Protein kinase enzymes in the human vagina-relation to key mediators of the cyclic AMP and cyclic GMP pathways.

Aside from phosphodiesterase (PDE) isoenzymes, protein kinases (cAK=cyclic AMP-binding protein kinase, cGK=cyclic GMP-binding protein kinase) have also been identified as important receptors for cyclic nucleotides. A significance of protein kinases in the control of the function of the male and female reproductive tract has been suggested; however, up until today, only a few approaches have addressed these enzymes in female genital tissues. The present study aimed to investigate by means of biochemical and immunohistochemical methods the expression of cAK and cGK. The distribution of cAK(I) and cGK(I) in relation to the vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and PDE type 4 (PDE4) was also evaluated. Cytosolic supernatants prepared from specimens of vaginal wall smooth muscle or epithelium were subjected to anion exchange chromatography and the activities of cAK and cGK(I) measured. To evaluate the distribution of cAK(I) and cGK(I) in relation to VIP, CGRP and PDE4, immunohistochemistry was conducted in sections of the human vaginal wall (full-wall specimens). Activities representing cGK(I) and cAK(I) were resolved from the chromatography column. Staining specific for cAK(Iα) was identified in both vascular and non-vascular vaginal smooth musculature, immunoreactivity for cGK(Iß) was observed in the smooth muscle and endothelium of small arteries interspersing the sections. cAK(Iα)-positive vessels were found innervated by slender varicose nerve fibers presenting the expression of VIP and CGRP. These arteries also expressed PDE4. Localization of cAK and cGK in close relation to key mediators of the cyclic AMP (PDE4, VIP) and cyclic GMP (CGRP) pathways indicate that both signaling systems may synergistically work together in human vaginal tissue.

Effects of endopeptidase inhibition on the relaxation response of isolated human penile erectile tissue to vasoactive peptides.

Peptides, such as CNP, CGRP and VIP, are involved in the function of male penile erectile tissue. Tissue levels of said peptides are controlled by the endopeptidase enzymes. Theoretically, the inhibition of the degradation of CNP, CGRP and/or VIP should result in an enhancement in penile smooth muscle relaxation. The effects were investigated of CNP or VIP (0.1 nm-1 µm), without and following pre-exposure of the tissue to a threshold concentration of the endopeptidase inhibitor KC 12615 (10 µm, for 20 min), on the reversion of tension induced by means of electrical field stimulation. Drug effects on the production of cyclic AMP/GMP were also evaluated. Neither KC 12615, CNP and VIP nor the combination of CNP plus KC 12615 or VIP plus KC 12615 increased the response of the tissue to EFS. While no effects were observed of a pre-exposure of the tissue to KC 12615 on the production of cyclic AMP in the presence of VIP, an enhancement was registered in the accumulation of cyclic AMP in the presence of CNP plus KC 12615. Further studies are indicated to investigate whether endopeptidase inhibitors might tend to be more effective in tissues affected by a decreased local production of vasoactive peptides.

Expression and distribution of the transient receptor potential cationic channel ankyrin 1 (TRPA1) in the human vagina.

The transient receptor potential cationic channel type A1 (TRPA1), belonging to a superfamily of cationic membrane channels, has been suggested to act as mechano- and pain sensor and, thus, to play a role in neurotransmission in the human body, including the urogenital tract. While the expression of TRPA1 has been investigated in a variety of tissues, up until today, no study has addressed the expression and distribution in the female genital tract. The present study aimed to investigate the expression and distribution of TRPA1 protein in human vaginal tissue. Reverse transcriptase PCR (RT-PCR) was applied in order to identify messenger ribonuleic acid specifically encoding for TRPA/A1. The distribution of TRPA1 in relation to the neuronal nitric oxide synthase (nNOS) and the signaling peptide calcitonin gene-related peptide (CGRP) was examined by means of immunohistochemical methods (double-antibody technique, laser fluorescence microscopy). RT-PCR analysis revealed the expression of mRNA encoding sequences specific for TRPA in the vaginal wall and epithelium. Immunostaining related to TRPA1 was observed in the basal epithelium and in slender varicose nerve fibers transversing the subepithelial and stromal space of the vaginal sections. In addition, these fibers presented immunoreactivity specific for nNOS or CGRP. The smooth musculature of the vaginal wall and small vessels interspersing the tissue did not present signals related to TRPA1. The findings indicate that TRPA1 might be involved in afferent neurotransmission in the vagina and work synergistically together with the nitric oxide/cyclic guanosine monophosphate pathway.

