Cellular uptake of coumarin-6 under microfluidic conditions into HCE-T cells from nanoscale formulations.

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.

Relationship of temperament, growth, carcass characteristics and tenderness in beef steers.

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.

Cloning and functional expression of the rat alpha(2) subunit of soluble guanylyl cyclase.

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.

NO-cGMP signaling molecules in cells of the rat molar dentin-pulp complex.

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.

Expression and tissue localization of soluble guanylyl cyclase in the human placenta using novel antibodies directed against the alpha(2) subunit.

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.

Effect of MgATP on pinacidil-induced displacement of glibenclamide from the sulphonylurea receptor in a pancreatic beta-cell line and rat cerebral cortex.

1. The effects of blockers and openers of K+ channels on binding of [3H]-glibenclamide to microsomes obtained from a pancreatic beta-cell line (HIT-T15) or rat cerebral cortex were examined. 2. The blockers quinine, chlorpromazine and thiopentone and the openers cromakalim [(+/- ) 6-cyano-3,4-dihydro-2,2-dimethyl-trans-4-(2-oxo-1- pyrrolidyl)-2H-benzo[b]pyran-3-ol] and minoxidil sulphate did not significantly interact with the sulphonylurea receptor of HIT-cells both at phosphorylating (presence of MgATP) and dephosphorylating (absence of MgATP) conditions. 3. In the absence of MgATP, pinacidil (200-500 microM) did not significantly displace [3H]-glibenclamide binding to microsomes from HIT-cells. The displacement of [3H]-glibenclamide binding was strongly enhanced by MgATP and was due to a decrease in the number of high affinity binding sites for glibenclamide. 4. MgATP enhanced pinacidil-induced inhibition of [3H]-glibenclamide binding to microsomes from rat cerebral cortex. 5. The effect of MgATP on pinacidil-induced inhibition of [3H]-glibenclamide binding was maintained after solubilization of the membranes from HIT-cells or rat cerebral cortex. 6. It is concluded that the sulphonylurea receptor is regulated not only by sulphonylureas but also by the K+ channel openers, diazoxide and pinacidil, and by protein phosphorylation. The binding sites for sulphonylureas and these K+ channel openers are not identical, but appear to be located at a single protein or at tightly associated proteins.

Expression of nitric oxide-sensitive guanylyl cyclase subunits in human corpus cavernosum.

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.

Soluble guanylyl cyclase is localized at the neuromuscular junction in human skeletal muscle.

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.

Glyceryl trinitrate treatment up-regulates soluble guanylyl cyclase in rat dura mater.

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.

Subunits of soluble guanylyl cyclase in rat and guinea-pig sensory ganglia.

Soluble guanylyl cyclase is a heterodimeric (alpha, beta) enzyme generating the second messenger, cGMP, upon activation by the gaseous messenger, nitric oxide. The occurrence and distribution of alpha 1-, alpha 2-, beta 1- and beta 2-subunits were investigated in trigeminal and dorsal root ganglia on the mRNA and the protein level. Reverse transcription PCR analysis demonstrated mRNA coding for alpha 1-, alpha 2-, and beta 1-subunits in guinea-pig trigeminal and dorsal root ganglia. In agreement with these data, immunoreactivity to the alpha 1-subunit was found in satellite and Schwann cells, while alpha 2-subunit immunoreactivity was localized to axons of large diameter. The distribution of the beta 1-subunit could not be studied on the protein level since the antiserum was ineffective in immunohistochemistry. However, previous studies and the RT-PCR data argue in favour of alpha 1/beta 1-and alpha 2/beta 1-heterodimerization and colocalization. In both species, beta 2-subunit immunoreactivity was confined to neuronal perikarya, primarily of large diameter. Although these results were obtained with two different antibodies directed against different epitopes, the corresponding mRNA could not be detected by RT-PCR analysis. The reason for this discrepancy remains unclear, at present, but could be explained by a variant beta 2- or highly homologous as yet unidentified beta-subunit. This study demonstrates the presence of soluble guanylyl cyclase in sensory ganglia with a differential, cell type-specific distribution of the individual subunits.

Contribution of the renin-angiotensin system to subsensitivity of soluble guanylyl cyclase in TGR(mREN2)27 rats.

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.

The beta 2 subunit of nitric oxide-sensitive guanylyl cyclase is developmentally regulated in rat kidney.

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.

Irreversible inflammation is associated with decreased levels of the alpha1-, beta1-, and alpha2-subunits of sGC in human odontoblasts.

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.