The association of frontal recess anatomy and mucosal disease on the presence of chronic frontal sinusitis: a computed tomographic analysis.

OBJECTIVES: The frontal sinus has the most complex and variable drainage routes of all paranasal sinus regions. The goal of this study was to identify these anatomical factors and inflammation areas relating to chronic frontal sinusitis by comparing radiological presentations in patients with and without frontal sinusitis. METHODS: All adult patients with chronic rhinosinusitis who had received computed tomography (CT) scans of the nasal cavities and paranasal sinuses between October 2010 and September 2011. Logistic regression analysis was used to compare the distribution of various frontal recess cells and surrounding inflammatory conditions in patients with and without frontal sinusitis. RESULTS: Analysis of 240 sides of CT scans was performed with 66 sides excluded. The opacification of the frontal recess and sinus lateralis demonstrated a strong association with an increased presence of frontal sinusitis by multiple logistic regression models. CONCLUSION: Opacification of the frontal recess and sinus lateralis was found to be associated with a significantly increased risk of frontal sinusitis and developing severe blockage of drainage pathways. It provides evidence that mucosal inflammation disease in these two areas is a very important factor leading to chronic frontal sinusitis.

Efficacy of fexofenadine in isolated rat tracheas.

OBJECTIVES: Histamine is an important chemical mediator in both nasal and bronchial inflammation in patients with allergic rhinitis and asthma. The effect of histamine receptor-1 antagonists on nasal mucosa in vivo is well known, however, the effect on tracheal smooth muscle has rarely been explored. The purpose of this study was to determine the effects of fexofenadine on isolated tracheal smooth muscle in vitro. METHODS: Six tracheal strips were used for each experiment, and one untreated strip served as a control. We examined the effectiveness of fexofenadine on isolated rat tracheal smooth muscle by testing the effect on: 1) tracheal smooth muscle resting tension; 2) contraction caused by 10E-6 M methacholine as a parasympathetic mimetic; and 3) electrically induced tracheal smooth muscle contractions. RESULTS: The results indicated that addition of methacholine caused the trachea to contract in a dose-dependent manner. The addition of fexofenadine at a dose of 10E-4 M elicited a significant relaxation response compared to 10E-6 M methacholine-induced contraction. There were no detectable changes in the peak tension of electrical field stimulation-induced contractions in the fexofenadine group. CONCLUSION: High concentrations of fexofenadine had an anti-cholinergic effect. In addition to diminishing histamine-mediated allergic symptoms, fexofenadine may have a potentially therapeutic implication in alleviating asthma-related symptoms due to reducing methacholine-induced contractions of tracheal smooth muscle though these aspects were not studied.

Feedback inhibition of macrophage tumor necrosis factor-alpha production by tristetraprolin.

Tumor necrosis factor-alpha (TNF-alpha) is a major mediator of both acute and chronic inflammatory responses in many diseases. Tristetraprolin (TTP), the prototype of a class of Cys-Cys-Cys-His (CCCH) zinc finger proteins, inhibited TNF-alpha production from macrophages by destabilizing its messenger RNA. This effect appeared to result from direct TTP binding to the AU-rich element of the TNF-alpha messenger RNA. TTP is a cytosolic protein in these cells, and its biosynthesis was induced by the same agents that stimulate TNF-alpha production, including TNF-alpha itself. These findings identify TTP as a component of a negative feedback loop that interferes with TNF-alpha production by destabilizing its messenger RNA. This pathway represents a potential target for anti-TNF-alpha therapies.

Evidence that tristetraprolin binds to AU-rich elements and promotes the deadenylation and destabilization of tumor necrosis factor alpha mRNA.

Mice deficient in tristetraprolin (TTP), the prototype of a family of CCCH zinc finger proteins, develop an inflammatory syndrome mediated by excess tumor necrosis factor alpha (TNF-alpha). Macrophages derived from these mice oversecrete TNF-alpha, by a mechanism that involves stabilization of TNF-alpha mRNA, and TTP can bind directly to the AU-rich element (ARE) in TNF-alpha mRNA (E. Carballo, W. S. Lai, and P. J. Blackshear, Science 281:1001-1005, 1998). We show here that TTP binding to the TNF-alpha ARE is dependent upon the integrity of both zinc fingers, since mutation of a single cysteine residue in either zinc finger to arginine severely attenuated the binding of TTP to the TNF-alpha ARE. In intact cells, TTP at low expression levels promoted a decrease in size of the TNF-alpha mRNA as well as a decrease in its amount; at higher expression levels, the shift to a smaller TNF-alpha mRNA size persisted, while the accumulation of this smaller species increased. RNase H experiments indicated that the shift to a smaller size was due to TTP-promoted deadenylation of TNF-alpha mRNA. This CCCH protein is likely to be important in the deadenylation and degradation of TNF-alpha mRNA and perhaps other ARE-containing mRNAs, both in normal physiology and in certain pathological conditions.

