Biosynthesis and axonal transport of rat neurohypophysial proteins and peptides.

35S-cysteine injected adjacent to the supraoptic nucleus (SON) of the rat is rapidly incorporated into proteins. These 35S-cysteine-labeled proteins in the SON (1-24 h after injection) were separated by polyacrylamide gel electrophoresis, and the distribution of radioactive proteins on the gels was analyzed. 1 h after injection, about 73% of the radioactivity appeared in two peaks (both about 20,000 mol wt). With time, these peaks (putative precursors of neurophysin) decreased, as a 12,000 mol wt peak (containing two distinct neurophysins) increased in radioactivity. Both the 20,000- and 12,000-mol wt proteins are transported into the axonal (median eminence) and nerve terminal (posterior pituitary) regions of the rat hypothalamo-neurohypophysial system. Conversion of the larger precursor protein to the smaller neurophysin appears to occur, in large part, intra-axonally during axonal transport. Six distinct 35S-cysteine-labeled peptides (less than 2500 mol wt), in addition to arginine vasopressin and oxytocin, are also synthesized in the SON and transported to the posterior pituitary where they are released together with labeled neurophysin by potassium depolarization in the presence of extracellular calcium. These data provide support for the hypothesis that the neurohypophysial peptides (vasopressin and oxytocin) and neurophysins are derived from the post-translational clevage of protein precursors synthesized in the SON, and that the conversion process can occur in the neurosecretory granule during axonal transport.

Stimulated vasopressin synthesis by a fetal hypothalamic factor.

The hypothalamo-neurohypophyseal complex (HNC) of the fetal guinea pig shows a dramatic increase in its content of vasopressin and neurophysin between the 40th and 55th days of gestation. The values for radioimmunoassayable hormone and binding protein are at day 40, 2 milliunits and less than 0.1 microgram; and at day 55, about 100 milliunits and 10 micrograms, respectively. Isotope incorporation experiments with organ cultures of the fetal HNC taken prior to the 35th day of gestation added additional confirmation of the inability of the hypothalamic neurosecretory cells to synthesize vasopressin or neurophysin at this time. However, by the 45th day, similar organ cultures show a vigorous incorporation of labeled amino acids into both hormone and binding protein. Furthermore, the HNC of the 45-day-old fetus apparently contains a factor that stimulates specifically the biosynthesis of vasopressin and neurophysin in HNC cultures from the adult guinea pig. This factor is not detectable in either cortex or liver of the 45-day-old fetus or in the fetal HNC taken prior to or after the period of exponential rise (40th to 55th days) of hormone and binding protein.

Neurophysin biosynthesis: conversion of a putative precursor during axonal transport.

[35S]Cysteine injected adjacent to the supraoptic nucleus of the rat is rapidly incorporated into a 20,000-dalton protein that, in time, is converted to a 12,000-dalton labeled protein, neurophysin. This putative precursor of neurophysin appears to be synthesized in the supraoptic nucleus and transformed to neurophysin and related peptides during axonal transport to the neurohypophysis.

Pancreatic Neuroendocrine Tumor Producing Insulin and Vasopressin.

The objective of the study is to report a rare case of pancreatic neuroendocrine tumor (pNET) producing insulin and vasopressin. We describe the clinical presentation and management of a metastatic pNET with refractory hypoglycemia and progressive severe hyponatremia. A 52-year-old patient had abdominal pain leading to the diagnosis of a tumor that was initially presumed to be splenic in origin. Investigations ultimately identified a pancreatic mass that on biopsy proved to be a pNET. Eventually, he developed extensive liver metastases, and with tumor progression, he manifested hypoglycemia and severe hyponatremia. He was managed with multiple therapies including somatostatin analogue, peptide-receptor-radionuclide-therapy (PRRT), diazoxide, and everolimus; none of these therapeutic modalities was successful in controlling functional and structural progression of the tumor. Ultimately, the pNET proved fatal and autopsy confirmed widely metastatic disease that stained strongly and diffusely for vasopressin, a feature not seen in the previous liver biopsy. This case illustrates the challenges of diagnosis and management of aggressive insulin-producing pNETs and highlights the potential concomitant ectopic production of vasopressin leading to refractory hyponatremia.

Neurophysin biosynthesis in vitro in oat cell carcinoma of the lung with ectopic vasopressin production.

