Homologous regulation of estrogen receptor subtypes in goldfish (Carassius auratus).

While considerable information is available on the physiological effects of estrogen, much less is known about the regulation of estrogen receptor (ER) subtypes, particularly in non-mammalian vertebrates. Using goldfish as primary experimental model, we investigated sex- and tissue-specific homologous regulation of ER subtypes (ERalpha, ERbetaI, and ERbetaII) by estradiol in vivo, in the liver and gonads. Treatment with estradiol, significantly upregulated transcript levels for all three types of ERs (ERalpha, ERbetaI, and ERbetaII) in the goldfish ovary and testis. In the goldfish liver, treatment with estradiol significantly increased ERalpha, ERbetaI transcript levels without affecting ERbetaII. In all cases increased ER transcript level was correlated with increased ER protein level determined by Western blot analysis, although we are not able to distinguish between ER subtypes. The results provide strong support for the hypothesis that homologous regulation of ERs is tissue- and gender-specific, and may be a mechanism for estrogen-mediated regulation of reproduction in goldfish.

Defects in cAMP-pathway may initiate carcinogenesis in dividing nerve cells: a review.

The mechanisms of carcinogenesis in nervous tissues are not well understood. It is now established that adenosine 3,',5'-cyclic monophosphate (cAMP)-pathway plays a crucial role in initiating differentiation in transformed and embryonic cells of neuronal and glial origin. Therefore, we propose that defects in the cAMP-pathway may initiate the first phase of carcinogenesis (immortalization). Subsequent genetic abnormalities in oncogenes, anti-oncogenes or other cellular genes individually or in combination may lead to transformation (cancer phenotype). This hypothesis is derived from the fact that an elevation of the cAMP level in murine NB cells induces terminal differentiation in many of these cells in spite of the fact that they are highly aneuploid. Additional changes in cAMP-regulated genes responsible for initiating differentiation may make these cells resistant to cAMP or may make the cAMP-effect on differentiation reversible. Indeed, cAMP-resistant cells exist in NB cell populations, and the cAMP-effect on differentiation is reversible in glioma cells. Identification of genes that initiate, promote and maintain terminal differentiation and those which prevent differentiation following elevation of cAMP in NB cells may increase our understanding of the mechanisms of carcinogenesis. This review illustrates the following: (a) historical background leading to the discovery of cAMP as an inducer of differentiation in nerve cells; (b) identification of potential sites in cAMP-pathway that may play a crucial role in initiating the first phase of carcinogenesis (immortalization) and potential gene targets in immortalized cells whose alterations may cause neoplastic transformation of nerve cells. It is interesting to note that the cAMP pathway remains responsive to an elevated cAMP level in inducing differentiation in NB cells in spite of chromosomal anomalies and genetic changes associated with the maintenance of a cancer phenotype.

Pros and cons of antioxidant use during radiation therapy.

Radiation therapy is one of the major treatment modalities in the management of human cancer. While impressive progress like more accurate dosimetry and more precise methods of radiation targeting to tumor tissue has been made, the value of radiation therapy in tumor control may have reached a plateau. At present, two opposing hypotheses regarding the use of antioxidants during radiation therapy have been proposed. One hypothesis states that supplementation with high doses of multiple micronutrients including high dose dietary antioxidants (vitamins C and E, and carotenoids) may improve the efficacy of radiation therapy by increasing tumor response and decreasing some of its toxicity on normal cells. The other hypothesis suggests that antioxidants (dietary or endogenously made) should not be used during radiation therapy, because they would protect cancer cells against radiation damage. Each of these hypotheses is based on different conceptual frameworks that are derived from results obtained from specific experimental designs, and thus, each may be correct within its parameters. The question arises whether any of these concepts and experimental designs can be used during radiation therapy to improve the management of human cancer by this modality. This review has analyzed published data that are used in support of each hypothesis, and has revealed that the current controversies can be resolved, if the results obtained from one experimental design are not extrapolated to the other. This review has also discussed the scientific rationale for a micronutrient protocol that includes high doses of dietary antioxidants (vitamin C, vitamin E succinate and natural beta-carotene) which can be used adjunctively with radiation therapy.

Molecular composition of 4-aminopyridine-sensitive voltage-gated K(+) channels of vascular smooth muscle.

