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1.
Mol Pharm ; 12(1): 34-45, 2015 Jan 05.
Article in English | MEDLINE | ID: mdl-25412253

ABSTRACT

UNLABELLED: In vitro studies of ocular bioavailability of active pharmaceutical ingredients (API) from colloidal drug delivery systems do not consider physiological shear stress generated by eyelid wiping and tear flow. The present study introduces a live cell imaging approach which enables the investigation of model drug uptake from various formulations under shear stress by using custom-made microchannels for the cultivation of human corneal epithelial cells (HCE-T). Coumarin-6 (C-6) was used as a model API incorporated into solid lipid nanoparticles and liposomes, and as an aqueous crystalline suspension. Confocal laser scanning microscopy visualized C-6 uptake into HCE-T cells in a time-resolved manner with an applied shear stress of 0.1 Pa. Static conditions were also studied for comparative purposes. Additionally, solid lipid nanoparticles (SLN) were labeled with a fluorescent phospholipid to check whether C-6 uptake was associated with SLN incorporation into the cells. RESULTS: Intact SLN were not incorporated into the cells, i.e., C-6 was passively redistributed from SLN to lipophilic cellular compartments. C-6 was enriched up to a given limit in HCE-T cells within 5 min of contact with the dispersions both under static and under flow conditions. The C-6 delivery rate from liposomes was superior to that from SLN whereby the suspension exhibited the lowest rate. C-6 release rates were comparable for static and flow conditions. Alternate flushing with formulations and buffer revealed that cells accumulated C-6. The results suggest that combining microfluidics with live cell imaging provides a valuable option for in vitro studies of ocular drug delivery.


Subject(s)
Cornea/drug effects , Coumarins/chemistry , Epithelial Cells/drug effects , Nanoparticles/chemistry , Thiazoles/chemistry , Biological Availability , Cell Line , Cell Line, Tumor , Cell Survival , Cornea/metabolism , Crystallization , Drug Delivery Systems , Drug Design , Epithelial Cells/cytology , Eye/drug effects , Fluorometry/methods , Humans , Lipids/chemistry , Liposomes/chemistry , Liposomes/metabolism , Micelles , Microfluidic Analytical Techniques , Microfluidics , Microscopy, Confocal , Microscopy, Electron, Transmission , Microscopy, Fluorescence
2.
J Dent Res ; 90(4): 517-22, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21212316

ABSTRACT

The nitric oxide (NO) receptor enzyme soluble guanylate cyclase (sGC) contains one prosthetic heme group as an αß heterodimer, and two heterodimer isoforms (α(1)ß(1), α(2)ß(1)) were characterized to have enzyme activity. To test the irreversible inflammation-dependent regulation of sGC in odontoblasts, we incubated decalcified frozen sections of healthy and inflamed human third molars with antibodies against ß-actin, nitrotyrosine, inducible nitric oxide synthase (iNOS), α(1)-, ß(1)-, and α(2)-subunits of sGC and analyzed them at protein levels by quantitative immunohistochemistry. The irreversible inflammation induced an increase in the signal intensities for nitrotyrosine and iNOS and a decrease for the α(1)-, ß(1)-, and α(2)-subunits of sGC in odontoblasts. Inflammatory mediators, reactive oxygen, and nitrogen species may impair the expression of the α(1)-, ß(1)-, and α(2)-subunits in odontoblasts. The decrease of sGC at the protein level in inflamed odontoblasts is compatible with a critical role for sGC to mediate biological effects of NO in health.


Subject(s)
Dental Caries/enzymology , Guanylate Cyclase/analysis , Odontoblasts/enzymology , Pulpitis/enzymology , Receptors, Cytoplasmic and Nuclear/analysis , Actins/analysis , Adolescent , Adult , CD11b Antigen/analysis , CD3 Complex/analysis , Dental Caries/pathology , Dental Pulp/enzymology , Dental Pulp/pathology , Dentin/enzymology , Dentin/pathology , Humans , Immunohistochemistry , Inflammation , Inflammation Mediators/analysis , Isoenzymes/analysis , Microscopy, Confocal , Nitric Oxide Synthase Type II/analysis , Odontoblasts/metabolism , Pulpitis/pathology , Reactive Nitrogen Species/analysis , Reactive Oxygen Species/analysis , Soluble Guanylyl Cyclase , Tyrosine/analogs & derivatives , Tyrosine/analysis , Young Adult
3.
Meat Sci ; 81(3): 433-8, 2009 Mar.
Article in English | MEDLINE | ID: mdl-22064279