The incorporation of labeled compounds into neurophysins of a transplantable human oat cell carcinoma of the lung with ectopic vasopressin production was studied in vitro. Neurophysins in cell extracts and in incubation media were isolated by immunoprecipitation and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. When cells were incubated with L-[35S]cysteine for 12 h, SDS-polyacrylamide gel electrophoresis of the immunoprecipitates from cell extract and medium resolved two forms of neurophysins with apparent molecular mass of 10,000 (10K) and 20,000 (20K). Both forms of [35S]-neurophysins were completely displaced from the immunoprecipitates by excess human neurophysin. Incubation of cells with L-[35S]cysteine and D-[3H]-glucosamine hydrochloride revealed that glucosamine was incorporated into the 20K neurophysin region, but not into 10K species. To observe the kinetics of labeling of the two forms of neurophysins, cells were incubated with L[35S]cysteine for varying periods of time. After short labeling periods, most of the radioactivity resided in 20K species, which plateaued after 1 h, whereas 10K neurophysin progressively increased in its height. When cells were chased with unlabeled cysteine after the exposure to a short pulse of labeling, 20K neurophysin peak gradually decreased with an apparent initial half-life of 1 h. In contrast, the label in 10K neurophysin steadily increased, which exceeded the former by 3 h of chase. Analysis of 20K neurophysin in cell extract by isoelectric focusing on polyacrylamide gel demonstrated that it was principally composed of a protein with an apparent isoelectric point (pI) of 5.7. These results suggest that neurophysin is synthesized in ectopic vasopressin-producing tumors by post-translational processing from a glycosylated proneurophysin with an apparent molecular mass of 20,000 daltons and a pI of 5.7.

Nature of the immunoreactive neurophysins in ectopic vasopressin-producing oat cell carcinomas of the lung. Demonstration of a putative common precursor to vasopressin and neurophysin.

In an attempt to delineate the nature of the immunoreactive neurophysins in oat cell carcinomas of the lung with ectopic vasopressin production, tumor neurophysins were characterized by gel filtration and by electrophoresis. In all of the five tumor tissues, activities of both vasopressin and nicotine-stimulated neurophysin (NSN) determined by radioimmunoassay were demonstrated. A small amount of oxytocin as well as estrogen-stimulated neurophysin was detected in three of the tissues. When tissue extract was subjected to Sephadex G-50 gel filtration in 0.2 N acetic acid, the major portion of immunoreactive NSN emerged in the fractions corresponding to the molecular size of 10,000. The migration pattern of NSN in these fractions on electrophoresis was qualitatively the same as that of NSN extracted from human posterior pituitary glands. In addition to this major neurophysin, immunoreactive NSN with the molecular size of 20,000 was consistently demonstrated in three tumor extracts. This high molecular weight form of neurophysin represented 6.5--8.7% of total NSN immunoactivities in each tumor extract and its elution profile was not changed when analyzed under denaturating conditions in 6 M guanidine hydrochloride. On electrophoresis, it migrated near the gamma globulin region; however, the peak was broad suggesting that it consists of more than two different molecular populations. A substantial portion of the high molecular weight NSN appears to be a glycoprotein judging from its binding to concanavalin A. When the high molecular weight from of neurophysin was incubated with trypsin, essentially all of the activities were converted into NSN with the molecular size of 10,000. Moreover, an equimolar amount of vasopressin was liberated after the treatment, the elution pattern of which closely resembled that of synthetic arginine vasopressin. When a lower concentration of trypsin was used, some of the 20,000-dalton neurophysin exhibited activities of both NSN and vasopressin. Since the antivasopressin serum used in this study appeared to be directed toward the ring portion side of vasopressin, these results suggest that this 20,000-dalton neurophysin is, in all probability, a common precursor to vasopressin and neurophysin, and that vasopressin may be located in the middle of the precursor molecule.

Hyponatremia in rats induces downregulation of vasopressin synthesis.

Hyponatremia due to inappropriate secretion of vasopressin is a common disorder in human pathophysiology, but vasopressin synthesis during hypoosmolality has not been investigated. We used a new method to quantitate synthesis of vasopressin in rats after 3, 7, and 14 d of hyponatremia induced by administering dDAVP (a vasopressin agonist) and a liquid diet. Vasopressin synthesis was completely turned off by 7 d. Vasopressin mRNA levels in the hypothalamus paralleled the reduction in synthesis and were reduced to levels of only 10-15% of the content in control rats. When hyponatremia was corrected by withdrawal of dDAVP, vasopressin mRNA slowly returned to normal over 7 d. The observation that vasopressin synthesis can be so completely turned off leads to several conclusions: under normal physiological conditions the neurohypophysis is chronically upregulated; there must be an osmotic threshold for initiation of vasopressin synthesis (and release); the large store of hormone in the posterior pituitary is essential for vasopressin to be available during times of decreased synthesis; and, finally, some nonosmolar stimulus for synthesis must be present during clinical disorders when vasopressin is secreted (and synthesized) despite hypoosmolality.