Voltage-gated K(+) channels (Kv) play a critical role in regulating arterial tone by modulating the membrane potential of vascular smooth muscle cells. Our previous work demonstrated that the dominant 4-aminopyridine (4-AP)-sensitive, delayed rectifier Kv current of rabbit portal vein (RPV) myocytes demonstrates similar 4-AP sensitivity and biophysical properties to Kv1alpha-containing channels. To identify the molecular constituents underlying the 4-AP-sensitive Kv current of vascular myocytes, we characterized the expression pattern of Kv1alpha subunits and their modulatory Kvbeta subunits in RPV. The mRNAs encoding pore-forming subunits Kv1.2, Kv1.4, and Kv1.5 were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), whereas Kv1.1, Kv1.3, and Kv1.6 transcripts were undetectable. Kvbeta1.1, beta1.2, beta1.3, beta2.1, and beta2.2 messages were expressed, whereas Kvbeta3.1 and beta4 mRNAs were undetected by RT-PCR. Kv1.2, Kv1.4, Kv1.5, Kvbeta1.2, beta1.3, and beta2.1 proteins were detected in RPV by Western blotting and/or immunocytochemistry of freshly isolated myocytes. We provide the first evidence, from coimmunoprecipitation studies, for the formation of heteromultimeric Kv channel complexes composed of Kv1.2, Kv1.5, and Kvbeta1.2 subunits in vascular smooth muscle.

Heteromultimeric Kv1.2-Kv1.5 channels underlie 4-aminopyridine-sensitive delayed rectifier K(+) current of rabbit vascular myocytes.

The molecular identity of vascular delayed rectifier K(+) channels (K(DR)) is poorly characterized. Inhibition by 4-aminopyridine (4-AP) of K(DR) of rabbit portal vein (RPV) myocytes was studied by patch clamp and compared with that of channels composed of Kv1.5 and/or Kv1.2 subunits cloned from the RPV and expressed in mammalian cells. 4-AP block of K(DR) was pulse-frequency dependent, required channel activation, and was associated with a positive shift in voltage dependence of activation. 4-AP caused a voltage-dependent reduction in mean open time of K(DR). Relief of 4-AP block of whole cell currents during washout required channel activation and was unaffected by voltage. Homotetrameric Kv1.5 channels did not exhibit the shift in voltage dependence of activation exhibited by the native channels. In contrast, Kv1.2 channels displayed a shift in voltage dependence of activation, and this characteristic was also evident during 4-AP treatment when Kv1.2 was coexpressed with Kv1.5 or coupled to Kv1.5 in a tandem construct to produce heterotetrameric [Kv1.5/Kv1.2](2) channels. K(DR) currents were not sensitive to charybdotoxin, which blocks homotetrameric Kv1.2 channels. The findings of this study (1) indicate that vascular K(DR) are inhibited by 4-AP via an open-state block mechanism and trapping of the drug within the pore on channel closure and (2) provide novel evidence based on a comparison of functional characteristics that indicate the dominant form of vascular K(DR) channel complex in RPV involves the heteromultimeric association of Kv1.2 and Kv1.5 subunits.

Scientific rationale for using high-dose multiple micronutrients as an adjunct to standard and experimental cancer therapies.

We have hypothesized that high-dose multiple micronutrients, including antioxidants, as an adjunct to standard (radiation therapy and chemotherapy) or experimental therapy (hyperthermia and immunotherapy), may improve the efficacy of cancer therapy by increasing tumor response and decreasing toxicity. Several in vitro studies and some in vivo investigations support this hypothesis. A second hypothesis is that antioxidants may interfere with the efficacy of radiation therapy and chemotherapy. This hypothesis is based on the concept that antioxidants will destroy free radicals that are generated during therapy, thereby protecting cancer cells against death. None of the published data on the effect of antioxidants in combination with radiation or chemotherapeutic agents on tumor cells supports the second hypothesis. Scientific rationale in support of a micronutrient protocol to be used as an adjunct to standard or experimental cancer therapy is presented.

Differentiation genes: are they primary targets for human carcinogenesis?