Mitogens stimulate the rapid nuclear to cytosolic translocation of tristetraprolin, a potential zinc-finger transcription factor.

Tristetraprolin (TTP) is the prototype of a group of potential transcription factors that contain two or more unusual CCCH zinc fingers. TTP is encoded by the immediate-early response gene Zfp-36, which is rapidly induced in fibroblasts in response to insulin and other growth factors. Indirect evidence suggests that TTP might function as an inhibitory transcription factor. The present studies evaluated the effect of mitogens on the subcellular localization of TTP using Western blotting of cellular nuclear and cytosolic fractions. In NIH/3T3 mouse fibroblasts that constitutively express TTP, 70% of the protein was located in the nucleus of quiescent, serum-deprived cells. Immunoreactive TTP began to increase in the cytosolic compartment within 1 min of serum stimulation of the cells; this increase in cytosolic protein was essentially complete within 5 min of serum stimulation (81% of total) and was accompanied by a commensurate decrease in nuclear TTP. This translocation was complete well before the increase in TTP synthesis that occurred after serum stimulation. Similar experiments in cells expressing a mutant TTP, in which the major mitogen-activated protein kinase site (serine 220) had been mutated to alanine, revealed normal nuclear to cytosolic translocation after serum stimulation, indicating that phosphorylation of this site is not necessary for this translocation to occur. These results suggest that TTP is rapidly modified in response to mitogens so that it is rapidly released from the nucleus to the cytosol, or that proteins retaining TTP in the nucleus are modified to release it into the cytosol. Thus, TTP's proposed function as a transcription factor, possibly an inhibitory one, may be regulated in cells in part by a novel mechanism, i.e. that of rapid, mitogen-stimulated translocation out of the cellular nucleus.

100% rapid rescreening for quality assurance in a quality control program in a public health cytologic laboratory.

OBJECTIVE: To verij5 the efficacy of the quality control (QC) program in a cytologic laboratwy with a rapid rescreening (RR) protocol. STUDY DESIGN: RR, according to the Turret RR method, of all samples initially screened as negative at the Laboratory of Cytology, Adolfo Lutz Institute, was performed. The slides were reviewed for 60 seconds. Suspect smears were fully checked by 2 reviewers to determine the final diagnoses. A total of 2954 sequential cytologic results were considered in this study. Of the 2954, 2568 (86.9%) were considered initially negative according to our internal QC, and these cases underwent RR. Also, 10% were randomly selected from these negative cases for full reviewing. The internal QC in our laboratory includes review of cases selected according to clinical and cytomorphologic criteria. RESULTS: Among the 2954 total cases, QC detected 386 (13%) atypias with final diagnoses reported according to The Bethesda System 2001 as follows: 82 (2.18%) low grade squamous intraepithelial lesions (LSILs), 35 (1.18%) high grade squamous intraepithelial lesions (HSILs), 2 (0.06%) squamous cell carcinomas, 105 (3.5%) atypical cells of undetermined significance (ASC-US), 4 (0.12%) atypical endocervical cells (AECs) and 158 (5.3%) unsatisfactory samples. RR of 2568 smears initially considered negative selected 194 (7.5%) slides. Of the 194, 146 (75.3%) were negative, 28 (14.4%) ASC-US, 5 (2.6%) AEC, 1 (0.5%) LSIL and 14 (7.2%) unsatisfactory. Full review of a 10% random fraction of the 2568 cases interpreted as negative did not detect lesions but did detect 5 (1.95%) unsatisfactory samples. CONCLUSION: Internal QC used in our laboratory based on clinical and cytomorphologic criteria to select cases for review proved to be an efficient method of detecting HSIL and cervical cancer. The consensus basis of this program strongly limits the false positive and false negative rates and also provides subjects with continuing education. One hundred percent RR is more efficient than 10% full reviewing in detecting cervical abnormalities.

Cloning and characterization of two yeast genes encoding members of the CCCH class of zinc finger proteins: zinc finger-mediated impairment of cell growth.