Synthesis and structure-activity relationships of trisubstituted phenyl urea derivatives as neuropeptide Y5 receptor antagonists.

1-((1R,2R)-2-Hydroxy-1-methyl-2-phenylethyl)-1-methyl-3-(4-phenoxyphenyl)urea (1) was identified as a hit from the screening of the neuropeptide Y5 (NPY5) receptor. This lead was optimized for in vitro potency by changing the stereochemistry, the phenylethyl segment, the urea portion, and the 4-phenoxyphenyl group on the molecule. Over 40 analogues of 1 were prepared to study the structure-activity relationship for this series. The most potent compounds in this class have IC(50)s less than 0.1 nM at the NPY5 receptor (e.g., 40f, 44a, and 47). To determine the functional activity for this series of compounds, selected analogues were tested in a cellular assay measuring forskolin-induced cyclic AMP accumulation in 293 cells transfected with the human NPY5 receptor. All urea analogues tested in the functional assay acted as antagonists (e.g., 1, 32, 40a, and 44e).

Structure-activity relationships of a series of pyrrolo[3,2-d]pyrimidine derivatives and related compounds as neuropeptide Y5 receptor antagonists.

Neuropeptide Y (NPY) has been shown to play an important role in the regulation of food intake and energy balance. Pharmacological data suggests that the Y5 receptor subtype contributes to the effects of NPY on appetite, and therefore a Y5 antagonist might be a useful therapeutic agent for the treatment of obesity. In attempts to identify potential Y5 antagonists, a series of pyrrolo[3, 2-d]pyrimidine derivatives was prepared and evaluated for their ability to bind to Y5 receptors in vitro. We report here the synthesis and initial structure-activity relationship investigations for this class of compounds. The target compounds were prepared by a variety of synthetic routes designed to modify both the substitution and the heterocyclic core of the pyrrolo[3,2-d]pyrimidine lead 1. In addition to identifying several potent Y5 antagonists for evaluation as potential antiobesity agents, a pharmacophore model for the human Y5 receptor is presented.

Enzymatic generation of the amino terminus of the beta-amyloid peptide.

The major pathological change in Alzheimer's disease is the deposition of 39-42-amino acid beta-amyloid peptide (BAP) in the brain. Since BAP begins at the aspartate residue (Asp1, or codon 672 of the amyloid precursor protein (APP)770 transcript), the ability of several proteases to cleave the peptide bond methionine-Asp1 (M/D) was evaluated by using peptides and recombinant APP molecules as substrates. Cathepsin G and chymotrypsin cleave the synthetic peptide HSEVKMDAEF at M/D under acidic conditions, whereas cleavage at lysine-methionine (K/M) predominates when the pH is alkaline. Trypsin and cathepsins B, D, and L are unable to cleave the synthetic peptide at M/D. Peptide SEVNLDAEF, representing the mutation found in early onset Alzheimer's disease families from Sweden, is cleaved by cathepsin G and chymotrypsin at leucine-aspartate (L/D). Incubation of cathepsin G with soluble protease nexin-2 obtained from recombinant APP (APP-REP) derivatives resulted in proteolytic cleavage at or near the amino terminus of BAP. Cathepsin G-mediated cleavage was also observed in the domain representing the amino terminus of BAP when mature plasma membrane-associated APP-REP molecules were used as substrates. Our results strongly suggest the involvement of a chymotrypsin-like serine protease in the generation of the amino terminus of BAP beginning at Asp1.