ABSTRACT

Relationships of temperament evaluated at different production stages with growth, carcass characteristics and beef tenderness were determined in Bonsmara crossbred steers managed under commercial managent. Temperament was evaluated at weaning and at initiation of the finishing phase. Steers from a Roswell, NM ranch (n=156) and a Cline, TX ranch (n=21) were stratified at fall weaning by weight and source and randomly allotted to winter ryegrass at Uvalde or Overton, TX followed by feeding in a commercial feedlot near Batesville, TX. Cattle were observed for temperament (escape velocity, EV, m/s; pen and chute temperament score, PTS and CTS) at weaning and upon entry to the feedlot. Cattle were harvested at approximately 7 mm 12th rib fat. Carcass data was taken approximately 36 hrs post-mortem and 2.5cm thick steaks were removed from the 13th rib for Warner-Bratzler shear force (WBS) determination. The only measures of temperament significantly related to performance were EV and PTS. Weaning EV appeared to be more related to feedlot ADG (r=-0.26, P<0.003), ribeye area (r=-0.37, P<0.0008), yield grade (r=0.29, P<0.01) and WBS, r=0.27, P<0.005) than did the later measures of temperament. However, in-feedlot EV was associated with feedlot weights (r=-0.28, P<0.0004). Results of this research suggest temperament, particularly at weaning, is related to feedlot performance, carcass merit, and beef tenderness at a low to moderate level and evaluation of this trait may be a helpful management tool.

4.
J Dent Res ; 84(7): 618-23, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15972589

ABSTRACT

By the formation of cyclic guanosine 3',5'-monophosphate (cGMP), nitric oxide (NO)-sensitive enzyme-soluble guanylate cyclase (sGC) plays a receptor role for NO within the NO-cGMP signaling cascade, which is involved in vasodilatation and neurotransmission. The hypothesis that NO-cGMP signaling molecules modulate cells of the dentin-pulp complex was investigated in rat molars by histochemical, immunohistochemical, immuno-ultrastructural, and organ bath techniques. NO synthase (NOS) I-III, the sGC alpha(2)-subunit/beta(1)-subunit, and cGMP were detected in odontoblasts and blood vessels. NOS I, sGC alpha(2), and cGMP were identified in nerve fibers. Treatment of rat molars with the NO donor NONOate (10(-5) M) increased cGMP staining intensities in blood vessels and odontoblasts, while NO synthase inhibitor L-NAME (10(-4) M) attenuated intensity of the reaction products for cGMP, suggesting an effect of endogenous NO on sGC. These correlations of patterns and alterations of cGMP staining intensities after treatment with the NO donor or NO inhibitor might represent an NO-sGC-cGMP signaling-dependent modulation of odontoblasts, blood vessels, and nerve fibers in the dentin-pulp complex.


Subject(s)
Cyclic GMP/metabolism , Dental Pulp/enzymology , Dentin/enzymology , Nitric Oxide/metabolism , Odontoblasts/enzymology , Animals , Dental Pulp/ultrastructure , Dentin/ultrastructure , Guanylate Cyclase/metabolism , Immunohistochemistry , Male , Molar/cytology , Molar/enzymology , Molar/ultrastructure , Nitric Oxide Synthase/metabolism , Odontoblasts/ultrastructure , Rats , Rats, Wistar , Signal Transduction/physiology , Tissue Distribution
5.
Neuroreport ; 12(18): 3993-6, 2001 Dec 21.
Article in English | MEDLINE | ID: mdl-11742226

ABSTRACT

Nitric oxide (NO) is a key molecule in vascular headaches and the dura mater has been implicated as a tissue where vascular headache develops. Here we demonstrate expression, enzyme activity and cellular distribution of the intracellular receptor for NO, soluble guanylyl cyclase (sGC), in rat dura mater. Subcutaneous treatment of rats with the NO-donor glyceryl trinitrate (GTN) induced an increase of sGC expression and activity in dural blood vessels after 20-30 min. It has previously been shown that GTN induces headache in normal subjects after 20-30 min. Our findings suggest that an up-regulation of the NO target enzyme contributes to the pathogenesis of GTN-induced headache explaining the subacute rather than acute onset of symptoms.


