Arterial expression of 5-HT(2B )and 5-HT(1B )receptors during development of DOCA-salt hypertension

BACKGROUND: 5-hydroxytryptamine (5-HT)(2B )and 5-HT(1B )receptors are upregulated in arteries from hypertensive DOCA-salt rats and directly by mineralocorticoids. We hypothesized that increased 5-HT(2B )and 5-HT(1B )receptor density and contractile function would precede increased blood pressure in DOCA-high salt rats. We performed DOCA-salt time course (days 1, 3, 5 and 7) studies using treatment groups of: DOCA-high salt, DOCA-low salt, Sham and Sham-high salt rats. RESULTS: In isolated-tissue baths, DOCA-high salt aorta contracted to the 5-HT(2B )receptor agonist BW723C86 on day 1; Sham aorta did not contract. The 5-HT(1B )receptor agonist CP93129 had no effect in arteries from any group. On days 3, 5 and 7 CP93129 and BW723C86 contracted DOCA-high salt and Sham-high salt aorta; Sham and DOCA-low salt aorta did not respond. Western analysis of DOCA-high salt aortic homogenates revealed increased 5-HT(2B )receptor levels by day 3; 5-HT(1B )receptor density was unchanged. Aortic homogenates from the other groups showed unchanged 5-HT(2B )and 5-HT(1B )receptor levels. CONCLUSION: These data suggest that functional changes of 5-HT(2B )but not 5-HT(1B )receptors may play a role in the development of DOCA-salt hypertension

Drug Delivery: Enabling Technology for Drug Discovery and Development. iPRECIO® Micro Infusion Pump: Programmable, Refillable, and Implantable

Successful drug delivery using implantable pumps may be found in over 12,500 published articles. Their versatility in delivering continuous infusion, intermittent or complex infusion protocols acutely or chronically has made them ubiquitous in drug discovery and basic research. The recent availability of iPRECIO®, a programmable, refillable, and implantable infusion pump has made it possible to carry out quantitative pharmacology (PKPD) in single animals. When combined with specialized catheters, specific administration sites have been selected. When combined with radiotelemetry, the physiologic gold standard, more sensitive and powerful means of detecting drug induced therapeutic, and/or adverse effects has been possible. Numerous application examples are cited from iPRECIO® use in Japan, United States, and Europe with iPRECIO® as an enabling drug delivery device where the refillable and programmability functionality were key benefits. The ability to start/stop drug delivery and to have control periods prior dosing made it possible to have equivalent effects at a much lower dose than previously studied. Five different iPRECIO® applications are described in detail with references to the original work where the implantable, refillable, and programmable benefits are demonstrated with their different end-points

Modification of Proteins by Norepinephrine is Important for Vascular Contraction

Norepinephrine (NE) is thought to mediate its effects through G-protein coupled receptors. However, previous studies have shown that NE and another primary amine, serotonin, also have the ability to exert effects in a receptor-independent manner. We hypothesized that the enzyme transglutaminase II (TG II) has the ability to modify proteins with NE and that this modification is physiologically relevant. As our model we used rat aortic and vena cava tissues, two tissues that depend on NE to modulate vascular tone. Immunohistochemical and immunocytochemical staining showed that NE and TG II are present in smooth muscle cells of these tissues. Western analysis shows aorta and vena cava homogenate proteins are recognized by an antibody raised against NE conjugated to bovine serum albumin (NE-BSA). NE and α-actin colocalize in cultured aorta and vena cava smooth muscle cells. Freshly dissociated smooth muscle cells from these vessels were able to take up NE-biotin. In isolated tissue baths, inhibition of TG II with cystamine (0.5 mM) completely abolished NE-induced contraction in the aorta but only attenuated the receptor-independent contractant KCl (max contraction to 100 mM KCl in cystamine treated = 88.8 ± 7.0% of vehicle treated, p < 0.05). In the vena cava, contraction to NE was abolished with 0.1 mM cystamine and KCl contraction was attenuated (max contraction to 100 mM KCl in cystamine treated = 54.8 ± 7.0% of vehicle treated, p < 0.05). Taken together, these results show that vascular smooth muscle cells take up and utilize NE for the modification of proteins, and that this modification may play an important role in vascular contraction

Integration of Mitogen-Activated Protein Kinase Kinase Activation in Vascular 5-Hydroxytryptamine 2A Receptor Signal Transduction 1