In spite of extensive research in molecular carcinogenesis, genes that can be considered primary targets in human carcinogenesis remain to be identified. Mutated oncogenes or cellular growth regulatory genes, when incorporated into normal human epithelial cells, failed to immortalize or transform these cells. Therefore, they may be secondary events in human carcinogenesis. Based on some experimental studies we have proposed that downregulation of a differentiation gene may be the primary event in human carcinogenesis. Such a gene could be referred to as a tumor-initiating gene. Downregulation of a differentiation gene can be accomplished by a mutation in the differentiation gene, by activation of differentiation suppressor genes, and by inactivation of tumor suppressor genes. Downregulation of a differentiation gene can lead to immortalization of normal cells. Mutations in cellular proto-oncogenes, growth regulatory genes, and tumor suppressor genes in immortalized cells can lead to transformation. Such genes could be called tumor-promoting genes. This hypothesis can be documented by experiments published on differentiation of neuroblastoma (NB) cells in culture. The fact that terminal differentiation can be induced in NB cells by adenosine 3',5'-cyclic monophosphate (cAMP) suggests that the differentiation gene in these cells is not mutated, and thus can be activated by an appropriate agent. The fact that cAMP-resistant cells exist in NB cell populations suggests that a differentiation gene is mutated in these cancer cells, or that differentiation regulatory genes have become unresponsive to cAMP. In addition to cAMP, several other differentiating agents have been identified. Our proposed hypothesis of carcinogenesis can also be applied to other human tumors such as melanoma, pheochromocytoma, medulloblastoma, glioma, sarcoma, and colon cancer.

State-dependent block of rabbit vascular smooth muscle delayed rectifier and Kv1.5 channels by inhibitors of cytochrome P450-dependent enzymes.

The effects of the cytochrome P450 inhibitors clotrimazole, ketoconazole, and 1-aminobenzotriazole (1-ABT) on native delayed rectifier (K(DR)) and cloned Kv1.5 (RPV Kv1.5) K+ channels of rabbit portal vein (RPV) myocytes were determined using whole-cell and single channel patch-clamp analysis. Clotrimazole reduced K(DR) and RPV Kv1.5 whole-cell current with respective Kd values of 1.15 +/- 0.39 and 1.99 +/- 0.6 microM. Clotrimazole acted via an open state blocking mechanism based on the following: 1) the early time course of K(DR) current activation was not affected, but inhibition developed with time during depolarizing steps and increased the rate of decay in current amplitude; 2) the inhibition was voltage-dependent, increasing steeply over the voltage range of K(DR) activation; and 3) mean open time of RPV Kv1.5 channels in inside-out patches was decreased significantly. Ketoconazole reduced K(DR) current amplitude with a Kd value of 38 +/- 3.2 microM. However, ketoconazole acted via a closed (resting) state blocking mechanism: 1) K(DR) amplitude was reduced throughout the duration of depolarizing steps and the rate of decay of current was unaffected, 2) there was no voltage dependence to the block by ketoconazole over the K(DR) activation range, and 3) ketoconazole did not affect mean open time of RPV Kv1.5 channels in inside-out membrane patches. 1-ABT between 0.5 and 3 mM did not affect native K(DR) or RPV Kv1.5 current of rabbit portal vein myocytes. Clotrimazole and ketoconazole, but not 1-ABT, suppress vascular K(DR) channels by direct, state-dependent block mechanisms not involving the modulation of cytochrome P450 enzyme activity.

Peripartum cardiomyopathy: echocardiogram to predict prognosis.

Peripartum cardiomyopathy is an uncommon complication of human pregnancy that threatens both the mother and fetus with maternal congestive heart failure. Clinicians must be aware of this problem in order to provide prompt diagnosis and effective treatment that will insure a favorable return of normal left ventricular function.

Alpha tocopheryl succinate, retinoic acid and polar carotenoids enhanced the growth-inhibitory effect of a cholesterol-lowering drug on immortalized and transformed nerve cells in culture.

