stimulation of phospholipase C. The translocated alpha-PKC in the particulate fraction from TRH- or PDBu-treated cultures was not solubilized with Triton X-100. In comparable studies using an immunofluorescence assay, alpha-PKC immunofluorescence remained in detergent-insoluble preparations from TRH- and PDBu-stimulated, but not resting cells. The association of activated alpha-PKC with chelator- and detergent-insoluble material suggested that activated alpha-PKC may be associated with membrane and cytoskeletal components.     

Synthesis and Tumor-promoting Activities of 12-Epi-phorbol-12,13-dibutyrate

12-Epi-phorbol-12,13-dibutyrate (1), the C12-epimer of the most frequently used phorbol ester probe, phorbol-12,13-dibutyrate (PDBu), has been synthesized from phorbol in 9 steps in order to investigate the structural requirements for tumor-promoting activity. Compound 1 showed about 100-fold weaker in vitro biological activities related to in vivo tumor promotion, Epstein-Barr virus early antigen (EBV-EA)-inducing ability, superoxide (O₂ ^-) generation-inducing ability, and binding to the protein kinase C (PKC) regulatory domain surrogate peptides. The results indicated that the β-stereochemistry at position 12 of the phorbol skeleton is important for optimal activity. Binding selectivity to each PKC C1 domain of 1 was almost equal to that of PDBu.     

Comparison of the effects of TRH and D-Ala2-metenkephalinamide on hippocampal electrical activity and behavior in the unanesthetized rat

Administration of TRH into the lateral ventricle of unanesthetized rats produced increases in the incidence of hippocampal theta (5.9–9.1 Hz) rhythm, locomotor activity and shaking behavior. The increase in theta rhythm produced by TRH was brief (<5 min) and was coincident with a brief, large increase in locomotor activity. Intracerebroventricular injection of either TRH or D-Ala²-metenkephalinamide (D-Ala²-ME) also induced episodes of shaking behavior. Shakes induced by D-Ala²-ME were associated with the occurrence of hippocampal epileptiform activity whereas those caused by TRH occurred in the absence of any recorded abnormalities in hippocampal activity. These results suggest that the increase in hippocampal theta rhythm after TRH is secondary to the increase in locomotor activity and, that in contrast to enkephalins, shaking behavior caused by TRH may not be related to an action on the electrographic activity of the hippocampus.     

Protein Kinase C in Rat Brain Is Altered by Developmental Lead Exposure

The absence of learning-related redistribution of hippocampal protein kinase C (PKC) has been correlated with impairment of learning performance induced by developmental lead (Pb) exposure. This study was designed to examine whether the properties of brain PKC are altered by chronic Pb exposure during development. Two-tenth percent Pb acetate was administered to pregnant and lactating dams and then administered to weanlings in drinking water until postnatal day (PN) 56. Effects of Pb on translocation of PKC were studied in brain slices prepared from hippocampus. When the slices were treated with 0.33 M phorbol-12, 13-dibutyrate (PDBu) for 15 min, a significant increase in PKC activity was observed in the membrane fraction of hippocampal slices from Pb-exposed rats, suggesting that chronic Pb exposure potentiates PDBu-activated PKC translocation. Data obtained from saturation binding assays in the frontal cortices of Pb-exposed rats showed a decrease in the dissociation constant (K_D) in both membrane and cytosolic PKC. A decrease in the total binding sites (B_max) of [³H]PDBu binding was only observed in membrane PKC. Furthermore, developmental Pb exposure decreased PKC-, but not PKC-, -II, and - in the membrane fraction of the hippocampus and the frontal cortex. These results indicate that chronic Pb exposure during development increases phorbol ester binding affinity, enhances phorbol ester-induced translocation of PKC, and down-regulates membrane PKC, mainly PKC-.     