Subject(s)
Dura Mater/drug effects , Dura Mater/enzymology , Guanylate Cyclase/metabolism , Nitroglycerin/pharmacology , Vasodilator Agents/pharmacology , Animals , Dura Mater/blood supply , Endothelium, Vascular/enzymology , Endothelium, Vascular/ultrastructure , Female , Guanylate Cyclase/analysis , Headache/metabolism , Male , Microscopy, Immunoelectron , Rats , Rats, Sprague-Dawley
6.
J Biol Chem ; 276(33): 30737-43, 2001 Aug 17.
Article in English | MEDLINE | ID: mdl-11406623

ABSTRACT

Previously characterized mammalian soluble guanylyl cyclases form alpha/beta heterodimers that can be activated by the gaseous messenger, nitric oxide, and the novel guanylyl cyclase modulator YC-1. Four mammalian subunits have been cloned named alpha(1), beta(1), alpha(2), and beta(2). The alpha(1)/beta(1) and alpha(2)/beta(1) heterodimeric enzyme isoforms have been rigorously characterized. The role of the beta(2) subunit has remained elusive. Here we isolate a novel variant of this subunit and show that the beta(2) subunit does not need to form heterodimers for catalytic activity because enzyme activity can be measured when it is expressed alone in Sf9 cells. In analogy to the beta(3) subunit recently isolated from the insect Manduca sexta, activity was dependent on the presence of 4 mm free Mn(2+). The EC(50) values for the NO-donor diethylamine/NO were shifted to the left by 1 order of magnitude as compared with the alpha(1)/beta(1) heterodimeric form. In the presence of the detergent Tween, NO sensitivity of beta(2) was abolished, but the enzyme could be activated by protoporphyrin IX, indicating removal of a prosthetic heme group and exchange for the heme precursor. We suggest that the beta(2) subunit is the first mammalian NO-sensitive guanylyl cyclase lacking a heterodimeric structure.


Subject(s)
Guanylate Cyclase/metabolism , Nitric Oxide/physiology , Amino Acid Motifs , Amino Acid Sequence , Animals , Cell Line , Dimerization , Enzyme Activation , Guanylate Cyclase/chemistry , Manganese/pharmacology , Molecular Sequence Data , Protein Subunits , Rats , Spodoptera
7.
Biochem Biophys Res Commun ; 283(4): 883-7, 2001 May 18.
Article in English | MEDLINE | ID: mdl-11350067

ABSTRACT

Inhaled nitric oxide (NO) is known to influence the contractile state of pulmonary arteries most likely by activation of soluble guanylyl cyclase (sGC) in smooth muscle cells. However, the cellular distribution of sGC has not been determined empirically, due to a lack of specific antibodies. Here, we describe a novel antibody directed against the beta1 subunit of sGC to study the cellular distribution of sGC in lung during development. Using the novel antibody, the enzyme was demonstrated in fetal, neonatal, and adult lungs by Western blot, showing maximum expression in neonatal lung. These data were confirmed by measurements of sGC activity. In pulmonary arteries of fetal lung sGC-beta1 immunoreactivity was present in smooth muscle cells and absent in endothelial cells. With postnatal development an increase in immunoreactivity in endothelial cells and a reciprocal decrease in smooth muscle cells was apparent. The reported changes in sGC expression likely contribute to the known age-dependent differences in response to inhaled NO.