ABSTRACT Vascular 5-Hydroxytryptamine 2A (5-HT 2A ) receptor signaling and contraction has been associated with the activation of L-type calcium channels, phospholipase C (PLC) and, as we previously demonstrated, tyrosine kinase activation. We hypothesize the 5-HT 2A receptor activates all three pathways independently to elicit contraction and that one of the tyrosine kinases activated by 5-HT is mitogen-activated protein kinase kinase (MEK). Endothelium-denuded rat thoracic aorta was mounted into isolated tissue baths for measurement of isometric contractile force. 5-HT, ␣-methyl-5-HT and 2,5-dimethoxy-4-iodoamphetamine all contracted the rat aorta, whereas the 5-HT 2A receptor antagonist ketanserin (30 nM) blocked contraction to 5-HT. The tyrosine kinase inhibitor genistein (5 M) shifted contraction to 5-HT, ␣-methyl-5-HT and DOI ϳ10-fold to the right, whereas daidzein (5 M), the inactive isomer of genistein, was unable to shift 5-HT-induced contraction. PD098059 (10 M), an inhibitor of MEK, shifted contraction to 5-HT ϳ7-fold to the right. We next examined the integration of tyrosine kinase activation in 5-HT 2A receptor signaling. 5-HT-induced contraction was reduced individually by the PLC inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC; 100 M) or the Ca ϩϩ channel inhibitor nifedipine (50 nM); the remaining response to 5-HT was reduced by further addition of either genistein or PD098059. When nifedipine and NCDC were used in combination, a part of the contraction to 5-HT remained; this contraction was further reduced by genistein or PD098059. In cultured aortic smooth muscle cells, 5-HT (0.01-100 M) stimulated tyrosyl-phosphorylation of 42-and 44-kDa proteins identified as Erk MAPKs; this phosphorylation was reduced by PD098059 (10 M). Neither nifedipine nor NCDC reduced 5-HT (1 M)-stimulated Erk MAPK tyrosylphosphorylation, but the combination of nifedipine, NCDC and PD098059 abolished 5-HT (1 M)-stimulated Erk MAPK tyrosyl-phosphorylation. Taken together, these studies indicate that stimulation of a vascular 5-HT 2A receptor activates Ca ϩϩ channels and PLC as well as MEK to cause rat aortic contraction and that MEK activation is at least partially independent of the two pathways classically associated with 5-HT 2A receptors

Inability of Serotonin to Activate the c-Jun N-terminal Kinase and p38 Kinase Pathways in Rat Aortic Vascular Smooth Muscle Cells

BACKGROUND: Serotonin (5-HT, 5-hydroxytryptamine) activates the Extracellular Signal-Regulated Kinase (ERK)/ Mitogen-Activated Protein Kinase (MAPK) pathways, in vascular smooth muscle cells. Parallel MAPK pathways, the c-Jun N-terminal Kinase (JNK) and p38 pathway, are activated by stimulators of the ERK/MAPK pathway. We hypothesized that 5-HT would activate the JNK and p38 pathways in rat vascular smooth muscle cells. RESULTS: Results were determined using standard Western analysis and phosphospecific JNK and p38 antibodies. No significant activation by 5-HT (10(-9) – 10(-5) M; 30 min) of the JNK or p38 pathways, as measured by protein phosphorylation, was observed in any of these experiments. These experiments were repeated in the presence of the serine/threonine phosphatase inhibitor okadaic acid (1 uM) and the tyrosine phosphatase inhibitor sodium orthovanadate (1 uM) to maximize any observable signal. Even under these optimized conditions, no activation of the JNK or p38 pathways by 5-HT was observed. Time course experiments (5-HT 10(-5) M; 5 min, 15 min, 30 min and 60 min) showed no significant activation of JNK after incubation with 5-HT at any time point. However, we detected strong activation of JNK p54 and p46 (5- and 7 fold increases in bands p54 and p46, respectively over control levels) by anisomycin (500 ng/ml, 30 min). Similarly, a JNK activity assay failed to reveal activation of JNK by 5-HT, in contrast to the strong stimulation by anisomycin. CONCLUSION: Collectively, these data support the conclusion that 5-HT does not activate the JNK or p38 pathways in rat vascular smooth muscle cells

Preferential Myosin Heavy Chain Isoform B Expression May Contribute to the Faster Velocity of Contraction in Veins versus Arteries

Smooth muscle myosin heavy chains occur in 2 isoforms, SMA (slow) and SMB (fast). We hypothesized that the SMB isoform is predominant in the faster-contracting rat vena cava compared to thoracic aorta. We compared the time to half maximal contraction in response to a maximal concentration of endothelin-1 (ET-1; 100 nM), potassium chloride (KCl; 100 mM) and norepinephrine (NE; 10 µM). The time to half maximal contraction was shorter in the vena cava compared to aorta (aorta: ET-1 = 235.8 ± 13.8 s, KCl = 140.0 ± 33.3 s, NE = 19.8 ± 2.7 s; vena cava: ET-1 = 121.8 ± 15.6 s, KCl = 49.5 ± 6.7 s, NE = 9.0 ± 3.3 s). Reverse-transcription polymerase chain reaction supported the greater expression of SMB in the vena cava compared to aorta. SMB was expressed to a greater extent than SMA in the vessel wall of the vena cava. Western analysis determined that expression of SMB, relative to total smooth muscle myosin heavy chains, was 12.5 ± 4.9-fold higher in the vena cava compared to aorta, while SMA was 4.9 ± 1.2-fold higher in the aorta than vena cava. Thus, the SMB isoform is the predominant form expressed in rat veins, providing one possible mechanism for the faster response of veins to vasoconstrictors

Cyclooxygenase, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase MAPK, Rho kinase, and Src mediate hydrogen peroxide-induced contraction of rat thoracic aorta and vena cava.