Oxytocin synthesis and oxytocin receptor expression by cell lines of human small cell carcinoma of the lung stimulate tumor growth through autocrine/paracrine signaling.

The objective of the present work was to investigate the existence of an oxytocin (OT)-mediated autocrine/paracrine signaling upon small cell carcinoma of the lung (SCCL) cell growth. In that view, OT receptor (OTR) expression, concomitant with OT synthesis and secretion, was evidenced on three different SCCL cell lines (DMS79, H146, and H345) and related to the vasopressin (VP) system. Specific OT, VP, OTR, V1a VP receptor (V1aR), and V1b/V3 VP receptor (V1bR/V3R) transcripts were identified by reverse transcription-PCR in all cell lines studied. Binding of 125I-(d(CH2)(5)(1), Tyr(Me)(2),Thr(4),Orn(8),Tyr(9)-NH2)-vasotocin (OVTA) was observed on all SCCL cell lines, with a K(d) (dissociation constant) ranging from 0.025-0.089 nM, depending on the cell line and the analytical method. Selectivity of 125I-OVTA binding was confirmed by displacement curves obtained with various OTR and VP receptor agonists and antagonists (OT, OVTA, L-371,257, VP, F180). Immunocytochemistry identified cellular OT and VP, and peptide secretion was measured in supernatants of SCCL cultures. [3H]Thymidine incorporations, applied on H345 cells, demonstrated a dose-dependent mitogenic effect of exogenous OT (1 and 100 nM) that was abolished by the OTR antagonist OVTA. A decrease of proliferation was also observed with OVTA alone, showing a functional mitogenic effect of tumor-derived OT. Taken together, these observations demonstrate the existence of a functional OT-mediated autocrine/paracrine signaling actively implicated in growth and development of SCCL tumors. Furthermore, these findings point to the potential of OT antagonists for development as therapeutic agents for the treatment of SCCL.

Biosynthesis of vasopressin, oxytocin, and neurophysins: isolation and characterization of two common precursors (propressophysin and prooxyphysin).

[35S]Cysteine-labeled putative precursors for vasopressin-associated neurophysin (NP-VP) and oxytocin-associated neurophysin (NP-OT) were isolated from the supraoptic nuclei (SONs) of normal rats. Homozygous Brattleboro rats were deficient in one of these precursors, the NP-VP precursor. Direct support for the hypothesis that OT and its NP- and VP and its NP are synthesized from two separate macromolecular common precursors was obtained by limited proteolysis of the precursors with trypsin and identification of the fragments as NPs, VP, or OT by a number of criteria (14). In this paper, these common precursors designated propressophysin (precursor for NP-VP and arginine VP) and prooxyphysin (precursor for NP-OT and OT) were further characterized. Propressophysin was specifically bound by Concanavalin-A-Sepharose and was eluted by 0.2 M alpha-methyl mannoside, showing that it is glycosylated. In contrast, prooxyphysin does not appear to be glycosylated. Using [3H]fucose as label injected near the SONs, two proteins (mol wt, approximately 10,000) were identified, which appear to be derived from propressophysin, that are rapidly transported to the posterior pituitary of normal rats. These two proteins were absent in homozygous Brattleboro animals. Both propressophysin and prooxyphysin were bound by a NP-Sepharose affinity support. The binding was different from that of arginine VP or OT to NP, since it was independent of pH and was hydrophobic in nature. In addition to prooxyphysin, the SONs of homozygous Brattleboro rats also contained other [35S]cysteine-labeled protein (mol wt, 20,000) composed of a NP-like protein and NP-binding peptides. The tryptic peptides derived from these proteins were very different in their chromatographic (high performance liquid chromatography) properties than the tryptic peptides derived from propressophysin and prooxyphysin. This protein ('X') was not bound by a Concanavalin-A-Sepharose affinity column, although it had chromatographic properties similar to propressophysin on Sephadex G-75.