Flow threshold for enhanced phorbol ester binding in the ischemic gerbil brain

The correlation between regional phorbol ester binding and cerebral blood flow (CBF) was evaluated in the gerbil brain after 2-hour unilateral common carotid artery occlusion [³H]phorbol 12, 13-dibutyrate (PDBu) was used as a specific ligand for estimating the translocation of protein kinase C (PKC), and CBF was determined by the [¹⁴C]iodoantipyrine method. A quantitative autoradiographic method permitted concurrent measurement of these two parameters in the same brain. In the ischemia group of the animals, statistically significant, inverse correlations were noted between the CBF and PDBu binding in the hippocampus (CA₁ and CA₃ regions and dentate gyrus), the caudate-putamen and lateral nuclei of the thalamus. In these regions, the PDBu binding increased progressively as CBF fell below 35–40 ml/100 g/min. On the other hand, the PDBu binding in the cerebral cortices did not show any significant changes even when CBF was decreased to below 35 ml/100 g/min. The above data suggest that (1) the translocation of PKC to the cell membrane may be regionally specific in response to ischmia and may remain in the regions particularly vulnerable to ischemia such as the hippocampus, caudate-putamen and lateral nuclei of the thalamus in the early ischemic phase; (2) the threshold of CBF below which PKC begins to translocate to the cell membrane in the above regions, may be 35–40 ml/100 g/min in 2-hour ischemia.     

Up-modulation of phorbol dibutyrate receptors by carbachol and arachidonic acid in rat prostatic epithelial cells

Phorbol dibutyrate (PDBu) binding to rat prostatic epithelial cells has been measured as an indirect determination of protein kinase C in this cell system. Analysis of [³H]PDBu binding using competitive displacement demonstrated a single class of PDBu receptors with a Kd=141 nM and a binding capacity of 4.8 pmol PDBu bound/mg cell protein. Raising cytosolic Ca²⁺ levels by redistribution of intracellular Ca²⁺ after cell treatment with carbachol or arachidonic acid (which also affects the bulk biophysical properties of the cell membrane) resulted in up-regulation of the available number of PDBu receptors. These results appear to be a consequence of PKC translocation from the cytosolic compartment to the plasma membrane after a cytosolic Ca²⁺ increase, confirming previous results in other cell systems.     

Modulation of protein kinase C by adenosine: Involvement of adenosine A1 receptor-pertussis toxin sensitive nucleotide binding protein system

The objective of this study was to determine whether adenosine A₁ or A₂ receptor was responsible for the regulation of protein kinase C (PKC) in porcine coronary artery and its coupling to G-protein. Endothelium denuded arterial rings were incubated with PDBu (200nM) in the presence or absence of adenosine receptor agonists and antagonists for 1 day. Following incubation, the arterial rings were contracted with increasing concentrations of endothelin-1 (ET-1) (10^–10–10^–7M). Arteries incubated with PDBu alone failed to produce contraction in response to ET-1. On the contrary, inclusion of A₁ receptor agonist ENBA at 10^–9M in the incubation media with PDBu protected against the PDBu induced blunting of the ET-1 contractions by 50%. Incubation with ENBA alone increased ET-1 dependent contractions by about 2 fold. Inclusion of A₁ receptor antagonist, N0861 at 10^–6 M along with PDBu and ENBA, completely blocked the protective effect of ENBA against the PDBu induced attenuation of ET-1 contractions. N0861 also completely blocked the increase in ET-1 contractions in the arterial rings incubated with ENBA alone. Another A₁ receptor antagonist DPCPX also produced similar results as N0861. On the contrary, arterial rings incubated with relatively specific A₂ receptor agonist CGS 21680 at 10^–4M did not produce any protection against PDBu induced blunting of the ET-1 contractions. Incubation with CGS 21680 alone also did not significantly alter the ET-1 contractions. Interestingly, inclusion of A₂ receptor antagonist DMPX at 10^–4M in the incubation media along with CGS 21680 mimicked the effects of ENBA alone i.e. produced protection against PDBu and enhanced ET-1 contractions. Incubation of the arteries with ENBA alone caused an accumulation of PKC levels, whereas, incubation with CGS 21680 had no significant effect on PKC levels. To study the coupling of adenosine receptor with G-protein, the tissue was incubated for one day with cholera (CT) or pertussis toxin (PT) in the presence or absence or ENBA and PDBu as described above. Incubation with PT blocked the protective effect of ENBA against PDBu as well as the elevation of ET-1 response when incubated with ENBA alone. On the contrary, incubation with CT did not produce any significant effect on ENBA responses. These results indicate that PKC is modulated by adenosine via A₁ adenosine receptors and through a PT sensitive G-protein.This work was supported by National Heart, Lung and Blood Institute Grant HL-27339.     