Subject(s)
Aging/metabolism , Guanylate Cyclase/metabolism , Nitric Oxide/metabolism , Pulmonary Artery/enzymology , Amino Acid Sequence , Animals , Antibody Specificity , Guanylate Cyclase/immunology , Immunohistochemistry , Molecular Sequence Data , Rats , Rats, Wistar
8.
Neuroreport ; 12(5): 979-81, 2001 Apr 17.
Article in English | MEDLINE | ID: mdl-11303772

ABSTRACT

Soluble guanlylyl cyclase (sGC) seems to be involved in mechanisms for rapid translation of electrical and chemical signals at the neuromuscular junction. To explore the cellular localization of the alpha2, alpha1 and beta1 subunits of sGC, we studied normal and denervated human muscle biopsies immunohistochemically using antibodies directed against the alpha2 and alpha1/beta1 subunits of sGC and performed double labellings with alpha-bungarotoxin. Confocal imaging could localize the alpha2 and alpha1/beta1 subunits of sGC at neuromuscular junctions and vessels and the subunits remained concentrated at neuromuscular junctions following denervation. The presence of sGC at neuromuscular junctions and at vessels suggests sGC could serve as a postsynaptic second messenger for fine tuning of nerve-muscle interaction and dynamic regulation of intramuscular blood flow.


Subject(s)
Guanylate Cyclase/metabolism , Muscle, Skeletal/enzymology , Neuromuscular Junction/enzymology , Aged , Bungarotoxins/pharmacology , Female , Humans , Immunohistochemistry , Male , Microscopy, Confocal , Middle Aged , Muscle Denervation , Muscle, Skeletal/ultrastructure , Neuromuscular Diseases/enzymology , Neuromuscular Diseases/pathology , Neuromuscular Junction/ultrastructure , Second Messenger Systems/physiology
9.
Chemistry ; 7(5): 1133-42, 2001 Mar 02.
Article in English | MEDLINE | ID: mdl-11303873

ABSTRACT

We have studied the ligand behavior of racemic isovalinate (iva) and valinate (val) towards zinc(II) and calcium(II). The following solid metal amino acidates were obtained from aqueous solutions: Zn3Cl2(iva)4 (1), Zn3Cl2(val)4 (2). Zn(val)2 (3), Zn(iva)2 x 2H2O (4), Zn(iva)2 x 3.25H2O (5), Zn(iva)2 (6), Ca(iva)2x xH2O (7), and Ca(val)2 x H2O (8). Except for complex 3, these were hitherto unknown compounds. The conditions under which they formed, together with current ideas of the conditions on early Earth, support the assumption that alpha-amino acidate complexes of zinc and calcium might have belonged to early Earth's prebiotic chemical inventory. The zinc isovalinates 1, 4, and 5 were characterized by X-ray crystal structure analyses. Complex 1 forms a layer structure containing four- and five-coordinate metal atoms, whereas the zinc atoms in 4 and 5 are five-coordinate. Compound 5 possesses an unprecedented nonpolymeric structure built from cyclic [Zn6(iva)12] complexes, which are separated by water molecules. The thermolyses of solids 1. 3, and 8 at 320 degrees C in an N2 atmosphere yielded numerous organic products, including the cyclic dipeptide of valine from 3 and 8. Condensation, C-C bond breaking and bond formation, aromatization, decarboxylation, and deamination reactions occurred during the thermolyses. Such reactions of metal-bound a-amino acidates that are abiotically formed could already have contributed to an organic-geochemical diversity before life appeared on Earth.

10.
J Clin Endocrinol Metab ; 86(2): 909-12, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11158065

ABSTRACT

The cytoplasmic or soluble forms of guanylyl cyclase (sGC) are heme-containing heterodimeric enzymes that are regulated by nitric oxide (NO) and carbon monoxide (CO). These gaseous messenger molecules are produced in the human placenta and are potential regulators of vasodilation and trophoblast invasion. The alpha(2)-subunit of sGC has only recently been shown to naturally occur in placental extracts. In the present study, two novel antibodies directed against different epitopes of the alpha(2) subunit, were generated. Western Blot analysis confirmed the presence of a 82 kDa protein, identical with alpha(2) protein overexpressed in Sf9 cells. According to RNase protection analysis the alternatively spliced alpha(2i) variant was absent from human placenta. Immunohistochemical analysis showed the presence of alpha(2) protein in syncytiotrophoblast and villous and umbilical blood vessels, which are known sites of NO production. Strong expression was observed in the extravillous (intermediate) trophoblast, where the expression of CO-generating hemeoxygenases has recently been documented. Localization of alpha(2) subunit expression suggests a role for sGC in mediating the actions of both NO and CO. The novel antibodies characterized in the present study will be powerful tools to further elucidate the role of the NO/CO/cGMP signaling pathways in pathologic states such as preeclampsia and intrauterine growth retardation.