Putative precursors of vasopressin, oxytocin, and neurophysins in the rat hypothalamus.

The biosynthesis of [arginine8]vasopressin (AVP) and oxytocin (OT) was studied, and the results obtained have been compared with a study of their associated neurophysins (NP), AVP-NP and OT-NP. Rat hypothalamic extracts obtained at acid pH were subjected to Sephadex G-75 gel filtration chromatography and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Fractions of gel chromatography effluent and extract of SDS-PAGE gel slices were subjected to RIA for immunoreactive precursors of AVP, AVP-NP, OT, and OT-NP using specific antisera fro each molecule. Tritiated bovine serum albumin, ovalbumin, and cytochrome c were used as internal standards during gel filtration chromatography and SDS-PAGE. Molecular weight estimates obtained for precursors of AVP were more than 70K, 31K, 13K, 5K, and less than 5K (AVP) by G-75 chromatography and more than 70K, 39K, 15K, and less than 5K (AVP) by SDS-PAGE. Molecular weight estimates obtained for precursors of AVP-NP were more than 70K, 35K, 24K, and 12K (AVP-NP) by G-75 chromatography and 19K and 10K (AVP-NP) by SDS-PAGE. Molecular weight estimates of OT were more than 70K, 35K, 19K, 8K, and less than 5K (OT) by G-75 chromatography and 60K, 35K, 19K, 9K, and less than 5K (OT) by SDS-PAGE. Precursors of OT-NP were estimated to be more than 70K, 17-18K, and 10K (OT-NP) by G-75 chromatography and 28K, 18K and 10K (Ot-NP) by SDS-PAGE. These studies show that there are small differences in precursor processing among AVP, AVP-NP, and OT, OT-NP, but allow for the existence of common precursors for AVP and AVP-NP and for OT and OT-NP.

Biosynthesis of neurophysin proteins in the dog and their isolation.

Neurophysin (Np) is generally found in close association with vasopressin and oxytocin in the hypothalamo-neurohypophyseal complex. Dog neurophysin I and II have been isolated from fresh and frozen posterior pituitaries. The proteins were characterized on the basis of disc electrophoresis, immunological properties, amino acid composition and partial sequence determination. The amino terminal sequence of dog Np I is Ala-Ala-Leu-Asp-Leu-Asp-Val-Arg-Gln-Cys-Leu-Pro-Cys-Gly-Pro-Gly-Gly-Gln-Gly-while that of dog Np-II is Ala-Met-Ser-Asp-Leu-Glu-Leu. The dog Np I appears to be metabolically less stable than Np II. Isotope experiments with [35S]cystine or 3H-labeled amino acids using a design of "in vitro pulse and in vitro chase" as well as "in vivo pulse and in vivo chase," added further confirmation of the capability of the hypothalamic neurosecretory cells to synthesize concomitantly precursors of Np and vasopressin. The radioactively labeled precursors were converted to Np-like protein and vasopressin, both of which were isolated.

Characterisation of an intermediate in neurophysin biosynthesis in the guinea pig.

In addition to oxytocin (OT), vasopressin (AVP) and their respective neurophysins (NPs), another [35S]cysteine incorporating component is present in the guinea pig neurohypophysis. Gel filtration and Con A affinity chromatography revealed that this component was larger than NP and was glycosylated. NP-immunoreactivity was assessed using antisera which distinguish the OT- and AVP-related NPs. Whilst the anti-NP antiserum detected only one component (guinea pig NP), the anti-NP antiserum detected both NP and the glycosylated 35S-labelled component. These results suggest that a significant amount of NP in guinea pig neural lobes bears a glycopeptide extension and represents a partially processed form of the AVP precursor in this species.

Onset of neurophysin self-association upon neurophysin/neuropeptide hormone precursor biosynthesis.

The potential of the common biosynthetic precursor of neurophysin and neuropeptide hormones to self-associate has been assessed by quantitative affinity chromatographic analysis. The precursor form, with the hormone sequence in the amino terminal region and assumed able to interact intramolecularly with the hormone binding site of the neurophysin domain of the folded precursor, exhibits an affinity for neurophysin-agarose which is intermediate between those of unliganded neurophysin and non-covalently hormone-liganded neurophysin. The results lead to a prediction that neurophysin self-association is established upon precursor synthesis and prior to limited proteolysis of the precursor to release mature neurophysin and hormone components. Such self-association could play a role in packaging of the precursor into secretory granules and in regulating subsequent precursor processing events within the granules.