Galanin reduces PDBu-induced protein phosphorylation in rat ventral hippocampus.

The effect of galanin (GAL) on basal and phorbol-12,13-dibutyrate (PDBu) induced protein phosphorylation in rat ventral hippocampal miniprisms was investigated. GAL (0.5, 1 and 2 microM) inhibited PDBu stimulation in a concentration-dependent manner without altering basal protein phosphorylation. This inhibitory effect was prevented by the GAL antagonist galantide. GAL did not affect either the activity of protein kinase C (PKC) from rat brain or basal phosphorylation in ventral hippocampal hippogenates, suggesting that it did not directly modulate PKC activity. Depolarization of miniprisms from ventral hippocampi by 18 mM K+ prevented the effect of GAL on PDBu-induced phosphorylation. The results indicate that GAL indirectly regulates neuronal protein phosphorylation by a GAL receptor-mediated action.     

Central administration of thyrotropin-releasing hormone and histidyl-proline-diketopiperazine disrupts the acquisition of a food rewarded task by a non-aversive action

The effects of thyrotropin-releasing hormone (TRH) and its metabolites on operant behaviour have rarely been explored. In this study, the effects of intracerebroventricular (icv) administration of TRH and histidyl-proline-diketopiperazine (DKP), a metabolite of TRH, on the acquisition of a food-rewarded lever-press task were compared with saline-treated controls. TRH and DKP severely retarded the acquisition of lever pressing. The effects of systemically administered D-amphetamine were also examined in order to test whether this result was due to any stimulant properties of these peptides. These results suggest that stimulatory effects do not adequately account for impaired acquisition. The possibility that the disruption of learning was due to an aversive effect of icv administration of these peptides was tested by means of a conditioned place paradigm. Neither peptide induced an avoidance of the environment with which it had previously been paired. Several possible reasons for the peptides' adverse effect on learning are discussed, including the possibility that TRH and DKP act on attentional mechanisms.     

Lithium Decreases Membrane-Associated Protein Kinase C in Hippocampus: Selectivity for the α Isozyme

We investigated the effects of lithium on alterations in the amount and distribution of protein kinase C (PKC) in discrete areas of rat brain by using [³H]phorbol 12, 13-dibutyrate quantitative autoradiography as well as western blotting. Chronic administration of lithium resulted in a significant decrease in membrane-associated PKC in several hippocampal structures, most notably the subiculum and the CA1 region. In contrast, only modest changes in [³H]phorbol 12, 13-dibutyrate binding were observed in the various other cortical and subcortical structures examined. Immunoblotting using monoclonal anti-PKC antibodies revealed an isozyme-specific 30% decrease in hippocampal membrane-associated PKC α, in the absence of any changes in the labeling of either the β_(I/II) or γ isozymes. These changes were observed only after chronic (4 week) treatment with lithium, and not after acute (5 days) treatment, suggesting potential clinical relevance. Given the critical role of PKC in regulating neuronal signal transduction, lithium's effects on PKC in the limbic system represent an attractive molecular mechanism for its efficacy in treating both poles of manic-depressive illness. In addition, the decreased hippocampal membrane-associated PKC observed in the present study offers a possible explanation for lithium-induced memory impairment.     

Central somatostatinergic regulation of growth hormone secretion in dwarf chickens.

1. Basal circulating growth hormone (GH) concentrations in sex-linked-dwarf (SLD) chickens were unaffected by the intracerebroventricular (icv) injection of 10, 50 or 100 micrograms somatostatin (SRIF). 2. The GH response to systemic thyrotropin-releasing hormone (TRH; 10 micrograms/kg, iv) was, however, 'paradoxically' enhanced 20 min after icv SRIF administration. 3. A lower dose (1.0 micrograms) of SRIF had no effect on basal or TRH-induced GH release. 4. High-titre SRIF antisera (4 microliters) also had no acute effect on basal plasma GH concentrations, but augmented the GH response to TRH challenge. 5. SRIF would appear to act at central sites to modulate stimulated GH secretion in SLD chickens.     