Members of the CCCH zinc finger (Zf) protein family have in common two or more repeats of a novel Zf motif consisting of Cys and His residues in the form Cx8Cx5Cx3H [where x is a variable amino acid (aa)]. We used a degenerate polymerase chain reaction (PCR) strategy to clone members of this gene family from Saccharomyces cerevisiae. The deduced aa sequences encoded by these genes, designated CTH1 and CTH2, share 46% overall identity and 59% similarity, largely due to the two highly conserved Zf domains. We found readily detectable expression of a 1.4-kb mRNA encoding Cth1p. The 1.1-kb mRNA encoding Cth2p was barely detectable under normal growth conditions; however, disruption of CTH1 resulted in at least a threefold increase in CTH2 mRNA accumulation. No change in phenotype was detected following disruption of CTH1 and CTH2, either singly or together. In contrast, overexpression of the CTH genes or one of the related mammalian genes, tris-tetraprolin (TTP), caused delayed entry of cell cultures into exponential growth, and a decrease in final cell density. Removal of the Zf domain of Cth1p by truncation or deletion completely reversed this slow growth phenotype, indicating that it was mediated through this highly conserved structural motif.

Identification of four CCCH zinc finger proteins in Xenopus, including a novel vertebrate protein with four zinc fingers and severely restricted expression.

Tristetraprolin (TTP), the prototype of a class of CCCH zinc finger proteins, is a phosphoprotein that is rapidly and transiently induced by growth factors and serum in fibroblasts. Recent evidence suggests that a physiological function of TTP is to inhibit tumor necrosis factor alpha secretion from macrophages by binding to and destabilizing its mRNA (Carballo, E., Lai, W.S., Blackshear, P.J., 1998. Science, 281, 1001-1005). To investigate possible functions of CCCH proteins in early development of Xenopus, we isolated four Xenopus cDNAs encoding members of this class. Based on 49% overall amino acid identity and 84% amino acid identity within the double zinc finger domain, one of the Xenopus proteins (XC3H-1) appears to be the homologue of TTP. By similar analyses, XC3H-2 and XC3H-3 are homologues of ERF-1 (cMG1, TIS11B) and ERF-2 (TIS11D). A fourth protein, XC3H-4, is a previously unidentified member of the CCCH class of vertebrate zinc finger proteins; it contains four Cx8Cx5Cx3H repeats, two of which are YKTEL Cx8Cx5Cx3H repeats that are closely related to sequences found in the other CCCH proteins. Whereas XC3H-1, XC3H-2, and XC3H-3 were widely expressed in adult tissues, XC3H-4 mRNA was not detected in any of the adult tissues studied except for the ovary. Its expression appeared to be limited to the ovary, oocyte, egg and the early embryonic stages leading up to the mid-blastula transition. Its mRNA was highly expressed in oocytes of all ages, and was enriched in the animal pole cytosol of mature oocytes. Maternal expression was also seen with the other three messages, suggesting the possibility that these proteins are involved in regulating mRNA stability in oocyte maturation and/or early embryogenesis.

The NIEHS Xenopus maternal EST project: interim analysis of the first 13,879 ESTs from unfertilized eggs.

The sequencing of expressed sequence tags (ESTs) from Xenopus laevis has lagged behind efforts on many other common experimental organisms and man, partly because of the pseudotetraploid nature of the Xenopus genome. Nonetheless, large collections of Xenopus ESTs would be useful in gene discovery, oligonucleotide-based knockout studies, gene chip analyses of normal and perturbed development, mapping studies in the related diploid frog X. tropicalis, and for other reasons. We have created a normalized library of cDNAs from unfertilized Xenopus eggs. These cells contain all of the information necessary for the first several cell divisions in the early embryo, as well as much of the information needed for embryonic pattern formation and cell fate determination. To date, we have successfully sequenced 13,879 ESTs out of 16,607 attempts (83.6% success rate), with an average sequence read length of 508 bp. Using a fragment assembly program, these ESTs were assembled into 8,985 'contigs' comprised of up to 11 ESTs each. When these contigs were used to search publicly available databases, 46.2% bore no relationship to protein or DNA sequences in the database at the significance level of 1e-6. Examination of a sample of 100 of the assembled contigs revealed that most ( approximately 87%) were comprised of two apparent allelic variants. Expression profiles of 16 of the most prominent contigs showed that 12 exhibited some degree of zygotic expression. These findings have implications for sequence-specific applications for Xenopus ESTs, particularly the use of allele-specific oligonucleotides for knockout studies, differential hybridization techniques such as gene chip analysis, and the establishment of accurate nomenclature and databases for this species.