Subject(s)
Guanylate Cyclase/analysis , Guanylate Cyclase/genetics , Placenta/enzymology , Amino Acid Sequence , Animals , Antibody Specificity , Blotting, Western , Cell Line , Cytosol/enzymology , Epitopes/chemistry , Epitopes/immunology , Female , Guanylate Cyclase/chemistry , Humans , Immunohistochemistry , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/immunology , Placenta/cytology , Pregnancy , Protein Subunits , Reverse Transcriptase Polymerase Chain Reaction , Spodoptera , Transfection
11.
Naunyn Schmiedebergs Arch Pharmacol ; 364(6): 573-6, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11770014

ABSTRACT

We have recently shown that nitric oxide activates the beta2 subunit of soluble guanylyl cyclase. In the present study, we show developmental regulation of this subunit. Analysis of mRNA expression by RT-PCR and RNase protection analysis in kidneys revealed no expression of the beta2 subunit in neonatal and strong expression in adult rats. A reciprocal regulation with much lower expression levels was observed in rat lung. Further examination of kidneys from 3, 6, 16, 22, 25, 31 and 36-day-old rats showed that significant expression appears between postnatal day 16 and 22. Isolation of the rat beta2 promoter by genome walking and cloning into a reporter gene vector showed promoter activity for the sense but not the antisense construct providing an in vitro assay for further analysis of the developmental beta2 subunit regulation.


Subject(s)
Kidney/enzymology , Kidney/growth & development , Receptors, Cytoplasmic and Nuclear/biosynthesis , Animals , Animals, Newborn , Guanylate Cyclase , Lung/enzymology , Lung/growth & development , Molecular Sequence Data , Nitric Oxide/biosynthesis , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley , Sequence Analysis, DNA/methods , Soluble Guanylyl Cyclase
12.
Biochim Biophys Acta ; 1494(3): 286-9, 2000 Dec 01.
Article in English | MEDLINE | ID: mdl-11121588

ABSTRACT

Nitric oxide-sensitive guanylyl cyclase is a heterodimeric enzyme consisting of one alpha and one beta subunit. Here, we clone the first alpha(2) subunit ortholog and functionally express the cDNA in Sf-9 cells. Our data indicate a high degree of conservation of the primary sequence and functional activity of the rat alpha(2) subunit.


Subject(s)
Guanylate Cyclase/genetics , Amino Acid Sequence , Animals , Cell Line , Cloning, Molecular , Conserved Sequence , Guanylate Cyclase/biosynthesis , Guanylate Cyclase/chemistry , Isoenzymes/genetics , Kidney/metabolism , Male , Molecular Sequence Data , Nitric Oxide/pharmacology , Rats , Rats, Sprague-Dawley , Solubility
13.
Phys Rev Lett ; 84(25): 5716-21, 2000 Jun 19.
Article in English | MEDLINE | ID: mdl-10991038

ABSTRACT

We report the results of a search for a W' boson produced in pp collisions at a center-of-mass energy of 1.8 TeV using a 107 pb-1 data sample recorded by the Collider Detector at Fermilab. We consider the decay channel W'-->&munumu and search for anomalous production of high transverse mass munumu lepton pairs. We observe no excess of events above background and set limits on the rate of W' boson production and decay relative to standard model W boson production and decay using a fit of the transverse mass distribution observed. If we assume standard model strength couplings of the W' boson to quark and lepton pairs, we exclude a W' boson with invariant mass less than 660 GeV/c2 at 95% confidence level.