Phosphorylation of rod outer segment proteins modulates phosphatidylethanolamine N-methyltransferase and phospholipase A2 activities in photoreceptor membranes

The activities of enzymes involved in lipid metabolism—phospholipase A₂ (PLA₂) and phosphatidylethanolamine N-methyltransferase (PE N-MTase)—were found to be differently affected by pre-incubation of rod outer segments (ROS) under protein phosphorylating or dephosphorylating conditions. Exposure to cAMP-dependent protein kinase (PKA), under dark or light conditions, produced a significant increase in PE N-MTase activity, whereas PLA₂ activity decreased. Under standard protein kinase C (PKC) phosphorylating conditions in light, PE N-MTase activity was stimulated and PLA₂ activity was not affected. When the assays were performed in the dark, both enzymatic activities were unaffected when compared to the corresponding controls. Incubation of ROS membranes in light in the presence of PKC activators phorbol 12,13-dibutyrate (PDBu) and dioctanoylglycerol (DOG) resulted in the same pattern of changes in enzyme activities as described for standard PKC phosphorylating condition. Pre-incubation of membranes with the PKC inhibitor H-7 reduced the stimulation of PDBu on PE N-MTase activity, and had no effect on PLA₂ activity in ROS membranes incubated with the phorbol ester. Pre-treatment of isolated ROS with alkaline phosphatase resulted in decreased PE N-MTase activity and produced a significant stimulation of PLA₂ activity under dark as well as under light conditions when compared to the corresponding controls. These findings suggest that ROS protein phosphorylation and dephosphorylation modulates PE N-MTase and PLA₂ activities in isolated ROS, and that these activities are independently and specifically modulated by particular kinases. Furthermore, dephosphorylation of ROS proteins has the opposite effect to that produced by protein phosphorylation on the enzymes studied.     

Central regulation of gastric acid secretion by platelet-activating factor in anesthesized rats

PAF has been implicated in the pathogenesis of acute gastric injury. When given peripherally, PAF induces severe gastric mucosal damage. PAF metabolizing enzymes are present in the brain but the central effects of PAF on the stomach are unknown. We have investigated in the rat the gastric secretion and gross mucosal integrity in response to intracerebroventricular (icv) PAF and compared it with that to icv TRH, a known central gastric secretagogue. Gastric acid output was markedly increased by TRH (171.6 +/- 26.3 mumol/h mean +/- SE) and by 20 micrograms/kg/h iv pentagastrin (107.6 +/- 23.6) when compared to controls receiving icv vehicle (20.2 +/- 7.5; p less than 0.01 for both). In contrast, acid output decreased after icv PAF (13.5 +/- 7.5). Furthermore, icv PAF markedly inhibited acid output stimulated by iv pentagastrin (45.1 +/- 7.03; p less than 0.05). Morphological studies showed acute gastric mucosal erosions after icv TRH and no damage was observed after icv PAF or vehicle. Thus, icv PAF reduces pentagastrin stimulated acid output and does not alter gastric mucosal integrity, whereas icv TRH stimulates acid secretion and induces gastric injury. The opposite effects of PAF and TRH suggests the existence of a gastric modulatory system at the central level.     

Thyrotropin releasing hormone: autonomic effects upon cardiorespiratory function in endotoxic shock

These studies define potential sites and mechanisms by which thyrotropin releasing hormone (TRH) stimulates cardiorespiratory function in normotensive rats as well as in rats subjected to endotoxic shock. Changes in mean arterial pressure, pulse pressure, heart rate, and respiratory rate were determined in conscious animals following injection of TRH into the lateral, third, or fourth ventricular spaces. Injections of TRH into the third ventricular space resulted in a greater increase in cardiorespiratory variables than did fourth ventricular injection. In endotoxin-treated rats, the cardiorespiratory effects of intracerebroventricular (icv) TRH and its analog MK 771 were assessed. TRH and MK 771 were shown to act within the brain to reverse endotoxic shock hypotension; at the doses used, the pressor effects of these two tripeptides were achieved through selectively different actions upon heart rate and pulse pressure. Adrenal demedullated and sham-operated control rats subjected to endotoxic shock were injected with icv and intravenous (iv) TRH in order to evaluate the potential involvement of sympatho-medullary function in cardiorespiratory responses. The cardiovascular effects of icv TRH were dependent upon adrenal medullary integrity; effects of iv TRH were not. Doses of iv TRH which effectively reverse shock neither altered nociceptive latencies nor interfered with analgesic responses to morphine. Collectively, these studies reinforce the potential therapeutic utility of TRH and its analogs in the treatment of shock and indicate potential sites and mechanisms which mediate these salutary effects.     