Calcium binding protein (calbindin-D28k) and glutamate decarboxylase gene expression after kindling induced seizures.

In order to determine whether calcium binding protein (calbindin-D28k or CaBP) and glutamate decarboxylase (GAD) may be involved in the process underlying the generation of seizure activity, changes in CaBP protein and mRNA and in GAD mRNA were examined in the kindling model of epilepsy. Following amygdaloid (AK) and commissure (CK) kindling significant decreases in the concentration of CaBP of 20% and 30%, respectively, were specifically observed in the hippocampal formation. However, using a cDNA specific to mammalian CaBP, Northern analysis of poly(A+) RNA and slot blot analysis of total RNA revealed no changes in the levels of CaBP mRNA in hippocampus, subcortical area (including amygdala, substantia nigra and striatum) or cerebellum of rats sacrificed 30 min, 1 h, 6 h or 24 h after the last kindled seizure. Similarly when these blots were reprobed with a cDNA specific to mammalian GAD, no changes in GAD gene expression were observed. However, fos gene expression was markedly enhanced at 1 h after seizure. We also tested whether changes in CaBP or GAD mRNA could be detected at any of the various stages of the kindling process. Slot blot analysis of cortex, subcortical structures and hippocampus revealed no changes in CaBP or GAD mRNA during the course of commissure kindling. In situ hybridization studies with GAD and CaBP 35S-labeled antisense probes also indicated no obvious changes upon visual analysis of autoradiographs. However, when silver grains were counted, significant changes in GAD mRNA in individual cells in hippocampus and substantia nigra were noted after kindling induced epilepsy. Our results indicate that, unlike fos gene expression, prominent alterations in GAD and CaBP mRNA in gross brain regions (as measured by slot blot and Northern blot analyses) are not observed in the kindling process. However, our in situ hybridization studies suggest that changes in GAD mRNA in individual cells may be involved in the process underlying kindling induced seizure activity.

The biological activity of atrial natriuretic factor cleaved by endoprotease 3.4.24.11.

Ring cleavage of atrial natriuretic peptide (ANF) between Cys7 and Phe8 by endoprotease 3.4.24.11 yields X-ANF. Since endoprotease 3.4.24.11 may contribute to ANF metabolism in vivo, the present study determined if X-ANF exhibits reduced biological activity in comparison to the parent molecule.

Regulation of proenkephalin by Fos and Jun.

Fos and Jun form a heterodimeric complex that associates with the nucleotide sequence motif known as the AP-1 binding site. Although this complex has been proposed to function as a transcriptional regulator in neurons, no specific target gene has yet been identified. Proenkephalin mRNA increased in the hippocampus during seizure just after an increase in c-fos and c-jun expression was detected. Fos-Jun complexes bound specifically to a regulatory sequence in the 5' control region of the proenkephalin gene. Furthermore, c-fos and c-jun stimulated transcription from this control region synergistically in transactivation assays. These data suggest that the proenkephalin gene may be a physiological target for Fos and Jun in the hippocampus and indicate that these proto-oncogene transcription factors may play a role in neuronal responses to stimulation.

Nerve growth factor-induced differentiation in PC-12 cells is blocked by fos oncogene.

Rat pheochromocytoma (PC-12) cells respond to nerve growth factor (NGF) by cessation of cell division and by expression of several properties resembling those of differentiated sympathetic neurons. Within minutes after addition, NGF rapidly stimulates transient expression of c-fos. To investigate the possible role of c-fos in NGF-induced differentiation, activated mouse c-fos genes were introduced into PC-12 cells by electroporation. Constitutive expression of fos inhibited NGF-induced differentiation although transfected cells harbored intact NGF receptors. Dibutyryl cyclic AMP (db cAMP) and basic fibroblast growth factor (b-FGF)-induced differentiation were also inhibited. Transcriptional activation of c-fos, c-jun, and ornithine decarboxylase (ODC) by NGF was down-regulated, whereas expression of egr-1 was unaffected in PC-12 fos clones. These results suggest that deregulated expression of fos can interfere with the normal role of NGF in neuronal differentiation.