Expression of isotocin-neurophysin mRNA in developing zebrafish.

Neurohypophysial peptides are important regulators of homeostasis, reproduction and behavior. We have sequenced a zebrafish cDNA representing isotocin-neurophysin (IT-NP) mRNA. The developmental expression pattern of zebrafish IT-NP mRNA was determined by whole-mount in situ hybridization histochemistry. At 32 h post fertilization (hpf) no IT-NP mRNA is detected. However, by 36 hpf, staining for IT-NP mRNA is detected in a tight bilateral cluster of cells located in the anterior hypothalamus. The IT-NP mRNA expression pattern remains remarkably stable throughout further development at least until 120 hpf.

Heterologous biosynthesis and processing of preprovasopressin in Neuro2A neuroblastoma cells.

To obtain a model for the sorting and processing of preprovasopressin (preproVP), rat VP cDNA was transfected in murine Neuro2A neuroblastoma cells, which do not express VP. The precursor of VP was expressed and processed into the authentic VP gene products VP, neurophysin (NP) and glycopeptide (GP) as determined with reversed phase HPLC and radioimmunoassay. In addition, Neuro2A-specific forms of NP and GP were observed, which may be produced in the constitutive secretory pathway in these cells.

Conversion and release of an intermediate in vasopressin-neurophysin biosynthesis in the guinea-pig.

Guinea-pig neural lobes contain appreciable amounts of neurophysin with a glycopeptide extension (NPGP) which may represent a partially processed form of the arginine vasopressin (AVP) precursor. We have now studied the turnover and release of the NPGP component using a combination of in-vivo radiolabel incorporation and high pressure liquid chromatography. Measurement of the neural lobe content of 35S-labelled peptides at various times after hypothalamic injection of [35S]cysteine demonstrated that the oxytocin-related products accumulated more rapidly than the AVP-related products. The relative amounts of [35S]cysteine incorporated into NPGP and the AVP-related neurophysin (NPavp) changed markedly with time after in-vivo labelling. In-vitro incubation of neurosecretory granules prepared from neural lobes 4h after radiolabel injection produced a time- and temperature-dependent conversion of NPGP to NPavp. Incubation at 37 degrees C for 4h produced a 30% decrease in [35S]NPGP with a concomitant increase in [35S]NPavp, whilst there were no changes in the other 35S-labelled components. In-vitro stimulation of radiolabelled neural lobes by 56mM-K+ evoked a Ca++-dependent release of NPGP as well as the other expected neurosecretory components, and the amount of NPGP released reflected its neural lobe content. We conclude that the NPGP component found in guinea-pig neural lobes is a biosynthetic intermediate, most of which is further processed to NPavp. However, some NPGP may also be secreted from the neural lobe in an intact form.

Mesotocin gene expression in the diencephalon of domestic fowl: cloning and sequencing of the MT cDNA and distribution of MT gene expressing neurons in the chicken hypothalamus.

This study focuses on the structure and expression of the mesotocin (MT) gene in the chicken hypothalamus. Using an anchored and nested RT-PCR strategy, combined with circular RACE-PCR, the full length sequence of the chicken MT cDNA was obtained. The cDNA and derived amino acid sequences conformed to the structure of the oxytocin-like gene family. However, unlike most mammalian species, there does not appear to be frequent gene conversion between the MT and AVT cDNA sequences. A single specific hybridization signal of 1.2 kb was detected by Southern analysis of chicken genomic DNA, indicating only a single gene copy in the chicken genome. Northern analysis of hypothalamic RNA revealed a single band at approximately 0.6 kb. Using the same probe for in situ hybridization histochemistry, MT-mRNA was demonstrated to be predominantly localized in the parvocellular, magnocellular and periventricular subgroups of the paraventricular nucleus and, when compared to the distribution of neurons containing arginine-vasotocin (AVT)-mRNA in the same region, with far fewer neurons expressing the MT gene in the lateral subgroups. Only few and scattered neurons expressing the MT gene were found in the ventral and external subgroups of the supraoptic nucleus in which many neurons contain AVT transcripts, as demonstrated in consecutive sections. In all nuclei investigated, the intensity of AVT and MT hybridization signals per cell was approximately equal. No specific labelling for MT-mRNA was found in the bed nucleus of the stria terminalis, nor the nucleus accumbens. Using immunocytochemical detection of AVT and in situ hybridization for neurons expressing MT-mRNA, some neurons were found to contain both AVT and MT gene products in the paraventricular nucleus but not in the supraoptic nucleus.