OBJECTIVE: Statins (cholesterol lowering drugs) with a closed-ring structure (lovastatin, simvastatin and mevastatin) and an open-ring structure (pravastatin and fluvastatin) are currently used in the management of cardiac disease. Lovastatin and simvastatin inhibit the growth of tumor cells; however, the studies on the effect of a statin in combination with micronutrients such as alpha-tocopheryl succinate (alpha-TS), 13-cis retinoic acid (RA) and polar carotenoids (PC) have never been performed. The primary objective of this study was to investigate the effect of mevastatin alone and in combination with the above micronutrients on the growth of mouse neuroblastoma (NB) cells and rat immortalized dopamine (DA) neurons in culture. In addition, a comparative efficacy of mevastatin and pravastatin on the growth of NB cells was studied. METHODS: Cells were treated with mevastatin in combination with individual antioxidants, alpha-TS, RA and polar carotenoids, 24 hours after plating. Fresh growth medium and agents were changed at two days after treatment, and the viability in control and experimental groups was determined at three days after treatment by MTT assay. Each experiment was repeated three times with triplicate samples per treatment. Growth in experimental groups was expressed as % of untreated cells. RESULTS: Mevastatin inhibited the growth of neuroblastoma (NB) cells and immortalized, non-tumorigenic dopamine (DA) neurons in culture in a dose-dependent manner. Immortalized DA neurons were more sensitive to mevastatin than NB cells. Pravastatin at similar concentrations was ineffective in inhibiting the growth of NB cells. Mevastatin in combination with alpha-TS, RA or PC was more effective in reducing the growth of NB and DA neurons than the individual agents. CONCLUSIONS: Statins with a closed-ring structure can inhibit the growth of established cancer cells as well as immortalized cells (equivalent to pre-malignant lesion), whereas statins with an open-ring structure may be ineffective. A combination of a statin having a closed-ring structure with alpha-TS, RA and PC may be one of the potentially useful anti-cancer agents for prevention and treatment strategies.

Comparison of the pharmacological properties of EDHF-mediated vasorelaxation in guinea-pig cerebral and mesenteric resistance vessels.

In the presence of L-NNA (100 microM), indomethacin (10 microM) and ODQ (10 microM), acetylcholine induced a concentration-dependent vasorelaxation of guinea-pig mesenteric and middle cerebral arteries precontracted with cirazoline or histamine, but not with high K(+), indicating the contribution of an endothelium-derived hyperpolarizing factor (EDHF). In cerebral arteries, charybdotoxin (ChTX; 0.1 microM) completely inhibited the indomethacin, L-NNA and ODQ-insensitive relaxation; iberiotoxin (IbTX, 0.1 microM), 4-aminopyridine (4-AP, 1 mM), or barium (30 microM) significantly reduced the response; in the mesenteric artery, ChTX and IbTX also reduced this relaxation. Glibenclamide (10 microM) had no affect in either the mesenteric or cerebral artery. Neither clotrimazole (1 microM) nor 7-ethoxyresorufin (3 microM) affected EDHF-mediated relaxation in the mesenteric artery, but abolished or attenuated EDHF-mediated relaxations in the cerebral artery. AM404 (30 microM), a selective anandamide transport inhibitor, did not affect the vasorelaxation response to acetylcholine in the cerebral artery, but in the mesenteric artery potentiated the vasorelaxation response to acetylcholine in an IbTX, and apamin-sensitive, but SR 141816A-insensitive manner. Ouabain (100 microM) almost abolished EDHF-mediated relaxation in the mesenteric artery, but enhanced the relaxation in the cerebral artery whereas the addition of K(+) (5 - 20 mM) to precontracted guinea-pig cerebral or mesenteric artery induced further vasoconstriction. These data suggest that in the guinea-pig mesenteric and cerebral arteries different EDHFs mediate acetylcholine-induced relaxation, however, EDHF is unlikely to be mediated by K(+).

Inhibition by protein kinase C of the K(NDP) subtype of vascular smooth muscle ATP-sensitive potassium channel.

ATP-sensitive K(+) channels (K(ATP)) contribute to the regulation of tone in vascular smooth muscle cells. We determined the effects of protein kinase C (PKC) activation on the nucleoside diphosphate-activated (K(NDP)) subtype of vascular smooth muscle K(ATP) channel. Phorbol 12,13-dibutyrate (PdBu) and angiotensin II inhibited K(NDP) activity of C-A patches of rabbit portal vein (PV) myocytes, but an inactive phorbol ester was without effect, and pretreatment with PKC inhibitor prevented the actions of PdBu. Constitutively active PKC inhibited K(NDP) in I-O patches but was without effect in the presence of a specific peptide inhibitor of PKC. PdBu increased the duration of a long-lived interburst closed state but was without effect on burst duration or intraburst kinetics. PdBu treatment inhibited K(NDP), but not a 70-pS K(ATP) channel of rat PV. The results indicate that the K(NDP) subtype of vascular smooth muscle K(ATP) channel is inhibited by activation of PKC. Control of K(NDP) activity by intracellular signaling cascades involving PKC may, therefore, contribute to control of tone and arterial diameter by vasoconstrictors.

Cicatricial pemphigoid with an upper airway lesion.

