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Richard D Bukoski

Publications and source records attributed to Richard D Bukoski.

9 recordsLinked to original sources
PubMed2006

Adventitial neuronal somata.

Confocal analysis of the whole-mount rat mesenteric branch arteries (MBA) revealed nucleated structures with axonal processes which immunostained for calcitonin gene-related peptide (CGRP). Immunocytochemistry ruled out the possibility that these were immune elements (macrophages and mast or dendritic cells) in close proximity with nerve fibers. To test our hypothesis that beta-CGRP is expressed in the rat MBA, we performed RT-PCR using total RNA isolated from the mesenteric artery arcade and intron spanning primers designed to amplify 188 bp of the beta-CGRP and 333 bp of alpha-CGRP cDNA. The PCR yielded an amplicon of the predicted size which was cloned into the pCR 3.1 vector. DNA sequence analysis of the insert showed 100% homology with the beta-CGRP cDNA, indicating that mRNA encoding beta-CGRP is expressed in the vessel. To learn whether neuronal cell bodies are located in the adventitia of MBA, we performed a limited collagenase digestion of isolated segments and plated the resulting cells in Ham's F12 medium with 10% horse serum on polyornithine-coated cover glasses. The medium was replaced after 48 h with Ham's F12 nutrient mixture containing N2 supplement. This resulted in a mixed population of fibroblasts, a small number of smooth muscle cells and a subset of cells that sprouted axons and immunostained positively for neuronal cell adhesion molecule and CGRP antigens. Fibroblasts and smooth muscle cells did not label with these antibodies. These data demonstrate, for the first time, that a population of adventitial neuronal somata (termed ANNIES), possibly of sensory nerve origin, is located in small mesenteric arteries.

Animals↗
PubMed2004

Endocannabinoids acting at cannabinoid-1 receptors regulate cardiovascular function in hypertension.

BACKGROUND: Endocannabinoids are novel lipid mediators with hypotensive and cardiodepressor activity. Here, we examined the possible role of the endocannabinergic system in cardiovascular regulation in hypertension. METHODS AND RESULTS: In spontaneously hypertensive rats (SHR), cannabinoid-1 receptor (CB1) antagonists increase blood pressure and left ventricular contractile performance. Conversely, preventing the degradation of the endocannabinoid anandamide by an inhibitor of fatty acid amidohydrolase reduces blood pressure, cardiac contractility, and vascular resistance to levels in normotensive rats, and these effects are prevented by CB1 antagonists. Similar changes are observed in 2 additional models of hypertension, whereas in normotensive control rats, the same parameters remain unaffected by any of these treatments. CB1 agonists lower blood pressure much more in SHR than in normotensive Wistar-Kyoto rats, and the expression of CB1 is increased in heart and aortic endothelium of SHR compared with Wistar-Kyoto rats. CONCLUSIONS: We conclude that endocannabinoids tonically suppress cardiac contractility in hypertension and that enhancing the CB1-mediated cardiodepressor and vasodilator effects of endogenous anandamide by blocking its hydrolysis can normalize blood pressure. Targeting the endocannabinoid system offers novel therapeutic strategies in the treatment of hypertension.

Amidohydrolases↗
PubMed2003

Molecular cloning and characterization of a rat sensory nerve Ca2+-sensing receptor.

A full-length cDNA encoding a Ca2+-sensing receptor (CaSR) expressed in rat dorsal root ganglia (DRG) was identified using rapid amplification of 5'-cDNA ends and primer extension and then cloned into the plasmid vector pCR3.1. The DNA sequence of the DRG CaSR was 99.9% homologous with published rat kidney CaSR in the coding region and 247 bp upstream of the start site but showed little homology 5' to this site, which maps to exonic junction I/II, supporting the hypothesis that CaSR message arises as a splice variant and showing tissue-to-tissue heterogeneity. Western blot revealed a doublet of 140 and 160 kDa in a thyroparathyroid preparation and a single 140-kDa band in DRG. Deglycosylation using N-glycanase increased the mobility of CaSR protein from both DRG and thyroparathyroid, whereas endo-H was without effect, indicating that the DGR CaSR is a mature form of the receptor. A DRG CaSR-pEGFP fusion product was constructed, and when transfected into HEK-293 cells, it was distributed at the cell membrane and resulted in extracellular Ca2+ (0.5-3 mM)-evoked increases in intracellular Ca2+, which in some instances exhibited oscillatory behavior. We conclude that DRG CaSR cDNA arises from tissue-specific alternative splicing of a single gene, that the amino acid sequence of DRG CaSR is homologous to other known CaSRs, and that the DRG CaSR undergoes differential posttranslational processing relative to the thyroparathyroid CaSR and is functionally active when transfected into a human-derived cell line.