Heterologous expression of human vasopressin-neurophysin precursors in a pituitary cell line: defective transport of a mutant protein from patients with familial diabetes insipidus.

Familial hypothalamic diabetes insipidus is an autosomal dominant disorder characterized by deficient vasopressin synthesis. Different point mutations in the vasopressin-neurophysin (VP-NP) precursor gene have been found in affected families. In a Dutch kindred, a single G to T transversion in the NP-encoding exon B of one allele converts the highly conserved glycine 17 to a valine residue. In order to examine whether this point mutation affects the processing and transport of the VP-NP precursor, the normal (HV2) and mutant (MT6) vasopressin cDNAs were stably expressed in the mouse pituitary cell line AtT20. The normal precursor was correctly glycosylated and processed, and NP was detected in the culture medium. Secretion of NP was stimulated by 8-bromo-cAMP, indicating that the normal precursor was targeted to the regulated secretory pathway. In contrast, the mutant precursor was synthesized, but processing and secretion were dramatically reduced. The mutant precursor was core-glycosylated but remained endoglycosidase H-sensitive, suggesting that the protein did not reach the trans-Golgi network. These results were supported by immunocytochemical studies. In HV2 cells, NP derived from the precursor was concentrated in the tips of the cell processes where secretory granules accumulate. In MT6 cells, NP staining was restricted to the endoplasmic reticulum (ER) as determined by colocalization with an ER-resident protein, BiP. These results suggest that the mutation within the conserved part of NP alters the conformation of the precursor and thus triggers its retention in the ER.

The presence and in vivo biosynthesis of fragments of CPP (the C-terminal glycopeptide of the rat vasopressin precursor) in the hypothalamo-neurohypophyseal system.

The existence and rate of formation of fragments of the 39-residue C-terminal glycopeptide of propressophysin (CPP1-39) was investigated in the hypothalamo-neurohypophyseal system. Newly-prepared antisera to CPP were used to confirm the existence of smaller C-terminal fragments derived from CPP1-39. Radiolabelled fucose was injected into rats in vivo into the area of the supraoptic nucleus, and the labelled peptides formed in the neurohypophysis were examined at various time intervals up to five weeks after the injection. The products derived from the neurohypophyseal hormone precursors were separated by high-performance liquid chromatography. The level of the major immunoreactive C-terminal fragment (CPP22-39) was constant and represented about 5% of the intact CPP1-39 in the neurohypophysis. The appearance of newly-synthesized N-terminal fragment of CPP1-39 occurred only after 3 or 4 days. This fucose labelled fragment increased slowly thereafter until it reached the same level as the CPP C-terminal fragment immunoreactivity between 21 and 28 days after injection. The results suggest that CPP1-39 is extremely stable in the hypothalamo-neurohypophyseal neurons, and that the cleavage at Arg21-Leu22 is a delayed proteolytic event in the magnocellular neurons of the SON.

Factors regulating the production of vasopressin-associated human neurophysin by small-cell carcinoma of the lung: evaluation by computer-enhanced quantitative immunocytochemistry.

Expression of the vasopressin gene appears to be a property common to all small-cell lung tumours. For some cultures of small-cell lung carcinoma (SCCL), Northern and Western Blot analyses have revealed that expression of this gene and its protein products are regulated by cAMP and glucocorticoids. In this study, these evaluations have been extended by examining the production of vasopressin-associated human neurophysin (VP-HNP) by computer-enhanced quantitative immunocytochemistry in a classical cell-line (H69) of SCCL, and defining the amount of protein in cells by area of positive staining above an arbitrarily set threshold. Intracellular cAMP was raised by incubating cells with either 8,Br-cAMP (0.5 mM) and IBMX (0.5 mM), or with forskolin (25 microM) and IBMX (0.5 mM). Both of these treatments caused a significant increase in the amount of positive VP-HNP immunoreactivity in the cells, an increase that was further enhanced by simultaneous administration of dexamethasone (0.1 microM). Addition of dexamethasone alone, however, caused a significant decrease in VP-HNP levels. Results confirm earlier findings from Western Blot analysis revealing the influence these agents have on production of vasopressin gene-related proteins by H69 cells, and indicate that computer-enhanced quantitative immunocytochemistry can be effectively used to provide a suitable index of this production.