14.
Eur J Pharmacol ; 403(1-2): 27-35, 2000 Sep 01.
Article in English | MEDLINE | ID: mdl-10969140

ABSTRACT

Soluble guanylyl cyclase activity and its stimulation by diethylamineNONOate was measured in aortae from hypertensive TGR(mREN2)27 rats (TGR) and Sprague-Dawley controls. Superoxide dismutase was added in vitro to evaluate the contribution of oxidative breakdown of nitric oxide (NO) by superoxide anions. Expression of soluble guanylyl cyclase was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Basal and stimulated soluble guanylyl cyclase activity was significantly reduced in TGR rats, addition of superoxide dismutase had no effect. Expression of soluble guanylyl cyclase subunits was not different between strains. The independent contribution of hypertension and the overactive renin-angiotensin system to soluble guanylyl cyclase subsensitivity was assessed after normalization of TGR's blood pressure by the Ca(2+)-channel blocker amlodipine or the angiotensin converting enzyme-inhibitor enalapril. Soluble guanylyl cyclase activity in TGR was slightly increased by amlodipine and almost completely restored by enalapril. In conclusion, TGR showed desensitized vascular soluble guanylyl cyclase, depending on their overactive renin-angiotensin system.


Subject(s)
Guanylate Cyclase/metabolism , Hypertension/physiopathology , Renin-Angiotensin System/physiology , Aging/physiology , Amlodipine/pharmacology , Analysis of Variance , Animals , Animals, Genetically Modified , Aorta, Thoracic/drug effects , Aorta, Thoracic/enzymology , Blood Pressure/drug effects , Calcium Channel Blockers/pharmacology , Cyclic GMP/metabolism , Dose-Response Relationship, Drug , Enalapril/pharmacology , Guanylate Cyclase/genetics , Hydrazines/pharmacology , Hypertension/genetics , Male , Nitrogen Oxides , RNA, Messenger/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Solubility
16.
Biochem Biophys Res Commun ; 271(1): 64-9, 2000 Apr 29.
Article in English | MEDLINE | ID: mdl-10777682

ABSTRACT

Soluble or nitric oxide (NO) stimulated guanylyl cyclases are obligate heme-containing heterodimers (alpha/beta). We report the full-length cDNA of the human ortholog of the rat beta(2)-subunit from human kidney. A database search yielded matches of the 3' non-coding sequence with previously unassigned expressed sequence tags from kidney and stomach signet ring cell carcinoma. PCR comparison of cDNA from stomach signet ring cell carcinoma and normal stomach tissue demonstrated beta(2) subunit expression in cancer but not in normal tissue. On the cDNA level a frameshift deletion of one nucleotide was present in the novel human sequence which was confirmed on the genomic DNA level. In four closely related nonhuman primate species the frameshift deletion was absent while analysis of genomic DNA from different ethnic backgrounds revealed the uniform presence of the frameshift deletion in the human population.


Subject(s)
Frameshift Mutation , Guanylate Cyclase/chemistry , Guanylate Cyclase/genetics , Stomach Neoplasms/chemistry , Stomach Neoplasms/genetics , Aged , Amino Acid Sequence , Animals , Base Sequence , Blotting, Southern , Carcinoma, Signet Ring Cell/genetics , Cloning, Molecular , DNA, Complementary/metabolism , Expressed Sequence Tags , Gorilla gorilla , Humans , Kidney/metabolism , Macaca mulatta , Male , Molecular Sequence Data , Myocardium/metabolism , Polymerase Chain Reaction , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid
17.
Andrologia ; 32(1): 55-9, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10702869

ABSTRACT

The messenger molecule cyclic guanosine monophosphate (cGMP) is produced by different isoforms of the enzyme guanylate cyclase (GC). Natriuretic peptides (ANP and CNP) bind to and activate particulate GCs, whereas NO and CO activate a soluble form of GC. The specific relevance of the cGMP system for reproductive functions has been recently demonstrated by the successful use of sildenafil (Viagra), an inhibitor of cGMP-specific phosphodiesterase type 5, for the treatment of erectile dysfunction. In the testis, cGMP signal transduction pathways are involved in a variety of local functions, based on autocrine or paracrine effects. In particular, cGMP has been suggested to influence motility in spermatozoa, development of testicular germ cells, relaxation of peritubular lamina propria cells, testosterone synthesis in Leydig cells and dilatation of testicular blood vessels. The physiological significance of cGMP accumulation in Scrtoli cells is not yet clear. Taken as a whole, the evidence suggests that cGMP-mediated processes might influence both the potentia coeundi within the penis and the potentia generandi at various levels within the testis.