Cicatricial pemphigoid is an unusual mucocutaneous disease that is characterized by subepidermal blister formation involving the oral and conjunctival membranes. The oral lesions are expressed as erythema and induration and have rarely been associated with upper airway obstruction. We report the case of a patient with dyspnea and an abnormal flow-volume loop who was found to have subglottic compromise due to cicatricial pemphigoid. Immunosuppressive therapy improved his symptoms and air flow.

Relative sensitivity of undifferentiated and cyclic adenosine 3',5'-monophosphate-induced differentiated neuroblastoma cells to cyclosporin A: potential role of beta-amyloid and ubiquitin in neurotoxicity.

Cyclosporin A is routinely used in transplant therapy following allogeneic or xenogeneic tissue transplantation to prevent rejection. This immunosuppressive drug is also neurotoxic; however, its mechanisms of action for neurotoxicity are poorly understood. Undifferentiated and cyclic adenosine 3',5'-monophosphate (cAMP)-induced differentiated neuroblastoma (NB) cells were used as an experimental model to study the toxicity of cyclosporin A. Results showed that cyclosporin A promoted the outgrowth of neurites and inhibited the growth of undifferentiated NB cells. When cyclosporin A was added simultaneously with RO20-1724, an inhibitor of cyclic nucleotide phosphodiesterase, or with prostaglandin E1, a stimulator of adenylate cyclase, it markedly enhanced the growth inhibitory and differentiation effects of these cAMP-stimulating agents. In addition, cyclosporin A added to cAMP-induced differentiated NB cells caused dose-dependent degeneration of these cells as evidenced by the vacuolization of cytoplasm and the fragmentation of nuclear and cytoplasmic materials; however, neurites remained intact. Cyclosporin A alone did not alter the intensity of cell immunostaining for ubiquitin or beta-amyloid peptide (amino acids 1-14) (Abeta1-14); however, it enhanced the intensity of staining for both ubiquitin and Abeta in cells that were treated with cAMP-stimulating agents. The intensity of staining of amyloid precursor protein (amino acids 44-63) (APP44-66) did not change in any treated group, suggesting that the increase in Abeta staining is due to increased processing of APP to Abeta. We propose that one of the mechanisms of cyclosporin A-induced neurotoxicity involves increased levels of Abeta and ubiquitin.

Multiple antioxidants in the prevention and treatment of Alzheimer disease: analysis of biologic rationale.

The etiology of Alzheimer disease (AD) is not well understood; therefore, neither prevention strategies nor long-term effective treatment modalities are available for this disease. Based on laboratory and clinical studies, it appears that reactive oxygen species (ROS) and reactive nitrogen species (RNS) that are generated extracellularly and intracellularly by various mechanisms are among the major intermediary risk factors that initiate and promote neurodegeneration in idiopathic AD. Therefore, multiple antioxidant supplements could be useful in the prevention of AD, and as an adjunct to standard therapy in the treatment of AD. The products of inflammatory reactions such as prostaglandins (PGs; PGE1 and PGA1), free radicals, cytokines, and complement proteins are neurotoxic. Nonsteroidal antiinflammatory drugs (NSAIDs), which inhibit the synthesis of PGs, reduce the rate of deterioration of cognitive functions in patients with advanced AD. Cholinergic drugs are routinely used in the treatment of AD to improve cognitive functions. Therefore, we propose that a combination of multiple antioxidants and NSAIDs may be more beneficial in the prevention of AD, and that this combination taken together with cholinergic drugs may be more effective in the treatment of AD than the individual agents alone. We also hypothesize that, in idiopathic AD, epigenetic components of neurons such as mitochondria, membranes, other membranous structures, and protein modifications--rather than the genes of neurons--are the primary targets for the action of neurotoxins including free radicals. In some familial AD, mutations in amyloid precursor protein and presenilins are associated with the risk of early onset of this disease; however, their mechanisms of action are not fully understood.

Multiple antioxidants in the prevention and treatment of Parkinson's disease.