Dynamic alterations occur in the levels and composition of transcription factor AP-1 complexes after seizure.

Seizure causes a rapid and protracted increase in transcription factor AP-1 levels in the brain. The composition of AP-1 nucleoprotein complexes changes with time after seizure as a result of the sequential appearance and disappearance of Fos and several Fos-related proteins. Although these changes occur over an 8 hr time period, they are triggered by 15 min of seizure. Alterations in the levels and composition of transcription factors may represent one of the molecular mechanisms underlying neuronal adaptation.

Glutamate receptor agonists increase the expression of Fos, Fra, and AP-1 DNA binding activity in the mammalian brain.

Administration of convulsant doses of Metrazole (pentylenetetrazol) and picrotoxin, as well as maximal electroshock, results in a rapid but transient increase in c-fos mRNA in mouse brain. Elevation of c-fos mRNA is followed by the accumulation and subsequent disappearance of Fos, the protein encoded by c-fos. In addition, immunoblots reveal the induction of two additional proteins that are antigenically related to Fos (Fra, Fos-related antigens). Fos and the various Fra appear and disappear in a staggered manner over an 8 hour period, such that at longer times after stimulation the brain contains no Fos but relatively large amounts of Fra (the latter being designated here by their apparent molecular weights, Fra-46K and Fra-35K). Previous studies have established that Fos, as well as several Fra, contribute to transcription factor AP-1 nucleoprotein complexes along with Jun, the product of the jun proto-oncogene. The appearance in brain of Fos and Fra coincides with a protracted increase in total AP-1 DNA binding activity, indicating that all the Fos-like proteins can participate in AP-1 complexes. Furthermore, the molecular composition of these complexes alters with time after stimulation. The induction of c-fos by Metrazole is blocked or attenuated by known anticonvulsants such as diazepam and valproate as well as the N-methyl-D-aspartate (NMDA) receptor antagonists, 2-amino-5-phosphonovaleric acid (APV) and MK-801. This suggests that fos induction might involve stimulation of a glutamate receptor. This conclusion was strengthened by the observations that two glutamate receptor agonists, kainic acid and NMDA, induced c-fos expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of protease 3.4.24.11 as the major atrial natriuretic factor degrading enzyme in the rat kidney.

We have identified a metalloendoprotease from rat kidney cortex that cleaves the cysteine-phenylalanine bond (Cys7-Phe8) within the 17 amino acid ring structure of atrial natriuretic factor (ANF). Cleavage at this site represents the major ANF degradative activity in rat kidney, and is inhibited by the known metalloendoprotease inhibitors, thiorphan, phosphoramidon and zincov with IC50 values in the nanomolar range. Since these are specific inhibitors of protease 3.4.24.11, both protease 3.4.24.11 and ANF degrading activities were monitored during purification. Both activities copurified at each chromatographic step. Furthermore, purified protease 3.4.24.11 cleaved ANF specifically at the Cys7-Phe8 bond. It is concluded from this work that the major ANF degrading enzyme in rat kidney is protease 3.4.24.11.

1,25-Dihydroxyvitamin D3 treatment results in increased choline acetyltransferase activity in specific brain nuclei.