Developmental expression of MARCKS and protein kinase C in mice in relation to the exencephaly resulting from MARCKS deficiency.

The roles of protein kinase C and its substrates in development are poorly understood. Recently, we disrupted the mouse gene for a major cellular substrate for protein kinase C, the MARCKS protein (Proc. Natl. Acad. Sci. USA, 92, 944-948, 1995). The resulting phenotype consisted of universal perinatal lethality, agenesis of the corpus callosum and other forebrain commissures, and neuronal ectopia and other cortical and retinal lamination disturbances. These mice also had high frequencies of exencephaly (25% overall, 35% in females). In the present study, we have examined the normal expression of MARCKS and the various isozymes of protein kinase C at the time of cranial neural tube closure, in an attempt to correlate MARCKS expression in time and anatomical location with the exencephaly characteristic of MARCKS deficiency. Failure of neural tube closure occurred at various sites in the cranial neural tube, suggesting a cellular functional defect that was not limited to a specific location. Non-exencephalic MARCKS-deficient embryos appeared to be anatomically normal on embryonic day (E) 8.5-9.5. MARCKS and PKC alpha were expressed at the plasma membrane of the neuroepithelial cells comprising the future neural tube, as well as in the surface ectoderm and underlying mesenchyme. Endogenous protein kinase C species, comprising either or both alpha and delta, were capable of phosphorylating MARCKS in intact E8.5 embryos. Thus, MARCKS is expressed at the plasma membranes of the specific cell types involved in cranial neurulation; its deficiency presumably results in a still-to-be-elucidated functional defect in these cells that leads to exencephaly in a high proportion of cases.

Interaction of 4-aminopyridine with [3H]phencyclidine receptors in rat brain homogenates.

The effect of 4-aminopyridine and its analogs on the specific binding of [3H]phencyclidine was investigated in rat brain homogenates. 4-Aminopyridine (4-AP) and 3,4-diaminopyridine displaced [3H]phencyclidine binding, while 3-aminopyridine was without effect. The concentrations of 4-AP required for inhibition of binding increased with increasing the ligand concentration, and the resultant Dixon plots indicated a competitive type of interaction. However, 4-AP also accelerated the dissociation rate of the ligand-receptor complex, suggesting that the effect of 4-AP on phencyclidine receptors in the brain might not be purely competitive.

Posthumous sperm utilization in men presenting for sperm banking: an analysis of patient choice.

The question remains as to whether or not men would agree to posthumous sperm use for pregnancy initiation. Often, these individuals' lives are suddenly interrupted and prior consent is rarely given. Therefore, post-mortem retrieval or use of these spermatozoa remains controversial and the incidence of consent for post-mortem sperm use is not clear. Men who bank spermatozoa, however, represent a cohort that can be examined for frequency of consent for post-mortem sperm use. We performed a retrospective chart review for 364 patients presenting for sperm banking at a single institution from 2009 to 2011. Banked specimens represented either ejaculated or surgically retrieved spermatozoa. Demographic information was obtained for each patient and men were grouped by reason for sperm banking, relationship and paternity status, and consent for post-mortem sperm use. The frequency of post-mortem consent was determined within each group. Men were grouped based on reason for banking, including infertility ('Infertility') or malignancy prior to treatment ('Cancer'). Mean ± SD age of the infertility and cancer groups were 40.1 ± 9.9 years and 27.1 ± 9.6 years, respectively. Of the 364 men, 85.9% provided consent for post-mortem sperm use. In the infertility group, 87.4% of men consented. Of these, 92.9% men in a relationship and 62.5% single men consented. Regarding paternity status, 64.7% men with and 56.6% men without children consented. Within the cancer cohort, 83.8% men consented. Of men <18 years old and ≥18 years old, 65.2 and 85.8% consented, respectively. Relationship status yielded 93.2% men in relationships and 79.4% single men consenting. Paternity status in the cancer group yielded 95.8% with and 82.4% men without children consenting. In summary, most men presenting for sperm banking provided consent for post-mortem sperm use, irrespective of reason for banking. Men who are in a relationship or who are fathers were more likely to agree to post-mortem sperm use.

Akt1 deficiency modulates reward learning and reward prediction error in mice.