Subject(s)
Cyclic GMP/physiology , Testis/physiology , Animals , Humans , Leydig Cells/physiology , Male , Second Messenger Systems , Sertoli Cells/physiology , Spermatozoa/physiology , Testis/blood supply
18.
Biochem Pharmacol ; 59(6): 713-7, 2000 Mar 15.
Article in English | MEDLINE | ID: mdl-10677588

ABSTRACT

The muscles of the corpus cavernosum of the penis relax in response to stimulation of non-adrenergic, non-cholinergic nerves or nitric oxide (NO)-donating drugs to elicit erection. It is generally assumed that NO mediates this effect via activation of soluble guanylyl cyclase and a subsequent increase in cyclic guanosine 3', 5'-monophosphate concentration. However, there are no data on the expression of this enzyme in human corpus cavernosum. The purpose of the present study was the molecular characterization of NO-sensitive guanylyl cyclase in human corpus cavernosum. RNA was extracted from tissue samples obtained from seven patients undergoing penile prosthetic surgery or correction of penile deviation. Reverse transcriptase-polymerase chain reaction (RT-PCR) with specific primers for the subunits of NO-sensitive guanylyl cyclase was performed, and PCR products were subcloned and sequenced. Specific amplification products encoding the alpha(1), beta(1), alpha(2), and beta(2) subunits were detected. In addition, we isolated a transcript encoding a novel variant beta(2) subunit. To test whether this novel transcript arises by alternative splicing or whether it is encoded by a separate gene, a 4000-bp clone of the corresponding genomic DNA sequence was isolated. Sequence analysis suggests that the novel beta(2) variant arises by alternative splicing from the same gene as the beta(2) subunit on chromosome 13. In conclusion, our findings suggest the presence of different subunit mRNAs of NO-sensitive guanylyl cyclase in human corpus cavernosum.


Subject(s)
Guanylate Cyclase/biosynthesis , Nitric Oxide/metabolism , Penis/metabolism , Amino Acid Sequence , Guanylate Cyclase/genetics , Humans , Male , Molecular Sequence Data , Penis/enzymology , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid
20.
Biochem J ; 335 ( Pt 1): 125-30, 1998 Oct 01.
Article in English | MEDLINE | ID: mdl-9742221

ABSTRACT

Soluble guanylyl cyclase (sGC), the target enzyme of the signalling molecule NO, contains one prosthetic haem group and consists of an alpha and a beta subunit. So far, only the alpha1beta1 heterodimer has been shown to exist in different cells and tissues, and most biochemical studies of sGC have been performed with the alpha1 beta1 heterodimer. Here we demonstrate for the first time the natural occurrence of the alpha2 subunit on the protein level. The alpha2 subunit co-precipitated with the beta1 subunit from human placenta, showing the existence of the alpha2 beta1 isoform in vivo. The new enzyme was expressed in and purified from cells from the Spodoptera frugiperda ovary cell line Sf 9. Spectral analysis showed that the alpha2 beta1 heterodimer contains a prosthetic haem group revealing the same characteristics as the haem in the alpha1 beta1 form. The kinetic properties of both isoforms and sensitivity towards NO were indistinguishable. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of sGC, abolished NO-stimulated activity of both heterodimers. The new NO-independent activator, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), increased the maximal NO-stimulated activity of the new isoform, caused a leftward-shift in the NO concentration-response curve and turned CO into an effective activator, as it did for the alpha1 beta1 heterodimer (200-fold activation). In summary, the differences in primary structure of both alpha subunits are contrasted by their functional similarity. Further studies will be needed to elucidate the physiological purpose of the new isoform.


Subject(s)
Guanylate Cyclase/metabolism , Isoenzymes/metabolism , Animals , Chromatography, Affinity , Enzyme Inhibitors/pharmacology , Guanylate Cyclase/antagonists & inhibitors , Heme/metabolism , Humans , Indazoles/pharmacology , Isoenzymes/antagonists & inhibitors , Molecular Sequence Data , Nitric Oxide/metabolism , Oxadiazoles/pharmacology , Placenta/enzymology , Polymerase Chain Reaction , Quinoxalines/pharmacology , Solubility , Spodoptera
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