To better understand the role of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in brain function, the level of calcium-binding protein (CaBP) and the activities of choline acetyltransferase (CAT) and monoamine oxidase were measured in discrete brain nuclei of vitamin D-deficient and -replete male rats. The nuclei sampled were those in which receptors for 1,25-(OH)2D3 and/or vitamin D-dependent CaBP have been localized. Significant elevations in CAT activity were found in the arcuatemedian eminence and in the bed nucleus of the stria terminalis of rats made vitamin D replete by eight daily ip injections of 100 or 200 ng 1,25-(OH)2D3 as well as by constant intraventricular (ivi) infusion of 25 ng 1,25-(OH)2D3 for 7 days. The percent increase ranged from 12-45% and was related to the ip dose administered. Constant ivi of 2 mM CA2+ or 125 ng 25-hydroxyvitamin D3/day for 7 days did not alter CAT activity. No significant changes in monoamine oxidase or CaBP in discrete brain nuclei were observed with vitamin D repletion. Since the arcuate-median eminence of the hypothalamus is an important regulatory site in the neuroendocrine control of reproduction, serum testosterone was measured. Serum testosterone levels were abnormally low in the vitamin D-deficient animals, but increased 2- to 5-fold to normal values in those rats made vitamin D replete by constant ivi of 25 ng 1,25-(OH)2D3 or by ip injection of 100 or 200 ng 1,25-(OH)2D3. Patterns of serum LH paralleled those for testosterone. Our results suggest that 1,25-(OH)2D3 effects cholinergic activity in several discrete brain regions and may play a role in the neuroendocrine regulation of certain aspects of anterior pituitary gland function.

Purification of radioiodinated photoactivable glucagon by isocratic high-performance liquid chromatography.

2-Nitro-4-azidophenylsulphenyl-glucagon, a specific photoaffinity label for glucagon receptors, was synthesized and radioactively labelled with 125I. The radio-labelled peptide was purified from the reaction mixture by high-performance liquid chromatography in one step by isocratic elution from a C18 column with 20.4% n-propanol in 10 mM phosphate buffer (pH 2.5) as eluent. This glucagon derivative can be used to attach a label specifically to the glucagon receptor. The binding ability of the photoaffinity derivative was tested on isolated intact rat hepatocytes. Compared with a Kd of 3 nM for unmodified monoiodinated glucagon, the Kd value of the photoaffinity labelled monoiodinated glucagon tracer was 7 nM.

Vitamin D-dependent rat renal calcium-binding protein: development of a radioimmunoassay, tissue distribution, and immunologic identification.

A sensitive double antibody RIA has been developed for the 28,000 mol wt rat renal vitamin D-dependent calcium-binding protein. Using this assay, concentrations of calcium-binding protein (CaBP) as low as 30 ng can be measured. The assay is precise (intraassay variability, 5.0%) and reproductible (interassay variability, 8.2%). Measurements of renal CaBP by RIA showed a good correlation with measurements of CaBP by the chelex resin assay and by polyacrylamide gel analysis by densitometric tracing using a purified CaBP marker. The concentration of CaBP in the vitamin D-replete rat kidney is 7.3 +/- 1.0 (mean +/- SEM) micrograms/mg protein. In vitamin D-deficient rats the level of renal CaBP is 2.6 +/- 0.3 micrograms/mg protein. Tissue distribution of immunoreactive rat renal CaBP showed the highest concentration of CaBP in the rat cerebellum (38.3 +/- 5.1 micrograms/mg protein). Lower concentrations of immunoreactive CaBP were detected in several other rat tissues. No immunoreactive CaBP was detected in rat or human serum. In necropsy human kidney and cerebellum, high levels of immunoreactive CaBP were also detected (1.5 +/- 0.1 and 27.3 +/- 2.1 micrograms/mg protein, respectively). When extracts of rat kidney and brain and human cerebellum and kidney were assayed at several dilutions, immunodisplacement curves parallel to that of pure renal CaBP were observed, indicating immunochemical similarity. Fractionation of extracts of rat cerebellum, human kidney, and human cerebellum on Sephadex G-100 revealed immunoreactivity and calcium-binding activity in the 28,000 mol wt region similar to rat kidney.