Animals↗
PubMed2003

Renal interstitial Ca2+ during sodium loading of normotensive and Dahl-salt hypertensive rats.

It is now established that cells in many tissues including renal epithelial cells and perivascular sensory nerves have mechanisms that monitor and respond to the concentration of Ca2+ in the interstitial compartment [Ca2+(ISF)]. We tested the hypothesis that high Na+ intake alters renal [Ca2+(ISF)] and that the response is altered in salt-sensitive hypertensive versus normotensive rats. Male Wistar (W), Dahl salt-resistant (DR), and Dahl salt-sensitive (DS) rats were fed diets containing 0.45% or 8% NaCl for 7 days beginning at 8 to 10 weeks of age. Systolic blood pressure (BP) was measured before and at the end of the 7-day period. During the last 12 h the animals were placed in metabolic cages for urine collection. They were then anesthetized and renal [Ca2+(ISF)] was determined using in situ microdialysis. Feeding 8% NaCl caused a significant increase in systolic BP only in DS. The 8% NaCl also caused a significant increase in renal urinary Na+ excretion in all groups, had no effect on renal Ca2+ excretion in W or DS and significantly increased urinary Ca2+ excretion in DR. When fed 0.45% NaCl, renal [Ca2+(ISF)] was lower in W and DR compared with DS. Feeding 8% NaCl significantly increased [Ca(ISF)] in W, had no effect on this parameter in DR, and significantly decreased [Ca2+(ISF)] in DS ([Ca2+(ISF)] for DS on 0.45% NaCl = 1.89 +/- 0.15 v 8% NaCl = 1.08 +/- 0.07 mmol/L, n = 6 to 12, P <.05). These results indicate that Na+ loading significantly alters renal [Ca2+(ISF)]; that the response of DS is disturbed relative to controls, and are consistent with the hypothesis that Na+ loading can alter cell function by modulating [Ca2+(ISF)].

Animals↗
PubMed2003

Selective ligands and cellular effectors of a G protein-coupled endothelial cannabinoid receptor.

The cannabinoid analog abnormal cannabidiol [abn-cbd; (-)-4-(3-3,4-trans-p-menthadien-[1,8]-yl)-olivetol] does not bind to CB(1) or CB(2) receptors, yet it acts as a full agonist in relaxing rat isolated mesenteric artery segments. Vasorelaxation by abn-cbd is endothelium-dependent, pertussis toxin-sensitive, and is inhibited by the BK(Ca) channel inhibitor charybdotoxin, but not by the nitric-oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester or by the vanilloid VR1 receptor antagonist capsazepine. The cannabidiol analog O-1918 does not bind to CB(1) or CB(2) receptors and does not cause vasorelaxation at concentrations up to 30 microM, but it does cause concentration-dependent (1-30 microM) inhibition of the vasorelaxant effects of abn-cbd and anandamide. In anesthetized mice, O-1918 dose-dependently inhibits the hypotensive effect of abn-cbd but not the hypotensive effect of the CB(1) receptor agonist (-)-11-OH-Delta(9)-tetrahydrocannabinol dimethylheptyl. In human umbilical vein endothelial cells, abn-cbd induces phosphorylation of p42/44 mitogen-activated protein kinase and protein kinase B/Akt, which is inhibited by O-1918, by pertussis toxin or by phosphatidylinositol 3 (PI3) kinase inhibitors. These findings indicate that abn-cbd is a selective agonist and that O-1918 is a selective, silent antagonist of an endothelial "anandamide receptor", which is distinct from CB(1) or CB(2) receptors and is coupled through G(i)/G(o) to the PI3 kinase/Akt signaling pathway.

Animals↗
PubMed2002

Normal perivascular sensory dilator nerve function in arteries of Zucker diabetic fatty rats.