In contemporary reinforcement learning models, reward prediction error (RPE), the difference between the expected and actual reward, is thought to guide action value learning through the firing activity of dopaminergic neurons. Given the importance of dopamine in reward learning and the involvement of Akt1 in dopamine-dependent behaviors, the aim of this study was to investigate whether Akt1 deficiency modulates reward learning and the magnitude of RPE using Akt1 mutant mice as a model. In comparison to wild-type littermate controls, the expression of Akt1 proteins in mouse brains occurred in a gene-dosage-dependent manner and Akt1 heterozygous (HET) mice exhibited impaired striatal Akt1 activity under methamphetamine challenge. No genotypic difference was found in the basal levels of dopamine and its metabolites. In a series of reward-related learning tasks, HET mice displayed a relatively efficient method of updating reward information from the environment during the acquisition phase of the two natural reward tasks and in the reverse section of the dynamic foraging T-maze but not in methamphetamine-induced or aversive-related reward learning. The implementation of a standard reinforcement learning model and the Bayesian hierarchical parameter estimation show that HET mice have higher RPE magnitudes and that their action values are updated more rapidly among all three test sections in T-maze. These results indicate that Akt1 deficiency modulates natural reward learning and RPE. This study showed a promising avenue for investigating RPE in mutant mice and provided evidence for the potential link from genetic deficiency, to neurobiological abnormalities, to impairment in higher-order cognitive functioning.

Behavioral phenotyping of v-akt murine thymoma viral oncogene homolog 1-deficient mice reveals a sex-specific prepulse inhibition deficit in females that can be partially alleviated by glycogen synthase kinase-3 inhibitors but not by antipsychotics.

Schizophrenia is a severe mental illness with a strong genetic predisposition. Accumulating evidence from human genetics and animal studies suggest v-akt murine thymoma viral oncogene homolog 1 (Akt1) might contribute to susceptibility for schizophrenia. In contrast to inconclusive findings in human genetic studies, a mutant mouse model is a simplified and alternative approach to determining the biological functions of AKT1 and its possible role in the pathogenesis of schizophrenia. In study 1, a comprehensive battery of behavioral tests was performed on both male and female mice. The results of behavioral phenotyping did not reveal significant differences between genotypes or sexes, except increased time of immobility in the tail suspension test and acoustic prepulse inhibition (PPI) deficits in Akt1-knockout females. On the basis of the observed PPI deficit, in study 2a, neuromorphological alterations were examined with morphometric analysis of green fluorescent protein (GFP)-labeled pyramidal neurons in the auditory cortex of female mice. The results indicated abnormalities in the architecture and complexity of the neurons of mutant females compared with those of the controls. In study 2b, potentially effective pharmacological treatments were explored to mitigate the observed PPI deficits in females. Antipsychotics (either raclopride (3 mg/kg) or clozapine (3 mg/kg)) did not alleviate observed PPI deficits in Akt1-knockout females but it was partially normalized by 8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT, 5 mg/kg) and SB216763 (2.5 mg/kg). These findings imply the importance of AKT1 in some behavioral phenotypes and dendritic morphology in the auditory cortex of female mice, and they also suggest that subjects with Akt1 deficiency are insensitive to antipsychotic drugs, whereas glycogen synthase kinase-3 (GSK3) inhibitors could have therapeutic potential for the treatment of acoustic PPI deficits.

Interactions of CCCH zinc finger proteins with mRNA: tristetraprolin-mediated AU-rich element-dependent mRNA degradation can occur in the absence of a poly(A) tail.

The CCCH family of tandem zinc finger proteins has recently been shown to promote the turnover of certain mRNAs containing class II AU-rich elements (AREs). In the case of one member of this family, tristetraprolin (TTP), absence of the protein in knockout mice leads to stabilization of two mRNAs containing AREs of this type, those encoding tumor necrosis factor alpha (TNFalpha) and granulocyte-macrophage colony-stimulating factor. To begin to decipher the mechanism by which these zinc finger proteins stimulate the breakdown of this class of mRNAs, we co-transfected TTP and its related CCCH proteins into 293 cells with vectors encoding full-length TNFalpha, granulocyte-macrophage colony-stimulating factor, and interleukin-3 mRNAs. Co-expression of the CCCH proteins caused the rapid turnover of these ARE-containing mRNAs and also promoted the accumulation of stable breakdown intermediates that were truncated at the 3'-end of the mRNA, even further 5' than the 5'-end of the poly(A) tail. To determine whether an intact poly(A) tail was necessary for TTP to promote this type of mRNA degradation, we inserted the TNFalpha ARE into a nonpolyadenylated histone mRNA and also attached a histone 3'-end-processing sequence to the 3'-end of nonpolyadenylated interleukin-3 and TNFalpha mRNAs. In all three cases, TTP stimulated the turnover of the ARE-containing mRNAs, despite the demonstrated absence of a poly(A) tail. These studies indicate that members of this class of CCCH proteins can promote class II ARE-containing mRNA turnover even in the absence of a poly(A) tail, suggesting that the processive removal of the poly(A) tail may not be required for this type of CCCH protein-stimulated mRNA turnover.