ABSTRACT In hypertension, blood vessels exhibit increased reactive oxygen species production that may alter vascular tone. We previously observed that H 2 O 2 contracted rat thoracic vena cava under resting tone and aorta contracted with KCl. In arteries but not veins, H 2 O 2 -induced contraction required extracellular Ca 2ϩ influx. Because of this difference in Ca 2ϩ utilization, we hypothesized that signaling pathways mediating H 2 O 2 -induced contraction in vena cava under resting tone differed from those mediating did not reduce aortic or venous H 2 O 2 -induced contraction. p38 MAPK, Erk MAPK, and src inhibition did not reduce aortic or venous contraction to the TXA 2 receptor agonist U46619 (9,11-dideoxy-9␣,11␣-methanoepoxy PGF 2␣ , 1 M), whereas rho kinase inhibition significantly reduced aortic and venous contraction to U46619, and PI3-K inhibition reduced venous contraction to U46619. Our data suggest that, in rat thoracic aorta and vena cava, a COX-derived metabolite is one important mediator of H 2 O 2 contraction, possibly via rho kinase activation, and that H 2 O 2 -induced contraction via p38 and Erk MAPK probably occurs independently of TXA 2 receptor activation

Enhancement of sorghum grain yield and nutrition: A role for arbuscular mycorrhizal fungi regardless of soil phosphorus availability

Societal Impact Statement Sorghum is an important cereal crop that provides calories and nutrients for much of the world's population, and it is often grown with low fertiliser input. Optimising the yield, nutritive content and bioavailability of sorghum grain with minimal input is of importance for human nutrition, and arbuscular mycorrhizal (AM) fungi have previously shown potential to assist in this. Across sorghum genetic diversity, AM fungi improved the yield, nutrition and zinc and iron bioavailability of grain in a low phosphorus soil. Thus, food production systems that effectively manage AM fungi may improve consumer outcomes. Summary Sorghum is a C4 cereal crop that is an important source of calories and nutrition across the world, predominantly cultivated and consumed in low- and middle-income countries. Sorghum can be highly colonised by arbuscular mycorrhizal (AM) fungi, and the plant-fungal association can lead to improvements in biomass and nutrient uptake. High-throughput phenotyping allows us to non-destructively interrogate the ‘hidden’ effects of AM fungi on sorghum growth and phenology. Eight genetically diverse sorghum genotypes were grown in a soil amended with 2 or 20 mg P kg−1 and inoculated with an AM fungal culture of Rhizophagus irregularis. High-throughput phenotyping uncovered the ‘hidden’ effects of AM fungi on growth and phenology, while grain biomass, nutrition, Zn and Fe bioavailability and root AM colonisation was determined after destructive harvest. Sorghum plants colonised by AM fungi generally performed better than non-AM control plants, with greater yield, harvest indices, and grain P, Zn and Fe contents. During the early growth stages, AM colonisation led to temporary growth depressions. There were also AM fungal and P fertilisation effects on sorghum time-of-flowering. The sorghum genotype with the highest AM colonisation could barely produce grain when non-inoculated. The two genotypes that failed to mature had very low AM colonisation. Generally, the genetically diverse sorghum genotypes were highly responsive to AM colonisation and produced more grain of greater nutritive quality when colonised, without adverse consequences for micronutrient bioavailability

The chemerin knockout rat reveals chemerin dependence in female, but not male, experimental hypertension

Measures of the adipokine chemerin are elevated in multiple cardiovascular diseases, including hypertension, but little mechanistic work has been done to implicate chemerin as being causative in such diseases. The chemerin knockout (KO) rat was created to test the hypothesis that removal of chemerin would reduce pressure in the normal and hypertensive state. Western analyses confirmed loss of chemerin in the plasma and tissues of the KO vs. wild‐type (WT) rats. Chemerin concentration in plasma and tissues was lower in WT females than in WT males, as determined by Western analysis. Conscious male and female KO rats had modest differences in baseline measures vs. the WT that included systolic, diastolic, mean arterial and pulse pressures, and heart rate, all measured telemetrically. The mineralocorticoid deoxycorticosterone acetate (DOCA) and salt water, combined with uninephrectomy as a hypertensive stimulus, elevated mean and systolic blood pressures of the male KO higher than the male WT. By contrast, all pressures in the female KO were lower than their WT throughout DOCA‐salt treatment. These results revealed an unexpected sex difference in chemerin expression and the ability of chemerin to modify blood pressure in response to a hypertensive challenge.—Watts, S. W., Darios, E. S., Mullick, A. E., Garver, H., Saunders, T. L., Hughes, E. D., Filipiak, W. E., Zeidler, M. G., McMullen, N., Sinal, C. J., Kumar, R. K., Ferland, D. J., Fink, G. D. The chemerin knockout rat reveals chemerin dependence in female, but not male, experimental hypertension. FASEB J. 32, 6596–6614 (2018). www.fasebj.orgPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154357/1/fsb2fj201800479.pd