BACKGROUND: Type II diabetes in humans is associated with pathology of both the cardiovascular and peripheral sensory nervous systems. Because abnormal vasodilator responses have been reported in animals of type II diabetes and perivascular sensory nerves are a source of vasodilator substances, we tested the hypothesis that sensory nerve-dependent relaxation is abnormal in arteries of the Zucker diabetic fatty (ZDF) rat model of type II diabetes. METHODS: The ZDF rats and genetic controls were studied at 26 weeks of age. Tail-cuff systolic blood pressure (BP) was measured, serum was obtained for chemical determinations, and mesenteric branch arteries were isolated for wire myograph analysis and confocal-based measurement of calcitonin gene-related peptide (CGRP) positive nerve density. RESULTS: No differences in BP were detected. Serum glucose, triglycerides, and cholesterol were significantly elevated in ZDF. Sensory nerve-dependent vasodilation was assessed by measuring relaxation of phenylephrine preconstricted arterial segments to cumulative addition of divalent calcium ion (Ca2+) or capsaicin. Neither Ca(2+)-nor capsaicin-induced relaxation were different in ZDF versus control (maximal ZDF response to Ca2+ = 64% +/- 2% v 59% +/- 4%; ED50 for Ca2+ = 3.7 +/- 0.5 mmol/L v 3.2 +/- 0.5 mmol/L; n = 5, P = not significant [NS]; maximal ZDF response to capsaicin = 68% +/- 9% v 74% +/- 4%; ZDF ED50 = 3.8 +/- 0.5 nmol/L v 9.8 +/- 7 nmol/L; n = 5, P = NS). In contrast, the maximal relaxation response to acetylcholine was impaired in ZDF (maximal ZDF response = 83% +/- 5% v 94% +/- 2%, n = 4, P = .039; ED50 for acetylcholine = 8.1 +/- 2.9 nmol/L for ZDF v 33.5 +/- 18.2; n = 4 per group, P = .086). The CGRP positive nerve density was not different between groups. CONCLUSIONS: Blood pressure, perivascular sensory nerve CGRP content, and dilator function is normal in the ZDF model of type II diabetes, whereas endothelium-dependent relaxation is impaired.

Acetylcholine↗
PubMed2002

CB(1) receptor antagonist SR141716A inhibits Ca(2+)-induced relaxation in CB(1) receptor-deficient mice.

Mesenteric branch arteries isolated from cannabinoid type 1 receptor knockout (CB(1)(-/-)) mice, their wild-type littermates (CB(1)(+/+) mice), and C57BL/J wild-type mice were studied to test the hypothesis that murine arteries undergo high sensitivity Ca(2+)-induced relaxation that is CB(1) receptor dependent. Confocal microscope analysis of mesenteric branch arteries from wild-type mice showed the presence of Ca(2+) receptor-positive periadventitial nerves. Arterial segments of C57 control mice mounted on wire myographs contracted in response to 5 micromol/L norepinephrine and responded to the cumulative addition of extracellular Ca(2+) with a concentration-dependent relaxation that reached a maximum of 72.0 +/- 6.3% of the prerelaxation tone and had an EC(50) for Ca(2+) of 2.90 +/- 0.54 mmol/L. The relaxation was antagonized by precontraction in buffer containing 100 mmol/L K(+) and by pretreatment with 10 mmol/L tetraethylammonium. Arteries from CB(1)(-/-) and CB(1)(+/+) mice also relaxed in response to extracellular Ca(2+) with no differences being detected between the knockout and their littermate controls. SR141716A, a selective CB(1) antagonist, caused concentration-dependent inhibition of Ca(2+)-induced relaxation in both the knockout and wild-type strains (60% inhibition at 1 micromol/L). O-1918, a cannabidiol analog, had a similar blocking effect in arteries of both wild-type and CB(1)(-/-) mice at 10 micromol/L. In contrast, 1 micromol/L SR144538, a cannabinoid type 2 receptor antagonist, or 50 micromol/L 18alpha-glycyrrhetinic acid, a gap junction blocker, were without effect. SR141716A (1 to 30 micromol/L) was also assessed for nonspecific actions on whole-cell K(+) currents in isolated vascular smooth muscle cells. SR141716A inhibited macroscopic K(+) currents at concentrations higher than those required to inhibit Ca(2+)-induced relaxation, and appeared to have little effect on currents through large conductance Ca(2+)-activated K(+) channels. These data indicate that arteries of the mouse relax in response to cumulative addition of extracellular Ca(2+) in a hyperpolarization-dependent manner and rule out a role for CB(1) or CB(2) receptors in this effect. The possible role of a nonclassical cannabinoid receptor is discussed.

Acetylcholine↗