Dexmedetomidine-induced Contraction Involves Phosphorylation of Caldesmon by JNK in Endothelium-denuded Rat Aortas

Caldesmon, an inhibitory actin binding protein, binds to actin and inhibits actin-myosin interactions, whereas caldesmon phosphorylation reverses the inhibitory effect of caldesmon on actin-myosin interactions, potentially leading to enhanced contraction. The goal of this study was to investigate the cellular signaling pathway responsible for caldesmon phosphorylation, which is involved in the regulation of the contraction induced by dexmedetomidine (DMT), an alpha-2 adrenoceptor agonist, in endothelium-denuded rat aortas. SP600125 (a c-Jun NH₂-terminal kinase [JNK] inhibitor) dose-response curves were generated in aortas that were pre-contracted with DMT or phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator. Dose-response curves to the PKC inhibitor chelerythrine were generated in rat aortas pre-contracted with DMT. The effects of SP600125 and rauwolscine (an alpha-2 adrenoceptor inhibitor) on DMT-induced caldesmon phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) were investigated by western blot analysis. PDBu-induced caldesmon and DMT-induced PKC phosphorylation in rat aortic VSMCs was investigated by western blot analysis. The effects of GF109203X (a PKC inhibitor) on DMT- or PDBu-induced JNK phosphorylation in VSMCs were assessed. SP600125 resulted in the relaxation of aortas that were pre-contracted with DMT or PDBu, whereas rauwolscine attenuated DMT-induced contraction. Chelerythrine resulted in the vasodilation of aortas pre-contracted with DMT. SP600125 and rauwolscine inhibited DMT-induced caldesmon phosphorylation. Additionally, PDBu induced caldesmon phosphorylation, and GF109203X attenuated the JNK phosphorylation induced by DMT or PDBu. DMT induced PKC phosphorylation in rat aortic VSMCs. These results suggest that alpha-2 adrenoceptor-mediated, DMT-induced contraction involves caldesmon phosphorylation that is mediated by JNK phosphorylation by PKC.     

Ubenimex (Bestatin), an aminopeptidase inhibitor, modulates protein kinase C in K562 cells.

Ubenimex (Bestatin) is a potent inhibitor of aminopeptidases (APase) including APase N (EC 3.4.11.2), a widely distributed membrane-bound metalloprotease. Binding of Ubenimex (UBX) to cells has been implicated in a variety of its biological activities, while little evidence has yet been provided as to any subsequent mechanisms of intracellular signal transduction. We now examined the possible involvement of protein kinase C (PKC), a key regulator in transmembrane signaling. Human leukemia K562 cells were cultured in the presence or absence of UBX (1 to 50 micrograms.ml-1, 1 to 72 h), and the subcellular distribution as well as phorbol-12, 13-dibutyrate (PDBu)-induced redistribution of PKC activities were assessed. The membrane-bound enzymatic activity tended to increase in the presence of UBX, while a significant loss of the activity was demonstrable upon subsequent exposure to PDBu (100 nM, 10 min) in both the cytosolic and membrane fractions. Specific binding of [3H]PDBu to intact K562 cells was also down-modulated with UBX concentration- and time-dependently, suggesting loss of PKC enzyme protein on the cell surface. Western blot analysis of the total cell extracts disclosed no appreciable alteration in the amount of PKC protein. APase inhibition with UBX was observable independently of PKC modulation. The present findings were discussed with reference to the possible differential mechanisms of PKC-mediated regulation of cellular responses depending on cell types.     