Multiple binding affinities of N-methylscopolamine to brain muscarinic acetylcholine receptors: differentiation from M1 and M2 receptor subtypes.

The properties of the specific binding of the muscarinic receptor ligands [3H]quinuclidinyl benzilate and N-[3H]methylscopolamine in rat brain were compared. The specific binding of both ligands was affected equally by heat, phospholipase A2 and trypsin. N-[3H]methylscopolamine labeled only a fraction of the total muscarinic receptors recognized by [3H]quinuclidinyl benzilate in different brain areas and in the heart. Evidence is presented that N-[3H]methylscopolamine, in fact, binds to a subpopulation of [3H]quinuclidinyl benzilate binding sites. The distribution of the high-affinity binding sites of N-[3H]methylscopolamine did not show a different tissue dependence as compared to the total receptor population, and did not parallel the distribution of the pirenzepine-sensitive M1 receptor subtype. Similarly, the affinity of both [3H]quinuclidinyl benzilate and N-[3H]methylscopolamine varied from one tissue to another by a maximum of 2-fold. Although (-)-quinuclidinyl benzilate competed for the specific binding of [3H]quinuclidinyl benzilate in different tissues according to the law of mass-action, N-methylscopolamine showed an anomalous interaction with two binding sites. The low-affinity binding sites of N-methylscopolamine showed saturability of [3H]quinuclidinyl benzilate binding and stereoselectivity. When the binding characteristics of these N-methylscopolamine-inaccessible binding sites of [3H]quinuclidinyl benzilate in the brain were investigated further, it was found that N-methylscopolamine bound exclusively with a single low affinity, whereas pirenzepine still interacted with two receptor populations incorporated in these sites. It is concluded from several lines of evidence that the heterogeneity of binding of N-methylscopolamine to muscarinic receptors does not represent an interaction with the muscarinic M1 and M2 receptor subtypes defined by pirenzepine. Thus, the unique binding profile of pirenzepine to muscarinic receptors cannot be explained merely on the basis of its hydrophilic nature.

Regulation of [3H]phorbol-12,13-dibutyrate binding sites in mouse neuroblastoma cells: simultaneous down-regulation by phorbol esters and desensitization of their inhibition of muscarinic receptor function.

The binding characteristics of [3H]phorbol-12,13-dibutyrate ([3H]PDBu) in mouse neuroblastoma N1E-115 cells were studied. The specific binding of [3H]PDBu to intact cells was saturable and to a homogeneous class of binding sites, with a Kd of 21 nM. Phorbol 12-myristate-13-acetate and PDBu competed for [3H]PDBu binding whereas 4 alpha-phorbol did not. The binding of [3H]PDBu to the cells was selective, as it was not affected by several agents that interact with various neurotransmitter receptors in N1E-115 cells. The density of the phorbol ester binding site decreased as the cell passage increased, although the Kd of [3H]PDBu binding remained relatively constant. Upon exposure of the cells to 100 nM PDBu for 1 hr at 37 degrees C, a translocation of the binding sites from the cytosol to the particulate fraction was observed. A similar pretreatment of the cells with 1 mM carbamylcholine, however, was ineffective. The specific binding of [3H]PDBu was down-regulated in both a time- and a concentration-dependent fashion by exposure of the cells to PDBu. When the cells were treated with 100 nM PDBu for 24 hr, the maximum binding site density of [3H]PDBu was decreased to 47% of control, with no change in the Kd. Recovery of [3H]PDBu binding after exposure to the phorbol ester for 24 hr was slow and incomplete, and was dependent on protein synthesis. The down-regulation of [3H]PDBu binding after pretreatment of the cells with PDBu for 24 hr was accompanied by an attenuation of the ability of phorbol 12-myristate-13-acetate to inhibit carbamylcholine-induced cyclic GMP formation as well as inositol phosphates accumulation in these cells, indicating desensitization of protein kinase C function.