Parkinson's disease (PD) is one of the major progressive neurological disorders for which no preventative or long-term effective treatment strategies are available. Epidemiologic studies have failed to identify specific environmental, dietary or lifestyle risk factors for PD except for toxic exposure to manganese, meperidine (Demerol, the "designer drug" version of which often contains a toxic byproduct of the synthesis, 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine [MPTP]), and some herbicides and pesticides. The search for genetic risk factors such as mutation, overexpression or underexpression of nuclear genes in DA neurons in idiopathic PD has not been successful as yet. Polymorphism in certain genes appears to be a risk factor, but there is no direct evidence for the causal relationship between polymorphism and increased risk of PD. In familial PD, mutation in the alpha-synuclein gene is associated with the disease, but a direct role of this gene in degeneration of DA neurons remains to be established. Although mutations in the Parkin gene has been associated with autosomal recessive juvenile Parkinson's disease, the role of this gene mutation in causing degeneration of DA neurons has not been defined. We have reported that in hereditary PD, a mutation in the alpha-synuclein gene may increase the sensitivity of DA neurons to neurotoxins. We hypothesize that, in idiopathic PD, epigenetic (mitochondria, membranes, protein modifications) rather than genetic events are primary targets which, when impaired, initiate degeneration in DA neurons, eventually leading to cell death. Although the nature of neurotoxins that cause degeneration in DA neurons in PD is not well understood, oxidative stress is one of the intermediary risk factors that could initiate and/or promote degeneration of DA neurons. Therefore, supplementation with antioxidants may prevent or reduce the rate of progression of this disease. Supplementation with multiple antioxidants at appropriate doses is essential because various types of free radicals are produced, antioxidants vary in their ability to quench different free radicals and cellular environments vary with respect to their lipid and aqueous phases. L-dihydroxyphenylalanine (L-dopa) is one of the agents used in the treatment of PD. Since L-dopa is known to produce free radicals during its normal metabolism, the combination of L-dopa with high levels of multiple antioxidants may improve the efficacy of L-dopa therapy.

Adenosine 3',5'-cyclic monophosphate increases processing of amyloid precursor protein (APP) to beta-amyloid in neuroblastoma cells without changing APP levels or expression of APP mRNA.

Beta-Amyloid (Abeta), a 39-43 residue peptide generated by splicing of the amyloid precursor protein (APP), is one of the major components of senile plaques which are the hallmark of Alzheimer's disease (AD); and therefore, a role of Abeta in neuronal degeneration has been proposed. The factors which regulate the levels of Abeta have not been fully identified. Since an elevation of the intracellular levels of adenosine, 3', 5'-cyclic monophosphate (cAMP) in neuroblastoma cells (NB) induces terminal differentiation, and since these differentiated NB cells undergo spontaneous degeneration, the role of cAMP in the regulation of Abeta levels in these cells have been investigated. In order to determine the specificity of the effect of cAMP on nerve cells, rat glioma cells (C-6) were investigated in a similar manner. Results showed that an elevation of the levels of cAMP in NB cells enhances the intensity of Abeta immunostaining without changing the levels of APP or APP mRNA. This suggests that the rate of processing of APP to Abeta increases following an elevation of cAMP level in NB cells. Data also revealed that an elevation of cAMP level in glioma cells did not alter the intensity of staining with APP or Abeta.

Cyclosporin A regulates the levels of cyclophilin A in neuroblastoma cells in culture.

Cyclophilin A (CyP-A), a member of a highly conserved family of proteins, immunophilins, is the major intracellular receptor for the immunosuppressive drug, cyclosporin A (CsA). CyP-A is widely expressed in many tissues, but is found in the highest concentration in brain tissues and may perform critical neuronal functions. CsA is a known neurotoxin. Therefore, understanding the regulation of CyP-A levels in nerve cells, particularly by CsA, is important. We have utilized murine neuroblastoma (NB) cells as an experimental model to investigate this issue. Our results show that CsA alone was sufficient to induce morphological differentiation in undifferentiated NB cells and to increase CyP-A levels as determined by immunostaining. However, inducing terminal differentiation by elevating adenosine 3',5'-cyclic monophosphate (cAMP) levels using either 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO20-1724), an inhibitor of cyclic nucleotide phosphodiesterase, or prostaglandin E1 (PGE1), a stimulator of adenylate cyclase, was not sufficient to increase CyP-A levels. CsA was required to increase CyP-A levels in both RO20-1724- and PGE1-induced differentiated NB cells. Increases in CyP-A levels, however, occurred without any change in the expression of the CyP-A gene as determined by reverse-transcriptase polymerase-chain reaction analysis using (CyP-A)-specific primers. These results suggest that CsA regulates the level of its own binding protein, CyP-A, in both undifferentiated and cAMP-induced differentiated NB cells in culture.