Saccharomyces cerevisiae DNA repair processes: an update

The effect of incubating T3-1 cells with phorbol 12,13-dibutyrate (PDBu) on the protein kinase C (PKC) isoform content (predominantly , and isoforms) was assessed by immunoblotting, enzyme activity assay and [³H]PDBu binding. After exposure to PDBu for 17 h the immunoreactivity detected for both PKC and PKC had disappeared from cytosol and had increased slightly in membranes. Immunoreactivity for PKC was present as two bands in cytosol; after PDBu treatment both bands decreased in intensity, the higher molecular weight band more than the lower. The lower molecular weight band corresponded with a component of constitutive PKC activity eluting from DEAE cellulose that was defined by inhibition of basal activity with GF 109203X or H7. Investigation of very short treatment times with PDBu using binding, immunoblot and activity measurements (in the presence/absence of Ca²⁺) indicated that translocation of PKC and was very rapid — detectable by 10 sec, maximal within minutes. Reduction of these isoforms in membranes took much longer, and was not apparent up to 150 min. The immunoblot data for PKC in cytosol showed no detectable effect of PDBu treatment on the low molecular weight band up to 150 min although it was reduced at 17 h. Translocation of the upper band was detectable at 10 sec but this band may have resulted from cross-reaction with other PKC isoforms. The constitutive activity and low molecular weight (authentic) PKC immunoreactivity were partially affected after long exposure only, suggesting an action of PDBu on PKC secondary to activation of the other PKC isoforms. An endogenous receptor agonist, luteinising hormone-releasing hormone (LHRH), was also used to assess by immunoblotting, translocation of the PKC isoforms. Although all the isoforms did translocate from cytosol to membrane fractions, they did so with distinctly different time courses: PKC moved more rapidly than PKC which appeared to translocate more quickly than PKC . After downregulation of the responsive PKC isoforms with PDBu, the remaining PKC was not translocated by LHRH. (Mol Cell Biochem 165: 65–75, 1996)     

Immunocytochemical evidence for PDBu-induced activation of RhoA/ROCK in human internal anal sphincter smooth muscle cells

Studies were performed to determine the unknown status of PKC and RhoA/ROCK in the phorbol 12,13-dibutyrate (PDBu)-stimulated state in the human internal anal sphincter (IAS) smooth muscle cells (SMCs). We determined the effects of PDBu (10(-7) M), the PKC activator, on PKCα and RhoA and ROCK II translocation in the human IAS SMCs. We used immunocytochemistry and fluorescence microcopy in the basal state, following PDBu, and before and after PKC inhibitor calphostin C (10(-6) M), cell-permeable RhoA inhibitor C3 exoenzyme (2.5 μg/ml), and ROCK inhibitor Y 27632 (10(-6) M). We also determined changes in the SMC lengths via computerized digital micrometry. In the basal state PKCα was distributed almost uniformly throughout the cell, whereas RhoA and ROCK II were located in the higher intensities toward the periphery. PDBu caused significant translocation of PKCα, RhoA, and ROCK II. PDBu-induced translocation of PKCα was attenuated by calphostin C and not by C3 exoenzyme and Y 27632. However, PDBu-induced translocation of RhoA was blocked by C3 exoenzyme, and that of ROCK II was attenuated by both C3 exoenzyme and Y 27632. Contraction of the human IAS SMCs caused by PDBu in parallel with RhoA/ROCK II translocation was attenuated by C3 exoenzyme and Y 27632 but not by calphostin C. In human IAS SMCs RhoA/ROCK compared with PKC are constitutively active, and contractility by PDBu is associated with RhoA/ROCK activation rather than PKC. The relative contribution of RhoA/ROCK vs. PKC in the pathophysiology and potential therapy for the IAS dysfunction remains to be determined.     

Modulation of cyclic AMP accumulation in GH3 cells by a phorbol ester and thyroliberin

4 beta phorbol-12, 13-dibutyrate (PDBu) stimulated cyclic AMP accumulation in GH3 pituitary tumour cells in the presence of isobutylmethylxanthine. This effect persisted after preincubation of cells with cholera or pertussis toxins. In contrast, vasoactive intestinal polypeptide (VIP)-stimulated cyclic AMP accumulation was inhibited by PDBu in a dose dependent fashion (IC50 = 5.1 nM). Thyroliberin (TRH) had a similar, but non-additive, stimulatory effect on cyclic AMP accumulation with PDBu, however it did not inhibit VIP stimulation. These results suggest that TRH may stimulate cyclic AMP accumulation through protein kinase C and that stimulation of adenylate cyclase by PDBu and TRH may occur distal to the guanine nucleotide binding regulatory proteins, Ns and Ni.